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Abstract

The application of natural dyes for staining of various biological tissues from an alternative source will decrease the expense for purchasing the synthetic dye and reduce their effects on human and environment. Therefore, the objective of this study was to investigate the extraction of natural dye from Cola nut (Cola acuminata) using various solvents and its staining property on the rat tissues. The cola nut was pulvized using pestle and mortar, 5gram was used to make 5% of aqueous extract. The rat tissues were processed for paraffin embedding technique and sectioned at 5 μm thicknesses. The sections were stained with haematoxylin and the extracts as secondary stain. The results showed that the natural extract from Cola acuminata stained the cytoplasm of various tissues with yellowish-brown colouration. This finding suggests that Cola acuminata can be used as an alternative dye for histological staining.
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Scientific Journal of Veterinary Advances (2012) 1(2) 33-37
Kola nut (cola acuminata) extract as a substitute to histological tissue stain
eosin
S.A. Shehua, M.L. Sonfadaa, A. Danmaigoroa, A.A. Umara, S.A. Henaa, I.M. Wiamb
aDepartment of Veterinary Anatomy, Usmanu Danfodiyo University, Sokoto
bDepartment of Veterinary Anatomy, University of Maiduguri, Maiduguri
*Corresponding author; Department of Veterinary Anatomy, Usmanu Danfodiyo University, Sokoto
A R T I C L E I N F O
Article history:
Received 14 July 2012
Accepted 19 July 2012
Available online 28 August 2012
Keywords:
Kolanut extract
Substitute
Histological tissue
Eosin
A B S T R A C T
The application of natural dyes for staining of various biological
tissues from an alternative source will decrease the expense for
purchasing the synthetic dye and reduce their effects on human and
environment. Therefore, the objective of this study was to
investigate the extraction of natural dye from Cola nut (Cola
acuminata) using various solvents and its staining property on the rat
tissues. The cola nut was pulvized using pestle and mortar, 5gram
was used to make 5% of aqueous extract. The rat tissues were
processed for paraffin embedding technique and sectioned at 5 μm
thicknesses. The sections were stained with haematoxylin and the
extracts as secondary stain. The results showed that the natural
extract from Cola acuminata stained the cytoplasm of various tissues
with yellowish-brown colouration. This finding suggests that Cola
acuminata can be used as an alternative dye for histological staining.
© 2012 Sjournals. All rights reserved.
1. Introduction
In histology, there are two types of dyes, Natural dyes obtained from natural sources and Synthetic dyes
produced through chemical reactions (Avwioro, et al., 2005; Carleton, et al., 1976).The synthetic dyes are very
efficient, but they are hazardous to human and animal health (Bhuyan, et al., 2004). Thus, the searching of new
natural dyes for histological staining that are eco-friendly and biodegradable necessary.
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Original article
S.A. Shehu et al. / Scientific Journal of Veterinary Advances (2012) 1(2) 33-37
34
Cola is a genus of about 125 species of trees, native to the tropical rainforest of Africa (Benjamin, 1991).
There are several kinds of cola seeds derived from different species, but Cola vera and Cola acuminata are mostly
used and preferred for medicinal purposes (Ayensu, 1978). The seeds are chewed to curb hunger, allay thirst, and
enable people to work hard in hot conditions (Ayensu, 1978). Kola Nut seeds are considered a symbol of hospitality
and used in many social ceremonies such as marriage, birth and funerals. It was observed that those engage in
constant kola nut chewing have their gums and teeth stained (Cheek, 2002). However, staining with Cola
acuminata has not been investigated. Recent move to make use of local natural resources to minimize total
dependence on foreign, imported products in order to improve economic values of our local material has been
made. Positive result has been documented from the use of Lawsiana inermis and Hibiscus sabdoriffa to stain
histological tissue (Wiam, et al., 2006). Synthetic dyes are increasingly becoming expensive to our laboratories
where funding is limited. It is for these reasons that an alternative natural dye which is cheaper and bio-friendly is
been sort as potential natural histological dye.
2. Material and methods
The plant sample C. acuminata was purchased from Sokoto Central Market and was identified at the
herbarium of Department of Biological sciences, Faculty of Science, Usmanu Danfodiyo University Sokoto. The
Kola nut seed was mashed using perforated empty tin can and pulverized using Mortar and pestle. Five (5) grams
of the powder was weighed using Mettler’s balance and mixed in 100ml of distilled water and the extract was
kept for 24 hours, which was later sieved with cotton wool followed by a folder filter paper placed in a glass
funnel and Conical flask. The filtrate was labeled 5% aqueous extract of the C. acuminata.
2.1. pH Determination
Digital BMF/pH meter (England) was used for determination of pH value of dye.
2.2. Histological investigation
One apparently healthy rat was sacrificed and organs (Intestine, Liver, Kidney, Cardiac muscle and Lungs)
were removed and fixed in 10% buffered formalin for 72 hours to fix properly and processed for paraffin
embedding technique (Bancroft, 2002) and sectioned at 5 μm thickness. The technique for normal Haematoxylin
and Eosin (H & E) was followed.
The tissue slides were grouped and are deep in the two (2) different prepared solutions which are as
follows: A: 5% aqueous extract of C. acuminata, B: Eosin, which serve as the control group and lastly the
mounted tissues were covered with cover slip using Canada balsam mountant.
