Main Article Content

Shiny Jasphin
Archana Parampalli Raghavendra
Monica Charlotte Solomon
Arul Amuthan
Brij Mohan Kumar Singh
Nikitha Valerina Kairanna


Background: Many histological stains cause health hazards to technicians, pathologists, and researchers. The hazard-free and eco-friendly natural anthocyanins have the potential to be a new source for histological stains. This study aims to systematically review the use of plant products containing anthocyanin for histopathological diagnosis. Methods: A comprehensive literature search was done using suitable keywords on Wiley, PubMed, Scopus, Google Scholar, and Web of Science databases. A total of 30 articles were selected for systematic review, where data obtained from the studies were tabulated. Results: About 90% of the reviewed studies have proven that anthocyanin-containing plant products may be used as natural stains. Out of the 30 studies, 49% involved the use of Hibiscus extract, 11% utilised mulberry extract, 9% utilised pomegranate, another 9% involved rose, and the remaining ones utilised black plum, black rice, butterfly pea, the flame of woods, onion skin, and red poppy extracts. Almost 40% of the studies concluded that aqueous extracts are superior to alcohol ones, and 46% used either iron or alum as mordant. Conclusion: Natural stains containing anthocyanin could be a better alternative to synthetic histological stains. Further extensive studies should be conducted to observe the use of these stains in pathological diagnosis.


Download data is not yet available.

Article Details

How to Cite
Jasphin, S., Parampalli Raghavendra, A., Charlotte Solomon , M. ., Amuthan , A. ., Mohan Kumar Singh , B. ., & Valerina Kairanna , N. . (2023). ANTHOCYANINS: A HUE FOR HISTOLOGY - SYSTEMATIC REVIEW. Malaysian Journal of Science, 42(1), 106–125.
Review Articles
Author Biographies

Shiny Jasphin, Dept of Physiology, Melaka Manipal Medical College (Manipal Campus), Manipal Academy of Higher Education, Manipal, Karnataka, INDIA-576104.

Research scholar, Dept of Physiology

Archana Parampalli Raghavendra, Dept of Physiology, Melaka Manipal Medical College (Manipal Campus), Manipal Academy of Higher Education, Manipal, Karnataka, INDIA-576104.

Dept of Physiology

Monica Charlotte Solomon , Dept of Oral Pathology, Manipal College of Dental Sciences, Manipal, Manipal Academy of Higher Education, Manipal, Karnataka, INDIA-576104.

Dept of Oral Pathology

Arul Amuthan , Dept of Pharmacology, Melaka Manipal Medical College, (Manipal Campus), Manipal Academy of Higher Education, Manipal, Karnataka, INDIA-576104.

Dept of Pharmacology

Brij Mohan Kumar Singh , Dept. of Pathology, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal, Karnataka, INDIA-576104.

Dept. of Pathology

Nikitha Valerina Kairanna , Dept. of Pathology, Kasturba Medical College, Manipal, Manipal Academy of Higher Education, Manipal, Karnataka, INDIA-576104.

Dept. of Pathology


Abdullah, R., Lee, P. M., & Lee, K. H. (2010). Multiple colours and pH stability of floral anthocyanin extract: Clitoria ternatea. 2010 International Conference on Science and Social Research 254–258.

Abubakar, S., Usman, A. B., Etim, V., Nnadi, O., & Alaku, C. (2012). Application of Organic Dyes from Roselle calyx (Hibiscus sabdariffa linn) for Mycological Staining. Indian Journal of Innovations and Developments 1(9): 687–690.

Ademiluyi, A. O., Oboh, G., & Oluwaseun, A. J. (2013). Anthocyanin – Rich Red Dye of Hibiscus sabdariffa calyx Modulates Cisplatin-induced Nephrotoxicity and Oxidative Stress in Rats. International Journal of Biomedical Science 9(4) 243–248.

