Dual Staining Effect of Extracts Roselle (Hibiscus Sabdariffa L.) and Red Cabbage (Brassica Oleracea) on Wistar Rat Tissues

Background: Apart from the areas where natural dyes are used a lot, if staining studies on tissues increase, their use in pathology laboratory will become widespread. Two basic elements in cell tissues, the nucleus, and cytoplasm, should be stained and evaluated together for histopathological diagnosis. The most commonly used dye combinations for this diagnosis are Hematoxylin-Eosin dyes, also known as nuclei and cytoplasm dyes. Histological staining studies of natural origin did not reveal staining studies on the same tissue with the extracts of two plants. Methods and Results: In this study, roselle (Hibiscus sabdariffa L.) and red cabbage (Brassica oleracea) leaves were used as a source of natural dye and also liver and kidney tissues of Wistar albino rat as dye biomaterials. The nuclei and cytoplasm regions of rat kidney and liver tissues were stained in different colors with these plants and the use of metal salts. Relying upon hematoxylin-eosin should it ever be scarce will result in the scarcity of plant-based dyes on the market. Conclusion: Since a more colorful, different nucleus and cytoplasm staining was obtained compared to previous work with the same tissues. It is expected that the introduction of new natural dye alternatives where histological dye production is not widespread will contribute to the world’s economy and industry.


Introduction
Natural dyes, generally supposed to be a cheap, non-toxic, renewable and sustainable resource with minimal environmental impact, have attracted the attention of the scienti c community to use them in a variety of traditional and newly discovered application disciplines [1]. It is known that natural dyes have been used for coloring Textil materials [2]. However, there are also studies with natural dyes on the staining of tissues, which are a different discipline [3]. Biopsy specimens or tissue fragments are taken from animals or humans in medical and research laboratories. These are kept and processed with many chemicals in short periods in the pathology laboratory. The purpose of this process called tissue followup is to clean and polish the sections to be taken from the tissue samples and make them suitable for examination and staining. Tissue tracking consists of three basic stages.
Step 1 is the dehydration, which is the removal of water from the tissues. During the process, the tissues are kept in stepwise ethyl alcohol solutions from 70 % to absolute alcohol. In Step 2, the water is removed but the alcohol remains in the tissue. Tissues are held in toluene and chloroform, organic apolar solvents. In the third and nal step, the rigidity of the tissue is necessary for subsequent thin sectioning. For this purpose, melted para n tissues are left in cassettes. When the para n has hardened the tissue is ready for a microtome to cut through the para n. Tissue sections with para n are transferred to slides. Before using chemical and dye solutions, the para n must be dissolved [4,5].
The bark can be processed with a mix of hot water and urea and to obtain the dye called hematoxylin.
Hematoxylin is used in cell staining, after oxidation, it can be used in cell staining. It is available under different names since the durability of dye obtained when exposed to natural chemical oxidation and the color shades in the tissue are different. Metal salts known as mordant, alum, iron sulfate, tungsten, molybdenum, and lead salts can be used since the oxidized dye cannot stain the tissue alone. In particular, nucleus dyeing does not take place without iron sulfate [6][7][8].
Each medicinal laboratory has its own hematoxylin-eosin (HE) dual staining procedure that starts with xylene. For example, the HE dual staining method routinely used in Ankara University. This method is applied similarly in many laboratories around the World [9, 10]. Studies using water and ethanol extracts of Hibiscus sabdariffa in tissue dyeing are more numerous than dyeing with red cabbage leaves (RC). In these studies, Hibiscus sabdariffa L. (HS) extract and pink-light red staining were performed in the cytoplasm [11,12]. Red cabbage leaves extract was used as a drug dye [13]. In addition to tissue staining, there are many studies in the literature regarding the determination of biological activities of plant extracts [14][15][16][17].
In this study, based on the hematoxylin-eosin dual staining method, which is used routinely, studies were performed using alum and ferrous salts in red cabbage extract instead of hematoxylin and alum salt in Hibiscus sabdariffa extract instead of eosin. The colors given by hematoxylin and eosin in the nucleus and cytoplasm were taken as examples. An environmentally friendly, natural sourced dye combination was created.

Materials And Methods
Hibiscus sabdariffa L and red cabbage (Brassica oleracea) leaves were obtained from the local markets of Kayseri. Preparing of two plant extracts was taken place like below. All the chemicals were used in analytical pure. Alum (KAl SO 4 .12H 2 O) was used 5-10g/ in 50ml extract. FeSO 4 .5H 2 O was used 10-20 g/ in 50ml extract. DDW water.

