บทความวิชาการ

สัตวแพทยั มหานครสาร์์ JOURNAL OF MAHANAKORN VETERIINARY MEDIICIINE Available online: www.vet.mut.ac.th/journal_jmvm

การใช้สสกี ัดจากพืชในการย้อมสีเนื้อเยื่อ

อารยา สืบขาเพชรํ 1,# และณฐกาญจนั ์ นายมอญ2

1ภาควิชากายวิภาคศาสตร ์ คณะสัตวแพทยศาสตร์ มหาวิทยาลัยเทคโนโลยีมหานคร กรุงเทพฯ 10530 2ภาควิชาเทคนิคการสัตวแพทย์ คณะเทคนิคการสัตวแพทย์ มหาวิทยาลัยเกษตรศาสตร์ กรุงเทพฯ 10900

บทคัดย่อ: ปัจจุบันการศึกษาทางจุลกายวิภาคศาสตร์ได้มีการใช้สีย้อมมากมายหลายชนิดเพื่อย้อมเนื้อเยื่อต่างๆ ซึ่งขึ้นกับวัตถุประสงค์ในการศึกษา สีที่ใช้ย้อมเนื้อเยื่อโดยทั่วไปมีแหล่งที่มาทั้งจากแหล่งธรรมชาติและจากการ สังเคราะห์ ด้วยในปัจจุบันทั่วโลกให้ความสําคัญในการใช้สารต่างๆ ที่ไม่มีผลกระทบต่อสุขภาพของมนุษย์และ เป็นมิตรกับสิ่งแวดล้อม การใช้สีธรรมชาติจากพืชในการย้อมเนื้อเยื่อจึงได้รับความสนใจมากขึ้น เนื่องจากส่วน ต่างๆ ของพืชนั้นมีส่วนประกอบของสีอยู่มากมาย อีกทั้งยังมีราคาถูกกว่าและสามารถใช้งานได้อย่างยั่งยืนกว่า สีที่ได้จากการสังเคราะห์ รายงานฉบับนี้ได้ทวนสรุปโดยย่อเกี่ยวกับการแบ่งชนิดของสีธรรมชาติจากพืชในแง่ ของโครงสร้างทางเคมีของสี วิธีการสกัด มอร์แดนท์ และได้ยกตัวอย่างงานวิจัยที่ศึกษาการใช้สีสกัดจากพืชใน การย้อมเนื้อเยื่อชนิดต่างๆ ซึ่งอาจนํามาใช้เป็นข้อมูลพื้นฐานและเป็นทางเลือกในการศึกษาและพัฒนาการย้อม สีเนื้อเยื่อจากสีที่สกัดจากพืช

คําสําคัญ: สีย้อมจากพืช สีย้อมจากธรรมชาติ การย้อม เนื้อเยื่อสัตว์ #ผู้รับผิดชอบบทความ สัตวแพทย์มหานครสาร. 2557. 9(1): 63-78. E-mail address: [email protected]

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

Using Plant Extract for Histological

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 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 , methods of plant dye extraction, 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, , Staining, #Corresponding author J. Mahanakorn Vet. Med. 2014. 9(1): 63-78. E-mail address: [email protected]

Introduction are obtained from natural sources such as Histology is the study of cells and plants, , , clays and minerals tissues by using a light . The (Carleton et al., 1976). Plants and insects ability to visualize or identify histological known to be used in histological staining for structures is frequently enhanced through animal tissues are Haematoxylon the use of histological stains. They give campechianum (logwood), from which contrast to the tissue as well as highlighting stain is obtained and particular features of interest. There are two cacti, from which stain types of dyes that classified by their origins, is obtained (Egbujo et al., 2008), synthetic and natural dyes. The natural dyes respectively. The majority of natural dyes

