สััตวแพทย ์มหานครสาร · review of plant dye classification...

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สตวแพทยสตวแพทยมหานครสารมหานครสาร JJOOUURRNNAALL OOFF MMAAHHAANNAAKKOORRNN VVEETTEERRIINNAARRYY MMEEDDIICCIINNEE Available online: www.vet.mut.ac.th/journal_jmvm

การใชสสกดจากพชในการยอมสเนอเยอ

อารยา สบขาเพชร1,# และณฐกาญจน นายมอญ2

1ภาควชากายวภาคศาสตร คณะสตวแพทยศาสตร มหาวทยาลยเทคโนโลยมหานคร กรงเทพฯ 10530

2ภาควชาเทคนคการสตวแพทย คณะเทคนคการสตวแพทย มหาวทยาลยเกษตรศาสตร กรงเทพฯ 10900

บทคดยอ: ปจจบนการศกษาทางจลกายวภาคศาสตรไดมการใชสยอมมากมายหลายชนดเพอยอมเนอเยอตางๆ ซงขนกบวตถประสงคในการศกษา สทใชยอมเนอเยอโดยทวไปมแหลงทมาทงจากแหลงธรรมชาตและจากการสงเคราะห ดวยในปจจบนทวโลกใหความสาคญในการใชสารตางๆ ทไมมผลกระทบตอสขภาพของมนษยและเปนมตรกบสงแวดลอม การใชสธรรมชาตจากพชในการยอมเนอเยอจงไดรบความสนใจมากขน เนองจากสวนตางๆ ของพชนนมสวนประกอบของสอยมากมาย อกทงยงมราคาถกกวาและสามารถใชงานไดอยางยงยนกวาสทไดจากการสงเคราะห รายงานฉบบนไดทวนสรปโดยยอเกยวกบการแบงชนดของสธรรมชาตจากพชในแงของโครงสรางทางเคมของส วธการสกด มอรแดนท และไดยกตวอยางงานวจยทศกษาการใชสสกดจากพชในการยอมเนอเยอชนดตางๆ ซงอาจนามาใชเปนขอมลพนฐานและเปนทางเลอกในการศกษาและพฒนาการยอมสเนอเยอจากสทสกดจากพช

คาสาคญ: สยอมจากพช สยอมจากธรรมชาต การยอม เนอเยอสตว #ผรบผดชอบบทความ สตวแพทยมหานครสาร. 2557. 9(1): 63-78. E-mail address: [email protected]

Araya Suebkhampet and Nattakarn Naimon / J. Mahanakorn Vet. Med. 2014. 9(1): 63-78.

64

Using Dye Plant Extract for Histological Staining

Araya Suebkhampet1,# and Nattakarn Naimon2

1Department of Anatomy, Faculty of Veterinary Medicine, Mahanakorn University of Technology, Bangkok 10530, THAILAND

2Department of Veterinary Technology, Faculty of Veterinary Technology, Kasetsart University, Bangkok 10900, THAILAND

Abstract: Nowadays, many and various kinds of dyes are used for histological staining due to the purpose of study. They are generally from both natural and synthetic origins. With the worldwide concern over the use of human health and eco-friendly materials, the use of natural dyes from plants has gain interested since most of them contain plenty of dye from their parts. They are also cheaper and more sustainable than the synthetic dyes. A brief review of plant dye classification based on their chemical structures of major dye pigments, methods of plant dye extraction, mordants and some of its application in diverse tissues are presented in this report. These would be helpful to further study and development of alternative tissue stains from plant natural dyes for tissue staining.

Keywords: Plant dye, Natural dye, Staining, Animal histology #Corresponding author J. Mahanakorn Vet. Med. 2014. 9(1): 63-78. E-mail address: [email protected]

Introduction

Histology is the study of cells and tissues by using a light microscope. The ability to visualize or identify histological structures is frequently enhanced through the use of histological stains. They give contrast to the tissue as well as highlighting particular features of interest. There are two types of dyes that classified by their origins, synthetic and natural dyes. The natural dyes

are obtained from natural sources such as plants, insects, animals, clays and minerals (Carleton et al., 1976). Plants and insects known to be used in histological staining for animal tissues are Haematoxylon campechianum (logwood), from which haematoxylin stain is obtained and Dactylopius cacti, from which carmine stain is obtained (Egbujo et al., 2008), respectively. The majority of natural dyes


65

are extracted from plant parts such as roots, barks, leaves, berries and seeds and wood.