Tissue Supply
In this study, 1 Wistar albino female rat, weighing 150-250 g and raised at the Erciyes University Experimental and Clinical Research Center (DEKAM) was used. This study was approved by the Erciyes University ethics committee dated 16 November 2016 and numbered 16/144. The rat was kept in cages, at 21°C with normal daily organization, 12 h of light/dark, and was fed and watered appropriately. The rat was put to euthanized with ketamine + xylazine and its tissues were taken. All of the procedures were performed according to ethical guidelines [3].

Preparation of Hibiscus sabdariffa L. Extract
The dried of Hibiscus sabdariffa L. was ground to a dark red-black powder using a manual grinding machine (Waring, commercial). 50 g of the dry powder of the plant was weighed (Schimadzu bl 3200).
The powder was put in 100mL chloroform, boiled for ve minutes. Distilled water was added to the remained solution. This dye solution was used directly or stored in a refrigerator at 4°C and then was used for staining.

Preparation of Red Cabbage Leaves Extract
Red cabbage leaves were ground to a dark red-black powder with the use of a manual grinding machine (Waring, commercial). The dry powder of the plant was weighed at 50 g (Schimadzu bl 3200). The powder was left in 100mL water for 24 hours at room temperature. Then the extract (50 % w/v) was ltered two times with Whatman No.1 lter paper. The Filtrate was boiled until half of the water had evaporated. The remaining blue-purple colored ltrate was used as the dye solution. The ltrate was stored in the refrigerator at 4°C and then was used for staining [12,3].

Preparation of the Tissue Preparates
The tissue samples taken from the rats were xed in a 4% neutral buffered formaldehyde solution. After xing, routine histological tissue follow-up steps were performed. The tissue samples were rst dehydrated through an increasing series of ethyl alcohol made transparent with xylene, and then blocked by burial in para n. The 5 µm cross-sections taken from the tissue para n blocks for dyeing were then applied the staining procedure to see the effect of the Hibiscus sabdariffa L. and red cabbage leaves. The intensity of staining was evaluated as negative (−), weak (+), medium (++), and very intense (+++). 3 The best staining intensity (+++) in the evaluation of the staining result was considered as H&E stained tissue.
Others have been evaluated accordingly.

Depara nization of Tissue Preparation
Para n was removed from the preparate by the classical method. Preparates were left in an oven at 60 C for 30 minutes. They were removed from the oven and allowed to cool to room temperature for 3-5 min and put them into the xylene [8].
1. Immersed in Xylene for 3 min, and repeated three times 2. Immersed in E. Alcohol for 3 min, and repeated three times (from 70% Ethyl Alcohol to 96 %) 3. Washed with distilled water for 3min. 4. Immersed in Hematoxylin solution for 6-8min 5. Washed with distilled water for 3 min 6. Immersed in 1% Ammonia water for 2 min 7. Washed with distilled water for 3 min 8. Immersed in Eosin solution for 10 -12min 9. Washed with distlled water for 3 min 10. Immersed in E. Alcohol for 3 min, and repeated three times (from 96% Ethyl alcohol to 70%).

Phytochemical Analysis of extracts
The phytochemical analysis steps below were applied separately to the extract of Red cabbage obtained in water and the chloroform extract of Hibiscus sabdariffa [18].
Test for Saponin. 2 ml extract was placed in a test tube and shaken vigorously. The formation of stable foam was taken as an indication of the presence of saponin.
Test for Phenol and Tannin, 2 ml extract was mixed with 2 ml of 2 % solution of ferric chloride (FeCl 3 ). A blue-green or black coloration indicated the presence of phenol and tannin.
Test for Terpenoid (Salkowski's Test), 2 ml extract was mixed with 2 ml of chloroform. Then 2 ml of concentrated sulphuric acid was added carefully and shaken gently. A reddish-brown coloration of the interphase was formed to show positive results for the presence of terpenoid.
Test for Flavonoid (Zinc-Hydrochloride reduction test), 2 ml extract was mixed with zinc dust and concentrated hydrochloric acid was added to it dropwise. It gave red color after a few minutes indicating the presence of avonoid.
Test for Glycoside, 2 ml extract was mixed with 2 ml of glacial acetic acid containing 2 drops of 2 % FeCl 3 . The mixture was poured into another tube containing 2 ml of concentrated sulphuric acid. A brown ring at the interphase indicated the presence of glycoside [18].