The tissues were placed under a light microscope with x400 magnification and image of the tissue were
capture using a motic camera (Moticam 1000, 1.3 mega Pixel) and the photomicrograph were transferred to the
computer. The quality and stability of C. acuminata extract were examined for the intensity of nucleus and
cytoplasm staining.
3. Results and discussion
The pH of the aqueous extract of the Cola acuminata was 5.93 making it acidic. Giving by the theory of
neutralization reaction, acidic dyes stain cytoplasm while the basic dye, stain the neuleus, the pH of the extract
implies that the substance is an acidic, thus it choice of cytoplasmic stain. As shown on plates i x, the aqueous
extract has demonstrate the property of neutralization reaction.
In this research, Haematoxylin and aqueous C. auminata extracts stained the nucleus and cytoplasm of tissues with
bluish and yellowish coloration respectively. The intensity of their staining was not significantly different from
those stained with haematoxylin and eosin as shown in plate i - x
The results suggest that the dye from C. acuminata can be extracted by either acidic or neutral solvents, the acidity
and alkalinity of media do not affects the staining quality of C. acuminata extract, like in the Peterocarpus osun dye
as reported by Avwioro et al.,(2005).
Besides the pH of the extract, mordant has been reported to affect some stains (Avwioro et al., 2005). In some
studies of natural extract, Black plum (Syzygium cumini) mordant was used (Papawee, et al., 2011) However, in
S.A. Shehu et al. / Scientific Journal of Veterinary Advances (2012) 1(2) 33-37
35
case of this study, no mordant was used and as such no significant effect on the staining qualities of C. acuminata
extract was noticed. This is unlike most dyes used in histochemistry such as hematoxylin, which is first oxidized to
hematein and mordant is added before it can be used as a stain for tissues as reported by Bancroft, (2002) and
Avwioro et al., (2005).
The ability of a dye to stain specific tissue structures is determined by certain factors such as the electrostatic
attractions. Acidic structures (e.g. nucleus) are stained by basic dyes (e.g hematoxylin) while basic structures (e.g.
cytoplasm) are stained with acidic dyes (e.g. eosin) (Avwioro et al., 2005; Avwioro, O.G. 2002). In this study, the
natural dye from C. acuminata stained the cytoplasm of the tissues processed. The chemical components of C.
acuminata dyes may probably be the Hydro (H+) polar molecule in respect to the pH that is found to be 5.93. this
study were in agreement with the studies conducted by Avwioro et al., (2005) which showed that the red dye
stuffs obtained from Pterocarpus osun species were used in staining tissue section for histopathological diagnosis
of diseases. However, the nut of C. acuminata is reported to contain vitamin C, Gallic acid, tannins, anthocyanins
cyaniding glucoside, petunidin, malvidin and other components that act as antioxidant property for medicine and
cosmetic.
4. Conclusion
Cola acuminata extracts could be used to stain cytoplasm of body tissue with a yellowish-brown coloration,
while serving as an alternative natural dye for histological staining.
Plate i. Rat liver (Haematoxylin & Kola extract x400). Plate ii. Rat liver (Haematoxylin & Eosin x400).
Plate iii. Rat Intestine (Haematoxylin & Kola extract x400). Plate iv. Rat intestine (Haematoxylin & Eosin x400).
S.A. Shehu et al. / Scientific Journal of Veterinary Advances (2012) 1(2) 33-37
36
Plate v. Rat Kidney (Haematoxylin & Kola extract x400). Plate vi. Rat Kidney (Haematoxylin & Esoin x400).
Plate vii. Rat Lung (Haematoxylin & Kola extract x400). Plate viii. Rat Lung (Haematoxylin & Eosin x400).
Plate ix. Rat Cardaic Muscle (Haematoxylin & Kola extract x400). Plate x. Rat Cardaic Muscle (Haematoxylin & Eosin x400).
Acknowledgements
This study was supported by the Department of Veterinary Anatomy, Faculty of Veterinary Medicine, Usmanu
Danfodiyo University, Sokoto.
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(Syzgium cumini) Journal of the Microscopy Society of Thailand 4(1), 13 15.
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Histochemistry and tissue pathology Medicinal plants of West Africa
  • O G Avwioro
  • Nigeria
  • E S Ayensu
  • Michigan
  • F J Baker
  • R E Silverton
  • C J Palister
Avwioro, O.G., 2002. Histochemistry and tissue pathology, 1st ed., Claverianun press, Nigeria, Ayensu, E. S., 1978. Medicinal plants of West Africa, Reference Publication International, Michigan. Baker, F.J., Silverton, R.E., Palister, C.J., 2001. Introduction to Medical Laboratory, 7th edn., Bounty Press Limited, Ibadan, Nigeria, pp. 258-261.
  • A Shehu
A. Shehu et al. / Scientific Journal of Veterinary Advances (2012) 1(2) 33-37
Medicinal plants of West Africa, Reference Publication International
  • O G Avwioro
  • E S Ayensu
  • Michigan
  • F J Baker
  • R E Silverton
  • C J Palister
Avwioro, O.G., 2002. Histochemistry and tissue pathology, 1st ed., Claverianun press, Nigeria, Ayensu, E. S., 1978. Medicinal plants of West Africa, Reference Publication International, Michigan. Baker, F.J., Silverton, R.E., Palister, C.J., 2001. Introduction to Medical Laboratory, 7th edn., Bounty Press Limited, Ibadan, Nigeria, pp. 258-261.