The dyeing with natural dyes was one of the oldest techniques practiced by the ancient people such as wall paintings in the caves or textile dyeing. The first use of dye in histology credited to Antonie van Leeuwenhoek, the father of microbiology who worked with saffron, a natural dye extracted from saffron crocus. In the mid 1800s, amateur microscopists first used haematoxylin to stain cellular components (Titford M., 2005). Later scientists developed a wide range of haematoxylin staining techniques. The systemically studied dye for histology started in the second half of the 19th century by Weigert C, J Gerlach, P Ehrlich and H Gierke. By then coloring materials were mostly still natural origin like carmine, cochineal, haematoxylin and indigo. Gradually the use of the natural dyes has decreased due to the introduction of synthetic dyes (aniline dye from extract of coal tar) which first invention by William Henry Perkin in 1856. They widely applied in many fields as food, cosmetic and textile industries (Prabhu and Bhute, 2012). They are rapid stain with vast range of new colors, easy to use and commercially available. From then, the synthetic dyes together with those for histological staining have developed.

Many dyes, techniques and procedures are utilized to stain various types of animal tissues. At present, haematoxylin and eosin stain (H&E) is widely used as a light microscopical stain in histology and histopathology. It is combination of natural haematoxylin stain and the synthetic eosin stain. Haematoxylin stains the cell nuclei dark blue while eosin stains cell cytoplasm, most connective tissue fibers and matrices in varying shades of pink and red. Haematoxylin obtained from logwood tree that is native to the Mexico and northern Central America (Baker & Silverton, 1976), but is now mainly cultivated in the West Indies (Bancrot and Gamble, 2008). Haematoxylin can be prepared in numerous ways and applied to tissues from different sites. It has to be oxidized to haematein before using (Culling, 1974). Haematein can be produced from haematoxylin in two ways: natural oxidation and chemical oxidation. For natural oxidation, haematoxylin is exposed to light and air (Ehrlich’s and Delafield’s haematoxylin solution), and for chemical oxidation, sodium iodate (e.g. Mayer’s haematoxylin) or mercuric oxide (e.g. Harris’s haematoxylin) are used. Natural oxidation of haematoxylin seems to retain its staining ability longer than that of the chemical oxidation. However, it takes much longer time for preparation. Haematoxylin has also been


66

prepared synthetically (Morsingh and Robinson, 1970) but the natural dye is mostly used. Haematein is anionic charge, having a poor affinity for tissues without the presence of mordant. A mordant is a substance used to set dyes on tissue sections. The common mordants for haematoxylin are aluminium, iron and tungsten. The dye-mordant complex possesses a net positive charge and enables to bind to anionic tissue regions including nuclear chromatin. Mordant is regularly included in the dyeing protocols when natural dyes are used in order to fix or intensify the stains in cell or tissue staining. It is mainly a polyvalent metal ion that forms coordination complexes with certain dyes which then attaches to the tissues (Llewellyn, 2005). Different kinds of mordants give a different hue and stability of the dye in the tissues.