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

are extracted from plant parts such as roots, Many dyes, techniques and procedures barks, leaves, berries and seeds and wood. are utilized to stain various types of animal The with natural dyes was one tissues. At present, haematoxylin and eosin of the oldest techniques practiced by the stain (H&E) is widely used as a light ancient people such as wall paintings in the microscopical stain in histology and caves or dyeing. The first use of dye histopathology. It is combination of natural in histology credited to Antonie van haematoxylin stain and the synthetic eosin Leeuwenhoek, the father of microbiology stain. Haematoxylin stains the cell nuclei who worked with , a natural dye dark blue while eosin stains cell cytoplasm, extracted from saffron crocus. In the mid most connective tissue fibers and matrices in 1800s, amateur microscopists first used varying shades of and . haematoxylin to stain cellular components Haematoxylin obtained from logwood tree (Titford M., 2005). Later scientists developed that is native to the and northern a wide range of haematoxylin staining (Baker & Silverton, 1976), techniques. The systemically studied dye for but is now mainly cultivated in the West histology started in the second half of the Indies (Bancrot and Gamble, 2008). 19th century by Weigert C, J Gerlach, P Haematoxylin can be prepared in numerous Ehrlich and H Gierke. By then coloring ways and applied to tissues from different materials were mostly still natural origin like sites. It has to be oxidized to haematein carmine, cochineal, haematoxylin and . before using (Culling, 1974). Haematein can Gradually the use of the natural dyes has be produced from haematoxylin in two decreased due to the introduction of ways: natural oxidation and chemical synthetic dyes (aniline dye from extract of oxidation. For natural oxidation, coal tar) which first invention by William haematoxylin is exposed to light and air Henry Perkin in 1856. They widely applied in (Ehrlich’s and Delafield’s haematoxylin many fields as food, cosmetic and textile solution), and for chemical oxidation, industries (Prabhu and Bhute, 2012). They sodium iodate (e.g. Mayer’s haematoxylin) or are rapid stain with vast range of new colors, mercuric oxide (e.g. Harris’s haematoxylin) easy to use and commercially available. are used. Natural oxidation of haematoxylin From then, the synthetic dyes together with seems to retain its staining ability longer those for histological staining have than that of the chemical oxidation. developed. However, it takes much longer time for preparation. Haematoxylin has also been 65 Araya Suebkhampet and Nattakarn Naimon / J. Mahanakorn Vet. Med. 2014. 9(1): 63-78.

prepared synthetically (Morsingh and potential use in various purposes. It is Robinson, 1970) but the natural dye is interesting that over 2000 dyes are mostly used. Haematein is anionic charge, synthesized from various parts of plants, of having a poor affinity for tissues without the which only about 150 have been presence of . A mordant is a commercially exploited (Gulrajani, 1992). substance used to set dyes on tissue They are used for applying to different sections. The common mordants for substrates such as , paper, leather, haematoxylin are , and wood, hair etc. They are also widely used in tungsten. The dye-mordant complex cosmetic, food and pharmaceutical possesses a net positive charge and enables industries. A number of them own the to bind to anionic tissue regions including pharmaceutical properties and have been nuclear chromatin. Mordant is regularly used in traditional medicine. Curcumin, the included in the dyeing protocols when bright yellow dye of has been used natural dyes are used in order to fix or traditionally as a remedy for stomach and intensify the stains in cell or tissue staining. It liver ailments, as well as topically to heal is mainly a polyvalent metal ion that forms sores and revitalizes the skin (Chaturvedi, coordination complexes with certain dyes 2009). It has anti-oxidant, anti-inflammatory, which then attaches to the tissues anti-cancer and anti-septic properties (Llewellyn, 2005). Different kinds of (Chengaiah et al., 2010). Many other mordants give a different hue and stability common natural dyes such as lycopene (red of the dye in the tissues. dye) in tomatoes or lawsone (varies in color The use of natural dyes which are from orange to reddish brown) in are cheaper, eco-friendly and biodegradable has reported as antimicrobial agents (Siva, 2007; become a matter of significant important Habbal et al., 2011). due to the increased environmental In last 10 years, natural dyes have awareness and in order to avoid some been studied for their potential use in hazardous synthetic dyes to humans and various fields including histological staining. animals (Eom, et al., 2001). Several synthetic The present report describes the basic dyes (e.g. dyes with azo bonds nitro- or information about of plant dye classification amino-groups) cause allergic-like symptoms based on their chemical structures, methods or are carcinogenic (Ratna and Padhi, 2012). of the dye extraction, mordants and some of Therefore, alternative natural dyes have its application for tissue staining. These been currently more interested for their would be useful information to the

