Bioactive Compounds in Chestnut (Castanea sativa Mill.): Composition, Health-Promoting Properties, and Technological Applications

Abstract

Chestnut (Castanea sativa Mill.) is a Mediterranean staple food valued for its cultural heritage, gastronomic identity, nutritional profile, bioactivities, and socio-economic and environmental relevance. This narrative review synthesizes current knowledge on chestnut fruits and by-products, linking ecophysiology and genetic diversity to chemical composition and functionality. It summarizes the nutrient profile (high starch and dietary fiber; gluten-free; B vitamins; essential minerals; and favorable fatty acids) and the diversity of phytochemicals—particularly phenolic acids, flavonoids, and ellagitannins (e.g., castalagin and vescalagin)—that underpin antioxidant, anti-inflammatory, antimicrobial, anti-proliferative, and metabolic effects demonstrated across in vitro, cellular, and in vivo models. We compare conventional and green extraction strategies (e.g., hydroethanolic, ultrasound-/microwave-assisted, and supercritical and subcritical water), highlighting method-dependent yields, composition, and bioactivity, and the valorization of shells, burs, and leaves within circular bioeconomy frameworks. Technological applications span functional foods (gluten-free flours, beverages, and emulsions), nutraceuticals, and cosmetics (skin-protective and regenerative formulations), and active packaging/biopolymers with antioxidant and antimicrobial performance. We discuss sources of variability (cultivar, environment, maturation, and processing) affecting bioactive content and efficacy, and outline future directions. Finally, this review emphasizes the importance of university-facilitated co-creation with companies and consumers—within the framework of Responsible Research and Innovation—as a pathway to strengthen the economic valorization and full utilization of the chestnut value chain, enhancing its societal relevance, sustainability, and health-promoting potential.

1. Introduction

Castanea sativa Mill. is one of the oldest fruits of the Mediterranean Region, being native to this region, although it is nowadays cultivated in other locations [1]. It deserves special recognition not only for its nutritional and cultural value but also for its socio-economic and environmental importance [2]. Since ancient times, chestnuts have been a staple food in many regions, particularly mountainous ones, often replacing cereals, and continue to play a central role in Mediterranean gastronomic identity [1,3].

Nutritionally, chestnut fruits are gluten-free, being an ideal food for individuals with gluten sensitivity and intolerance, such as those with celiac disease [3,4]. Chestnuts have a high water, starch, and dietary fiber content, are low in lipids, provide a slow-release energy source, and contain minerals and vitamins (as detailed in the following sections) [5]. The fruit’s nutritional properties align with the principles of the Mediterranean Diet, recognized by UNESCO as Intangible Cultural Heritage of Humanity, and reinforce the chestnut’s role in promoting a healthy and balanced diet [6].

Beyond their intrinsic nutritional attributes, the composition and functional quality of chestnut fruits vary markedly across cultivars, geographical origins, and environmental conditions. Factors such as genotype, altitude, temperature, rainfall patterns, and soil characteristics can significantly influence starch content, sugar profile, mineral composition, and, especially, phenolic compound accumulation. These variations have been documented among European cultivars such as ‘Judia’, ‘Longal’, and ‘Martaínha’, which exhibit distinct phytochemical and physiological profiles. Moreover, chestnuts grown in colder or higher-altitude regions often present higher levels of polyphenols and flavonoids, enhancing their antioxidant potential and overall functional value [7,8].

In recent decades, growing interest has focused on the bioactive compounds present in chestnut fruits and by-products, given their potential health benefits [9]. Studies have identified a high abundance of phenolic compounds (e.g., phenolic acids, flavonoids, and condensed tannins), which confer antioxidant and anti-inflammatory properties [9,10] (detailed in the following sections). This potential has fostered the transformation of fruits into new food products, such as flours and frozen products, serving as raw materials for pasta, bread, cakes, beverages, and snacks, or for gastronomy, for consumers seeking healthy, functional foods [9,11,12]. The combination of nutritional value and bioactive compounds reinforces the understanding of chestnut fruits as a functional food [9].

Although Castanea sativa has been the subject of several review papers [4,13], most existing syntheses focus on isolated dimensions of the species, such as its nutritional composition, ecological importance, or selected classes of phenolic compounds. What is currently lacking is a comprehensive and integrative review that brings together the ecophysiological characterization of the species, its genetic and environmental determinants, detailed nutrient and phytochemical profiles, extraction and processing methodologies, and the full spectrum of bioactivities reported for chestnut fruits and by-products. Recent bibliometric analysis [14] highlights an exponential rise in publications related to Castanea sativa, but also reveals that studies on bioactive compounds, biomedical applications, and sustainable valorization strategies remain comparatively underexplored. Moreover, no existing review contextualizes chestnut within emerging frameworks, such as the circular bioeconomy, green extraction technologies, and innovation pathways relevant to the food, cosmetics, and pharmaceutical sectors. By addressing these gaps, the present review aims to provide an updated, interdisciplinary synthesis that supports both scientific advancement and the development of sustainable, value-added applications across the chestnut value chain.

Therefore, this review aims to provide a comprehensive overview of the bioactive compounds in chestnut fruits (C. sativa) and their by-products, highlighting their chemical diversity, extraction and processing methods, and biological activities. Attention is given to the influence of genetic, environmental, and technological factors on compound composition and functionality. The review also explores potential industrial applications in food, cosmetic, and pharmaceutical sectors, as well as the role of the chestnut value chain in promoting sustainability and circular economy principles. Through this review, we seek to identify current knowledge gaps, emerging research trends, and future perspectives on harnessing the full bioactive potential of chestnut for human health and sustainable development.

2. Ecophysiological Characterization of Castanea sativa

Castanea sativa is a Mediterranean species, thriving in regions with humid, pre-Atlantic bioclimates, corresponding to the environmental zones of the Anatolian, Lusitanian Mediterranean Mountains, and North Mediterranean [15].

The genus Castanea belongs to the Fagaceae family, together with Quercus L. and Fagus L. The evolutionary history of the Castanea genus dates back to the Tertiary period, with an origin in Eastern Asia and subsequent migration to the Western side towards Europe (Castanea sativa Mill.) and North America (Castanea dentata Borkh), and to the Eastern side to Japan and Korea (Castanea crenata Sieb. e Zucc) [16,17].

Genetic analyses of wild and cultivated populations have revealed a high degree of variability within C. sativa, reflecting historical dispersal and regional adaptation [18,19,20]. Three major European genetic clusters are recognized: Western, Central, and Eastern, with significant substructure in the Iberian Peninsula, where two centers of diversity occur in the Northwest and Central regions [19]. This diversity supports a broad ecological amplitude, allowing chestnut to thrive from the Atlantic to the continental Mediterranean zones, typically at altitudes between 300 and 1200 m [21,22].

2.1. Morphological and Anatomical Features

Chestnut is a deciduous tree with simple, alternate, and lanceolate leaves measuring 12–18 cm in length and 4–7 cm in width. The leaves are serrated, with 15–20 pairs of secondary veins clearly visible on the abaxial surface (Figure 1). According to Gomes-Laranjo et al. [23], Portuguese cultivars typically have leaves measuring ~15.5 cm in length and ~5.5 mm in width, with about 17 secondary veins.

The leaf’s upper surface contains a palisade mesophyll optimized for photosynthesis, whereas the lower surface includes a spongy parenchyma, which favors gas exchange. Among cultivars, leaf thickness ranges from 152 µm (“Amarelal”) to 265 µm (“Martaínha”), with “Longal” showing 196 µm. The ratio between palisade and spongy parenchyma varies between 1.31 and 1.98 [23].

Non-glandular trichomes are particularly dense in the “Martaínha” cultivar. They play an adaptive role by reducing transpiration and increasing light reflectivity [24]. Stomatal density varies between 298 and 468 stomata/mm² depending on cultivar [23]. Figure 1 illustrates the principal organs of the “Judia” cultivar (leaves, burrs, and nuts).

2.2. Ecophysiological Adaptation

Chestnut exhibits anisohydric behavior, maintaining photosynthetic activity as water potential decreases. Optimal photosynthesis (A₁₀₀ ≈ 9–11 µmol CO₂/m²s) occurs at stem water potentials between −1.2 and −0.5 MPa, with 50% reduction at −1.7 MPa and 90% reduction at −1.25 MPa [25]. The capacity to tolerate negative water potential illustrates its hydroplasticity.

Maximum photosynthetic rate is typically reached at ~25 °C, declining by half at 37 °C [26]. The light compensation point is about 60 µmol/m²s, and 90% of the maximum rate is achieved at 1300 µmol/m²s PPFD (photosynthetic photon flux density) [25]. These parameters characterize C. sativa as a half-shade species adapted to moderate light and temperature regimes.

Physiological studies demonstrate that photosynthetic rate and stomatal conductance are strongly linked to leaf temperature and light intensity, with a 42% reduction observed in “Judia” when water potential dropped from 0.6 to 1.7 MPa [27]. This pattern reflects cultivar-dependent drought tolerance, confirming the species’ anisohydric nature.

