Exploring the Staining Potential of Zingiber Officinale Extracts (Ginger) as a Natural Alternative to Eosin in Haematoxylin & Eosin Staining: A Cross-sectional Study
Correspondence Address :
Mitulkumar Rameshbhai Prajapati,
Associate Professor, Department of Oral Pathology and Microbiology, Ahmedabad Dental College and Hospital, Near Science City, Bhadaj, Ahmedabad, Gujarat, India.
E-mail: opadc08@gmail.com
Introduction: Most synthetic dyes are carcinogenic, and chronic exposure to these dyes has an impact on the health of laboratory technicians and pathologists. Eosin, a widely used synthetic dye in routine histopathological staining, poses potential risks. Zingiber officinale contains phenols and several colouring compounds that have the ability to stain tissues.
Aim: To explore and compare the staining efficacy of ginger extract as a natural dye with synthetic eosin dye.
Materials and Methods: This cross-sectional study was conducted at the Oral Pathology Department of Ahmedabad Dental College and Hospital from April 2023 to June 2023. Fresh rhizomes of Zingiber officinale were collected and air-dried. A staining solution of Zingiber officinale was obtained by dissolving 25g of powder in 90% alcohol. This solution was used to stain 30 sections of oral biopsy specimens. The stained slides were evaluated by two independent observers using various parameters such as nuclear and cytoplasmic detail, overall histologic appearance, intensity, and contrast. Statistical significance was determined using the Chi-square test.
Results: Z.officinale (Ginger) stain the cytoplasm of the cell and connective tissue elements with pale eosin colour, yellowish golden to the RBCs and deep brownish to the bony tissue. Statistical analysis comparable staining intensity, contrast, nuclear staining, cytoplasmic staining and overall histologic appearance between the two groups with p-values of 0.531, 0.917, 1.000, 0.924 and 0.7003, respectively.
Conclusion: Z.officinale (Ginger) can be utilised as a natural alternative to eosin in routinely used Hematoxylin and Eosin staining.
Histopathology, Natural histopathological stain, Plant extracts, Zingiberaceae
Nature has provided us with many resources, and one of them is colour (1). Stains are dyes that colour tissues to facilitate the optical differentiation of tissue components. Hematoxylin and eosin are globally used stains in histopathology. Hematoxylin is a natural dye, while eosin is a synthetic dye (2). Synthetic dyes, despite their effectiveness, have been reported to pose risks to human health. Additionally, the scientific community has experienced repeated shortages of raw materials for stains, especially during times such as pandemics. The development and use of non-toxic, environmentally friendly dyes have garnered significant attention in laboratories worldwide (3).
Impaired respiratory function, airway diseases, and conditions like emphysema are some of the toxic effects of eosin. Direct exposure to open cuts, abraded or irritated skin can lead to systemic injury with harmful effects. Furthermore, eosin can irritate the skin, eyes, and mucous membranes, leading to conditions like chelitis, stomatitis, and dermatitis (4).
The rhizome of Zingiber officinale, a member of the Zingiberaceae family, has a deep yellow colour. Zingiber officinale, also known as ginger, is commonly used as a whole medicinal plant, as a spice in cooking, in beverage manufacturing, and for making tea. Flavonoids, a polyphenolic compound, contribute to its acidic nature, and colouring compounds that impart colour are found in Zingiber officinale (5). To our knowledge, there are only a few studies available that explore the staining potential of ginger. Ajileye AB et al., concluded that the natural histological dye extracted from Z. officinale can effectively stain both muscle fibers and the cytoplasm of a tissue section (6). Sudhakaran A et al., conducted a study to assess the staining efficacy and shelf life of Z. officinale and found it to be considerably longer compared to Curcuma longa (5). The current study emphasises the innovative application of ginger extract in histopathology as a natural alternative to eosin and aimed to evaluate the staining characteristics of ginger extract when used as a counterstain for hematoxylin in Hematoxylin and Eosin staining.
During a three-month period from April 2023 to June 2023, this cross-sectional study was conducted at the Oral Pathology Department of Ahmedabad Dental College and Hospital, Ahmedabad, Gujarat, India. The study received approval from the Institutional Ethics Committee, with the number ADC/IHEC/27.
Inclusion criteria: Well-fixed pathologically diagnosed benign tissues with adequate tissue structure were included in the study.
Exclusion criteria: Biopsies that were not properly fixed, processed, or had inadequate tissue structure, as well as, normal and malignant tissues, were excluded.
From the archives of the Department of Oral and Maxillofacial Pathology, 30 paraffin-embedded tissue blocks of oral biopsy specimens were retrieved. Two sections with a thickness of 5 μm were prepared from each tissue block.
Procedure
Steps in the dye preparation of Zingiber officinale (Ginger) dye: Fresh rhizomes of Zingiber officinale were collected from the local market of Ahmedabad, cut into pieces, air-dried, and then ground into a powder using a mechanical grinder. A total of 25 g of Zingiber officinale powder was diluted in 100 mL of 90% alcohol. The prepared solution was left for 24 hours and then filtered using Whatman’s filter paper No.1 to collect the supernatant fluid (5). The supernatant fluid was obtained using a micropipette and kept in a coupling jar for staining (Table/Fig 1).
Group-I (Control Group): 30 slides were stained with conventional Haematoxylin and eosin staining method (H&E) (Table/Fig 2) (7).
Group-II (Experimental Group): 30 slides were stained using Hematoxylin and Ginger (H&G) (5).
Procedure for staining with Zingiber officinale (5): After dewaxing the tissue sections on a hot plate, they were cleared in xylene for 15 minutes. Hydration was done using 80% alcohol, followed by rinsing with water. The sections were stained with Harris hematoxylin for five minutes and then rinsed in running water. Differentiation was performed using 1% Acid alcohol for 2-3 seconds, followed by bluing for 15 minutes in running water. The sections were counterstained with the prepared Zingiber officinale stain for eight minutes, rinsed in water, cleared in xylene, and mounted.