66 Araya Suebkhampet and Nattakarn Naimon / J. Mahanakorn Vet. Med. 2014. 9(1): 63-78. researchers who are searching for the new temperature and pH which depend on the natural dyes for tissue staining. properties of particular dye components (Prabhu and Bhute, 2012). After extraction, Classification of natural dyes from plants the extracts are generally filtered through Natural dyes can be classified in various filters such as cheesecloth, different ways such as based on their or paper filter. The filtrates may be chemical structure, source, hue and method freshly used (normally aqueous extract) or of application, etc. The plant dye further evaporated of solvent, washing and classification described in this review is drying to get purified dye. There are mainly based on their chemical structures which are four methods used in extraction of natural divided into 7 major groups (Siva, 2007; dyes (Samanta and Konar, 2011). Samanta and Konar, 2011; Prabhu and Bhute, 2012) as presented in Table1. 1) Aqueous extraction This method is has long been used Methods of plant dye extraction for natural dye extraction for a certain of Various parts of plants such as roots, time. The dye components in dried plant stems, leaves, flowers, fruits and seeds are powder are extracted in water at boil or used for dye extraction. Some plants may particular temperature. Then, the extract is have more than one color depending on cooled down and filtered. The dye solution which part of the plant one uses. The color is carried out under varying conditions as yield and shade of the color a plant mentioned above in order to get the produces vary according to time of the year optimal extraction condition. The optimum the plant is picked, how it is grown, soil condition is determined by studying the conditions, etc. Before extraction, the parts optical density value at definite wavelength of plants are collected and generally shade for the extracted solution using UV-Visible dried in air or sun dried. Then the grinding is absorbance spectrophotometer (Samanta carried out to break down the material into and Konar, 2011). The filtrate is further very small pieces or powder using the applied for dyeing. Here are some studies manual or electric grinding machines. that used the aqueous extract from plants Optimum conditions of extraction are for animal tissue staining. Natural dyes were determined by varying extraction parameters extracted from kujarat flowers (Hibiscus such as type of solvents, time of extraction, sadariffa) in aqueous medium and were ratio between plant material and solvent, used to replace of eosin stain in kidney from 67 Araya Suebkhampet and Nattakarn Naimon / J. Mahanakorn Vet. Med. 2014. 9(1): 63-78. :

(Lady's tinctoria wood

part used tinctorium cordifoli: wood Example of plants: Indigofera suffruticosa : leaves (madder): wood flowers Bedstraw): leaves Indigofera Rubia Galium verum

Red Blue color Range of

o g Indi 9,10-Anthraquinone Chemical structures

are

variety which

A dyes .

Dactylopius . These dyeing

dye dye

.

Almost all the red color textile

carmine carmine

for

the

achieve used

to

dye Basic information

prepare

to

oldest dye used for textile dyeing A variety

used

the conjugation is

, ) ) is used to prepare the is

of plants have provided indigo, but most natural indigo was obtained from those in the genus Indigofera characterized by good light fastness. natural dyes are based on the anthraquinoid structure. Indigo is the native in tropical areas. At present, almost all indigo produced is synthetic. It the blue of jeans. that dyes are fused three benzanoid rings Anthraquinone possess conjugation to achieve color These dyes are The cochineal, an example of They are mordant dyes. known anthraquinone dye from insects ( coccus

dyes

dyes Indigoid dyes Major groups Anthraquinone Anthraquinone Table 1 Classification of plant dyes

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

:

(black : leaves leaves : ) inermis

species

part used

Example of plants: Myrobalan (henna): leaves Lawsonia inermis walnut): shells of walnut fruits Tagetes Tagetes species ( wood wood Cotinus coggygria (marigold): flowers Terminalia chebula

k blac color Yellow Yellow Orange Range of Brownish-

Jl Flavone Lawsone Isoflavone Chemical structures

is , are

group

that .

