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PHARMACEUTICAL STUDY
Year : 2018  |  Volume : 39  |  Issue : 4  |  Page : 220-225  

Natural stains Zingiber officinale Roscoe (ginger) and Curcuma longa L. (turmeric) – A substitute to eosin


Department of Oral Pathology and Microbiology, SDM College of Dental Sciences and Hospital, Dharwad, Karnataka, India

Date of Web Publication5-Jul-2019

Correspondence Address:
Dr. Kaveri Hallikeri
Department of Oral Pathology and Microbiology, SDM College of Dental Sciences and Hospital, Dharwad - 580 009, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ayu.AYU_232_17

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   Abstract 


Background: Stained slides are of the utmost source of information in pathology. The routine stains utilize synthetic dyes which pose various health hazards which necessitate the importance of natural extracts as a histological stain. Aims: The aim of this study is to compare the efficacy of ginger and turmeric extracts as histological dyes in tissues keeping eosin as standard. Objectives: The objective of this study is to compare the staining potential, intensity, specificity, and shelf life of ginger and termuric extract dyes. Materials and Methods: Extracts of fresh rhizomes of Zingiber officinale Roscoe and Curcuma longa L. were obtained by dissolving them in alcohol, which was used to stain sections of 25 cases. Observer 1 compared the staining intensity, while observers 2 and 3 assessed preset parameters at regular intervals and subjected to Kappa statistics and Mann–Whitney U-test. Results: Z. officinale and C. longa solutions had pH 4–5 and temperature 24°C–27°C. Z. officinale showed better staining intensity and specificity to the cytoplasm and basic components of connective tissue as compared to C. longa. Crispness (P = 0.01) and background staining (P = 0.05) showed a significant difference. Shelf life of Z. officinale was better than C. longa. Conclusion: Z. officinale gave a better staining than C. longa and was found to be closest to eosin and is easily available, cost-effective and biodegradable.

Keywords: Curcuma longa, eosin, natural stains, Zingiber officinale


How to cite this article:
Sudhakaran A, Hallikeri K, Babu B. Natural stains Zingiber officinale Roscoe (ginger) and Curcuma longa L. (turmeric) – A substitute to eosin. AYU 2018;39:220-5

How to cite this URL:
Sudhakaran A, Hallikeri K, Babu B. Natural stains Zingiber officinale Roscoe (ginger) and Curcuma longa L. (turmeric) – A substitute to eosin. AYU [serial online] 2018 [cited 2019 Jul 19];39:220-5. Available from: http://www.ayujournal.org/text.asp?2018/39/4/220/262156




   Introduction Top


Stains are an integral part of the laboratory techniques in histology. Among the enormous number of dyes used in histopathology, hematoxylin (H) and eosin (E) dyes remain to be the gold standard for routine staining. Hematoxylin is a natural dye obtained from the barks of Haematoxylon campechianum, while eosin is a synthetic dye.[1] Most of the stains used in histology are synthetic dyes. Furthermore, the chemicals used for procedures prior to staining use chemical agents. Preparation of these dyes also requires treatment with chemical agents to finally formulate the end product, i.e., the dye available to us in the laboratories.[2]

Synthetic dyes are often efficient but may display hazards to human and animal health. This has led to awareness about these hazards and also to an extent the withdrawal of some of them.[3] Frequent exposure to these chemicals constantly affects the health of the laboratory technicians, pathologist and mainly those working in the laboratory. Moreover, many developing countries cannot meet the increasing costs of synthetic dyes. Not only are synthetic dyes expensive but they are also nonbiodegradable.

Various studies have demonstrated the use of natural dyes for use in plant as well as animal histological studies and have found satisfactory results in comparison to the synthetic stains used.[2],[4],[5] Despite various shortcomings with the use of natural stains, they can still be sought as an analogous to the conventional stains used.[6] However, continuum of studies using natural dyes in human tissues is very narrow and very minimal data in this regard is available in the literature.

Hence, a need for the substitution of synthetic dyes with natural extracts of plants that are both eco-friendly and cost-effective has gained importance.

Based on the aforementioned, the extracts of Zingiber officinale (G) and Curcuma longa L (T) were used as a histological dye to stain various tissues as an alternative to routinely used eosin. In the present study, the staining potential of Z. officinale (ginger) and C. longa L (turmeric) was compared. The staining intensity and specificity of these dyes were compared with the routine hematoxylin and eosin (H and E) staining. Finally, the efficacy and shelf life of these dyes were compared with each other.