2.3. Physiological Indicators

The pigment composition of C. sativa leaves varies with genotype (

Table 1. Average pigment composition in leaves of three main Portuguese cultivars. Adapted from [23,26].

Cultivar	Chlorophyll (μg/cm2)	Carotenoids (μg/cm2)	Total (μg/cm2)	Chla/Chlb	Chl/Car
Judia	113.3	29.3	142.6	3.60	3.87
Longal	103.0	26.3	129.3	4.01	3.92
Martaínha	85.7	19.5	105.2	4.35	4.39

). Average total chlorophyll content is about 110 µg/cm², with Judia reaching 142.6 µg/cm² and a Chl a/b ratio of 3.6, indicating adaptation to lower light intensity than the others. “Longal” and “Martaínha” show higher ratios (>4.0), associated with exposure to whole sunlight [23,26]. Carotenoids contribute to photoprotection by quenching reactive oxygen species under high irradiance.

Mineral nutrient content in leaves averages 31.7 g/kg dry weight (DW) for macronutrients and 1.12 g/kg DW for micronutrients, with nitrogen accounting for roughly 44% of total mineral composition [23]. This balanced nutrient profile sustains chlorophyll synthesis and enzymatic activity.

Castanea sativa is rich in secondary metabolites with potential bioactivities (see Section 3). The composition varies among leaves, bark, wood, flowers, hulls, burs, and edible nuts, and several classes of compounds, such as polyphenols, triterpenes and sterols, and volatile compounds (aroma and flavor) can be found (see Section 3). Temperature and water regime also affect biochemical composition. Phenolic content is influenced by environmental variables such as altitude, rainfall, and temperature [7]. Chestnuts from colder regions exhibit higher total polyphenol and flavonoid content, enhancing antioxidant capacity [7,28]. The secondary metabolites, mainly tannins, flavonoids, and phenolic acids, play a role in photoprotection and stress mitigation [29].

3. Nutrient and Chemical Profile of Chestnut Fruits

The growing interest in chestnuts (Castanea sativa) stems in part from various studies describing the potential health benefits of consuming them, whether fresh or processed. The edaphoclimatic conditions, as well as the agrobiodiversity of the chestnut, influence the characteristics of the chestnut fruit, such as its sensory properties (e.g., aroma, flavor, and texture), and its composition, which influences its nutritional characteristics [30]. In addition to common nutrients (e.g., lipids, proteins, and carbohydrates), compounds resulting from secondary metabolism also have high nutraceutical potential [30,31]. Next, we describe the primary nutrients in chestnuts reported in the recent literature, as well as the phytochemicals resulting from secondary metabolism relevant to health.

3.1. Main Nutrient Compounds

Compared to other nuts, fresh sweet chestnuts have a high moisture content (~50%) and a high dietary fiber content (4–10%) [8,32]. The main composition of this fruit is starch (~30%). It presents lipid (2–4%) and low protein (2–7%) content. Still, it is a source of free amino acids, including the essential amino acids leucine, lysine, phenylalanine, valine, and threonine, although in low amounts [32]. Regarding the lipid content, the presence of monounsaturated fatty acids, such as oleic acid (C18:1 n-9, cis-9; omega-9) and palmitoleic acid (16:1 n-7, omega-7), and polyunsaturated fatty acids (PUFA), such as linoleic acid (18:2 n-6 or 18:2 cis-9,12, an omega 6) and linolenic acid [α-linolenic acid (ALA), 18:3 n-3 or 18:3 cis-9,12,15, an omega-3], being these PUFA essential fatty acids [31,33,34],

Table 2. Identification and concentration of main nutrient compounds, protein, lipids, vitamins, minerals, and carbohydrates are described in the chestnut (Castanea sativa) fruit.

Compound Class	Compound Subclass	Compound	Concentration	Ref.
		Moisture	~50% FW	[8]
Protein			2–7% FW	[32]
Lipids	Total Fat		1.4–3.0 g/100 g FW	[13]
	Fatty acids (per 100 g FW)	Monounsaturated (total)	0.78 g	[31,34]
		Oleic acid (C18:1 n-9)	0.749 g	[31]
		Palmitoleic acid (16:1 n-7)	0.021 g
		Polyunsaturated (total)	0.894 g	[31,34]
		Linoleic acid (18:2 n-6)	0.798 g	[31]
		α-Linolenic acid (18:3 n-3)	0.095 g
Carbohydrates	Total carbohydrates (DW)		70–80 g/100 g DW	[8]
	Fiber		4–10% FW	[8,32]
	Individual soluble sugars (per 100 g DW)	Total soluble sugars	51–57 g	[30]
		Glucose	0.18–3.15 g	[8,32]
		Fructose	0.27–3.17 g
		Maltose	0.17–1.58 g
		Sucrose	9.36–29.89 g
Vitamins		Vitamin C	0–6.87 mg/100 g, DW	[30]
			40.2 mg/100 g FW	[13,34]
		Folates	58 µg/100 g FW
		Niacin	1.1 mg/100 g FW
		Pantothenic acid	0.48 mg/100 g FW
		Riboflavin	0.02 mg/100 g FW
		Thiamin	0.14 mg/100 g FW
		Vitamin A	26 IU/100 g FW
Minerals	Macrominerals	Potassium	484 mg/100 g FW	[32]
			377–789 mg/100 g DW	[8]
		Phosphorus	38 mg/100 g FW	[34]
			96.5 to 179 mg/100 g DW	[8]
		Magnesium	30 mg/100 g FW	[34]
			58.7 to 101 mg/100 g DW	[8]
		Calcium	19 mg/100 g FW	[34]
			26.9–103 mg/100 g DW	[8]
	Trace minerals	Cooper	0.418 mg/100 g FW	[34]
		Iron	0.94 mg/100 g FW
		Manganese	0.336 mg/100 g FW
		Zinc	0.49 mg/100 g FW
		Sodium	2 mg/100 g FW
			0.65 to 6.90 mg/100 g DW	[8]

Note: The compound’s amount is presented as described in the respective references. Abbreviations: DW: dry weight, FW: fresh weight.

. Chestnuts contain several vitamins, namely the water-soluble B vitamins, such as folates (vitamin B9), niacin (vitamin B3), pantothenic acid (vitamin B5), thiamine (vitamin B1), riboflavin (vitamin B2); and ascorbic acid (vitamin C) [13] which are essential micronutrients with key biological functions (Table 2 and Figure 2). Chestnut fruit also contains a variety of minerals, inorganic micronutrients, that have relevant roles acting as enzyme cofactors or being part of enzymes’ catalytic activity and that participate in other relevant cellular processes [8,35], such as copper (Cu), zinc (Zn), iron (Fe), and manganese (Mn) Table 2.

3.2. Main Phytochemicals

Chestnut fruits are also a source of phytochemicals produced through secondary metabolism, with polyphenols among the most relevant classes, known for their diverse bioactivities [36,37].

Table 3. Main phenolic compounds present in chestnut (Castanea sativa) fruit.

Compound Subclass	Compound	Concentration	Ref.
	Hydroxycinnamic acids (caffeic, coumaric, ferulic, and chlorogenic)	4.06 ± 0.03 to 62.89 ± 0.22 mg/100 g, DW	[30]
		0.91 ± 0.15 to 4.44 ± 0.21 mg/kg FW	[39]
	Hydroxybenzoic acids (gallic acid and derivatives)	86.44 ± 13.01 to 185.54 ± 11.40 mg/kg FW
	Gallic acid	23.30 ± 0.07 mg/kg DW	[38]
	Neochlorogenic acid	0.15 ± 0.01 mg/kg DW
	Chlorogenic acid	0.94 ± 0.07 mg/kg DW
	p-Hydroxybenzoic acid	1.43 ± 0.10 mg/kg DW
	Caffeic acid	3.15 ± 0.01 mg/kg DW
	Vanillic acid	9.35 ± 0.04 mg/kg DW
	Syringic acid	0.26 ± 0.01 mg/kg DW
	p-Coumaric acid	6.73 ± 0.03 mg/kg DW
	Ferulic acid	3.37 ± 0.01 mg/kg DW
Flavonoids	Flavonols (hyperoside, isoquercitrin, quercetin, quercitrin, and rutin)	0 to 8.62 ± 0.03 (mg/100 g, DW)	[30]
		9.64 ± 1.37 to 78.97 ± 2.03 mg/kg FW	[39]
	Catechins (catechin and epicatechin)	5.71 ± 0.90 to 39.40 ± 0.34(mg/100 g, DW)	[30]
		4.56 ± 0.01 to 74.17 ± 0.13 mg/kg DW	[38]
	Procyanidin A2	0.42 ± 0.02 mg/kg DW
	Procyanidin B2	8.67 ± 0.01 mg/kg DW
	Rutin	1.59 ± 0.03 mg/kg DW
	Isoquercitrin	2.17 ± 0.01 mg/kg DW
	Hyperoside	2.91 ± 0.01 mg/kg DW
	Quercitrin	0.08 ± 0.01 mg/kg DW
	Myricetin	0.60 ± 0.01 mg/kg DW
	Kaempferol-3-glucoside	0.12 ± 0.01 mg/kg DW
	Quercetin	0.22 ± 0.01 mg/kg DW
	Isorhamnetin	0.07 ± 0.01 mg/kg DW
	Kaempferol	0.98 ± 0.01 mg/kg DW
	Phloridzin	6.82 ± 0.01 mg/kg DW
	Phloretin	2.09 ± 0.03 mg/kg DW
Tannins	Castalagin and vescalagin	5.44 ± 0.09 to 24.79 ± 0.43 mg/100 g, DW	[30]
	Ellagic acid	-	[10]
		26.73 ± 0.02 mg 100/g DW	[30]
		11.11 ± 0.01 mg/kg DW	[38]

