# Bioactive Compounds, Nutritional Traits, and Antioxidant Properties ofArtocarpus altilis (Parkinson) Fruits: Exploiting a Potential Functional Food for Food Security on the Comoros Islands

Authors: Toilibou Soifoini; Dario Donno; Victor Jeannoda; Ernest Rakotoniaina; Soule Hamidou; Said Mohamed Achmet; Noe Rene Solo; Kamaleddine Afraitane; Cristina Giacoma; Gabriele Loris Beccaro
Journal: Journal of Food Quality
 (2018)

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## Abstract

Comoros Union presents a considerable biodiversity of food resources that are neglected or still not valorised, as breadfruit. This study aimed to evaluating nutritional and nutraceutical traits ofArtocarpus altilis (Parkinson) Fosberg by characterizing its main bioactive compounds, nutritional traits, and antioxidant properties in order to contribute to the development of traditional and innovative uses of this species as functional food (e.g., infant flour). Bioactive compound composition, antioxidant properties, protein and sugar content, lipids, fibre, and macro- and microelements were observed in these fruits after a specific drying process. Breadfruit showed positive nutritional traits. The main identified phenolic groups were cinnamic acids (with a maximum of 51.88 ± 2.63 mg/100 gDW for chlorogenic acid) and tannins. The highest value of antioxidant activity was 6.40 ± 1.02 mmol·Fe2+/kgDW. This preliminary phytochemical investigation may provide a contribution to the identification and quantification of lead compounds responsible for traditional nutritional and therapeutic claims.


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## Body

## 1. Introduction

In the Comoros Union, as in all the developing countries, malnutrition and food insecurity affect a very large percentage of the population [1, 2]. Despite government efforts to combat hunger and malnutrition, they remain among the leading causes of mortality of children ranging from 0 to 5 years of age.Food insecurity in the Comoros Union is at a troubling level due to poverty. According to the Global Hunger Index 2011 report, the International Food Policy Research Institute (IFPRI) reported an increase in poverty of nearly 17%, placing the country 73rd of 81 countries surveyed. The IFPRI statistics reveal a troubling nutritional situation: 46% of the Comorians are undernourished, and children under 5 years of age have a mortality rate estimated at 10.4%, with 22% of cases being underweight deaths [3].The country is bursting with a significant diversity of food resources, but they are naturally not exploited or neglected [4]. This is the case of the breadfruit tree (Artocarpus altilis), which has fruits rich in both starch and nutrients. The breadfruit tree (Artocarpus altilis (Parkinson) Fosberg) [5] is a tropical plant belonging to the Moraceae family whose seedless fruits are rich in starch. The breadfruit tree is a large monoecious tree native to the Pacific Islands with abundant white latex in different plant parts, and the tree can reach 8 to 20 m in height [6, 7]. It grows exclusively in tropical environments at temperatures between 15 and 40°C [8, 9]. The breadfruit tree produces seasonal syncarp fruit twice a year during a period of 4 to 6 months; the main harvest occurs from June to September and a secondary one from December to February [10]. In the Comoros Union, some breadfruit trees near households produce fruit all year round. The productivity of the breadfruit tree depends on the environmental conditions (soil type, rainfall, and sunshine). The breadfruit tree (Artocarpus altilis (Parkinson) Fosberg) is native to the Pacific, where it was cultivated and used 3000 years before being repopulated in other tropical and subtropical regions. Breadfruit has long been an important base crop and the main component of traditional agroforestry systems in Oceania, where many varieties are grown [11]. Currently, breadfruit is grown in 86 countries, and 26 other countries have ecological conditions appropriate for its cultivation. Several varieties have been recorded largely from the Pacific islands, which is the case for 132 cultivars in Vanuatu [12] and 130 cultivars in Pohnpei [13]. Outside of the Pacific area, breadfruit diversity is limited to 10 cultivars of Polynesian origin. These trees were collected by British and French settlers in order to introduce them to their respective colonies of Mauritius and the Caribbean [7, 14]. These few cultivars then spread to both Central and South America, Africa, India, South-east Asia, Indonesia, and Sri Lanka, as well as to Madagascar and the Maldives and Seychelles. Nearly all the breadfruit trees in West Africa may originate from a single introduction to a botanical garden in Guinea in 1899 [15]. However, this idea has never been scientifically studied in depth.The breadfruit tree is known locally as “fouryapa.” Its cultivation is simple and does not require special efforts. As a plant that does not produce seed, it is only propagated vegetatively. Its cultivation is by suckers (root projections), root cuttings, or grafting of mature branches.The breadfruit tree is a multipurpose species that provides food, medicine, building materials, and feed. Breadfruit is a versatile food, with different types of culinary preparations. At the mature stage, breadfruit can be steamed, baked, or fried [16, 17]. It can also be transformed into flour or used for making cakes [18]. Breadfruit can be stored either by drying or by fermentation [19]. The breadfruit tree is also used for nonfood purposes. Its latex and bark are used as traditional medicine to treat sprains, sciatica, and skin diseases [20–25]. The trunk is used for construction, and its leaves have traditionally been used to treat cirrhosis of the liver, hypertension, and diabetes [26]. The breadfruit has a high percentage of carbohydrates, mostly as starch, and a small amount of protein but of excellent quality [27–29]. The chemical analysis of breadfruit flour showed a high starch content (80.9 ± 0.9%), relatively high crude fibre and ash contents (1.6 ± 0.3% and 4.2 ± 0.3%, resp.), a low protein content (4.0 ± 0.5%) [30], and a very low lipid content (0.51 ± 0.05%). Depending on the cultivar, bread flour contains excellent minerals, such as calcium, iron, potassium, magnesium, phosphorus, and sodium, in wide-ranging concentrations (283–1491 mg/g calcium, 6.2–21.2 mg/g iron, 7.5–16.2 mg/g potassium, 630–2281 mg/g magnesium, 846–2379 mg/g phosphorus, and 70–843 mg/g sodium) [17]. In addition to carbohydrates, breadfruit is also a rich source of fibre, vitamin C, and flavonoids [11, 31]. While breadfruit is a valuable food resource, its current use is limited by the poor storage properties of its fresh fruits [7, 11, 32].This work aims at determining the chemical and nutritional potential of breadfruit of the Comoros Islands for its potential as a large-scale crop to guarantee both food security and the protection of biodiversity.