Agbede, M. B., Benard, S. A., Afolabi, O. O., Okoye, J. O., Bankole, J. K., Fowotade, A. A., Olutunde, O. A., & Muhammed, O. A. (2017). The Use of Hibiscus sabdariffa Extract as Nuclear Stain for Skin Morphology and Connective Tissue with Eosin Counterstain. Sokoto Journal of Medical Laboratory Science 2(4), 28–32.

Ajileye, A. B., Iteire, A. K., & Arigi, Q. B. (2015). Zingiber officinale (ginger) extract as a histological dye for muscle fibers and cytoplasm. International Journal of Medical Science and Public Health 4(10): 1445–1448.

Akinloye, A. J., & Olagoke, A. (2010). Screening of some indigenous herbal dyes for use in plant histological staining. Journal of Forestry Research 21(1): 81–84.

Ali, O.-H., Al-sayed, H., Yasin, N., & Afifi, E. (2016). Effect of Different Extraction Methods on Stablity of Anthocyanins Extracted from Red Onion peels (Allium cepa) and Its Uses as Food Colorants. Bulletin of the National Nutrition Institute 47(2): 196–219.

Anonymous. (2020). IARC Monographs on the Identification of Carcinogenic Hazards to Humans – International Agency for research on cancer. WHO.

Avwioro, G. O. (2002). Histochemistry and Tissue Pathology, pp.134-213, Abraka, Nigeria: University press.

Avwioro, G. O. (2010). Histochemistry and tissue pathology, principles and techniques, pp.561-568 Abraka, Nigeria: University press.

Awika, J. M., Rooney, L. W., & Waniska, R. D. (2005). Anthocyanins from black sorghum and their antioxidant properties. Food Chemistry 90(1–2): 293–301.

Bassey, R. B., Bakare, A. A., Peter, A. I., Oremosu, A. A., & Osinubi, A. A. (2012). Factors influencing extract of Hibiscus sabdariffa staining of rat testes. Biotechnic and Histochemistry 87(6): 403–407.

Bassey, Rosemary B., Osinubi, A. A., & Oremosu, A. A. (2012). Staining effect of Hibiscus sabdariffa extract on sperm cell morphology of Sprague-Dawley rats. Journal of Histotechnology 35(3): 110–113.

Benard, S., Afolabi, O., Fowotade, A., Okoye, J., Olutunde, O., & Bankole, J. (2017). Hibiscus-Van Gieson Stain for collagen fibers. African Journal of Cellular Pathology 9: 1–4.

Benard, S., Muhammed, A., Fowotade, A., Afolabi, O., & Olutunde, O. (2017). Iron-Roselle: A Progressive Nuclear Stain for Connective Tissue of Skin. International Journal of Health Research and Innovation 5(2): 25–31.

Benard, S A, Muhammaed, A., Fowotade A A, Afolabi O O, & Olutunde, O. A. (2015). Hibiscus sabdariffa extract as haematoxylin substitute in the histological demonstration of brain tissues. African Journal of Cellular Pathology 5: 32–35.

Benard, Solomon A. (2008). Iron-roselle: A progressive nuclear stain substitute for haematoxylin. Journal of Histotechnology 31(2): 57–59.

Bondoc, C. C. (2018). Curcuma longa Linn rhizome extract as an alternative stain for histological studies. Journal of Pharmacognosy and Phytochemistry 7(5): 3010–3017.

Bordoloi, B., Jaiswal, R., Siddiqui, S., & Tandon, A. (2017). Health Hazards of Special Stains. Saudi Journal of Pathology and Microbiology 2(5): 175–178.

Boulton, R. (2001). The Copigmentation of Anthocyanins and Its Role in the Color of Red Wine: A Critical Review. American Journal of Enology and Viticulture 52(2): 67–87.

Budak, M., & Budak, G. G. (2015). A new biochemical dye molecule and nuclear stain formulation in histotechnology diagnostics as an alternative to haematoxylin. Journal of Histotechnology 38(4): 113–121.