   Materials and Methods Top


The extracts of C. longa L and Z. officinale were obtained by standardized procedure given by the authors Ajiliye and Kumar et al., respectively.[4],[5] The extract was used to stain the routine biopsy samples received in the department of oral pathology and microbiology.

Steps in extraction of Zingiber officinale staining solution

Fresh rhizomes of Z. officinale were obtained and washed well to remove the dirt and other impurities. The rhizome was then peeled and cut into small pieces, after which 25 g of these pieces were mixed with 100 ml of 90% alcohol. After 24 h, this mixture was filtered to obtain 80 ml of extract of Z. officinale which was used for staining.

Steps in extraction of Curcuma longa L staining solution

Fresh rhizomes of C. longa were collected, cut into small pieces and dried. These pieces were finely powdered. Twenty grams of the powder was dissolved in 100 ml of 70% alcohol and left for 24 h. The supernatant was then separated and 70 ml of C. longa extract was obtained which was used for staining.

Sample collection

Paraffinized blocks of five cases, each of buccal mucosa, lichen planus, fibroma/pyogenic granuloma, salivary gland and squamous cell carcinoma, were obtained for the study. Three sets of tissue sections of about 3–5-μm thickness, from each case, were obtained using a semiautomatic soft-tissue microtome. One set of tissues was stained with routine H and E stain using standard staining procedure. The prepared solutions of Z. officinale and C. longa were used in place of eosin to stain total 25 tissues each following the standardization of the staining procedure.

The parameters assessed during staining were the pH of the stain, room temperature, concentration of the staining solution and the shelf life of the stained slides. The values are tabulated in [Table 1].
Table 1: Parameters of solutions assessed during the staining

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Staining procedure using Zingiber officinale

Tissue sections were dewaxed on a slide warmer and cleared in xylene for 15 min., there after it was hydrated in 80% alcohol and rinsed in water, then stained with Harris hematoxylin for 5 min and rinsed in running tap water. It was then differentiated in 1% acid alcohol for 2–3 s and bluing in running tap water for 15 min was done. It was thereafter, counterstained with 90% ethanolic extract of Z. officinale stain for 8 min. Sections were finally rinsed in water, cleared in xylene and mounted in DPX mountant.

Staining procedure using Curcuma longa L

Staining procedure which was similar to Z. officinale was followed till step 5, i.e., bluing, after which the sections were counterstained with 90% ethanolic extract of C. longa stain for 9 min, thereafter rinsed in water, dehydrated in 80% absolute alcohol, cleared in xylene and mounted.

Observer 1 assessed the staining intensity in the layers of the epithelium, the crispness of staining of the cytoplasm and nucleus and the different components of the connective tissue such as the collagen fibers, muscle, nervous tissue and blood vessels.

Tissues stained with Z. officinale and C. longa were compared simultaneously and scored as good and average based on the comparative staining results using the eosin stain as standard [Table 2].
Table 2: Staining of tissue including the epithelium and connective tissue component by observer 1

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Observers 2 and 3 were given a set of coded slides (to avoid bias) to observe and assess them based on the following preset qualitative parameters:

  1. Overall experience of viewing the slides over H and E – Good, average and bad
  2. Study of the tissue components – Yes/No
  3. Crispness of the stain – Yes/No
  4. Background staining observed – Yes/No
  5. Diagnosis of the lesion – Yes/No.


The same observers were asked to re-assess these slides after a period of 15 and 30 days to check any difference in intensity of the stained slides as compared to that observed immediately after staining.

Additional parameter was added for reassessment after 15 and 30 days:

  1. Intensity of staining – Average, good, faded and same as before.


The results based on the above mentioned criteria were tabulated for both Z. officinale and C. longa. The data was subjected by means of IBM SPSS Statistics 19 version, and interobserver variability was done using Kappa statistics. Level of significance was set a 5% (P < 0.05). Furthermore, the individual results of observers 2 and 3 for both the stains were analyzed using Mann–Whitney U-test. Ethical clearance was obtained from the Institutional Review Board for the study (IRB. No-2017/P/OP/54).


   Results and Observation Top


The pH of both the stains was between 4 and 5, while the room temperature recorded during staining ranged between 24°C–27°C.