The use of natural dyes which are cheaper, eco-friendly and biodegradable has become a matter of significant important due to the increased environmental awareness and in order to avoid some hazardous synthetic dyes to humans and animals (Eom, et al., 2001). Several synthetic dyes (e.g. dyes with azo bonds nitro- or amino-groups) cause allergic-like symptoms or are carcinogenic (Ratna and Padhi, 2012). Therefore, alternative natural dyes have been currently more interested for their

potential use in various purposes. It is interesting that over 2000 dyes are synthesized from various parts of plants, of which only about 150 have been commercially exploited (Gulrajani, 1992). They are used for applying to different substrates such as textiles, paper, leather, wood, hair etc. They are also widely used in cosmetic, food and pharmaceutical industries. A number of them own the pharmaceutical properties and have been used in traditional medicine. Curcumin, the bright yellow dye of turmeric has been used traditionally as a remedy for stomach and liver ailments, as well as topically to heal sores and revitalizes the skin (Chaturvedi, 2009). It has anti-oxidant, anti-inflammatory, anti-cancer and anti-septic properties (Chengaiah et al., 2010). Many other common natural dyes such as lycopene (red dye) in tomatoes or lawsone (varies in color from orange to reddish brown) in henna are reported as antimicrobial agents (Siva, 2007; Habbal et al., 2011).

In last 10 years, natural dyes have been studied for their potential use in various fields including histological staining. The present report describes the basic information about of plant dye classification based on their chemical structures, methods of the dye extraction, mordants and some of its application for tissue staining. These would be useful information to the


67

researchers who are searching for the new natural dyes for tissue staining. Classification of natural dyes from plants

Natural dyes can be classified in different ways such as based on their chemical structure, source, hue and method of application, etc. The plant dye classification described in this review is based on their chemical structures which are divided into 7 major groups (Siva, 2007; Samanta and Konar, 2011; Prabhu and Bhute, 2012) as presented in Table1.

Methods of plant dye extraction Various parts of plants such as roots,

stems, leaves, flowers, fruits and seeds are used for dye extraction. Some plants may have more than one color depending on which part of the plant one uses. The color yield and shade of the color a plant produces vary according to time of the year the plant is picked, how it is grown, soil conditions, etc. Before extraction, the parts of plants are collected and generally shade dried in air or sun dried. Then the grinding is carried out to break down the material into very small pieces or powder using the manual or electric grinding machines. Optimum conditions of extraction are determined by varying extraction parameters such as type of solvents, time of extraction, ratio between plant material and solvent,

temperature and pH which depend on the properties of particular dye components (Prabhu and Bhute, 2012). After extraction, the extracts are generally filtered through various filters such as cheesecloth, cotton wool or paper filter. The filtrates may be freshly used (normally aqueous extract) or further evaporated of solvent, washing and drying to get purified dye. There are mainly four methods used in extraction of natural dyes (Samanta and Konar, 2011). 1) Aqueous extraction

This method is has long been used for natural dye extraction for a certain of time. The dye components in dried plant powder are extracted in water at boil or particular temperature. Then, the extract is cooled down and filtered. The dye solution is carried out under varying conditions as mentioned above in order to get the optimal extraction condition. The optimum condition is determined by studying the optical density value at definite wavelength for the extracted solution using UV-Visible absorbance spectrophotometer (Samanta and Konar, 2011). The filtrate is further applied for dyeing. Here are some studies that used the aqueous extract from plants for animal tissue staining. Natural dyes were extracted from kujarat flowers (Hibiscus sadariffa) in aqueous medium and were used to replace of eosin stain in kidney from

Araya

68

Tabl

e 1

Clas

sifica

tion

of p

lant

dye

s

a Suebkham

Exam

ple

of p

lant

s:

part

used

Indi

gofe

ratin

ctor

ia

Rang

e of

colo

r

Blue

Chem

ical s

truct

ures

Ba

sic in

form

atio

n

Indi

gois

the

olde

stdy

eus

edfo

rtex

tile

dyein

gA

varie

ty

Maj

or g

roup

s

Indi

goid

dyes

mpet and N

Indi

gofe

ra ti

ncto

ria:

leav

es

Indi

gofe

ra

Blue

In

digo

is th

e ol

dest

dye

used

for t

extil

e dy

eing.

A va

riety

of p

lant

s ha

ve p

rovid

ed in

digo

, but

mos

t na

tura

l ind

igo

was

obta

ined

from

tho

se in

the

gen

us In

digo

fera

whi

ch

Indi

goid

dye

s

attakarn Na

suffr

utico

sa: l

eave

s

In

digo

nativ

e in

tro

pica

l ar

eas.