. this

ink

of

as

dye isoflavones

and

compounds and

popular fabrics )

and flavones most

of

Basic information the polyphenolic Continued ,

clothing

are for

(henna) derivatives

dye

Lawsone (henna) the most popular dye of this group is the following few days to a reddish brown. Juglone is another example of these dyes that has been used as a natural dye for clothing and fabrics and as ink Flavonoids Flavonoids are polyphenolic compounds that are isoflavones, catechins, anthocyanidins and chalcones. Most of the natural yellow colors are hydroxy and methoxymethoxy derivatives of flavones and isoflavones used to dye skin, fingernails, hair, wool and leather. used to dye skin, fingernails, hair, wool and leather. are orange after application but darken over Henna stains plants and are categorized, according to abundant in flavonols, flavones, flavanones, chemical structure, into

-

s s Classification of plant dyes ( hydroxy - Flavonoid Flavonoid Major groups Alpha napthoquinones Table 1

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

: :

chica

part used BignoniaBignonia chica Example of plants:

campechianum (logwood): wood Vaccinium myrtillus: berries Haematoxylon Anthocyanin: Anthocyanin: Carajurin: - leaves

color Range Range Range to blue to blue to to violet from red from red Range of

Haematine Anthocyanin Chemical structures 2-Phenylchromenium

of are

They structure

. commonly

member

The chemical

.

conditions flavonoids

in

lors is attributed to several the acidic

oxyl groups, the degree of lor changes with pH, pink or pink or lor changes with pH,

mber of monosaccharides mber of monosaccharides solutions in neutral solutions, blue to in neutral solutions, of in )

related

red

alkaline and member Basic information closely ,

Continued in are are

conditions yellow yellow in alkaline solutions The member of -

factors as the number of hydr factors as and the nu methoxylation Dihydropyrans are closely related in chemical structure use in histological staining. Haematein haematoxylin, exhibits indicator-like properties, being blue in aqueous alkaline conditions and red in acidic conditions They are Anthocyanidins Anthocyanidins are member of the flavonoids commonly molecules. The variation in the co Their co to the cation. attached greenish greenish red in acidic solutions, found in plants. They are glycosides of 2- glycosides plants. They are found in soluble are water which salts phenylchromenylium with anthocyanins (anthocyanidins includes anthocyanidines sugar), carajurin and delphinidine. to the flavones. The example of known dihydropyran is to the flavones. of derivative haematein which is an oxidized mordant dyes.

Classification of plant dyes ( dins: Anthocyanani Anthocyanani Major groups Dihydropyrans Table 1

70

Araya Suebkhampet and Nattakarn Naimon / J. Mahanakorn Vet. Med. 2014. 9(1): 63-78. albino mice (Hashim, 2006). Suebkhumpet compound in many analytical processes as and Sotthibandhu (2012) used aqueous well as extraction of plant natural dye has extract of butterfly pea flowers (Clitoria gained wide acceptance in recent years ternatea) to stain animal peripheral blood since this technique is safe for health, smears. inexpensive and harmonize with nature. SFE uses carbon dioxide as a solvent. This 2) Extraction by non-aqueous and other method is based on the enhanced solvating solvent assisted system power of gases above their critical point The solvents generally used for plant (Samanta and Konar, 2011). Cardoni et al. dye extraction are ethanol, methanol, (2000) studied the extraction of lycopene acetone, chloroform, ether, clove oil, etc. and β-carotene from ripe tomatoes using The dried material powder is weighed and SFE. The detail information and procedure soaked in solvent in different percentages of this method has been reported by and time durations. The crude dye extract Sapkale et al. (2010). can be used for tissue staining after extraction or can be further applied for 3) Extraction by acid or alkaline assisted solvent evaporation in order to concentrate system the dye solution before staining. This method adjusts the pH of the Alternatively, the dried plant powder is extraction by adding hydrochloric acid or soaked in solvent to allow effective in aqueous medium. The percolation, then the soaked powder is different pH in extraction protocols may give extracted in the solvent using Soxhlet differently results as percent yield and hue Extractor (Steam Heated Extractor). The of the extracted dye which due to the extract is then concentrated using rotary reaction of the chemical structure of the dye evaporator and may further drying in the to the pH variation. Samanta et al, 2007 drying oven (Okolie. 2008). Finally, the studied the extraction of color from jackfruit extract is obtained in powdered form which wood under various pH conditions (ranged will be dissolved in the solvent or buffer at between pH 4-12) and reported that the the desire concentrations before tissue optimum condition for the extraction is at staining. pH 11.0. Supercritical fluid extraction (SFE), an alternative to conventional solvent 4) Extraction by other methods extraction for separation of organic 4.1) Ultrasound assisted extraction