Cabrita, L., Fossen, T., & Andersen, Ø. M. (2000). Colour and stability of the six common anthocyanidin 3-glucosides in aqueous solutions. Food Chemistry 68(1): 101–107.

Cacace, J. E., & Mazza, G. (2003). Mass transfer process during extraction of phenolic compounds from milled berries. Journal of Food Engineering 59(4): 379–389.

Castañeda, A., Hernández, L. P., Elena, P., Rodríguez, J. A., & Galán-Vidal, C. A. (2009). Chemical studies of anthocyanins: A review. Food Chemistry 113(4): 859–871.

Charurungsipong, P., Tangduangdee, C., Amornraksa, S., Asavasanti, S., & Lin, J. (2020). Improvement of Anthocyanin Stability in Butterfly Pea Flower Extract by Co-pigmentation with Catechin. E3S Web of Conferences, 141(9).

Chomean, S., Nantabut, M., Kongtia, W., Saenguthai, K., & Kaset, C. (2019). Evaluation of natural dyes for human spermatozoa morphology assessment. Acta Histochemica 121(2): 227–233.

Cruz, P. E., De Vera, A. P., & Villa, A. D. (2018). The efficiancy of Santan flower (Ixora occinea Linn.) as an alternative stain to eosinY in Wright-Giemsa stain. LPU-Laguna Journal of Allied Medicine 3(1): 11–21.

Dapson, R., Horobin, R. W., & Kiernan, J. A. (2010). Hematoxylin shortages: Their causes and duration, and other dyes that can replace hemalum in routine haematoxylin and eosin staining. Biotechnic and Histochemistry 85(1): 55–63.

Dela, G., Or, E., Ovadia, R., Nissim-Levi, A., Weiss, D., & Oren-Shamir, M. (2003). Changes in anthocyanin concentration and composition in “Jaguar” rose flowers due to transient high-temperature conditions. Plant Science 164(3): 333–340.

do Carmo Brito, B. de N., da Silva Pena, R., Santos Lopes, A., & Campos Chiste, R. (2017). Anthocyanins of Jambolao (Syzygium cumini): Extraction and pH-Dependent Colour Changes. Journal of Food Science 82(10): 2286–2290.

Ebrahimi, M., & Parham, A. (2020). Using Herbal dyes as an alternative staining method for sperm evaluation. Veterinary Medicine and Science 6: 441-446.

Egbujo, E. C., Adisa, O. J., & Yahaya, A. B. (2008). A Study of the Staining Effect of Roselle (Hibiscus sabdariffa) on the Histologic Section of the Testis. International Journal of Morphology 26(4): 927–930.

Ekici, L. (2014). Effects of concentration methods on bioactivity and colour properties of poppy (Papaver rhoeas L.) sorbet, a traditional Turkish beverage. LWT - Food Science and Technology 56(1): 40–48.

Fossen, T., & Andersen, Ø. M. (2003). Anthocyanins from red onion, Allium cepa, with novel aglycone. Phytochemistry 62(8): 1217–1220.

Fuleki, T. (1971). Anthocyanins in red onion, Allium cepa. Journal of Food Science 36(1): 101–104.

Gopalkrishnan, B., & Chiranjeev, R. (2018). Pharmacognostical study of Ixora coccinea flower. Pharmacognosy Journal 10(5): 1042–1046.

Gowali, F. M. (1995). Picro-Hibiscin Stain for Degenerated Muscle Fibers. Laboratory Medicine 26(7): 470–473.

Hashim, E. A. (2006). The use of watery extract of kujarat flowers Hibiscus sabdariffa as a natural histological stain. Iraqi Journal of Medical Science 5(1): 29–33.

Hassimotto, N. M. A., Genovese, M. I., & Lajolo, F. M. (2007). Identification and characterisation of anthocyanins from wild mulberry (Morus nigra L.) growing in Brazil. Food Science and Technology International 13(1): 17–25.