At p

rese

nt,

alm

ost

all

indi

go

prod

uced

is sy

nthe

tic. It

is th

e bl

ue o

f blu

e jea

ns.

aimon / J. M

g

Mahanakorn

Rubi

a tin

ctor

ium

(mad

der):

wood

Red

Anth

raqu

inon

e dy

es a

re fu

sed

thre

e be

nzan

oid

rings

that

poss

ess

conj

ugat

ion

toac

hiev

eco

lor

Thes

edy

esar

e

Anth

raqu

inon

e

dyes

Vet. Med. 2

(mad

der):

woo

d

Rubi

a co

rdifo

li: wo

od

Galiu

m v

erum

(Lad

y's

9,1

0-An

thra

quin

one

poss

ess

conj

ugat

ion

to a

chiev

e co

lor.

Thes

e dy

es a

re

char

acte

rized

by

good

ligh

t fa

stnes

s. Al

mos

t al

l the

red

natu

ral

dyes

are

bas

ed o

n th

e an

thra

quin

oid

struc

ture

.

dyes

2014. 9(1): 6

Beds

traw)

: flo

wers

They

are

mor

dant

dye

s. Th

e co

chin

eal,

an e

xam

ple

of

know

n an

thra

quin

one

dye

from

in

sect

s (D

acty

lopi

us

cocc

us)

isus

edto

prep

are

the

carm

inedy

e

63-78.

cocc

us),

is us

ed to

pre

pare

the

carm

ine d

ye.

Araya

Tabl

e 1

Clas

sifica

tion

of p

lant

dye

s (Co

ntin

ued)

a Suebkham

Exam

ple

of p

lant

s:

part

used

Laws

onia

iner

mis

Rang

e of

colo

r

Oran

ge

Chem

ical s

truct

ures

Ba

sic in

form

atio

n

Laws

one

(hen

na)

the

mos

tpop

ular

dye

ofth

isgro

upis

Maj

or g

roup

s

Alph

ahy

drox

y

mpet and N

Laws

onia

iner

mis

(hen

na):

leav

es

Jugla

ns n

igra

(bla

ck

Oran

ge

Brow

nish

-

blac

k

Laws

one

Laws

one

(hen

na),

the

mos

t pop

ular

dye

of t

his g

roup

, is

used

to

dye

skin

, fin

gern

ails,

hair,

wool

and

lea

ther

.

Henn

a sta

ins a

re o

rang

e af

ter a

pplic

atio

n bu

t dar

ken

over

Alph

a-hy

drox

y-

napt

hoqu

inon

es

attakarn Na

waln

ut):

shel

ls of

waln

ut fr

uits

Laws

one

the

follo

wing

few

day

s to

a r

eddi

sh b

rown

. Jug

lone

is

anot

her e

xam

ple

of th

ese

dyes

that

has

bee

n us

ed a

s a

natu

rald

yefo

rclo

thin

gand

fabr

icsan

das

ink

aimon / J. M

Tage

tess

pecie

sYe

llow

J

l

natu

ral d

ye fo

r clo

thin

g and

fabr

ics a

nd a

s ink

.

Flav

onoi

dsar

epo

lyph

enol

icco

mpo

unds

that

are

Flav

onoi

ds

Mahanakorn

Tage

tes s

pecie

s

(mar

igold

): flo

wers

Term

inal

ia c

hebu

la

Yello

w Fl

avon

oids

ar

e po

lyph

enol

ic co

mpo

unds

th

at

are

abun

dant

in

plan

ts an

d ar

e ca

tego

rized

, ac

cord

ing

to

chem

ical

struc

ture

, int

o fla

vono

ls, f

lavo

nes,

flava

none

s,

Flav

onoi

ds

Vet. Med. 2

(Myr

obal

an):

leav

es

Cotin

us c

oggy

gria:

wood

Fl

avon

e iso

flavo

nes,

cate

chin

s, an

thoc

yani

dins

and

cha

lcone

s.