Note: The compound’s amount is presented as described in the respective references. Abbreviations: DW: dry weight; FW: fresh weight.

shows the most pertinent phenolics described in chestnut fruit, and some example molecules are shown in Figure 2. As observed in Table 3, the content in phenolic acids (e.g., gallic acid, vanillic acid, and p-coumaric acid) is higher than that of flavonoids [8,30]. Among the flavonoids, catechins, procyanidin B2, and phloridzin appear in higher amounts [30,38]. Considerable amounts of tannins, in particular ellagic, castalagin, and vescalagin, were also described [30,38]. The main bioactivities of these compounds are described in Section 6.

4. Bioactive Compounds in Chestnut By-Products

During the industrial processing of chestnuts, some components are discarded, such as the outer and inner shells; these by-products can account for 15% to 25% of the fruit’s total weight [40,41]. Other waste products from chestnuts include the leaves and the burs. To promote the circular economy and the valorization of waste and by-products, several studies report the extraction and characterization of polyphenols from chestnut shells, leaves, and burs. A summary of those bioactive compounds is presented in

Table 4. Main phenolic compounds present in chestnut (Castanea sativa) by-products and waste products.

Compound Subclass	Compound	Concentration	By-Product	Extraction Method	Ref.
Phenolic acids	Crenatin	0.90 ± 0.09 mg/100 g	Burs	MeOH	[10]
		15.77 ± 1.17 mg/100 g	Leaves
	Gallic acid	81.52 ± 0.99 mg/100 g DW	Leaves	UAE-MeOH	[43]
		7.24 ± 0.17 to 10.86 ± 0.092 mg/g	Leaves	MAC-EtOH, UAE-EtOH, MAE-w	[44]
		3.78 ± 0.056 to 14.84 ± 0.02 mg/g	Shells	MAC-EtOH, UAE-EtOH, MAE-w	[44]
		3.14 mg/g DW	Shells	MAE-EtOH	[40]
		257.56 ± 12.88 to 263.22 ± 13.16 mg/g DW	Shells	UAE-EtOH; MAE-w	[42]
		4.28 ± 0.03 to 17.55 ± 0.067 mg/g	Burs	MAC-EtOH, UAE-EtOH, MAE-w	[44]
	Protocatechuic acid	38.20 ± 1.91 mg/g DW	Shells	UAE-EtOH; MAE-w	[42]
	Vanillic acid	0.55 ± 0.03 mg/g DW	Shells	UAE-EtOH; MAE-w	[42]
	Syringic acid	0.02 ± 0.00 to 0.16 ± 0.01 mg/g DW	Shells	UAE-EtOH; MAE-w	[42]
	Methyl-gallate	19.07 ± 0.17 mg/100 g DW	Leaves	UAE-MeOH	[43]
	Neochlorogenic acid	2.29 ± 0.11 to 9.71 ± 0.49 mg/g DW	Shells	UAE-EtOH; MAE-w	[42]
	Caftaric acid	1.52 ± 0.08 to 8.13 ± 0.41 mg/g DW	Shells	UAE-EtOH; MAE-w	[42]
	Chlorogenic acid	1.01 ± 0.05 to 1.73 ± 0.09 mg/g DW	Shells	UAE-EtOH; MAE-w	[42]
	4-O-caffeyolquinic acid	0.76 ± 0.04 to 6.14 ± 0.31 mg/g DW	Shells	UAE-EtOH; MAE-w	[42]
	Caffeic acid	0.63 ± 0.03 to 0.66 ± 0.03 mg/g DW	Shells	UAE-EtOH; MAE-w	[42]
	p-Coumaric acid	0.32 ± 0.02 to 0.46 ± 0.02 mg/g DW	Shells	UAE-EtOH; MAE-w	[42]
	Ferulic acid	0.15 ± 0.01 to 0.22 ± 0.01 mg/g DW	Shells	UAE-EtOH; MAE-w	[42]
Flavonoids	Quercetin-3-O-β-D- -glucopyranoside	0.04 ± 0.002 mg/100 g	Burs	MeOH	[10]
		3.37 ± 0.12 mg/100 g	Leaves	MeOH
	Isorhamnetin-3-O-β-D-glucopyranoside	10.61 ± 0.29 mg/100 g	Burs	MeOH	[10]
		50.33 ± 1.87 mg/100 g	Leaves	MeOH
	Quercetin-3-O-α-L- -rhamnopyranoside	3.06 ± 0.24 mg/100 g	Leaves	MeOH	[10]
	Quercetin-3-O- -glucuronide	137.73 ± 4.19 mg/100 g DW	Leaves	UAE-MeOH	[43]
		1.12 ± 0.05 to 4.23 ± 0.32 mg/g	Leaves	MAC-EtOH, UAE-EtOH, MAE-w	[44]
		0.36 ± 0.004 to 1.66 ± 0.15 mg/g	Burs	MAC-EtOH, UAE-EtOH, MAE-w	[44]
	Quercetin-3-O- -rutinoside	0.33 ± 0.01 to 1.54 ± 0.07 mg/g	Leaves	MAC-EtOH, UAE-EtOH, MAE-w	[44]
		0.18 ± 0.004 to 2.67 ± 0.12 mg/g	Burs	MAC-EtOH, UAE-EtOH, MAE-w	[44]
	Quercetin-3-O- -glucoside	184.30 ± 4.02 mg/100 g DW	Leaves	UAE-MeOH	[43]
	Quercetin-O- -hexoside	0.25 ± 0.04 to 3.03 ± 0.12 mg/g	Leaves	MAC-EtOH, UAE-EtOH, MAE-w	[44]
		0.063 ± 0.001 to 1.18 ± 0.05 mg/g	Burs	MAC-EtOH, UAE-EtOH, MAE-w	[44]
	Kaempferol-3-O- -glucoside	61.60 ± 1.39 mg/100 g DW	Leaves	UAE-MeOH	[43]
		0.39 ± 0.04 to 6.98 ± 0.1 mg/g	Leaves	MAC-EtOH, UAE-EtOH, MAE-w	[44]
		0.1 ± 0.002 to 0.41 ± 0.01 mg/g	Burs	MAC-EtOH, UAE-EtOH, MAE-w	[44]
	Isorhamnetin-3-O- -glucoside	22.33 ± 0.64 mg/100 g DW	Leaves	UAE-MeOH	[43]
	Isorhamnetin-3-O- -rutinoside	0.93 ± 0.05 to 3.08 ± 0.2 mg/g	Leaves	MAC-EtOH, UAE-EtOH, MAE-w	[44]
		0.10 ± 0.005 to 0.83 ± 0.07 mg/g	Burs	MAC-EtOH, UAE-EtOH, MAE-w	[44]
	Myricetin-3- -glucoside	0.07 mg/g DW	Shells	MAE-EtOH	[40]
	Gallocatechin	0.76 mg/g DW	Shells	MAE-EtOH	[40]
	Catechin	0.15 mg/g DW	Shells	MAE-EtOH	[40]
		8.14 ± 0.41 to 10.35 ± 0.52 mg/g DW	Shells	UAE; MAE
	Epicatechin	0.03 mg/g DW	Shells	MAE-EtOH	[40]
		0.53 ± 0.03 mg/g DW	Shells	UAE-EtOH; MAE-w
Tannins	Chestanin	3.23 ± 0.06 mg/100 g	Burs	MeOH	[10]
		85.84 ± 2.18 mg/100 g	Leaves	MeOH
	Cretanin	16.75 ± 2.24 mg/100 g	Burs	MeOH	[10]
		0.186 ± 0.002 to 3.66 ± 0.17 mg/g	Burs	MeOH
		95.2 ± 7.21 mg/100 g	Leaves	MeOH
		1.01 ± 0.05 to 1.87 ± 0.13 mg/g	Leaves	MAC-EtOH, UAE-EtOH, MAE-w	[44]
	Ellagitannis	436.93 mg/100 g DW	Leaves	UAE-MeOH	[43]
	Gallotannins	13.51 ± 0.19 mg/100 g DW	Leaves	UAE-MeOH	[43]
	Ellagic acid	3.09 ± 0.25 mg/100 g	Burs	MeOH	[10]
		0.9 mg/g DW	Shells	MAE-EtOH	[40]
		0.33 ± 0.003 to 1.81 ± 0.04 mg/g	Shells	MAC-EtOH, UAE-EtOH, MAE-w	[44]
		7.97 ± 0.59 mg/100 g	Leaves	MeOH	[10]
		3.94 ± 0.20 to 4.04 ± 0.20 mg/g DW	Shells	UAE-EtOH; MAE-w	[42]
	Ellagic acid pentoside	15.59 ± 2.49 mg/100 g DW	Leaves	UAE-MeOH	[43]

Note: The values are presented as described by the authors in the respective references. Abbreviations: DW: dry weight; MAC-EtOH: maceration with ethanol; UAE-EtOH: ultrasound-assisted extraction with ethanol; UAE-MeOH: ultrasound-assisted extraction with methanol; MAE-w: microwave-assisted extraction with water; MAE-EtOH: microwave-assisted extraction with ethanol; MeOH: methanol.