Hiemori, M., Koh, E., & Mitchell, A. E. (2009). Influence of cooking on anthocyanins in black rice (Oryza sativa L. japonica var. SBR). Journal of Agricultural and Food Chemistry 57(5): 1908–1914.

Hinojosa-Gómez, J., San Martín-Hernández, C., Heredia, J. B., León-Félix, J., Osuna-Enciso, T., & Muy-Rangel, M. D. (2020). Anthocyanin Induction by Drought Stress in the Calyx of Roselle Cultivars. Molecules 25(7): 1555.

Hou, Z., Qin, P., Zhang, Y., Cui, S., & Ren, G. (2013). Identification of anthocyanins isolated from black rice (Oryza sativa L.) and their degradation kinetics. Food Research International 50(2): 691–697.

Ihuma J O, Asenge, G. H., Abioye, J. O. K., & Dick, S. K. (2012). Application of methonolic extracts from Hibiscus sabdariffa linn as a biological staining agent for some fungal species. International Journal of Plant Animal and Environmental Sciences 2(2): 254–259.

Jiang, Y., & Nie, W. J. (2015). Chemical properties in fruits of mulberry species from the Xinjiang province of China. Food Chemistry 174: 460–466.

Kamal, S. B. (2018). Staining of Leishmania donavani Promastigotes by natural flower dyes. Pakistan Journal of Biotechnology 15(2): 299–302.

Khoo, H. E., Azlan, A., Tang, S. T., & Lim, S. M. (2017). Anthocyanidins and anthocyanins: Coloured pigments as food, pharmaceutical ingredients, and the potential health benefits. Food and Nutrition Research 61(1).

Kumar, N., Mehul, J., Das, B., & Solanki, J. B. (2015). Staining of Platyhelminthes by herbal dyes: An eco-friendly technique for the taxonomist. Veterinary World 8(11): 1321–1325.

Kusculu, N. G., & Aslan, H. G. (2019). Evaluation of an extract of the Punica granatum flower as a biological stain of rat tissues: A preliminary study. Molecular Biology Reports 46(1): 581–585.

Kusculuo, N. G. (2018). Evaluation and comparison of staining effect of Punica granatum flower extract on testis and ovary of Wistar rats: First results. African Journal of Biotechnology 17(32): 989–993.

Kusculuo, N. G., Cucer, N., & Onal, A. (2017). Staining of Onion and Buccal Epithelial Cells with Onion Skin Extract | Open Access Journals. Research & Reviews: Research Journal of Biology 5(2): 1–6.

Kuskuluo, N. G., & Benli, H. (2017). Staining Effect of Pomegranate Flower Extract on Human Blood Cells: First Results. Journal of Environmental Science and Engineering 6:249–251.

Lahiani, A., Klaiman, E., & Grimm, O. (2018). Enabling Histopathological Annotations on Immunofluorescent Images through Virtualization of Haematoxylin and Eosin. Journal of Pathology Informatics, 9: 1.

Lakshmi, C. (2014). Food Colouring: The Natural Way. Research Journal of Chemical Sciences, 4(2): 87–96.

Lee, P. M., & Abdullah, R. (2011). Thermal Degradation of Blue Anthocyanin Extract of Clitoria ternatea Flower. 2nd International Conference on Biotechnology and Food Science 49–53.

Ma’aruf, S. Y., Mohammed, M. O., Muhammad, A. T., Okechi, O. O., Tsamiya, R. I., Abubakar, U., Mohammed, I., Umar, A., Sani, S. M., Kabir, H., Bello, B. A., Garba, S., & Abubakar, S. D. (2020). Staining Property of Alcoholic and Aqueous Hibiscus sabdariffa Extract in Demonstration of Selected Bacteria in Tissue Sections of Wistar Rats. Microbiology Research Journal International 30(2), 26–33.

