This study was designed to determine the staining potentials of aqueous extract of Ceratonia Siliqua bark adapted for the first time as a counter stain in Haematoxylin and Eosin staining reaction. The staining solution was used to stain liver, lung and kidney sections. Phytochemical screening of the extract revealed the presence of tannin (hennatonic acid or Lawsone), while results of the staining reactions showed that Ceratonia siliqua at a low pH, has intense staining qualities, confirming further that the effectiveness of a stain is influenced by the pH of its environment.Key words: Ceratonia siliqua, Extract, Haematoxylin and Eosin, Counterstain, General tissue structure

Ogbodo, LA

Okon, AU

Okpidu, EE

Onyenekwe, ECN

Oyadonghan, GP

English

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 Anthonio Research Center © 2012 151       Okpidu et al.., IJBAIR; 1(4): 151-154   International Journal of Basic, Applied and Innovative Research IJBAIR, 2012, 1(4): 151 - 154 www.antrescentpub.com  RESEARCH  PAPER  HISTOLOGICAL STUDY ON THE STAINING POTENTIALS OF AQUEOUS EXTRACT OF CERATONIA SILIQUA BARK *1Okpidu EE., 1Okon AU., 2Oyadonghan GP., 3Ogbodo LA., 3Onyenekwe ECN.   Department of 1Medical Laboratory Science (Histopathology unit), Ambrose Alli University Ekpoma, Edo State, Nigeria; 2Anatomy, Abia State University, Uturu, Abia State, Nigeria; 3Morbid Anatomy, University of Nigeria Teaching Hospital, Ituku-Ozalla, Enugu, Nigeria. *Corresponding Author: egmaok@yahoo.com  Received: 17th August, 2012  Accepted: 20th December, 2012  Published: 31st December, 2012  ABSTRACT  This study was designed to determine the staining potentials of aqueous extract of Ceratonia Siliqua bark adapted for the first time as a counter stain in Haematoxylin and Eosin staining reaction. The staining solutin was used to stain liver, lung and kidney sections. Phytochemical screening of the extract revealed the presence of tannin (hennatonic acid or Lawsone), while results of the staining reactions showed that Ceratonia siliqua at a low pH, has intense staining qualities, confirming further that the effectiveness of a stain is influenced by the pH of its environment.  Key words: Ceratonia siliqua, Extract, Haematoxylin and Eosin, Counterstain, General tissue structure. ______________________________________________ INTRODUCTION  Ceratonia siliqua, commonly known as the Carob tree and St. John's-bread, is a species of flowering evergreen shrub or tree in the pea family, Fabaceae. The tree grows up to 10 metres (33 ft) tall. It is native to the Mediterranean region including Southern Europe, Northern Africa, the larger Mediterranean islands, the Levant and Middle-East of Western Asia into Iran and the Canary Islands and Macaronesia (Battle and Tous, 1997). It is widely cultivated for its edible legume beans, and as an ornamental tree in gardens, it has a thick trunk with brown rough bark and sturdy branches (Missbah et al., 1996). The seeds, also known as locust beans, are used as animal feed. They are also the source of locust bean gum, a thicken ng agent used in numerous processed foods (Turnbull et al., 2006). The fruit contains leucodelphinidin, a colour ess chemical compound, tannins (in leaves and bark), galactan, pentosan, (in seeds), mucilage and starch (Turnbull et al., 2006). Extracts of leaves and bark have been recommended for the treatment of syphilis and venereal diseases, and seems to have a soothing effect on epilepsy (Turnbull et al., 2006).  On the other hand, a dye is a coloured substance that has an affinity to the substrate to which it is being applied (Zollinger, 2003). The greatest source of dyes has been from the plant kingdom, notably roots, berries, bark, leaves and wood, but only a few have ever been used on a commercial scale. There are two types of dyes: natural dyes- obtained from natural sources, and synthetic dyes -produced through chemical reactions (Carleton et al., 1976). Several natural dyes are used in histology, histochemistry, and histopathology. In fact the most important and commonly used histological dyes, is haematoxylin -a natural dye produced from the logwood, and Haematoxylon campechianum with Eosin (in combination), are used for the demonstration of general tissue structures (Avwioro, 2002).         