Local Dye-Hibiscus Sabdariffa Good As Haematoxylin Substitute For Nuclear Staining In Brain Tissues

Another breakthrough was recorded by Nigerian Histology researchers recently in their quest to find local alternatives to expensive, harmful and environmentally hostile imported chemicals and reagents used in the histology laboratories across Sub-Saharan Africa.
The title of the publication as reported by the African Journal Of Cellular Pathology is 'Hisbiscus Sabdariffa As Haematoxylin Substitute In Histological Demonstration Of Brain Tissues'.
The work authored by Benard Solomon  of the Histopathology Laboratory, Unilorin Teaching Hospital and other reputable researchers such as Muhammed A.O., of the Histopathology Department, usmanu danfodiyo University, Sokoto, Afolabi OO of the Department of Pathology, College of Health Sciences, University of Ilorin, Fowotade AA and Olutunde OA both of the Histopathology Laboratory, Unilorin Teaching Hospital, Kwara State, Nigeria examined the posibility of the dye extract substituting for heamatoxylin in nuclear staining of brain tissues.  The outcome was revolutionary and impactful as it may be the ultimate solution to the high cost of importing foreign reagents for use in the histopathology laboratory as it concerns the widely used haematoxylin.

While introducing the work, the authors submitted that 'Hibiscus sadariffa belong to the family
Malvaceae, which is commonly called roselle.  Roselle is cultivated in India, Malaysia, Tropics,
subtropics and Central America (Roselle, 1987,Durance et al., 1999). The plant is widely
grown in Nigeria and other sub-Saharan African countries as a crop used for demarcation of farm
plots. The aqueous extract of the dry red calyx is often prepared as a drink for refreshment
locally called ‘zobo’. Other uses of the calyces include its being an edible vegetable among the
Yoruba ethnic group of South-West, Nigeria and a natural food colorant. (Aballa & Siddig 1993:
Adegunloye et al., 1996). Egyptians use to drink hibiscus extract and call it karkadae, while in
Iraq, it is called red tea. Studies in Iraq suggest using of hibiscus extract in food industry, using
it as a syrup and coloring agent (Alzubaidi,1977). Hibiscus was found as a natural source
of pectin, which solidify jelly and ice cream preparation (Ali, 2000). Hashim, (2006) revealed in his work that Hibiscus sabdariffa is called kujarat flower in Iran. Hibiscus calyces
contain per 100gm of edible portion, calcium (1.263mg), niacin (3.765mg), riboflavin
(0.277mg) and iron (8.98mg) (Durance et al.,1999,. Chemical analysis in Iraq, (Muller et al.,
1992) of kujarat reported their values of 100m of Ca and 9.55mg of Fe per mg of dry matter.
Three water-soluble ploysaccharids have been isolated from flower buds of Hibiscus sabdariffa
(HIB, 1.2.3)(Muller et al., 1992). Medical uses of these flowers are wide. Infusion of calyces
are regarded as diuretic, choleretic, febrifugal, hypotensive, decreasing the viscosity of blood
and stimulating intestinal peristalsis (Roselle,1987). Other medical study proposed its effect
in protection from induced cytotoxicity and genoetoxicity by different mechanisms (Tesng
et al., 1996). Other researchers proposed its use as a natural stain. They used extract to stain blood film, fungi and plant tissue (Al –Sarraj et al., 1997: Al-Sarraj, 1997). It was also used as a
natural histological cytoplasmic stain (Hashim, 2006, and Abd-Alhafeez Ibnouf et al., 2014).
Dried calyces of Hibiscus have dark red pigment. Chemical components of this pigment
were reported in previous studies (Hashim, 2006). It contains flavonoids gossypetine,
hibiscetine and sabdaretine. The major pigment reported as hibiscin that was identified as
daphniphylline. Small amount of delphinidine 3- monoglucosides, cyanidin 3-monoglucosides
(chrysanthenin) and delphinidine are also present (Roselle, 1987). The use of hibiscus
extract of dry Roselle calyx in the histological demonstrations of nuclear staining in lymph
node, appendix, testis and renal tissues has already been reported (Benard, 2008, Egbujo et
al., 2008, Abd-Alhafeez Ibnouf et al., 2014). However, the nuclear staining effect of the
extract on brain tissue has yet to be known hence the justification for this study.'