Mat Nor, N. A., & Arof, A. K. (2016). On statistical analysis of factors affecting anthocyanin extraction from Ixora siamensis. Optical Materials 60: 462–466.

Mohandas, R., Ramani, P., Sherlin j, H., Gheena, S., Ramasubramanian, A., Don, K. R., Jayaraj, G., & Santhanam, A. (2019). Organic stains used in histopathology-A systematic review. Drug Invention Today 11: 426–432.

Moncada, M. C., Moura, S., Melo, M. J., Roque, A., Lodeiro, C., & Pina, F. (2003). Complexation of aluminum (III) by anthocyanins and synthetic flavylium salts: A source for blue and purple colour. Inorganica Chimica Acta 356: 51–61.

Moosazad, S., Ghajarbeigi, P., Mahmoudi, R., Shahsavari, S., Vahidi, R., & Soltani, A. (2019). Antibacterial and Antioxidant Properties of Colorant Extracted from Red Onion Skin. Journal of Chemical Health Risks, 9(3): 235–243.

Ozgen, M., Serce, S., & Kaya, C. (2009). Phytochemical and antioxidant properties of anthocyanin-rich Morus nigra and Morus rubra fruits. Scientia Horticulturae, 119(3): 275–279.

Patil, N., & Datar, A. G. (2015). Extraction, stability and separation of anthocyanins of Ixora coccinea linn. International Journal of Pharmacy and Pharmaceutical Sciences 7(3): 198–202.

Pedro, A. C., Granato, D., & Rosso, N. D. (2016). Extraction of anthocyanins and polyphenols from black rice (Oryza sativa L.) by modeling and assessing their reversibility and stability. Food Chemistry 191: 12–20.

Pirzadeh, M., Caporaso, N., Rauf, A., Shariati, M. A., Yessimbekov, Z., Khan, M. U., Imran, M., & Mubarak, M. S. (2021). Pomegranate as a source of bioactive constituents: A review on their characterization, properties and applications. Critical Reviews in Food Science and Nutrition 61(6):982-999.

Quina, F. H., Moreira, P. F., Vautier-Giongo, C., Rettori, D., Rodrigues, R. F., Freitas, A. A., Silva, P. F., & Maçanita, A. L. (2009). Photochemistry of anthocyanins and their biological role in plant tissues. Pure and Applied Chemistry 81(9): 1687–1694.

Rahimi, H. R., Arastoo, M., & Ostad, S. N. (2012). A comprehensive review of Punica granatum (Pomegranate) properties in toxicological, pharmacological, cellular and molecular biology researches. Iranian Journal of Pharmaceutical Research 11(2): 385–400.

Ramamoorthy, A., Ravi, S., Jeddy, N., Thangavelu, R., & Janardhanan, S. (2016). Natural alternatives for chemicals used in histopathology lab- A literature review. In Journal of Clinical and Diagnostic Research 10(11): EE01-EE04.

Saenguthai, K., Chomean, S., & Kaset, C. (2018). The evaluation of Oryza sativa L. (Black rice) extracts for detection of spermatozoa on the clothing and vaginal swab samples. Legal Medicine 35: 91–97.

Shaik, A., Naidu, K. K., & Panda, J. (2018). A review on anthocyanins: A promising role on phytochemistry and pharmacology. International Research Journal of Pharmacy 9(1): 1–9.

Shao, Y., Xu, F., Sun, X., Bao, J., & Beta, T. (2014). Phenolic acids, anthocyanins, and antioxidant capacity in rice (Oryza sativa L.) grains at four stages of development after flowering. Food Chemistry 143: 90–96.

Singh, R. (2017). Sources of natural dye-A critical review. International Journal of Engineering, Science and Mathematics 6(5): 180–186.

Siva, R. (2007). Status of natural dyes and dye-yielding plants in India. Current Science 92(7): 916–925.