Mos

t of

the

nat

ural

yel

low

colo

rs ar

e hy

drox

y an

d

met

hoxy

deriv

ative

soff

lavo

nesa

ndiso

flavo

nes

2014. 9(1): 6

wood

met

hoxy

der

ivativ

es o

f fla

vone

s and

isof

lavo

nes.

63-78.

69

Isofla

vone

Araya

70

Tabl

e 1

Clas

sifica

tion

of p

lant

dye

s (Co

ntin

ued)

a Suebkham

Exam

ple

of p

lant

s:

part

used

Haem

atox

ylon

Rang

e of

colo

r

Rang

e

Chem

ical s

truct

ures

Ba

sic in

form

atio

n

Dihy

drop

yran

sar

eclo

sely

rela

ted

inch

emica

lstr

uctu

re

Maj

or g

roup

s

Dihy

drop

yran

s

mpet and N

Haem

atox

ylon

cam

pech

ianu

m

(logw

ood)

: woo

d

Rang

e

from

red

to v

iole

t

Dihy

drop

yran

s ar

e clo

sely

rel

ated

in c

hem

ical

struc

ture

to t

he fl

avon

es. T

he e

xam

ple

of k

nown

dih

ydro

pyra

n is

haem

atein

wh

ich

is an

ox

idize

d de

rivat

ive

of

Dihy

drop

yran

s

attakarn Na

to b

lue

Ha

emat

ine

haem

atox

ylin,

use

in

histo

logic

al s

tain

ing.

Haem

atein

exhi

bits

indi

cato

r-like

pro

perti

es, b

eing

blue

in a

queo

us

alka

line

cond

itions

and

red

inac

idic

cond

itions

They

are

aimon / J. M

Anth

ocya

nin:

Rang

e

al

kalin

e co

nditio

ns, a

nd re

d in

acid

ic co

nditio

ns. T

hey

are

mor

dant

dye

s.

Anth

ocya

nidi

nsar

em

embe

rof

the

flavo

noid

sco

mm

only

Anth

ocya

nani

Mahanakorn

Anth

ocya

nin:

Vacc

iniu

m m

yrtil

lus:

berri

es

Rang

e

from

red

to v

iole

t

Anth

ocya

nidi

ns a

re m

embe

r of

the

fla

vono

ids

com

mon

ly

foun

d in

plan

ts.

They

ar

e gly

cosid

es

of

2-

phen

ylch

rom

enyl

ium

salts

wh

ich

are

wate

r so

lubl

e

Anth

ocya

nani

dins

:

Vet. Med. 2

Cara

jurin

:

- Bign

onia

chica

:

to b

lue

2-

Phen

ylch

rom

eniu

m

mol

ecul

es. T

he v

ariat

ion

in th

e co

lors

is at

tribu

ted

to se

vera

l

fact

ors

as t

he n

umbe

r of

hyd

roxy

l gro

ups,

the

degre

e of

met

hoxy

latio

n an

d th

e nu

mbe

r of

m

onos

acch

arid

es

2014. 9(1): 6

Bign

onia

chic

a:

leav

es

atta

ched

to th

e ca

tion.

The

ir co

lor c

hang

es w

ith p

H, p

ink

or

red

in a

cidic

solu

tions

, pur

ple

in n

eutra

l sol

utio

ns, b

lue

to

green

ish-y

ello

win

alka

line

solu

tions

The

mem

ber

of

63-78.

An

thoc

yani

n gre

enish

yello

w in

al

kalin

e so

lutio

ns.

The

mem

ber

of

anth

ocya

nidi

nes

inclu

des

anth

ocya

nins

(ant

hocy

anid

ins

with

suga

r), c

araju

rin a

nd d

elph

inid

ine.