Detailing the materials and methods used, the authors submitted that 'Dry, red calyces of H. sabdariffa were obtained at a local market in Ilorin, the capital of Kwara State in the North-Central region of Nigeria. They were subsequently ground using a Binatone blender to a fairly powdery form. To 10g of the ground red calyces of H. sabdariffa in a conical flask, 200mls of distilled water was
added and brought to boil to give the brilliant red colored extract which was immediately
allowed to cool and filtered to give a clear H. sabdariffa extract. The staining formula was
compounded as follows:
H. sabdariffa extract 100ml
NaCl 5.0g
10% ferric chloride solution 1.2ml
Glacial acetic acid 3.0ml
Tissue Preparation and Staining
10% formol saline fixed, paraffin embedded
tissues from the cerebellum, cerebrum, and pons
were sectioned at 4μm and stained with H&E
and Hibiscus/Eosin techniques. They were
subsequently dehydrated, cleared and mounted
with DPX. Photomicrographs of the sections
were also taken.'

Subsequently, they reported that the work resulted into the following ' The nucleus of the brain tissues was satisfactorily stained when compared with the standard haematoxylin and eosin control
sections. Nucleus stain dark-violet while red blood cells and cytoplasmic components stain light- pink.'

In their discussion, the authors stated that 'For long, histopathology laboratory resource
managers in developing countries has depended on expensive imported reagents for use in their
laboratories most especially haematoxylin. The loss of foreign currency exchange has been
much more pronounced in the economies of these countries over the past few decades due to
economic downturn leading to increased high cost of these reagents. The aforementioned
scenario has made the search for local alternatives to haematoxylin as nuclear stain a
compelling necessity. Earlier on, the use of extract of hibiscus sabdariffa (Iron-Roselle,) has
shown promising outcomes for nuclear staining (Benard, 2008, Egbujo et al., 2008). Aqueous
extract of H. sabdariffa was also used to stain mice tissues as substitute to eosin as a
cytoplasmic stain ( Hashim, 2006). Abd-Alhafeez and his colleagues, (Abd-Alhafeez
Ibnouf et al., 2014) also reported the use of pure aqueous extract of H. sabdariffa for the staining
of cytoplasmic contents as eosin substitute on formalin fixed, paraffin embedded renal tissues.
However, to the author’s knowledge, this study represents the first initiative of using H.
sabdariffa aqueous extract solution as nuclear
stain for brain tissues. Results show satisfactory staining of brain tissues with the Hibiscus/Eosin
method. The nucleus is stained dark-violet while the cytoplasm is stained light- pink. Red
blood cells stain pink-red. The nuclear staining obtained from the staining of cerebellum,
cerebrum, and pons of the brain shows similar results comparable with standard haematoxylin
and eosin to the extent that an uninformed observer will think that it is H and E. (Fig I&II).
Figure Ia, Ib and Ic show H& E staining of cerebrum, cerebellum and pons respectively
with nucleus staining purple, cytoplasm staining pink and red blood cells, red. Figure IIa, IIb and
IIc show Hibiscus/Eosin staining of cerebrum, cerebellum and pons respectively with nucleus
staining dark-violet, cytoplasm staining pink and red blood cells, pink. However, the lightpink
staining of the cytoplasm in the Hibiscus/Eosin technique is in contrast with the
intense pink staining seen in the standard H&E. This could be due to the blockage of reactive
sites in the cytoplasm due to the presence of acetic acid in the Hibiscus solution. This is in
line with an earlier observation made by Egbujo et al., (2008). It is worth of note that the Hibiscus extract formular used for this work has been used satisfactorily by Benard (2008) for lymph node and appendix with optimum staining achieved in 5 minutes. This has further established Iron-Roselle (Hibiscus extract solution) as a progressive nuclear stain. In addition, the staining time in Iron-Roselle is shorter compared to haematoxylin hence could be a rapid stain for general demonstration of structure of the brain. The distinct nuclear clarity and preservation of the histomorphology
of the brain is particularly impressive. Unlike as done with the haematoxylin staining,
differentiation in 1% acid alcohol is not necessary in the Hibiscus extract stained
sections. The Hibiscus extract /eosin technique shows great promise as a new technique in
histology for the demonstration of nuclear components in brain tissues. H. sabdariffa is natural, locally available, cheap, easy to prepare, safe and resistant to fading. The wider application of the H. sabdariffa solution in the demonstration of general tissue structures in combination with other dyes would be of great interest to researchers especially in the histological diagnoses of diseases.'

Conclusively, the researchers agreed that 'Hibiscus sabdariffa extract solution (ironroselle)
is suitable as haematoxylin substitute for the nuclear staining of brain tissues.'