Sridhara, S. U., Raju, S., Gopalkrishna, A. H., Haragannavar, V. C., Latha, D., & Mirshad, R. (2016). Hibiscus: A different hue in histopathology. Journal of Medicine Radiology Pathology and Surgery 2: 9–11.

Suabjakyong, P., Romratanapun, S., & Thitipramote, N. (2011). Extraction of Natural Histological Dye from Black Plum Fruit (Syzygium cumini). Journal of the Microscopy Society of Thailand 4(1): 13–15.

Sudhakaran, A., Hallikeri, K., & Babu, B. (2018). Natural stains Zingiber officinale Roscoe (ginger) and Curcuma longa L. (turmeric) – A substitute to eosin. AYU 39(4): 220–225.

Suebkhampet, A., & Sotthibandhu, P. (2012). Effect of using aqueous crude extract from butterfly pea flowers (Clitoria ternatae L.) as a dye on animal blood smear staining. Suranaree Journal of Science and Technology 19(1): 15–19.

Surendra, L., Nambiar, K. S., Haragannavar, V. C., Augustine, D., Sowmya, S. V., Babu, A., & Rao, R. S. (2018). Staining efficacy of rose extract in comparison with eosin stain: A histological study on oral tissues. World Journal of Dentistry 9(6): 500–504.

Suvarna, K., Layton, C., & Bancroft, J. (2013). Bancroft’s Theory and Practice of Histological Techniques (7th ed.). Churchill Livingston Elsiever.

Tousson, E., & Al-Behbehani, B. (2011). Black mulberries (Morus nigra) as a natural dye for animal tissues staining. Animal Biology 61(1): 49–56.

Tousson, E. M., & Al-Behbehani, B. (2010). Black Mulberries (Morus nigra) as a natural dye for nervous tissues staining. The Egyptian Journal of Experimental Biology (Zoology) 6(1): 159–164.

van der Kooi, C. J., & Stavenga, D. G. (2019). Vividly coloured poppy flowers due to dense pigmentation and strong scattering in thin petals. Journal of Comparative Physiology A: Neuroethology, Sensory, Neural, and Behavioral Physiology 205(3): 363–372.

Vankar, P. S. (2000). Chemistry of Natural Dyes. Resonance 5(10): 73–80.

Velickovic, J. M., Mitic, M. N., Arsic, B. B., Paunovic, D. Đ., Stojanovic, T., Veljkovic, J. N., Dimitrijevic, D. S., Stevanovic, S. D., & Kostic, D. A. (2019). HPLC Analysis of extracts of fresh petals of Papaver rhoeas L. Studia Universitatis Babes-Bolyai, Chemia 3: 239–247.

Verma, S., & Gupta, G. (2017). Natural dyes and its applications: A brief review. International Journal of Research and Analytical Reviews 4(4): 57–60.

Veuthey, T. V., Herrera, G. M., & Dodero, V. I. (2014). Dyes and stains: From molecular structure to histological application. Frontiers in Bioscience 19(1): 91–112.

Wahyuningsih, S., Wulandari, L., Wartono, M. W., Munawaroh, H., & Ramelan, A. H. (2017). The Effect of pH and Color Stability of Anthocyanin on Food Colorant. IOP Conference Series: Materials Science and Engineering 193(1).

Wan, H., Yu, C., Han, Y., Guo, X., Luo, L., Pan, H., Zheng, T., Wang, J., Cheng, T., & Zhang, Q. (2019). Determination of flavonoids and carotenoids and their contributions to various colours of rose cultivars (Rosa spp.). Frontiers in Plant Science 10: 123.

Yoshida, K., Kitahara, S., Ito, D., & Kondo, T. (2006). Ferric ions involved in the flower colour development of the Himalayan blue poppy, Meconopsis grandis. Phytochemistry 67(10): 992–998.

Zulfajri, M., & Muttakin. (2018). Activity analysis of anthocyanin from Syzygium cumini (L.) skeels as a natural indicator in acid-base titration. Rasayan J. Chem 11(1): 135–141.