Histopathological evaluation: Two blinded oral pathologists independently evaluated the slides and scored them according to the criteria used by Raju L et al., (Table/Fig 3) (8).
Statistical Analysis
The overall scores of the two observers were analysed using a Chi-square test, with statistical significance considered when the p-value was <0.05. International Business Management (IBM) Statistical Package for Social Sciences (SPSS) software was used to conduct all statistical analyses.
The authors observed that Z. officinale stained the tissue in a manner closer to eosin. Z. officinale stained the cytoplasm of the cells and connective tissue components like nerve bundles, muscle fibers, collagen fibers, and blood vessels with a pale eosin colour (Table/Fig 4). It imparted a yellowish-golden colour to the red blood cells and a deep brown colour to the bony tissue (Table/Fig 5).
Observer 1 scored 29 (96.67%) slides of H&E and 27 (90%) slides of H&G as having an adequate overall histologic appearance (Table/Fig 6). Observer 2 scored 27 (90%) slides of H&E and 25 (83.33%) slides of H&G as having an adequate overall histologic appearance (Table/Fig 7). Observer 1 scored 13 (43.33%) slides of H&E and 12 (40%) slides of H&G as excellent (Table/Fig 8). Observer 2 scored 13 (43.33%) slides of H&E and 14 (46.67%) slides of H&G as excellent (Table/Fig 8).
Cross-tabulation of scores showed no significant difference between H&E and H&G groups for staining intensity, contrast, cytoplasmic and nuclear staining, and overall histologic appearance for both observer 1 (Table/Fig 6) and observer 2 (Table/Fig 7) (p>0.05). The total scores between the H&E and H&G groups were comparable (p>0.05) (Table/Fig 8). The comparison of mean scores for staining intensity, contrast, cytoplasmic and nuclear staining, and overall histologic appearance showed no significant difference between the H&E and H&G groups, with p-values of 0.531, 0.917, 1.000, 0.924, and 0.703, respectively (Table/Fig 9).
Eosin, which is a synthetic Xanthene dye, is a highly effective stain but has harmful effects on both animal and human health. Constant exposure to chemicals from synthetic stains, such as eosin, can lead to health issues for pathologists, technicians, and others. Long-term exposure to eosin can result in cheilitis, stomatitis, dermatitis, and other health problems due to its irritating effects on the skin, eyes, and mucous membranes. In order to minimise health hazards, consumers are now more inclined to use natural dyes, as plant products are readily available (9). Natural dyes can be derived from various parts of plants such as leaves, flowers, fruits, barks, and roots. The search for biodegradable and safe dyes from natural sources has been prompted by the negative impact of chemicals and synthetic dyes on the environment (10). Additionally, the rising cost of synthetic dyes has made the use of cheaper naturally occurring dyes more popular, especially in developing countries (11). Natural dyes have been widely used in histology, histochemistry, and histopathology for diagnostic purposes (12). The flavonoids present in Z. officinale are polyphenolic compounds with an acidic nature that have the ability to release hydrogen and can stain the basic structure of cells (6).
Several researchers have conducted studies on natural dyes for use in histopathology. (Table/Fig 10) provides a comparison of parameters using different natural dyes in various studies, including the present study (5),(11),(12),(13),(14). To avoid bias, the slides were assessed by two independent observers, and they found that the quality of staining with Z. officinale did not interfere with the diagnostic process, and the stain did not fade even after a month. Z. officinale stained the cytoplasm and components of connective tissues like collagen fibers, muscle fibers, nerve bundles, and blood vessels, with a pale eosin colour and RBCs with a yellowish-orange shade, which was similar to the findings of Sudhakaran A et al., and Ajileye AB et al., (5),(6).
Sudhakaran A et al., found that ginger staining was superior to turmeric staining, and the p-values for overall histologic appearance, crispness of staining, background staining, accuracy for diagnosis, and intensity of stain were 0.364, 0.154, 0.071, 0.583, and 0.583, respectively (5). In the present study, the statistical analysis showed p-values for staining intensity (p=0.531), contrast (p=0.917), nuclear staining (p=1.000), cytoplasmic staining (p=0.924), and overall histologic appearance (p=0.7003) that were greater than 0.05. This suggests that ginger extract could be a potential alternative to eosin in histopathological staining procedures.
Limitation(s)
Due to the three-month duration of the present study, the durability of the stained slides was not evaluated. Additionally, the study did not include normal tissues or malignant neoplasms.
The staining potential of H&G was comparable to H&E, indicating that Z. officinale (Ginger) could be an efficient counterstain in place of eosin for demonstrating various structures in histopathology. It proves to be cost-effective, readily available, and biodegradable. However, further studies are needed to evaluate the utility of ginger in staining normal and malignant tissues, as well as to assess the durability of the stained slides.
DOI: 10.7860/JCDR/2023/66801.18628
Date of Submission: Jul 31, 2023
Date of Peer Review: Sep 08, 2023
Date of Acceptance: Sep 30, 2023
Date of Publishing: Oct 01, 2023
AUTHOR DECLARATION:
• Financial or Other Competing Interests: None
• Was Ethics Committee Approval obtained for this study? Yes
• Was informed consent obtained from the subjects involved in the study? Yes
• For any images presented appropriate consent has been obtained from the subjects. NA
PLAGIARISM CHECKING METHODS:
• Plagiarism X-checker: Aug 03, 2023
• Manual Googling: Sep 09, 2023
• iThenticate Software: Sep 23, 2023 (5%)
ETYMOLOGY: Author Origin
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