Egypt. J. Exp. Biol. (Zool.), 6(1): 159 – 164 (2010) © The Egyptian Society of Experimental Biology

RESEARCH ARTICLE

Ehab M. Tousson* Bahija Al-Behbehani**

BLACK MULBERRIES (MORUS NIGRA) AS A NATURAL DYE FOR NERVOUS TISSUES STAINING

ABSTRACT: Natural dyes have been used for staining INTRODUCTION wool, silk, carpet and cotton in addition to their uses in staining of tissue microscopical Dyes have been defined as intensely sections. The binding of dyes to textile coloured substances used for colouration products has been studied for many years, (Angelini et al., 1996). They are retained in but is still not fully understood. Less is known substances by physical adsorption, about the interaction between the dye and mechanical retention, the formation of tissue components. Black mulberry (Morus covalent chemical bonds or of complexes with nigra) fruits have strong staining activity and salts or metals, or by solution (Jacobson and a distinct flavour with juicy and acidic Wasileski, 1994). Stains are characterized by characteristics making them attractive for use their ability to absorb visible part of the in the processing industry in products such as electromagnetic spectrum (380-780 nm). For fruit juice, ice cream, jelly, and jam. This good staining property it has to have high study investigated a new staining method enough absorption coefficient (10 000 to 40 using black mulberry as natural source of dye 000 l.mol-1.cm-1). Natural dyes have been for nervous tissues. Whole brain of adult used for staining wool, silk, carpet and cotton Wistar rats (8-10 weeks old) were removed, (Dyer, 1977; Watson, 1991; Jacobson and immersed in 4% paraformaldehyde fixative, Wasileski, 1994; Mangan et al., 1995; and embedded in paraffin. Sections, 7-10 Angelini et al., 1996). Until the late nineteenth micrometre from brain hippocampus, cortex, century only the natural dyes were used for thalamus and hypothalamus were collected colouring weaving yarns. There are three and then stained by using the new method primary sources for natural dyes (plants, (dye extracted from black mulberry) and animals and minerals). The natural dyes come haematoxylin & eosin staining method for from roots, flowers, leaves, fruits and barks of control. By using the dye extracted from black plants, or from animal sources such as mulberry, neurobiologists and zoologists can cochineal and or mineral sources such as red make identification and differentiation soils. Regardless of the source, the natural between different brain parts and can also dyes can be broken down into two categories differentiate between nervous tissue cells. (substantive and adjective). Substantive dyes Staining with this dye can be an alternative to are also referred to as direct dyes, become high cost and time-consuming current chemically fixed to the fibre without the aid of chemical staining methods. any other chemicals or additives while the adjective dyes, referred to as mordant dyes, require the addition of a substance known as KEY WORDS: "mordant" to make the dyes colourfast Natural dyes, Mulberry, Nervous tissues, (Mangan et al., 1995). Most natural dyes are Brain, Rat. adjective dyes and the type of mordant used in the dyeing process affects the colour produced (Kirk et al., 1978; Jacobson and Wasileski, 1994). Dyers are able to get a CORRESPONDANCE: variety of colours and shades from the same Ehab M. Tousson* source depending on the type of material Zoology Department.Faculty of Science, Tanta used, the characteristic of local water, and University. Tanta, Egypt. the use of different mordants (Cannon and E-mail: [email protected] Cannon, 1994). For example, from pomegranate skin they can produce a range Bahija Al-Behbehani of colours from red to black by using different Science Department, College of Basic Education, PAAET, mordants. Kuwait The black mulberry (Morus nigra) trees are native from Egypt to Morocco in northern Africa and has been cultivated since ancient ARTICLE CODE: 19.01.10 ISSN: 2090 - 0511 On Line ISSN: 2090 - 0503 http://www.egyseb.org

