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September 14, 2022

Ohrid diamond – the dark horse of chestnuts?

Aesculus hippocastanum , is also called the 'Ohrid diamond', or horse/wild chestnuts. These plants grow in Ohrid, Republic of North Macedonia, at an altitude of 695 m. Various parts of this plant, such as the seeds, leaves, bark, and flowers, have been used in folk medicine and cosmetic preparations. Wild chestnuts are not just ornaments on the streets of Ohrid – in fact, they hide great healing properties. The Ohrid diamonds have an antioxidant potential and a stronger in vitro antimicrobial effect than some antibiotics. Research on the anti-cancer effect of this plant is ongoing.

Looking at the Ohrid diamond

The need to study biologically active compounds in plants, such as phenolic compounds, flavonoids, carbohydrates, is growing in importance in scientific research. These active compounds can have a medical effect on consumer’s health. Despite the chemical composition of wild chestnut  being well-known, there is still insufficient data on the antioxidant and antimicrobial activity shown by its seed and leaf extracts.

In response to this gap in knowledge, the aim of this research was to determine the antioxidant and antimicrobial activity of the wild chestnut, also known as the Ohrid diamond. The study also looked at whether water and ethyl alcohol are suitable solvents for chestnut extraction by preparation of aqueous and ethanolic extract from the seeds and leaves of wild chestnut.

Analysis and study

The study focused on the wild chestnut leaves and seeds. Extracts from these parts of the plant were produced in laboratory conditions and subsequently analysed to determine their full chemical and biological characterisation, including determining the following:

  • Any presence of toxic metals;
  • Total phenolic and flavonoids content;
  • Antioxidant potential (ability to capture DPPH radicals; ability to chelate iron ions; reducing ability; conjugated diene method);
  • Antimicrobial potential (disc-diffusion method; microdilution method).

Antioxidants in the Ohrid diamond

First of all, it was determined that there is no presence of heavy metals in either the leaf or seed extracts, indicating that the plant had not absorbed any pollutants. Furthermore, results showed a high content of phenolic and flavonoid compounds in the extracts. The seed extract gave much better results than the leaf extract. An extremely high antioxidant potential was found, of a level equal to some synthetic antioxidants used in the industry, such as BHT, alpha-tocopherol, or citric acid.

Antioxidants are important because of their ability to ‘catch’ free radicals (toxic substances) that are present in the human body, thereby performing ‘purification’ of the body. They are also important as preservatives in the food and pharmaceutical industries.

The ethanolic seed extract (57.10% – 85.25%) showed the highest ability to capture DPPH radicals when compared to the aqueous seed extracts (51.95% – 81.05%). On the other hand, ethanolic leaf extract showed better antioxidant activity (37.35% – 72.35%) compared to the leaf aqueous extracts (33.61% – 69.85%). The order of antioxidant capacity in terms of the chelating iron ions was: seed ethanolic extract (29.14% – 63.25%) > seed aqueous extract (27.19% – 57.15%) > leaf ethanolic extract (23.95% – 58.45%) > leaf aqueous extract (21.23% – 55.96%). Moreover, in terms of the conjugated diene method, the values ranged as follows: seed ethanolic extract (42.95% – 69.89%) > seed aqueous extract (39.26% – 67.65%) > leaf ethanolic extract (35.61% – 63.17%) > leaf aqueous extract (32.10% – 60.15%).

Antimicrobial effect

A strong antimicrobial effect of the seed extracts was also determined as part of the study. The ethanolic seed extract was characterised with the highest antimicrobial activity against most of the tested bacteria including Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes, Escherichia coli, and others, which was significant (p<0.05) compared to other tested extracts. However, aqueous seed extract showed significantly (p<0.05) higher antimicrobial activity against Enterococcus faecalis as well as Proteus vulgaris, compared to other tested extracts. It is important to note that ethanolic extracts from chestnut seed showed higher (p<0.05) antimicrobial activity against five tested bacteria (Staphylococcus aureus – 25.9 ± 0.07 mm; Listeria monocytogenes – 14.7 ± 0.02 mm; Escherichia coli – 11.8 ± 0.02 mm; Shigella sonnei – 11.5 ± 0.01 mm and Pseudomonas aeruginosa – 14.0 ± 0.03 mm) compared to tetracycline and chloramphenicol.

The efficacy of these extracts is of particular importance because it shows promise that the extracts could be used in pharmaceuticals. The wild chestnut extracts possess an antimicrobial effect on par with synthetic antibiotics, and do not confer resistance in the same way as traditional antibiotics.

However, it has not yet been proven that these extracts could be used as a medicine, but it does open up new opportunities for future scientific research.

Current research

Research into the properties of wild chestnut continues, with work being done on in vitro determination of the cytotoxic (anticancer) activity of the Ohrid diamond/wild chestnut extracts. Other extraction methods for precision targeting the desired components of this plant are being investigated.

Ohrid diamond wild chestnut extracts could have a variety of uses in the production of various beauty and pharmaceutical products; from the point of view of the food industry, these extracts could replace synthetic additives, lessening the consumer’s exposure to harmful chemicals.

Why is the Ohrid diamond different?

This research also included wild chestnuts from the Skopje and Strumica regions of the Republic of North Macedonia, but the results were unsatisfactory. It appears that chestnuts from the Ohrid region may be special due to the favourable climatic conditions, the altitude, the soil, and the unpolluted environment.

Given that it is known that synthetic antioxidants are detrimental to consumers’ health, and the fact that bacteria are becoming increasingly resistant to existing antibiotics, it is clear that we need new, natural components that are effective but safe to consumers.

Therefore, the antioxidant and antimicrobial activity identified in this study is a new starting point for future in vivo research. At the same time, Ohrid diamond wild chestnut extracts can be a suitable substitute for a number of synthetic additives in the food, pharmaceutical and cosmetic industries.


Dr. Monika Stojanova – University of Ss. Cyril and Methodius, Faculty of Agricultural Sciences and Food, Skopje, North Macedonia (;
Acad. Prof. Dr. Dragutin Djukic – University of Kragujevac, Faculty of Agronomy Čačak, Čačak, Serbia;
Prof. Dr. Marina Todor Stojanova – University of Ss. Cyril and Methodius, Faculty of Agricultural Sciences and Food, Skopje, North Macedonia;
Prof. Dr. Aziz Şatana – Erciyes University, Agriculture Faculty, Kayseri, Turkey;
Prof. Dr. Blazo Lalevic – University of Belgrade, Faculty of Agriculture, Belgrade, Serbia.



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Yuriy, K K, Arsene, M M J, et al (2021) Assessment of antimicrobial activity of ethanolic and aqueous extracts of Aesculus hippocastanum L. (horse chestnut) bark against bacteria isolated from urine of patients diagnosed positive to urinary tract infections.

Şeker, G K, (2019) Horse Chestnut. Chapter 3.26, Nonvitamin and Nonmineral Nutritional Supplements, 259-299.

Bonomo, M G, Cafaro C, et al (2018) Antimicrobial Activity, Antioxidant Properties and Phytochemical Screening of Aesculus hippocastanum Mother Tincture against Food-borne Bacteria. Letters in Drug Design & Discovery, 17, 48-56.

Written By

Monika Stojanova
Faculty of Agricultural Sciences and Food

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