72

albino mice (Hashim, 2006). Suebkhumpet and Sotthibandhu (2012) used aqueous extract of butterfly pea flowers (Clitoria ternatea) to stain animal peripheral blood smears. 2) Extraction by non-aqueous and other solvent assisted system

The solvents generally used for plant dye extraction are ethanol, methanol, acetone, chloroform, ether, clove oil, etc. The dried material powder is weighed and soaked in solvent in different percentages and time durations. The crude dye extract can be used for tissue staining after extraction or can be further applied for solvent evaporation in order to concentrate the dye solution before staining. Alternatively, the dried plant powder is soaked in solvent to allow effective percolation, then the soaked powder is extracted in the solvent using Soxhlet Extractor (Steam Heated Extractor). The extract is then concentrated using rotary evaporator and may further drying in the drying oven (Okolie. 2008). Finally, the extract is obtained in powdered form which will be dissolved in the solvent or buffer at the desire concentrations before tissue staining.

Supercritical fluid extraction (SFE), an alternative to conventional solvent extraction for separation of organic

compound in many analytical processes as well as extraction of plant natural dye has gained wide acceptance in recent years since this technique is safe for health, inexpensive and harmonize with nature. SFE uses carbon dioxide as a solvent. This method is based on the enhanced solvating power of gases above their critical point (Samanta and Konar, 2011). Cardoni et al. (2000) studied the extraction of lycopene

and β-carotene from ripe tomatoes using SFE. The detail information and procedure of this method has been reported by Sapkale et al. (2010).

3) Extraction by acid or alkaline assisted system This method adjusts the pH of the extraction by adding hydrochloric acid or sodium carbonate in aqueous medium. The different pH in extraction protocols may give differently results as percent yield and hue of the extracted dye which due to the reaction of the chemical structure of the dye to the pH variation. Samanta et al, 2007 studied the extraction of color from jackfruit wood under various pH conditions (ranged between pH 4-12) and reported that the optimum condition for the extraction is at pH 11.0. 4) Extraction by other methods

4.1) Ultrasound assisted extraction


73

This method is carried out by mixing dried and ground plant materials in solvent in a flask, which was then placed in an ultrasonic bath. The ultrasound applied for the extraction results in intermolecular tearing and surface scrubbing, causing plant tissue rupture and improving the release of intracellular substances into the solvent. The extraction is repeated two-three times before the extract is collected. Sivakumar et al. (2009) used ultrasound with 80W ultrasonic power for 3h contact time assisted enhancement in natural dye extraction from beetroot for industrial applications and natural dyeing of leather.

4.2) Enzyme assisted extraction This method uses enzymes (pectinase,

cellulose, protease, esterase etc.) for dye extraction. The enzymes are usually applied as a pre-treatment of plant materials before subjecting the plant material to solvent extraction. Various enzyme combinations are used to loosen the structural integrity of plant materials thereby enhancing the extraction of the desired color components. Conditions for optimum activity and selection of the right type of enzymes are essential to use them effectively for extraction (Sowbhagya and Chitra, 2010). Ultrasound assisted and enzyme assisted extraction can be used in combination in order to improve the extraction efficiency.

Mordants Natural dyes are mostly non-

substantive and must be applied by adding mordants which after combining with dye in the materials including the tissues, it forms an insoluble precipitate or lake and thus both the dye and mordant get fixed to become wash and light fast. Mordants are also often added to keep dyes from fading, or to brighten, deepen, or dull a color. They can be used before, during or after the dye bath due to the desired effect. There are different kinds of mordant as metallic mordants, tannin and oil mordants that have been generally used for fabric dyeing by natural dye for a long time. Similarly, they are also added in the histological staining protocols when the plant dyes are used (Al Tikritti and Walker, 1977; Avwioro et al., 2007). The metallic mordants are common used for tissue staining.