160 Egypt. J. Exp. Biol. (Zool.), 6(1): 159 – 164 (2010) times throughout the Mediterranean region of -Staining of experimental sections in the Asia and Europe (Berg, 2001; Ercisli, 2004; new stain of black mulberry fruits for 30-60 Ercisli and Orhan, 2007; Koca et al., 2008). minutes. Black mulberry fruits have strong staining - The stained sections were washed in activity and become dark red-black at running tap water for 2 minutes. maturity. The fruits of M. nigra have a distinct - Dehydration in 95% alcohol and then in flavour with juicy and acidic characteristics absolute alcohol, twice of 2 minutes each or making them attractive for use in the until excess eosin is removed. processing industry of products such as fruit juice, ice cream, jelly, and jam (Kafkas et al., - Clearing in xylene, and mounting in 2008). Ercişli and Orhan (2008) determined Canada balsam. the total phenolic content, antioxidant activity, - The stained sections were examined mineral content, and selected under microscope and a digital camera physicochemical properties of M. nigra (Cannon 620) captured images. genotypes. The main use of M. nigra in - Brightness and contrast of the images modern medicine is for the preparation of were adjusted using Adobe Photoshop syrup obtained from the ripe fruit employed to software. flavour or colour other medicines (Özgen et al., 2009). It is a dark violet or purple liquid, with a faint odour and a refreshing, sweet- RESULTS: acid taste (Koyuncu, 2004; Ercisli and Orhan, The results of the two staining methods 2008). In medicine, particular staining for are shown in figures 1-16. By use of this determination of astrocyte and neuron is method, we can identify the different brain performed with various stains such as parts and the cell types; differentiation immunocytochemical makers, Nissl stain, between neurons and astrocytes is possible. Bielschowsky, Golgi-Cox, antibody for neuron Staining of neurons and astrocytes in filaments, and PTAH, Cajal gold and Hortega cortex of cerebrum, hippocampus, thalamus lithium carbonate for astrocytes (Kiernan, and hypothalamus displays different patterns. 1999). Staining activity of black mulberry for It is observed that the dye gives a strong histological staining purpose has not been reaction with the granular cells of the examined. Therefore the objective of this cerebeller cortex that has strong response study is investigation of the staining activity of and the astrocytes of the cortex and black mulberry for histological purposes, hippocampus (Figs 3, 4, 7, & 8). On the other particularly for nervous tissues. hand, reaction between the dye and neurons of hypothalamic nuclei is poor (Figs 10& 12) MATERIAL AND METHODS: while the hippocampus and the cortex Processing of dye: neurons stained in dark brown. The astrocytes of lateral hypothalamus were stained in deep Approximately 50 g of black mulberry brown colour as a result of strong response fruits were collected and immersed in 200 ml mentioned above (Fig. 10). After staining with water, heated to just below boiling point and concentrated extract of black mulberry, immersed until the colour has been astrocytes of thalamus and hypothalamus are transferred from the dye solution. observed in faint colour, while their neurons Concentrated juice of black mulberry is cooled were observed in light brown (Figs 10, 12, 15, and filtered. & 16). Preparing histological samples: Whole brains of the rats were removed, immersed in 4% paraformaldehyde, dehydrated in ascending series of ethyl alcohol then cleared in xylene and embedded in paraffin. 7-10 µm sections from thalamus, hypothalamus, cortex and hippocampus of rat brains were collected and divided into two groups. The 1st group are the control sections that were stained with haematoxylin and eosin and the 2nd group is experimental sections that were stained by the new method. The staining procedure: - Deparaffinition of brain sections in xylene and hydration in descending series of ethyl alcohol down to water. Fig. 1. Histological architecture of rat cortex brain stained with H&E. 200 X.

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Tousson & Al-Behbehan, Black Mulberries (Morus Nigra) As a Natural Dye for Nervous Tissues Staining 161

Fig. 2. Histological architecture of rat cortex brain stained Fig. 6. Histological architecture of rat hippocampus stained with H&E. 400 X. with H&E. 400 X.

Fig. 3. Histological architecture of rat cortex brain stained Fig. 7& 8. Histological architecture of rat hippocampus with mulberry extract. 200 X. stained with mulberry extract. 200 X.

Fig. 4. Histological architecture of rat cortex brain stained Fig. 7& 8. Histological architecture of rat hippocampus with mulberry extract. 400 X. stained with mulberry extract. 200 X.

Fig. 5. Histological architecture of rat hippocampus stained Fig. 9. Histological architecture of rat hypothalamus stained with H&E. 200 X. with H&E showing the SON. 200 X.

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162 Egypt. J. Exp. Biol. (Zool.), 6(1): 159 – 164 (2010)

Fig. 10. Histological architecture of rat hypothalamus stained Fig. 14. Histological architecture of rat thalamus stained with with mulberry extract showing the SON. 200 X. H&E. 400 X.

Fig. 11. Histological architecture of rat hypothalamus Fig. 15. Histological architecture of rat thalamus stained with stained with H&E showing the SCN. 200 X. mulberry extract. 200 X.

Fig. 12. Histological architecture of rat hypothalamus stained Fig. 16. Histological architecture of rat thalamus stained with with mulberry extract showing the SCN. 200 X. mulberry extract. 200 X.