. As observed, the shells are a good source of phenolic acids, including gallic acid and protocatechuic acid [42], and flavonoids, including catechins [40]. The leaves are also a good source of phenolic acids, including crenatin [10], gallic acid, and methyl gallate [43], the most abundant. Quercetin derivatives are the main flavonoids in leaves (Table 4).

5. Extraction and Processing Techniques

In the extraction procedure, the most common methods use organic solvents to achieve higher yields and extract a wide range of molecules. However, the procedures vary widely across authors, making it challenging to compare results.

Cerulli et al. [10] obtained MeOH extracts of the chestnut leaves, burs, and fruit of Castanea sativa “Marrone di Roccadaspide”. For this procedure, the authors used an extraction method that sequentially used solvents of increasing polarity, starting with petroleum ether, then chloroform, and ending with methanol. After this, the extract was filtered, and the solvent evaporated. All samples were previously dried, but the plant material (g)/solvent (L) ratio varied. For the leaves, 40 g of plant material was used, and the extraction was carried out three times for each solvent (sequentially), using 0.6 L for 3 days each time. For the burs, 34.3 g of plant material was used, and 0.44 L of each solvent was used for 3 days, repeated only twice with petroleum ether and chloroform, but three times with methanol. Regarding the fruits, 100 g of dried fruit was used, and 0.4 L of each solvent was used for 3 days, three times. Nonetheless, the procedure yielded similar extraction yields: 11.87% (leaves), 11.60% (burs), and 13.26% (fruit). In addition, the resulting MeOH extract obtained from dried fruits was further processed using the n-butanol/water partition assay to remove free sugars [10]. Other authors use more straightforward, more environmentally friendly extraction methods, using water as the solvent and agitation at room temperature [45]. Another option is subcritical water extraction, which Pinto et al. [46] explored to optimize polyphenol extraction and maximize the antioxidant activity of chestnut shells. To achieve this, the authors employed response surface methodology. They investigated the effects of temperature and extraction time on the recovery of phenolic compounds and their antioxidant activity against various synthetic radicals, as well as those with biological relevance (e.g., superoxide) [46]. For subcritical water extraction, different plant-to-solvent ratios have been used, depending on the authors. For example, 10 g/100 mL [46] or 20 g/400 mL and 20 g/600 mL [47] have been used, highlighting the lack of uniformity across studies, which is likely to influence the bioactivities. The effect of the extraction method on the phytochemical composition of the resulting extracts is well established, mainly for polyphenols, as well as its relationship with various bioactivities, namely the antioxidant activity [48], which is addressed in the section below.

6. In Vitro Assessment of Radical Scavenging Activity

Antioxidant Activity: In Vitro Radical Scavenging, Cellular Antioxidant Activity, and In Vivo Studies

Various authors have addressed the antioxidant potential of extracts produced from chestnut-derived products, exploiting not only the bioactivity but also variations induced by the extraction method. As shown in Table 5, in addition to the chestnut fruit, various other by-products or production waste products have been tested for their antioxidant activity, including leaves or other parts usually considered non-edible components of chestnut husks.

Genovese et al. [45] reported the antioxidant potential of water and ethanolic extracts of the pellicle of the fruit obtained from Castanea sativa in Italy. The ethanolic extracts present a total phenolic content 2.4 times higher than the aqueous extract, and also a total flavonoid content 3.18 times higher. This was reflected in the antioxidant activity, as the ethanolic extracts showed higher radical-scavenging activity, as assessed by the DPPH and superoxide assays [45].

Also relevant is the comparison of different Castanea sativa cultivars to determine which cultivar has the highest potential as a source of antioxidant agents. A study performed by Barletta et al. [49] compared the antioxidant activity of hydroethanolic extracts of shells obtained from “Bastarda Rossa”, “Cecio”, and “Marroni” cultivars from Italy. The authors reported differences in FRAP and DPPH assays, with “Cecio” performing better than the other cultivars, likely associated with higher total phenolic and total flavanol content [49].

A relevant note is the wide range of radical scavenging assays used by different authors, and the variations in methodologies (e.g., extraction methods, radical scavenging methods, concentrations used, and units used to express the results), which limit the ability to compare results between authors.

Table 5. Radical scavenging activities of chestnut-derived products, by-products, and production waste.

Sample	Extract Type	Radical Scavenging Assay	Ref.
Burs	MeOH	DPPH: EC50 = 4.21 µg/mL; TEAC: 3.03 mg/mL; FRAP: 2.96 mmol ferric sulphate/g	[10]
Leaves	MeOH	DPPH: EC50 = 3.06 µg/mL; TEAC: 3.01 mg/mL; FRAP: 1.48 mmol ferric sulphate/g
Fruit	MeOH	DPPH: EC50 = 34.64 µg/mL; TEAC: 0.57 mg/mL; FRAP: 0.18 mmol ferric sulphate/g
Fruit pellicle	H2O	O2•− scavenging: ~40% inhibition at 172 µg/mL DPPH: ~50% inhibition at 172 µg/mL	[45]
	EtOH	O2•− scavenging: ~40% inhibition at 172 µg/mL DPPH: ~50% inhibition at 172 µg/mL
Shells	Subcritical water extraction	HOCl: IC50 = 0.79 µg/mL; O2•−: IC50 = 12.92 µg/mL; ROO•: 0.32 µmol TE/mg DW; DPPH: 815.66 mg trolox eq./g DW; ABTS: 901.16 mg ascorbic acid eq./g DW; FRAP: 7994.26 mg ferrous sulphate eq./g DW	[46]
Shells	Ultrasound-Assisted (Water) Extraction	DPPH: IC50 = 44.10 µg/mL; ABTS: IC50 = 65.40 µg/mL; FRAP: IC50 = 32.00 µg/mL; HOCl: IC50 = 0.70 µg/mL; O2•−: IC50 = 14.10 µg/mL; ROO•: 0.30 µmol TE/mg DW; •NO: IC50 = 0.10 µg/mL	[50]
Shells	EtOH: Water (70:30; v/v)	FRAP: between 4.56 and 7.04 mmol ascorbic acid eq./mg extract	[49]
Fruit peels	Subcritical water extraction	DPPH: 5.43 mmol trolox eq./g extract; Cu2+ chelating activity: 85.07 mmol EDTA eq./g extract	[47]

Notes: DPPH: 1,1-diphenyl-2-picrylhydrazyl; TEAC: trolox equivalent antioxidant capacity; FRAP: ferric-reducing antioxidant power; O₂^•−: superoxide radical; ROO^•: peroxy radical; HOCl: hypochlorous acid; ^•NO: nitric oxide radical; EDTA: ethylenediaminetetraacetic acid; eq.: equivalents. Results are listed as reported by the authors.

Table 5. Radical scavenging activities of chestnut-derived products, by-products, and production waste.

Sample	Extract Type	Radical Scavenging Assay	Ref.
Burs	MeOH	DPPH: EC50 = 4.21 µg/mL; TEAC: 3.03 mg/mL; FRAP: 2.96 mmol ferric sulphate/g	[10]
Leaves	MeOH	DPPH: EC50 = 3.06 µg/mL; TEAC: 3.01 mg/mL; FRAP: 1.48 mmol ferric sulphate/g
Fruit	MeOH	DPPH: EC50 = 34.64 µg/mL; TEAC: 0.57 mg/mL; FRAP: 0.18 mmol ferric sulphate/g
Fruit pellicle	H2O	O2•− scavenging: ~40% inhibition at 172 µg/mL DPPH: ~50% inhibition at 172 µg/mL	[45]
	EtOH	O2•− scavenging: ~40% inhibition at 172 µg/mL DPPH: ~50% inhibition at 172 µg/mL
Shells	Subcritical water extraction	HOCl: IC50 = 0.79 µg/mL; O2•−: IC50 = 12.92 µg/mL; ROO•: 0.32 µmol TE/mg DW; DPPH: 815.66 mg trolox eq./g DW; ABTS: 901.16 mg ascorbic acid eq./g DW; FRAP: 7994.26 mg ferrous sulphate eq./g DW	[46]
Shells	Ultrasound-Assisted (Water) Extraction	DPPH: IC50 = 44.10 µg/mL; ABTS: IC50 = 65.40 µg/mL; FRAP: IC50 = 32.00 µg/mL; HOCl: IC50 = 0.70 µg/mL; O2•−: IC50 = 14.10 µg/mL; ROO•: 0.30 µmol TE/mg DW; •NO: IC50 = 0.10 µg/mL	[50]
Shells	EtOH: Water (70:30; v/v)	FRAP: between 4.56 and 7.04 mmol ascorbic acid eq./mg extract	[49]
Fruit peels	Subcritical water extraction	DPPH: 5.43 mmol trolox eq./g extract; Cu2+ chelating activity: 85.07 mmol EDTA eq./g extract	[47]

Notes: DPPH: 1,1-diphenyl-2-picrylhydrazyl; TEAC: trolox equivalent antioxidant capacity; FRAP: ferric-reducing antioxidant power; O₂^•−: superoxide radical; ROO^•: peroxy radical; HOCl: hypochlorous acid; ^•NO: nitric oxide radical; EDTA: ethylenediaminetetraacetic acid; eq.: equivalents. Results are listed as reported by the authors.