The concentration of Z. officinale staining solution (checked by comparing the slide stained on the 1st day with that stained after a month) was found to be the same even after a month, while it was observed that the C. longa staining solution had dried up after 20 days and had to be replaced with a fresh solution [Table 1]. Observer 1 compared the staining intensity and crispness of staining between Z. officinale and C. longa-stained slides. It was found that Z. officinale had good staining intensity and crispness in contrast to C. longa, which was average as compared to the standard staining of eosin [Table 2].

Z. officinale stain imparted pale eosin color to the cytoplasm and connective tissue components such as collagen fibers, sebaceous gland, nerve bundles and blood vessel [Figure 1]. C. longa stained tissues had cytoplasm and connective tissue elements yellow to golden yellow [Figure 2]. A comparison of cytoplasmic staining in the layers of the epithelium showed slight background staining with C. longa [Figure 3]. In case of lichen planus, staining with Z. officinale showed better results than that of C. longa [Figure 4]. Furthermore, in cases of fibroma, a crisp staining was noticed with Z. officinale stain, while C. longa stained section showed a yellowish tinge [Figure 5]. Also, for salivary gland and squamous cell carcinoma cases, staining with Z. officinale showed better results and tissues were easier to identify than with the C. longa stained slides. [Figure 6] and [Figure 7].
Figure 1: Photomicrograph showing cytoplasmic stain of the epithelium (a, ×10), connective tissue components such as nerve bundles, muscle fibers (b, ×20), sebaceous gland (c, ×40), and blood vessel (d, ×20), using Z. officinale stain

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Figure 2: Photomicrograph showing cytoplasmic stain of the epithelium (a, ×10 and c, ×20) connective tissue components such as collagen fibers, blood vessels (b, ×20), and muscle fibers (d, ×20), using C. longa stain

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Figure 3: Photomicrograph showing comparison of cytoplasmic staining in layers of the epithelium using Z. officinale (a) and C. longa (b) stain at ×20

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Figure 4: Photomicrograph showing comparison of staining in case of lichen planus using Z. officinale (a) and C. longa (b) stain at ×20

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Figure 5: Photomicrograph of comparative staining of fibroma using Z. officinale (a) and C. longa (b) stain at ×20

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Figure 6: Photomicrograph showing comparison of staining in salivary gland acini using Z. officinale (a) and C. longa (b) stain at ×20

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Figure 7: Photomicrograph showing comparison of staining in squamous cell carcinoma using Z. officinale (a) and C. longa (b) stain at ×20

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Findings of preset parameters were assessed by observers 2 and 3. Both the observers found the staining of Z. officinale better and more closer to eosin than C. longa. Observations by both the observers were in agreement for parameters 1, 2, 4 and 6. The overall experience of viewing the newly stained slides was found to be good for Z. officinale, while it was not as good as eosin for C. longa. Furthermore, the observers had a disagreement on their observations for parameters 3 and 5, i.e. the background staining and aid of the stains in the diagnosis of the lesion [Table 3] and [Table 4].
Table 3: Parameters assessed by observers 2 and 3 in Zingiber officinale- stained slides immediately after staining the slides and after 15 and 30 days

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Table 4: Parameters assessed by observers 2 and 3 in Curcuma longa-stained slides immediately after staining the slides and after 15 and 30 days

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Interobserver Kappa statistics showed a fair agreement for Z. officinale, while a slight to moderate agreement was found for C. longa [Table 5]. Observer 2 noticed statistically significant difference in the assessment of background staining at baseline between the two stains (P = 0.05). Post 30 days, overall experience of viewing the slides over H and E, crispness of staining and background staining showed statistically significant difference among the stains (P = 0.01). Observer 3 noticed that at baseline, there was a statistically significant difference in the overall experience of viewing the slides over H and E (P = 0.05). A study of tissue components (P = 0.01) was observed between the two stains. After 15 days, overall experience of viewing the slides over H and E (P = 0.05), crispness of staining (P = 0.01) and background staining (P = 0.05) showed a statistically significant difference. After 30 days, revaluation showed statistically significant difference in background staining among the stains (P = 0.01) [Table 6].
Table 5: Inter-observer agreement of parameters for Zingiber officinale and Curcuma longa stains (Kappa statistics)

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Table 6: The difference in the quality of assessment of Zingiber officinale and Curcuma longa stains between the observers (P)

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   Discussion Top


Z. officinale and C. longa contain flavonoids, a polyphenolic compound making it acidic in nature and several coloring compounds which impart color.[5],[7] Similar to eosin stain, extracts of Z. officinale and C. longa stained the different components in the tissues in varying shades of yellow while RBCs in golden yellow color.[4],[5],[7]