DISCUSSION: Many staining methods in histology and neurobiology use natural dyes (Bancroft and Gamble, 2004). One of the most important natural dyes is hematoxylin that is extracted from the heart-wood of haematoxylin campechamum, the logwood tree of South and Central America (Angelini, 1996). In classification of dyes based on the methods of application, the extracted dye of mulberries fruits can be included in vital dyes, instance indigo in enzyme histochemistry. Also, based on combination of dyes with substrate, the dye is Fig. 13. Histological architecture of rat thalamus stained with progressive (Kiernan, 1999). The present H&E staining showing the SON. 200 X. study shows that dye extracted form black ISSN: 2090 - 0511 On Line ISSN: 2090 - 0503 http://www.egyseb.org

Tousson & Al-Behbehan, Black Mulberries (Morus Nigra) As a Natural Dye for Nervous Tissues Staining 163 mulberry could stain astrocytes and neurons than synthetic dyes. Synthetic dyeing in various parts of the rat brain. Through procedures may be polluting and certain this new method, histological identification diazo dyes are carcinogenetic. It is of various parts of the brain, and the necessary for the histopathologist to have comparison between neurons and astrocytes dyes that can be relied upon to give are possible. Staining activity of the dye is satisfactory and repeatable results. Black especially very effective on astrocytes. In mulberry produces bright colours in a other words, observation of staining variety of shades and not fade quicker as patterns in various areas of the brain other natural dyes when exposed to light. revealed that extracted dye of black Mulberries dye has some disadvantages mulberry have higher affinities for where unlike synthetic dyes that are created astrocytes than for neurons. It is necessary in a laboratory, Black mulberry dyes are for the histologist to have dyes that can be obtained from plants and are dependent on relied upon to give satisfactory and growing seasons. Less is known about the repeatable results. Utilizing the new dye interaction between dye and tissues, so we from black mulberry to impart stain onto recommend further studies to detect nervous tissues has a number of effective chemical dye extracts of advantages over other conventional mulberries that have staining activity in biological staining. Black mulberry dye-not particular sites and cells of the nervous as other natural dyes has limited tissue. This new method can be used as an availability. Almost all natural dyes as black alternative method for histological staining mulberries are free of azo compounds which in neuropathology and in clinical pathology are carcinogen and are more eco-friendly for research and education.

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164 Egypt. J. Exp. Biol. (Zool.), 6(1): 159 – 164 (2010)