Metalic mordants are polyvalent metal ions such as metal salts of aluminium, potassium, chromium, iron, copper and tin. The two most common metals used in natural dyeing are aluminium and ferric ions that having valences of three. Two types of bonds are involved in the reaction between a mordant dye and a mordant. One is covalent bond and the other is coordinate bond. Different mordants give different hue colors with the same dye. Metalic mordants are divided into two types as brightening


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and dulling mordants. Alum (potassium aluminum sulfate), potassium dichromate and stannous chloride are brightening mordants while copper sulphate and ferrous sulfate are dulling mordants Prabhu and Bhute, 2012). Although metallic mordants are effective, they are relatively toxic and environmentally pollutants. Thus, the natural mordants are considered. The sample of natural mordant is tannin which found in plant parts as Tara pods (Caesalpinia spinosa) and gallnuts from Rhus semialata. Ali et al. (2010) studied the effect of tannic acid and metallic mordants on the dyeing properties of natural dye extracted from Acacia nilotica bark.

Discussion and Conclusion The natural dyes have long been used

in various purposes including tissue staining. However, their application has reduced since the synthetic dyes were developed. Haematoxylin is an example of natural dye that widely used in histology, histopathology and histochemistry. At present, many commercial synthetic and some natural dyes used for tissue staining are available in the markets. However, the hazardous effects of synthetic dyes on human and environment caused scientists to concern about using the natural dyes instead of synthetic dyes. Therefore, alternative natural dyes have been studied for their potential use in

histological staining. There are many studies that investigated plant dye staining in diverse tissues as follow. The dried skin of pomegranate (Punica Granatum) fruit extract was used for rat brain staining and the yellow staining was detected in the astrocytes, neurons, red blood cells and elastic fibers (Gharravi et al., 2006). Bassey et al. (2011) stained the sections of the testes with the ethanolic extract of Curcuma longa. They used this extract as a counterstain for haematoxylin. Their results indicated that the dye distinctly stained the seminiferous epithelium and interstitium yellow that provided a good counter stain for haematoxylin. Kola nut (cola acuminata) extract was also used as a substitute to histological tissue stain eosin in rat tissues. The dye extract was detected in the cytoplasm of various tissues with yellow-brown color (Shehu et al., 2012). Suabjakyong et al. (2011) investigated the extraction of dye from black plum fruit (Syzygium cumini) using various solvents and its staining property on the rat hepatic tissue.

Suebkhumpet and Sotthibandhu (2012) used aqueous extract of butterfly pea flowers (Clitoria ternatea) to stain blood cells in different animal peripheral blood smears. They presented that the dye extract could stain and differentiate blood cells on the blood smears. Jan et al. (2011) studied


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the staining effect of dry extracted from dry leaves of henna (Lawsonia intermis) on histological sections of angiospermic stem. Okolie (2008) studied the staining of ova of intestinal parasites with extracts of Hibiscus Sabdariffa and Azadirachta Indica. The plant extract produced satisfactory staining in comparison to conventional methods used in identifying intestinal parasites on a preparation of stool sample. Ihuma et al. (2012) used methanolic extracts from Hisbiscus sabdariffa as a biological staining agent for some fungal species. Their results suggested that the dye extract could be use as a mycological stain. Al-Amura et al. (2012) studied the staining technique for helminthes by using red beet (Beta vulgaris) extract. They reported that stained helminthes were acquired a good stain with distinction their internal structures. Tousson and AL-Behbehani (2011) used black mulberries as a natural dye for tissue staining.

Although using natural dyes have many advantages, there still be some limitations that should be concerned. They are difficult to standardize the dye and its application because dyes collected from similar plants or natural sources vary due to climate, soil, maturity period and cultivation methods etc. Moreover, most natural dyes require mordants for fixing them on the stained tissues. The widely used metallic

mordants that applied in the dye solutions may cause health and disposal problems. Therefore, searching for the new natural mordants is an option for safer tissue staining. Many factors should be considered, if the natural dyes are used for tissue staining. One of them is the source of plants selected, it should be available in the endemic region in order to reduce the cost and to avoid raw materials insufficiency. The known chemical structures and properties of some plant dye extracts are also important and useful for determining the optimal extraction and tissue staining procedures. More detailed studies are needed to evaluate the potential and availability of natural dyes-yielding resources in many others plants. In addition, biotechnology and various fields of studies are required to increase the quantity and improve quality of plant natural dyes. More study about staining procedures of each dye plant still be required to establish new permanent preparations in the future.

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