Among comparable studies, the shell extracts reported by Lameirão et al. [50] showed similar IC50 values for scavenging HOCl, O₂^•−, and ROO^•, supporting the antioxidant potential of this chestnut by-product.

7. Assessment of Chestnut Fruit By-Products as Waste Bioactivities Using Cell-Based Assays and In Vivo Studies

7.1. Antioxidant Activity

Regarding antioxidant activity evaluated using biological models, although there are not many studies reporting this bioactivity, some authors have reported the potential of chestnut-derived extracts to mitigate oxidative stress. These studies are presented in Table 6.

In THP-1-XBlue-MD2-CD14 cells (derived from THP-1 (human acute monocytic leukemia cells) via transfection; expressing a NF-κB and AP-1 reporter gene encoding secreted embryonic alkaline phosphatase (SEAP) [51]) pre-exposed to 5 µg/mL of leaves, burs or fruit extracts for 1 h, and then treated with pyocyanin as oxidative agent, it was observed that all extracts reduced pyocyanin-induced oxidative stress. Burs extract showed the highest cellular antioxidant activity, reducing ROS by 74% (compared to the positive control), followed by leaves and fruit extracts, the latter presenting the lowest antioxidant activity [10].

Fruit peel extracts were also shown to reduce ROS levels in 3T3-L1 cells (fibroblasts, differentiated into adipocytes using high glucose, dexamethasone, insulin, and 3-isobutyl-1-methylxanthine (IBMX)) exposed to 75 µg/mL of extract, while not inducing cytotoxicity [47]. In TK6 cells (human lymphoblast) pre-exposed to a chestnut bark extract (commercially available, extraction not specified) and then treated with 100 µM of hydrogen peroxide (H₂O₂) as oxidant agent, it was shown that pre-exposure to the extract (12 and 24 µg/mL) reduced H₂O₂-induced oxidative stress [52]. In addition, it was also reported that 12 µg/mL of the bark extract was able to decrease mitomycin C-induced increase in reactive oxygen species, which could be correlated to the extract’s ability to counter mitomycin C mutagenicity. Also, 12 µg/mL of the bark extract induced low cytotoxicity in TK6 cells [52].

Using a model of streptozotocin-induced diabetes in Wistar rats, Jovanović et al. [53] reported that mice treated with a chestnut burs hydroethanolic extract (60 mg/kg; 4 weeks) showed reduced ROS and lipid peroxidation in the liver and kidney, when compared to rats only treated with streptozotocin. The effect was associated with an increased activity of MnSOD (manganese-dependent superoxide dismutase) and CuZnSOD (copper–zinc superoxide dismutase), as well as increased GSH/GSSG (reduced glutathione/oxidized glutathione) ratio [53]. In addition, the burs extract reduced streptozotocin-induced DNA damage and showed antiglycation activity (both in vivo and in vitro), thereby reducing N-(carboxymethyl)lysine (an advanced glycation end product; AGE) in both the liver and kidney [53]. This effect was correlated with reduced RAGE (receptor for advanced glycation end-products) and NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) protein levels in the liver and kidney, suggesting that the extract exerts its effect on the RAGE-NF-kB axis [53].

Table 6. Antioxidant activity of chestnut-derived products, by-products, and waste evaluated using in vitro and in vivo experimental models.

Sample	Extract Type	Experimental Model	Concentration	Observations	Ref.
Burs	MeOH	THP-1-XBlue-MD2-CD14 cells	5 µg/mL	Reduced pyocyanin-induced ROS	[10]
Leaves	MeOH
Fruit	MeOH
Burs	EtOH: Water (50:50; v/v)	Wistar rats (Streptozotocin-induced diabetes)	60 mg/kg	Reduced ROS and lipid peroxidation Increased MnSOD and CuZnSOD activity Increased GSH/GSSG ratio	[53]
Bark	Not specified	TK6 cells	12 µg/mL	Reduced H2O2-induced ROS; Reduced mitomycin C-induced ROS	[52]
Peels	Subcritical water extraction	3T3-L1 cells	75 µg/mL	Reduced ROS levels	[47]
Shells	Subcritical water extraction	Wistar rats	100 mg/kg	Increased SOD and GSH-Px activity in the liver, kidney, and serum	[54]
Wood distillate	Commercial extract; Pyrolysis	HaCaT cells	0.07% (v/v)	Reduced H2O2-induced ROS	[55]
		A431 cells
		NHDF cells
		HUVEC cells
Burs	Aqueous	RAW 264.7 cells	25, 50, and 100 µg/mL	Reduced LPS-induced ROS	[56]

Table 6. Antioxidant activity of chestnut-derived products, by-products, and waste evaluated using in vitro and in vivo experimental models.

Sample	Extract Type	Experimental Model	Concentration	Observations	Ref.
Burs	MeOH	THP-1-XBlue-MD2-CD14 cells	5 µg/mL	Reduced pyocyanin-induced ROS	[10]
Leaves	MeOH
Fruit	MeOH
Burs	EtOH: Water (50:50; v/v)	Wistar rats (Streptozotocin-induced diabetes)	60 mg/kg	Reduced ROS and lipid peroxidation Increased MnSOD and CuZnSOD activity Increased GSH/GSSG ratio	[53]
Bark	Not specified	TK6 cells	12 µg/mL	Reduced H2O2-induced ROS; Reduced mitomycin C-induced ROS	[52]
Peels	Subcritical water extraction	3T3-L1 cells	75 µg/mL	Reduced ROS levels	[47]
Shells	Subcritical water extraction	Wistar rats	100 mg/kg	Increased SOD and GSH-Px activity in the liver, kidney, and serum	[54]
Wood distillate	Commercial extract; Pyrolysis	HaCaT cells	0.07% (v/v)	Reduced H2O2-induced ROS	[55]
		A431 cells
		NHDF cells
		HUVEC cells
Burs	Aqueous	RAW 264.7 cells	25, 50, and 100 µg/mL	Reduced LPS-induced ROS	[56]

Filippelli et al. [55] evaluated the cellular antioxidant activity of wood distillate obtained from Castanea sativa biomass (pyrolyzed, commercially available), an underutilized waste that is receiving increased relevance. The wood distillate (0.07%; v/v) was able to reduce H₂O₂-induced ROS in HaCaT (non-tumoral human keratinocytes), A431 (human epidermoid carcinoma cells), HUVEC (non-tumoral human umbilical vein endothelial cells), and NHDF (non-tumoral human dermal fibroblasts) cell models [55]. However, it should be noted that 0.07% of the extract was cytotoxic to NHDF cells, as assessed by cleaved caspase 3 levels (a pro-apoptotic protein) [55].

7.2. Anti-Inflammatory Activity

Table 7 presents recent studies regarding the anti-inflammatory activity of chestnut-derived products and by-products. In addition to antioxidant activity, anti-inflammatory activity is highly sought after, and both are often addressed simultaneously. This is the case of the data reported by Filippelli et al. [55], which in addition to the cellular antioxidant activity, the authors also noted that the wood distillate reduced COX-2 (cyclooxygenase-2) and mPGES-1 (microsomal prostaglandin E synthase-1), ICAM-1 (intercellular adhesion molecule 1) and VCAM-1 (vascular cell adhesion molecule-1) protein levels.

A study focused on the anti-inflammatory activity produced by MeOH extracts of leaves, burs, and fruits showed a reduction in NF-kB activation in THP-1-XBlue-MD2-CD14 pre-exposed to 5 µg/mL of extracts for 1 h and then treated with LPS (lipopolysaccharides), when compared to the positive control (cells only treated with LPS). Leaves MeOH extracts exhibited the highest anti-inflammatory activity, reducing NF-kB activation by 65% and showing an effect greater than that of 1 µM prednisone (a corticosteroid used to treat inflammation) [10]. In addition, all extracts reduced nitric oxide (NO) production in LPS-stimulated J774.A1 cells (mouse macrophages), and pre-exposure to the leaf extracts resulted in the lowest NO levels (among extracts) [10], and thus provided insights into the role of chestnut leaf extracts in the modulation of the NF-kB pathway.