ASN-PH-020919     ISSN: 2315-5388   Anthonio Research Center © 2012 152       Okpidu et al.., IJBAIR; 1(4): 151-154  However, some synthetic dyes have been known to cause some hazards to human and animal health and have therefore been withdrawn. Also, synthetic dyes are increasingly becoming expensive and are no longer within the reach of many small laboratories in developing countries. In addition, some synthetic dyes are not enviro mentally friendly (Sewekow, 1988; Eom et al., 2001). It is for these reasons that an alternatively cheaper and bio-friendly natural dye extracted from Ceratonia siliqua, is been sort as a potential natural histological dye.  MATERIALS AND METHODS  Substance collection: Fresh bark of Ceratonia siliqua were collected from one of the carob trees in Otukpo, Otukpo Local Government Area of Benue State, Nigeria, and uthenticated by a botanist in the department of botany, Ambrose Alli University, Ekpoma, Edo State, Nigeria.  Substance preparation: They were rinsed in several changes of distilled water nd drained. The bark were cut into tiny bits of about 1cm in diameter and dried in an open air for 72 hours. About 3 kg of the bark was extracted by boiling with 1 liter of water and allowed to stand for 24h. The extract was filtered three times using Whatman’s Number one filter paper.  Preparation of sections: 3 human tissues, 3mm thick, were obtained from the liver, lungs and kidney at post mortem. They were fixed in 10% formol saline for 24hrs and processed for paraffin wax embedding with the automatic tissue processor (SAKURA FINETECH, Netherlands) by dehydrating through 70%, 90%, 95% and two changes of absolute ethanol for 90 min each. Clearing was achieved through changes of xylene twice for tw  hours each, infiltrating through two changes of paraffin wax at 70°C and embedded in paraffin wax. Sections were cut at 3 m with the rotary microtome (SAKURA FINE TECH, Nethrlands) and attached to slides and dried at 65°C for 45 min.  Staining Methods: Sections were dewaxed in 2 changes of xylene for 2 minutes each, hydrated through graded solutions of 100, 95% and 70% alcohol, and then rinsed with water. It was stained with Harris haematoxylin for 5 minutes, rinsed in water and differentiated in 1% acid alcohol for 2 to 60 seconds. The sections were blu d in running tap water and later stained with Ceratonia siliqua’s extract for 2-60 minutes. Sections were finally rinsed in water, dehydrated in grades of alcohol (70%, 80%, 95% and absolute), cleared in xylene, air-dried and mounted with dibuthylphthalate propylene xylene (DPX).  RESULTS  Extraction was completed within 24h. This gave a reddish brown colour. The sections were compared with haematoxylin and eosin technique. Ceratonia Siliqua bark extract stained collagen fibres, red blood cells and muscle fibres light-brown within 15 min. Haematoxylin and Eosin (H&E) stained tissue sections presented normal cytoarchitectural features characteristic for H & E stained tissues, while haematoxylin a d Ceratonia Siliqua’s bark extract (H& Ceratonia Siliqua’s bark extract) stained tissue sections presented poor cyt architectural distinctions (see figures 1A & B, 2A & B, and 3A & B).   Figure 1(A and B): Control section of human liver (H&E x100) showing normal characteristics of Haematoxylin and Eosin stain (3A), and a section of human liver (H & Ceratonia Siliqua’s bark extract x100) showing poor cytoarchitectural distinction (3B).  Anthonio Research Center © 2012 153       Okpidu et al.., IJBAIR; 1(4): 151-154    Figure 2 (A&B): Control section of Human Lung (H&E x100) showing normal tissue architecture (3A) and a section of human lungs (H & Cetatonia siliqua’s bark extract x100) showing poor cytoarchitectural distinction (3B).    Figure 3 (A&B): Control section of human Kidney (H&E x100) showing normal characteristics of Haematoxylin and Eosin stain (3A) and a section of human Kidney (H& Cetatonia siliqua’s bark extract x100 showing poor cytoarchitectural distinction (3B) DISCUSSION  Cellular structures are selectively stained by various natural and synthetic dyes. Some requires a combination of stains to demonstrate the presence of tissue structures. Available literature reveals that the pH status (acidity or alkalinity) of a dye as well as the use of a mordant, ffects the efficacy of some stains (Hoffman and Bauknecht, 1999). Our findings on C. siliqua bark extract show however, that the use of potassium alumini m alum as a mordant was not necessary because even simple aqueous solutions stained the tissues. This is unlike most dyes used in histochemistry, for example, haematoxylin, which s first oxidized to haematein and mordanted before it can be used as a stain for tissues. In fact, Elbadawi (1976) had stressed the need for the use of mordants in certain histochemical reactions and states that for the use of Verhoeff’s iron haematoxylin as stain for elastic fibres, ferric chloride use as a mordant was necessary.   