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

This method is carried out by mixing Mordants dried and ground plant materials in solvent Natural dyes are mostly non- in a flask, which was then placed in an substantive and must be applied by adding ultrasonic bath. The ultrasound applied for mordants which after combining with dye in the extraction results in intermolecular the materials including the tissues, it forms tearing and surface scrubbing, causing plant an insoluble precipitate or lake and thus tissue rupture and improving the release of both the dye and mordant get fixed to intracellular substances into the solvent. become wash and light fast. Mordants are The extraction is repeated two-three times also often added to keep dyes from fading, before the extract is collected. Sivakumar et or to brighten, deepen, or dull a color. They al. (2009) used ultrasound with 80W can be used before, during or after the dye ultrasonic power for 3h contact time bath due to the desired effect. There are assisted enhancement in natural dye different kinds of mordant as metallic extraction from beetroot for industrial mordants, tannin and oil mordants that have applications and natural dyeing of leather. been generally used for fabric dyeing by 4.2) Enzyme assisted extraction natural dye for a long time. Similarly, they This method uses enzymes (pectinase, are also added in the histological staining cellulose, protease, esterase etc.) for dye protocols when the plant dyes are used (Al extraction. The enzymes are usually applied Tikritti and Walker, 1977; Avwioro et al., as a pre-treatment of plant materials before 2007). The metallic mordants are common subjecting the plant material to solvent used for tissue staining. extraction. Various enzyme combinations are Metalic mordants are polyvalent metal used to loosen the structural integrity of ions such as metal salts of aluminium, plant materials thereby enhancing the potassium, chromium, iron, copper and tin. extraction of the desired color components. The two most common metals used in Conditions for optimum activity and natural dyeing are aluminium and ferric ions selection of the right type of enzymes are that having valences of three. Two types of essential to use them effectively for bonds are involved in the reaction between extraction (Sowbhagya and Chitra, 2010). a mordant dye and a mordant. One is Ultrasound assisted and enzyme assisted covalent bond and the other is coordinate extraction can be used in combination in bond. Different mordants give different hue order to improve the extraction efficiency. colors with the same dye. Metalic mordants are divided into two types as brightening 73 Araya Suebkhampet and Nattakarn Naimon / J. Mahanakorn Vet. Med. 2014. 9(1): 63-78. and dulling mordants. (potassium histological staining. There are many studies aluminum sulfate), potassium dichromate that investigated plant dye staining in and stannous chloride are brightening diverse tissues as follow. The dried skin of mordants while copper sulphate and ferrous (Punica Granatum) fruit extract sulfate are dulling mordants Prabhu and was used for rat brain staining and the Bhute, 2012). Although metallic mordants yellow staining was detected in the are effective, they are relatively toxic and astrocytes, neurons, red blood cells and environmentally pollutants. Thus, the elastic fibers (Gharravi et al., 2006). Bassey et natural mordants are considered. The al. (2011) stained the sections of the testes sample of natural mordant is tannin which with the ethanolic extract of Curcuma longa. found in plant parts as Tara pods They used this extract as a counterstain for (Caesalpinia spinosa) and gallnuts from Rhus haematoxylin. Their results indicated that semialata. Ali et al. (2010) studied the effect the dye distinctly stained the seminiferous of tannic acid and metallic mordants on the epithelium and interstitium yellow that dyeing properties of natural dye extracted provided a good counter stain for from Acacia nilotica bark. haematoxylin. Kola nut (cola acuminata) extract was also used as a substitute to Discussion and Conclusion histological tissue stain eosin in rat tissues. The natural dyes have long been used The dye extract was detected in the in various purposes including tissue staining. cytoplasm of various tissues with yellow- However, their application has reduced since brown color (Shehu et al., 2012). the synthetic dyes were developed. Suabjakyong et al. (2011) investigated the Haematoxylin is an example of natural dye extraction of dye from black plum fruit that widely used in histology, histopathology (Syzygium cumini) using various solvents and and histochemistry. At present, many its staining property on the rat hepatic commercial synthetic and some natural dyes tissue. used for tissue staining are available in the Suebkhumpet and Sotthibandhu markets. However, the hazardous effects of (2012) used aqueous extract of butterfly pea synthetic dyes on human and environment flowers (Clitoria ternatea) to stain blood caused scientists to concern about using the cells in different animal peripheral blood natural dyes instead of synthetic dyes. smears. They presented that the dye extract Therefore, alternative natural dyes have could stain and differentiate blood cells on been studied for their potential use in the blood smears. Jan et al. (2011) studied