Table 7. Anti-inflammatory activity of chestnut-derived products and by-products.

Sample	Extract Type	Experimental Model	Concentration	Observations	Ref.
Burs	MeOH	THP-1-XBlue-MD2-CD14 cells	5 µg/mL	Reduced LPS-induced NF-kB activation	[10]
Leaves
Fruit
Burs	MeOH	J774.A1 cells	5 µg/mL	Reduced LPS-induced NO production	[10]
Leaves
Fruit
Burs	MeOH-d4/H2O-d2 (50:50; v/v)	BV-2 cells	0.5 mg/mL	Reduced LPS-induced inflammation Decreased IL-1β and TNF-α expression Decreased NF-kB protein levels	[57]
Leaves
Wood distillate	Commercial extract; Pyrolysis	HUVEC cells	0.07% (v/v)	Reduced IL-1β/TNF-α-induced inflammation Reduced COX-2, mPGES-1, VCAM-1 and ICAM-1 levels	[55]
Buds	EtOH: Water (50:50; v/v)	Caco-2 cells	50 µg/mL	Reduced IL-1β/IFN-γ-induced inflammation Reduced CXCL-10, IL-8, MCP-1 and ICAM-1 levels Reduced NF-kB-driven transcription	[11]
Wood
Pericarp
Episperm
Burs	Aqueous	RAW 264.7 cells	100 µg/mL	Reduced LPS-induced nitric oxide production Reduced NF-kB activation Reduced iNOS protein level	[56]

Table 7. Anti-inflammatory activity of chestnut-derived products and by-products.

Sample	Extract Type	Experimental Model	Concentration	Observations	Ref.
Burs	MeOH	THP-1-XBlue-MD2-CD14 cells	5 µg/mL	Reduced LPS-induced NF-kB activation	[10]
Leaves
Fruit
Burs	MeOH	J774.A1 cells	5 µg/mL	Reduced LPS-induced NO production	[10]
Leaves
Fruit
Burs	MeOH-d4/H2O-d2 (50:50; v/v)	BV-2 cells	0.5 mg/mL	Reduced LPS-induced inflammation Decreased IL-1β and TNF-α expression Decreased NF-kB protein levels	[57]
Leaves
Wood distillate	Commercial extract; Pyrolysis	HUVEC cells	0.07% (v/v)	Reduced IL-1β/TNF-α-induced inflammation Reduced COX-2, mPGES-1, VCAM-1 and ICAM-1 levels	[55]
Buds	EtOH: Water (50:50; v/v)	Caco-2 cells	50 µg/mL	Reduced IL-1β/IFN-γ-induced inflammation Reduced CXCL-10, IL-8, MCP-1 and ICAM-1 levels Reduced NF-kB-driven transcription	[11]
Wood
Pericarp
Episperm
Burs	Aqueous	RAW 264.7 cells	100 µg/mL	Reduced LPS-induced nitric oxide production Reduced NF-kB activation Reduced iNOS protein level	[56]

Chiocchio et al. [57] reported the anti-inflammatory activity of leaves and burs hydro-methanolic extracts in LPS-stimulated BV-2 cells (mouse microglia). It was shown that both leaf and burs extracts were able to reduce LPS-induced cytotoxicity and reduce IL-1β and TNF-α transcription, and the effect was related to decreased NF-κB protein levels [57]. Also, studying chestnut by-products, Pozzoli et al. [11] evaluated the potential of hydroethanolic extracts obtained from buds, wood, pericarp, and episperm as anti-inflammatory agents at the intestinal level. The extracts effectively reduced CXCL-10 (C-X-C motif chemokine ligand 10), IL-8 (interleukin-8), MCP-1 (monocyte chemoattractant protein-1), and ICAM-1 levels in IL-1β/IFN-γ-stimulated Caco-2 cells (human colorectal adenocarcinoma; used as a model of intestinal epithelial cells). The effect was also correlated with an inhibition in the NF-kB pathway [11]. The extract obtained from the burs also reduced cytokine and ICAM-1 levels. Still, it did not affect NF-kB-driven transcription, suggesting that the inhibition of cytokine production may be achieved through the modulation of other pathways or that the anti-inflammatory activity is exerted downstream of NF-kB.

Also related to NF-kB pathway, Frusciante et al. [56] reported that 100 µg/mL of an aqueous burs extract reduced NF-kB activation in LPS-stimulated RAW 264.7 (mouse macrophages), observed as reduced p65 subunit protein levels, and also decreased expression of this subunit in the nucleus, which indicates the inhibition of this translocation to the nucleus, a key step in NF-kB pathway [56]. This resulted in reduced NO production, with the 100 µg/mL extract being more effective than 5 µg/mL of dexamethasone, due to reduced iNOS (inducible nitric oxide synthase) protein levels [56].

7.3. Anti-Tumoral Activity

Cerulli et al. [10] evaluated the cytotoxicity of leaves, burs, and fruit MeOH extracts in THP-1, THP-1-XBlue-MD2-CD14, and J774.A1 cell lines, and reported that the extracts were non-cytotoxic at 5 µg/mL [10]. Using aqueous and ethanolic fruit pellicles, a different study showed that the ethanolic extracts (with higher phenolic content) reduced MCF-7 cells (human breast adenocarcinoma) viability in a dose-dependent manner at concentrations ≥ 10 µg/mL, while the aqueous extracts only reduced cell viability significantly at concentrations ≥ 100 µg/mL [45]. Considering other by-products, the anti-proliferative activity of chestnut fruit shell extracts (obtained by subcritical water extraction) was evaluated in two intestinal cell lines, Caco-2 and HT-29-MTX (human colorectal adenocarcinoma cells treated with methotrexate (MTX)). Both cell lines responded similarly to the extracts, with a significant reduction in cell viability at concentrations ≥ 1 µg/mL [46].

Lameirão et al. [50] evaluated the anti-proliferative activity of a shell extract (ultrasound-assisted water extraction) in four cell lines: Caco-2, HT-29/MTX, HaCaT, and HFF-1 (human fibroblasts). Exposure to 0.1 µg/mL of extract reduced the cell viability of Caco-2 (68.8% cell viability) and HT-29/MTX (67% cell viability) [50], while not decreasing the viability of HaCaT and HFF-1 cells. HaCaT cells’ viability was reduced to 79.1% when exposed to 1 µg/mL. Still, a further increase in concentration (up to 1000 µg/mL) did not induce a significant decrease in viability. At the highest concentration tested, there was no significant difference between HaCaT, Caco-2, and HT-29/MTX cells’ viability. Regarding HFF-1 cells, no significant reduction in cell viability was observed at concentrations up to 1000 µg/mL [50].

Aiming to provide more detail regarding the molecular mechanisms underlying the anti-tumoral activity of chestnut-derived extracts, Cacciola et al. [58] assessed the anti-tumoral potential of aqueous extracts obtained from burned inner and outer chestnut shells. The anti-proliferative activity was evaluated in seven cell lines: DU 145 (human prostate carcinoma), PC-3 (human prostate adenocarcinoma), LNCaP (human prostate carcinoma), PNT2 (non-tumoral human prostate cells), HepG2 (human liver carcinoma), MCF-7 (human breast adenocarcinoma), and MDA-MB-231 (human breast adenocarcinoma). Interestingly, the various cell lines responded differently to the extract. Among the prostate cancer cell models, DU 145 was the most sensitive to the extract-induced cytotoxicity, with an IC₅₀ value of 35.78 µg/mL, followed by LNCaP (IC₅₀ = 54.88 µg/mL). In contrast, in non-tumoral cells, PNT2 exposed to the extracts showed no decrease in cell viability at concentrations up to 55.50 µg/mL, highlighting the potential of these extracts for the treatment of prostate cancer while not affecting non-tumoral cells. However, this effect is selective, as the extract’s anti-proliferative effect in PC-3 cells was less pronounced, reducing cell viability to ~75% at the highest concentration tested (100 µg/mL) [58]. The extract did not significantly reduce the cell viability of MDA-MB-231 or HepG2 cells in concentrations up to 100 µg/mL, and reduced the viability of MCF-7 cells to ~75% at this concentration [58]. Thus, the selective effect in two prostate cancer cells is apparent, and to further understand this effect, the authors performed an Annexin V/PI double staining assay in DU 145 cells exposed to 55.50 µg/mL or 100 µg/mL of extract, where an increase in cells undergoing apoptosis was observed [58].

Considering the anti-tumoral activity of extracts produced using by-products of different Castanea sativa cultivars, the anti-proliferative activity of hydroethanolic shell extracts obtained from three different cultivars was recently reported. The extracts reduced the viability of SaOS-2 cells (human osteosarcoma) in a dose- and time-dependent manner, but the effect was highly cultivar-dependent [49], highlighting the need to study further the anti-proliferative effects of the main compounds in chestnut-derived products to identify novel potential anti-tumoral molecules.