TLC analysis of extracts
The extracts of Hibiscus sabdariffa and red cabbage were subjected to thin layer chromatography solvent system. The fractionation patterns of the extract produced by different solvent mixtures. But, Ethyl acetate: Hexane (1:4) as optimal solvent produced sharp bands with good resolution. Red-pink medium intensity (++) staining was performed with the Hibiscus sabdariffa instead of eosin and also medium (++) purple-colored staining was done with red cabbage instead of hematoxylin in the nucleus. Microscopic examination results were shown in Table 1.

Results
The extracts of basil and alkanet plants instead of red cabbage were used for nucleus staining since they were purple-blue in color, but they did not give positive results (-) in staining. The cell cytoplasm was stained by chloroform extract of the Hibiscus sabdariffa L. When the extracts of the Hibiscus sabdariffa and red cabbage were applied to the tissues without using mordant, no positive results (-) were obtained (see Dyeing process 1). Photographs of negative tissue staining studies are not included in this study. But positive results were shown in Fig. 1 and Fig. 2. The result of phytochemical analysis (in Table 2) applied to red cabbage and Hibiscus sabdariffa extracts showed that the extracts had different chemical structures. This analysis showed that the nucleus was stained by the avonoid groups in red cabbage extract and the cytoplasm was stained by the phenolic structures in the Hibiscus sabdariffa extract.
In TLC analysis, different color discrimination was observed in Hibiscus sabdariffa and red cabbage extracts running in the same solvent. There are two plants and two different dye molecules as shown in Fig. 3. in the middle of the picture, the purple-blue dye of red cabbage was been leading. while the pink dye of Hibiscus sabdariffa was left behind in the rst left.

Discussion
There are many tissue staining studies done with the extract of Hibiscus sabdariffa plant. In the study conducted by Bassey et al, Hibiscus sabdariffa extract was used to investigate the sperm morphology of Sprague Dawley rats. Wistar rat testis and ovarian tissues were stained by an ethanol extract of Hibiscus sabdariffa. In this process which was applied directly to tissues at room temperature, and in1 hour the cytoplasm had light purple staining [12]. In polar solvent and untreated tissue without chemicals before dyeing and without mordant, the dye was not permanent and the dyeing process was long. In this study, permanent light red ++ (medium) staining occurred in the cytoplasm in a shorter time by using a metal salt in chloroform-water Hibiscus sabdariffa extract on tissues exposed to chemicals using routine H&E staining process steps (Dyeing process 2).
In another study, light and pale red staining occurred in the liver, kidney, heart, and lung tissues of Wistar albino rats with an aqueous extract without mordant of Hibiscus sabdariffa in the cytoplasm. 3 Thus, in this study, it was decided to apply the Hibiscus sabdariffa on the same tissues as a cytoplasm dye. Water, alcohol and chloroform extracts of basil, alkanet and red cabbage were used to stain the nucleus in hematoxylin color. Then, iron sulphate, tin chloride and alum salts were added to the extracts, it was seen that the aqueous extract of red cabbage including the salt pair (FeSO 4 . 5H 2 O + alum) stained the nuclei of the liver and kidney tissues. The details of the other plant extracts were not included. Because there was no strong dual staining until the red cabbage. The red cabbage solution containing (ferrous and alum) salt pair after waiting for 1 week in a dark place, the nucleus appeared, and purple color intensity of the nucleus was increased. After testing Hibiscus sabdariffa and red cabbage extracts in individual tissues, routine H&E staining procedure steps were used to provide a dye combination similar to hematoxylin-eosin. The presence of different avonoid dye groups in the dye solutions subjected to the same process has affected the dyeing results like shown in Fig. 3. Different terpenoid content of extracts was affected by the staining result. So, different regions on tissues were separately stained by two extracts. Thus, since the dye in the two extracts showed bounding with different places in the cell, Hibiscus sabdariffa extract stained cytoplasm. Red cabbage extract also stained the nucleus. Just like the difference in Hematoxylin and Eosin dye structures, they dyed different regions in the same tissue. In this study, a new natural dye pair (RC-HS) has been proposed instead of the Hematoxylin-eosin dye pair commonly used in medical laboratories. We hope that this will spread the use of natural dyes in medical elds outside of industrial areas.

Declarations Funding
No.