Furthermore, Hoffman and Bauknecht (1999) had observed that the ionic strength and pH of staining soluti ns often affect staining reactions. This however, was not observed with C. siliqua bark extract, as it stained at neutral, alkaline, and acidic media; though with a decrease in the quality of staining in the alkaline region. I  fact, there are other natural and synthetic dyes, which do not requi  the addition of an acid or a base. Eosin, commonly used as a counterstain for haematoxylin, is a typical example of such a stain. Nevertheless, Vickerstaff (1954) in staining eosinophils with congo red, states that ionic strength influences the staining reaction between sulphuric acid groups of the dye and the basic groups of the eosinophil granules.   According to Horobin, (2002), acidic dyes are anionic and will stain cationic or basic groups in tissues such as amino groups. Such dyes are mostly used to stain proteins in the cytoplasm and connective tissues. On the other hand, basic dyes are cationic and stain anionic or acidic materials such as carboxylates, sulphates (many complex  Anthonio Research Center © 2012 154       Okpidu et al.., IJBAIR; 1(4): 151-154  carbohydrates are sulphated) and phosphates (particul ly the phosphates in nucleic acids). Such basic dyes are used as nuclear stains and stains cytoplasmic carboxyl groups.   From the foregoing therefore, it is obvious that C. siliqua as a dye, can be said to posses some histological staining qualities and can therefore, be useful in histopathological investigations.  ACKNOWLEDGEMENT  We acknowledged the priceless support of all those (too numerous to mention), that contributed towards the success of this study.  REFERENCES  Avwioro, O.G. (2002): Histochemistry and tissue pathology. 1st ed. Claverianun press Nigeria. 134-213.  Batlle, I. and Tous, J. (1997). Carob tree. Ceratonia siliqua L. In: International Plant Genetic Resources Institute (IPGRI), Promoting the conservation and use of underutilized and neglected crops. (Prom Underused Crops) 17:1–92.  Carleton HM, Drumy RAB, Wallington EA, Cameron R. (1976). Histological technique.  4th edition. Oxford University Press. Lond. 108nnnnn.  Elbadawi, A. (1976) .Hexachrome modification of Movat’s stain. Stain Technol. 51: 249 – 253.  Eom, S., Shin, D., Yoon, K. (2001). Improving the dyeability of natural colourants on cotton by cationization. Ind J Fibre Text. Res.; 26: 425-431.  Hoffman, E.M. And Bauknecht, N. (1999). A dye bindig assay for the quantification of solution and cell-bound acidic polysaccharides produced by red algae. Anal. Biochem. 267 (2): 245 – 251.  Horobin, R.W. (2002) Theory of staining and its practical implications. In Theory and Practice of Histological Techniques, 5th Edn (Eds J.D. Bancroft and M. Gamble). Edinburgh: Churhill Livingstone.  Missbah EM., Aujjar, N., Belabed, A., Dessaux, Y., Filali-Maltouf, A. (1996). Characterization of rhizobia isolated from Carob tree (Ceratonia siliqua). Journal of Applied Microbiology; 80 (2): 165–73.  Sewekow, U. (1988). Natural dyes- an alternative to synthetic dyes. Melliand Textilberichte. 69 (4), 145-48.  Turnbull, L.A., Santamaria, L., Martorell, T., Rallo, J. and Hector, A. (2006): Seed size variability: from carob to carats. Biology Letters; 2 (3): 397–400.  Vickerstaff, T. (1954). The physical chemistry of dyeing, Olive and Boyd, London. p. 352.  Zollinger, H. (2003). Color Chemistry. Synthesis, Properties and Applications of Organic Dyes and Pigments, 3rd ed. Weinheim: Wiley-VCH.  AUTHOR(S) CONTRIBUTION  All the authors in this article contributed one way or the other to the success of this study and in the presentation of this paper. 

Histological study on the staining potentials of Aqueous extract of Ceratonia Siliqua bark

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Histological study on the staining potentials of Aqueous extract of Ceratonia Siliqua bark

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