Using a hydro-methanolic extract of Castanea sativa leaves, it was shown that the extract exhibited anti-tumoral activity against Caco-2, DLD-1 (human colorectal adenocarcinoma), and MCF-7, with IC₅₀ values ranging from 100 to 200 µg/mL. In contrast, for U87MG (human brain glioblastoma) and U251MG (human glioblastoma) cell lines, the obtained IC₅₀ values ranged between 200 and 300 µg/mL. The extract was significantly less effective against SK-MEL-28 (human skin melanoma), highlighting that the anti-tumor effect is highly dependent on the tumor type [43].

7.4. Cardioprotective Activity and Effect on Metabolic Indicators

Chestnut-derived extracts have demonstrated complementary effects on lipid and glucose metabolism, contributing to their overall metabolic benefits. Cravotto et al. [47] showed that supercritical water extract obtained from fruit peels exerted an antiadipogenic effect in 3T3-L1 cells by reducing intracellular triglyceride accumulation. In cells exposed to the extract (concentrations ≥ 20 µg/mL) during maturation, triglyceride accumulation decreased, although cell viability decreased [47]. In Wistar rats treated with a shell extract obtained by subcritical water extraction at 50 or 100 mg/kg, reduced triglyceride and low-density lipoprotein (LDL) cholesterol levels were observed, along with increased high-density lipoprotein (HDL) cholesterol levels [54], supporting the hypolipidemic potential of chestnut by-products.

Beyond lipid regulation, several studies indicate that Castanea sativa also exerts hypoglycaemic effects. In streptozotocin-induced diabetic Wistar rats, daily administration of a chestnut burs hydroethanolic extract (60 mg/kg; 4 weeks) significantly reduced blood glucose, glycated hemoglobin, cholesterol, and triglyceride levels, compared to rats treated only with streptozotocin [53]. In Section 7.1, the capacity of chestnut burs extract to reduce streptozotocin-induced oxidative stress in the liver and kidney is also reviewed, highlighting the wide range of health-promoting effects of this extract.

Additional mechanisms contributing to glucose regulation have been identified, such as the mild anti-diabetic activity of a hydro-methanolic extract obtained from the leaves, through inhibition of α-amylase and β-glucosidase, and anti-obesity activity, assessed as anti-lipase activity, suggesting a delay in carbohydrate digestion and glucose absorption [43]. Fruit shell extracts produced by subcritical water extraction showed mild but reproducible anti-α-amylase activity (~18% inhibition), which increased following simulated gastrointestinal digestion (~23%) [59]. Also, microwave-assisted extracts exhibited similar effects, inhibiting ~15% of α-amylase activity at 1 mg/mL [40]. Together, these findings point to enzyme inhibition as a relevant contributor to the hypoglycemic action of chestnut by-products.

Dietary supplementation studies also support a metabolic benefit. FVB/n mice (an inbred strain widely used for transgenic injection, which presents higher sensitivity to the Friend leukemia virus B [60]) fed a diet enriched with chestnut fruit (1.1%) showed reduced abdominal fat without signs of oxidative or DNA damage [12], suggesting an improvement in energy metabolism and adiposity that may indirectly minimize insulin resistance.

Overall, the combination of hypolipidemic, hypoglycemic, antioxidant, and enzyme-modulating activities highlights the potential of Castanea sativa fruit and its by-products as valuable functional ingredients and as a source of nutraceuticals targeting metabolic health. Research highlights both the nutritional value of chestnut kernels and the sustainable use of chestnut waste, offering health-promoting properties and supporting circular bioeconomy strategies. Chestnut shells and kernels are rich in polyphenols, flavonoids, tannins, vitamin E, and dietary fiber (see Section 3 and Section 4). These compounds exhibit potent antioxidant, anti-inflammatory, anticancer, hypolipidemic, hypoglycemic, neuroprotective, and antimicrobial activities, making chestnut by-products valuable for disease prevention and health promotion [49,54,61,62].

8. Technological and Industrial Applications of Chestnut Fruits, By-Products, and Waste Products

8.1. Chestnuts in Food Products and Functional Foods

Chestnut fruit is widely used to enrich flours, beverages, and food emulsions, enhancing nutritional value, antioxidant content, and sensory properties [34]. Chestnut flour is used in bread, cakes, muffins, cookies, and biscuits, often as a partial substitute for wheat flour (typically 10–50%). This increases fiber, minerals, and antioxidants, and is especially popular in gluten-free formulations [2,63,64,65]. Chestnut flour can be incorporated into gluten-free pasta, fresh pasta, and extruded snacks, enhancing antioxidant activity and imparting unique flavor and color. Substitution rates of up to 25% are typical in pasta and snacks [63,66,67,68,69]. Cascone et al. [67] blended chestnut flour with spelt and chickpea flours to create expanded (emulsified) snacks that retain bioactive compounds and desirable sensory properties.

Adding chestnut to traditional fermented rice beverages increases metabolite diversity, total polyphenol content, and antioxidant activity, while improving flavor and providing protection against oxidative stress in cell models [70]. Moreover, chestnut is used in traditional Mediterranean desserts, such as chestnut pudding, which can be formulated with or without added sugar, maintaining positive sensory and nutritional attributes [71].

Innovation in gastronomy led Lv et al. [72] to use chestnut flour in composite pastes for 3D and 4D food printing, combining with sodium alginate to create shape-changing snacks with improved texture and flavor.

Chestnut-derived ingredients are used in functional foods; enrichment of bakery products (e.g., cookies, pancakes) with chestnut shell extracts increases antioxidant content and sensory appeal [13,54]. Fermented chestnut products also show enhanced nutritional and functional properties [73]. In contrast, chestnut shell extracts are developed as supplements targeting oxidative stress, metabolic disorders, and gut health, with proven in vitro and in vivo bioactivity [49,74]. Chestnut extracts can also serve as natural antioxidants and antimicrobials, offering alternatives to synthetic preservatives [13,49,54,75].

Table 8. Chestnut by-products enhance food, supplement, and preservation applications, and chestnut-enriched flours, beverages, and emulsions for nutrition and function—examples of raw chestnut material and final commercialized products.

Application Area	Chestnut Component Used	Key Active Substances	Main Benefits	Example Product/Use	Commercial Example	Ref.
Functional bakery goods	Chestnut flour; chestnut shell extract	Polyphenols, flavonoids, fiber	Antioxidant enhancement, improved sensory profile, gluten-free	Cookies and pancakes	Cookies and crispbread with chestnut flour (e.g., Amisa®)	[13,62]
Nutraceuticals	Shell/bur extracts; leaf extracts	Polyphenols, tannins, tocopherols	Antioxidant and anti-inflammatory activity, oxidative stress reduction	Supplements, capsules, extracts, concentrated powders	Light Sweet Chestnut de Esencias Triunidad®	[49,74]
Food preservation	Shell/wood extracts; polysaccharides	Tannins, phenolics	Natural antioxidant and antimicrobial protection	Meat, bakery, dairy	Chestnut flowers as a substitute for SO2 in wines (patented [75]).	[13,49,76]
Dietary fiber source	Chestnut flour; shell fiber	Insoluble and soluble fiber, resistant starch	Gut health, metabolic regulation	Fiber-enriched foods	-	[13,61,76]
Bread, cakes, pasta	Chestnut flour (for recipe substitution)	Fiber, minerals, natural sugars	Higher dietary fiber, antioxidant potential, and improved flavor	Chestnut flour (10–50% substitution)	Organic Chestnut Tagliatelle (Pasta D’Alba®)	[63,64,65,66,68]
Snacks, extrudates	Chestnut flour blends	Polyphenols, fiber, carbohydrates	Improved texture, flavor, and nutritional quality	Extruded snacks, bars	-	[67,69]
Fermented beverages	Chestnut flour	Sugars, phenolics	Enhanced antioxidant capacity, flavor diversification, and fermentation substrate	Fermented beverages	Artisanal Judia beer and gin (JUDIA®)	[70]
3D/4D printed foods	Chestnut–alginate composite	Fiber, polysaccharides	Novel textures, personalized nutrition	4D-printed food matrices	-	[72]

summarizes chestnut by-products for food, supplement, and preservation applications, as well as chestnut-enriched flours, beverages, and emulsions for nutrition and function, all of which have already been studied by several authors.

Valorizing chestnut waste (shells and burs) reduces environmental impact and creates high-value products, aligning with circular bioeconomy principles. This approach supports food sustainability, waste reduction, and the development of new revenue streams for producers [13,40,49].

8.2. Cosmetic and Pharmaceuticals, Key Bioactive Compounds and Mechanisms

Chestnut and its by-products (shells, burs, leaves, and skins) are being developed into formulations for cosmetic and nutraceutical applications, with demonstrated antioxidant and regenerative (skin-repairing) properties.

Oil-in-water creams enriched with chestnut shell extracts have been developed for skin care. These formulations are rich in polyphenols (e.g., rutin and ellagitannins) and vitamin E, providing antioxidant, anti-inflammatory, and skin-repairing effects. They show good stability, skin compatibility, and consumer-appealing sensory properties [76]. Chestnut shell extracts also protect against collagen degradation and enhance skin hydration, supporting anti-aging and regenerative claims [76].