ﻋﺼﺎرة اﻟﺘﻮت اﻷﺳﻮد (Morus nigra) آﺼﺒﻐﺔ ﻃﺒﻴﻌﻴﺔ ﻟﺼﺒﻎ اﻷﻧﺴﺠﺔ اﻟﻌﺼﺒﻴﺔ إﻳﻬﺎب ﻃﻮﺳﻮن، *ﺑﻬﻴﺠﺔ اﻟﺒﻬﺒﻬﺎﻧﻰ *ﻗﺴﻢ ﻋﻠﻢ اﻟﺤﻴﻮان, آﻠﻴﺔ اﻟﻌﻠﻮم, ﺟﺎﻣﻌﺔ ﻃﻨﻄﺎ, ﻣﺼﺮ **ﻗﺴﻢ اﻟﻌﻠﻮم, آﻠﻴﺔ اﻟﺘﺮﺑﻴﺔ اﻻﺳﺎﺳﻴﺔ, اﻟﻜﻮﻳﺖ ﺗﺴﺘﺨﺪم اﻷﺻﺒﺎغ اﻟﻄﺒﻴﻌﻴﺔ ﻟﺼﺒﺎﻏﺔ اﻷﻧﺴﺠﺔ و اﻟﺴﺠﺎد ﺑﺼﺒﻐﺔ اﻟﻬﻴﻤﺎﺗﻮآﺴﻼﻳﻦ و اﻻﻳﻮﺳﻴﻦ آﻤﺠﻤﻮﻋﺔ ﺿﺎﺑﻄﺔ. واﻟﻘﻄﻦ ﺑﺎﻷﺿﺎﻓﺔ ﻻﺳﺘﺨﺪاﻣﻬﺎ ﻓﻰ ﺻﺒﻎ ﺑﻌﺾ اﻷﻧﺴﺠﺔ ﺑﺄﺳﺘﺨﺪام هﺬا اﻟﻨﻮع اﻟﺠﺪﻳﺪ ﻣﻦ اﻟﺼﺒﺎﻏﺔ ﺳﻴﺘﻤﻜﻦ ﻋﻠﻤﺎء اﻟﻤﺠﻬﺮﻳﺔ. ﺗﻢ دراﺳﺔ أﺗﺤﺎد اﻷﺻﺒﺎغ ﺑﺎﻻﻧﺴﺠﺔ ﻟﻌﺪة ﺳﻨﻴﻦ اﻟﺤﻴﻮان وﺑﻴﻮﻟﻮﺟﻴﺎ اﻻﻋﺼﺎب ﻣﻦ اﻟﺘﻌﺮف ﻋﻠﻰ اﻟﻤﻨﺎﻃﻖ دون ﻓﻬﻢ آﺎﻣﻞ ﻟﻨﻮﻋﻴﺔ هﺬة اﻟﻌﻼﻗﺔ ﻟﺬﻟﻚ ﻣﺎزال اﻟﻤﻌﺮوف اﻟﻤﺨﺘﻠﻔﺔ ﻟﻠﻤﺦ واﻟﺘﻤﻴﻴﺰ ﺑﻴﻨﻬﺎ آﻤﺎ أن هﺬﻩ اﻟﻄﺮﻳﻘﺔ اﻟﺠﺪﻳﺪة ﻋﻨﻬﺎ ﻗﻠﻴﻞ. اﻟﺘﻮت اﻷﺳﻮد (Morus nigra) ﻟﻪ ﻗﺪرة ﻋﺎﻟﻴﺔ ﻳﻤﻜﻨﻬﺎ أن ﺗﺤﻞ ﻣﺤﻞ اﻟﻄﺮق اﻟﺘﻘﻠﻴﺪﻳﺔ اﻟﻜﻴﻤﻴﺎﺋﻴﺔ ﻋﻠﻰ اﻟﺼﺒﺎﻏﻪ وﻟﻪ ﻧﻜﻬﺔ ﻣﻤﻴﺰة آﻌﺼﻴﺮ ﻓﻀﻼ ﻋﻦ ﺧﺼﺎﺋﺼﻪ اﻟﻤﺴﺘﻬﻠﻜﺔ ﻟﻠﻮﻗﺖ واﻟﻤﺎل. اﻟﺤﺎﻣﻀﻴﺔ ﻣﻤﺎ ﻳﺠﻌﻠﻪ ﺟﺬاﺑﺎ ﻟﻼﺳﺘﺨﺪام ﻓﻲ ﺻﻨﺎﻋﺔ ﺑﻌﺾ اﻟﻤﻨﺘﺠﺎت ﻣﺜﻞ ﻋﺼﻴﺮ اﻟﻔﻮاآﻪ ،اﻵﻳﺲ آﺮﻳﻢ ، اﻟﺠﻴﻠﻰ ، واﻟﻤﺮﺑﻰ. وﻧﺤﻦ ﻣﻦ ﺧﻼل أﺳﻠﻮب ﺟﺪﻳﺪ ﻗﻤﻨﺎ ﺑﺎﺳﺘﺨﺪام اﻟﺘﻮت اﻷﺳﻮد ﺑﺎﻋﺘﺒﺎرﻩ ﻣﺼﺪر ﻃﺒﻴﻌﻲ ﻟﻠﺼﺒﺎﻏﺔ ﻓﻲ ﺻﺒﺎﻏﺔ اﻟﻤﺤﻜﻤﻮن: اﻷﻧﺴﺠﺔ اﻟﻌﺼﺒﻴﺔ. ﺗﻢ ﺻﺒﻎ اﻣﺎآﻦ ﻣﺨﺘﻠﻔﺔ ﻣﻦ اﻟﻤﺦ أ.د. ﻣﻨﻴﺮ اﻟﺠﻨﺰوري ﻗﺴﻢ ﻋﻠﻢ اﻟﺤﻴﻮان، ﻋﻠﻮم ﻋﻴﻦ ﺷﻤﺲ ﺑﻮاﺳﻄﺔ اﻟﺼﺒﻐﺔ اﻟﻤﺴﺘﺨﺮﺟﺔ ﻣﻦ اﻟﺘﻮت اﻻﺳﻮد و ﻣﻘﺎرﻧﺘﻬﺎ أ.د. ﻣﺪﻳﺤﺔ ﻋﺸﺮي ﻗﺴﻢ ﻋﻠﻢ اﻟﺤﻴﻮان، ﺑﻨﺎت ﻋﻴﻦ ﺷﻤﺲ

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