Greater the concentration, more dye molecules are bound to tissue components and it may change with consequent use. In the present study, 25 g and 30 g of Z. officinale in 100 ml of 90% alcohol were used and staining intensity with 25 g was found better. Kumar et al. (2014) used different concentrations of C. longa and concentrations below 15 g did not yield good staining.[4] Thus, in the present study, 20 g of turmeric was dissolved in 70% alcohol. In a study by Avwioro et al. (2007), the extract of C. longa was purified using column chromatography and divided into fractions of varying colors. The constituents essential for staining were not assessed in his study. These fractions were dissolved in 100 ml of various solvents and mordants. They found optimum staining with 70% ethanol, whereas other solvents and mordants did not contributed to the quality of stain.[7]

Optimum pH for appropriate staining of eosin is around 4.3. Staining intensity drops significantly above a pH of 5, while staining becomes dark below a pH of 4.[8] The pH of the two dyes was acidic (pH – 4–5), due to the ability of their phenols to release hydrogen ion from the hydroxyl groups. The affinity of both the dyes toward the cytoplasm, collagen, and muscle fibers was confirmed in the present study and by Ajileye and Avwioro.[5],[7]

Temperature increases the rate of diffusion of the dye into tissues. In this study, the room temperature was noted to be within the range of 24°C–27°C, similar to the previous study.[7] Another factor influencing the staining intensity is the time. Following standardization, Z. officinale was used for 8 min and C. longa for 9 min.[5]

In addition, the slides were assessed by two observers to avoid bias and assess the use of these stains in histopathological diagnosis. Observers found that both the stains did not interfere in diagnosis and agreed unanimously that Z. officinale has good staining quality than C. longa. The same slides when assessed periodically, only fading of C. longa-stained slides was seen. Observers agreed on Z. officinale having an advantage over C. longa and these stains were similar to eosin.


   Conclusion Top


Both the stains showed staining potential similar to eosin, but Z. officinale gave a better staining than C. longa and was found to be in closest resemblance to eosin. The efficacy and shelf life of Z. officinale were considerably more than that of C. longa. Moreover, owing to the easy availability, cost-effectiveness, fewer hazards and biodegradable nature, Z. officinale holds an upper hand over eosin. Nonetheless, adapting to skills for viewing and diagnosing using this new stain can be time-consuming.

Acknowledgment

I would like to extend my sincere gratitude toward Dr. Kriti, Department of Public Health Dentistry, SDM College of Dental Sciences and Hospital, Dharwad, for her valuable help in our statistical analysis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Culling CF, Allison RT, Barr WT. Cellular Pathology Technique. 4th ed. Wellington: Butterworth and Co., Ltd.; 1985: 148-49.  Back to cited text no. 1
    
2.
Akinloye AJ, Illoh HC, Olagoke AO. Screening of some indigenous herbal dyes for use in plant histological staining. J For Res 2010;21:81-4.  Back to cited text no. 2
    
3.
Bhuyan R, Saikia CN. Isolation of colour components from native dye-bearing plants in Northeastern India. Bioresour Technol 2005;96:363-72.  Back to cited text no. 3
    
4.
Kumar S, Singh NN, Singh A, Singh N, Sinha RK. Use of Curcuma longa L. extract to stain various tissue samples for histological studies. Ayu 2014;35:447-51.  Back to cited text no. 4
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5.
Ajileye AB, Iteire AK, Arigi QB. Zingiber officinale (ginger) extract as a histological dye for muscle fibers and cytoplasm. Int J Med Sci Public Health 2015;10:1445-8.  Back to cited text no. 5
    
6.
Ramamoorthy A, Ravi S, Jeddy N, Thangavelu R, Janardhanan S. Natural alternatives for chemicals used in histopathology lab – A literature review. J Clin Diagn Res 2016;10:EE01-4.  Back to cited text no. 6
    
7.
Avwioro OG, Onwuka SK, Moody JO, Agbedahunsi JM, Oduola T, Ekpo OE, et al. Curcuma longa extract as a histological dye for collagen fibres and red blood cells. J Anat 2007;210:600-3.  Back to cited text no. 7
    
8.
Kumar GL, Kiernan JA, Gill GW. Special stains and H&E Education Guide. Staining methods: Nucleus & cytoplasm. 2nd edition. California: Dako North America; 2010. 24-25.  Back to cited text no. 8
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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