Eco-friendly extracts from chestnut shells and burs, standardized for high tannin and flavonoid content, have been formulated as cosmetic and pharmaceutical ingredients. In vivo and in vitro studies confirm their antioxidant activity, ability to reduce oxidative stress, and potential to modulate inflammation and metabolic parameters [49]. Selenized chestnut polysaccharides and fermented chestnut products further enhance antioxidant and regenerative effects [73,77]. Moreover, extracts from different parts of the chestnut (shells, burs, and leaves) can be combined to provide broader antioxidant and regenerative benefits [13].

8.3. Active Packaging and Biopolymers

Chestnut extracts, especially those derived from by-products such as shells, burs, and leaves, are increasingly used in active packaging and biopolymer films as natural preservatives and stabilizers. These applications leverage chestnut’s rich polyphenolic content for antioxidant and antimicrobial effects, supporting food preservation and sustainability. Chestnut extracts are incorporated into pullulan, chitosan, starch, and alginate-based films, enhancing their antioxidant and antimicrobial properties. These films exhibit high DPPH radical-scavenging activity (up to 94%) and effective antibacterial activity, particularly against Gram-positive bacteria such as Staphylococcus aureus and some Gram-negative strains [78,79,80,81,82,83,84,85].

Films containing chestnut extracts reduce spoilage microorganisms and mycotoxins in foods such as cheese and pasta, extend shelf life, and maintain food quality by inhibiting microbial growth and oxidative processes [82,86]. Chestnut extracts improve the mechanical, barrier, and UV-protective properties of biopolymer films, making them more robust and suitable for food packaging applications [79,80,81,83,85], as shown in

Table 9. Chestnut extracts in packaging applications.

Film Matrix	Sample	Key Benefits	Food Application	Ref.
Pullulan	Spiny burs	Antioxidant, antibacterial, UV barrier	General packaging	[79]
Chitosan	Shell, wood, extract	Antimicrobial, improved barrier	Cheese, pasta	[80,82,86]
Alginate	Extract	Enhanced mechanical/antimicrobial	General packaging	[81,84]
Pectin/gelatin/zein	Shell extract	Antioxidant, low permeability	Oil, general food	[85]

.

Polyphenols, tannins, and flavonoids in chestnut extracts disrupt bacterial membranes, inhibit microbial metabolism, and scavenge free radicals, providing both preservative and stabilizing effects [84,87]. Using chestnut by-products in packaging aligns with circular economy principles, reducing waste and reliance on synthetic additives [79,87].

8.4. Sustainable Extraction, Innovation, and Technology Transfer

Projects like FINNOVER demonstrate the technical and economic feasibility of creating new supply chains for chestnut bioactives, emphasizing green extraction and the development of standardized, value-added products from by-product streams [88,89]. The project aimed to create sustainable supply chains for the extraction and utilization of natural, biologically active molecules. It focused on developing a green extraction method and reuse strategy for botanical by-products to produce value-added products as an alternative to waste composting or incineration. The research specifically examined Castanea sp. bud derivatives and compared their phytochemical composition with by-products from the bud-waste management process. Using maceration and the greener Pulsed Ultrasound-Assisted Extraction (PUAE), the study found that PUAE yielded 160.42 mg/100 g FW of bioactive compounds, compared with 1276.17 mg/100 g FW in commercial products. Approximately 13% of the chemical content in the bud preparations was retained in the by-product extracts. This indicates that PUAE is a sustainable alternative to traditional waste management methods and could lead to new market products derived from bud-derivative processing waste [89].

The development and curation of comprehensive genomic datasets across multiple Castanea species have accelerated breeding, disease-resistance research, and genetic improvement. Publicly available databases and advanced analysis tools now support global research collaboration and technology transfer in chestnut genomics [90,91].

Efforts to relaunch the chestnut production chain emphasize integrated pest management, improved processing (especially drying and flour production), and the valorization of by-products. The recovery of polyphenols and other bioactive compounds from chestnut waste is highlighted as a sustainable and profitable strategy that supports environmental and economic goals [2]. These efforts support circular economy models and promote sustainable rural development.

9. Future Perspectives and Challenges

The European chestnut represents not only a key component of Mediterranean forest ecosystems but also a strategic crop for sustainable agri-food systems in southern Europe [92]. Its ecological resilience, cultural heritage, and potential for high-value products position it at the intersection of environmental adaptation, rural development, and circular bioeconomy [18,21]. However, climate change, the emergence of novel pathogens, and the fragmentation of traditional production systems demand an integrated, multidisciplinary response.

The greatest challenge for the future is finding a balance between productivity and ecosystem resilience. Chestnut growth, phenology, and nut quality are all at risk due to shifting patterns of precipitation and rising average temperatures [93,94,95]. Drought and heat waves, especially during flowering and nut development, cause physiological stress that lowers photosynthetic efficiency, accelerates senescence, and reduces yield stability [96,97]. In this context, advancing adaptive management strategies — encompassing irrigation scheduling, canopy regulation, soil mulching, and mycorrhizal inoculation — remains a scientific and technological necessity [93].

Another major challenge concerns market competitiveness and consumer perception. Chestnut fruit is part of European culinary culture, yet its global market remains seasonal, especially in certain areas. For future growth, processed foods need to be more diverse, storage and packaging technologies need to be more effective, and international branding needs to be stronger, grounded in health and functional claims. Future research and innovation in the agri-food sector should increasingly adopt collaborative models that bring together consumers, companies, and universities in co-creation processes. Such an approach promotes the development of products that are not only technologically and commercially viable but also socially responsible and aligned with real consumer needs. In this methodology proposed by the EIT Food RIS Consumer Engagement Labs Project [98], universities can act as facilitators and methodological anchors in these initiatives, ensuring scientific rigor, ethical soundness, and inclusiveness throughout the innovation cycle. By mediating dialogue between producers and users, academia helps bridge the gap between market-driven innovation and societal relevance. The integration of co-creation methodologies into research and development frameworks enables a more anticipatory, reflexive, and responsive innovation process—principles consistent with Responsible Research and Innovation [99]. Strengthening these collaborative ecosystems may foster sustainable and equitable innovation pathways in which bioactive compounds and horticultural products are developed not only for profit but also for public well-being and environmental sustainability.

Sustainability should also shape the future of post-harvest processing and product diversification. Using by-products such as shells, burs, and leaves can help make the economy carbon-neutral and create a real circular bioeconomy [47,100]. New green extraction technologies, such as supercritical CO₂, ultrasound-assisted, and subcritical water extraction, enable the recovery of polyphenols, tannins, and polysaccharides for use in nutraceuticals, cosmetics, and active packaging [89,100]. Integrating these techniques within local cooperatives or small processing units could transform waste into high-value bioactive ingredients, reinforcing rural economies while reducing environmental impact.

Chestnut production can be an example of a sustainable agroforestry system that combines food security, carbon sequestration, and rural cohesion, in line with the European Green Deal [101]. Horizon Europe and EIT Food are two European programs that are very important for creating innovation ecosystems that link academic research, business, and policy—a concept often described as the “Triple Helix” model of innovation [102]. Strengthening partnerships among universities, cooperatives, and technology centers will be critical to translate scientific advances into tangible socio-economic benefits [103].

In summary, the future of Castanea sativa in Portugal and Europe will depend on a synergistic approach that brings together plant physiology, biotechnology, digital innovation, and socio-economic policy. The main challenges ahead include adapting cultivation to climate change, preserving genetic diversity, enhancing product valorization and innovation, and ensuring environmental sustainability. By working across all these areas, the European chestnut tree will be able to solidify its dual identity as a cultural heritage species and a bioresource supporting resilient Mediterranean landscapes, a vision aligned with the principles of sustainable agroforestry.

10. Conclusions

Castanea sativa represents a multifunctional species of high nutritional, economic, and ecological relevance in Mediterranean ecosystems. Its production can be an example of a sustainable agroforestry system that combines food security, carbon sequestration, and rural cohesion, in line with the European Green Deal. Beyond its role as a traditional food, chestnut is increasingly recognized as a source of valuable bioactive compounds with demonstrated antioxidant, anti-inflammatory, antimicrobial, and metabolic properties. The valorization of chestnut fruits and by-products aligns with circular bioeconomy principles, supporting the sustainable use of natural resources and the creation of new value-added products for the food, cosmetic, and pharmaceutical industries. Future efforts should integrate green extraction technologies, standardized analytical methods, and interdisciplinary collaboration to optimize the recovery and application of bioactive compounds. Moreover, innovation driven by university–industry–consumer co-creation, within the framework of Responsible Research and Innovation, can foster the development of sustainable products that combine scientific soundness, market potential, and societal benefit. Altogether, advancing the chestnut value chain through sustainable innovation and stakeholder collaboration will contribute to human health promotion, rural development, and environmental resilience in Mediterranean landscapes.