74 Araya Suebkhampet and Nattakarn Naimon / J. Mahanakorn Vet. Med. 2014. 9(1): 63-78. the staining effect of dry extracted from dry mordants that applied in the dye solutions leaves of henna (Lawsonia intermis) on may cause health and disposal problems. histological sections of angiospermic stem. Therefore, searching for the new natural Okolie (2008) studied the staining of ova of mordants is an option for safer tissue intestinal parasites with extracts of Hibiscus staining. Many factors should be considered, Sabdariffa and Azadirachta Indica. The plant if the natural dyes are used for tissue extract produced satisfactory staining in staining. One of them is the source of plants comparison to conventional methods used selected, it should be available in the in identifying intestinal parasites on a endemic region in order to reduce the cost preparation of stool sample. Ihuma et al. and to avoid raw materials insufficiency. The (2012) used methanolic extracts from known chemical structures and properties of Hisbiscus sabdariffa as a biological staining some plant dye extracts are also important agent for some fungal species. Their results and useful for determining the optimal suggested that the dye extract could be use extraction and tissue staining procedures. as a mycological stain. Al-Amura et al. (2012) More detailed studies are needed to studied the staining technique for evaluate the potential and availability of helminthes by using red beet (Beta vulgaris) natural dyes-yielding resources in many extract. They reported that stained others plants. In addition, biotechnology and helminthes were acquired a good stain with various fields of studies are required to distinction their internal structures. Tousson increase the quantity and improve quality of and AL-Behbehani (2011) used black plant natural dyes. More study about mulberries as a natural dye for tissue staining procedures of each dye plant still staining. be required to establish new permanent Although using natural dyes have preparations in the future. many advantages, there still be some limitations that should be concerned. They References are difficult to standardize the dye and its Al-Amura, M. F.A., Hassen, Z.A. and AL- application because dyes collected from Mhanawi, B.H. 2012. Staining technique similar plants or natural sources vary due to for helminth parasites by use red beet climate, soil, maturity period and cultivation (Beta Vulgaris L.) extract. Bas. J. Vet. methods etc. Moreover, most natural dyes Res. 11(1): 283-292. require mordants for fixing them on the Ali, A., Ali, S., Saleem, H. and Hussain, T. stained tissues. The widely used metallic 2010. Effect of tannic and metallic 75 Araya Suebkhampet and Nattakarn Naimon / J. Mahanakorn Vet. Med. 2014. 9(1): 63-78.

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