## 2. Materials and Methods

### 2.1. Material and Harvest Site

The study material consisted of two fruit samples ofArtocarpus altilis (Parkinson) that were harvested in October 2016 in the same region but at two different sites (P1—A and P2—B). The samples were collected in the southern region of Grande Comore (or Ngazidja in Shikomor, the Comorian language), near the city of Ouziouani (Figure 1). The geographical coordinates are 11°53′37″S and 43°25′24″E for site P1—A (250 m above the sea level) and 11°53′36″S and 43°25′27″E for site P1—B (254 m above the sea level) (in degrees, minutes, and seconds, resp.). Selected sampling sites are the most accessible areas in the southern region of Ngazidja where breadfruit plants grow, thanks to specific pedoclimatic conditions.Figure 1
Aerial view of sampling sites (village of Ouziouani in Grande Comore). A: sample P1; B: sample P2.


### 2.2. Solvents and Chemicals

Sodium carbonate, Folin–Ciocalteu phenol reagent, sodium acetate, citric acid, potassium chloride, hydrochloric acid, iron(III) chloride hexahydrate, 2,4,6-tripyridyl-S-triazine, and 1,2-dihydrochloride-phenylenediamine (OPDA) were used for analyses. All polyphenolic and terpene standards, potassium dihydrogenphosphate, phosphoric acid, methanol, and HPLC-grade acetonitrile were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetic acid, ethanol, organic acids, and HPLC-grade formic acid were purchased from Fluka BioChemika (Buchs, Switzerland). The disodium salt of ethylenediaminetetraacetic acid was purchased from AMRESCO (Solon, OH, USA). Sodium fluoride was purchased from Riedel-de Haen (Seelze, Germany). Cetyltrimethylammonium bromide (cetrimide), ascorbic acid (AA), and dehydroascorbic acid (DHAA) were purchased from Extrasynthese (Genay, France). Milli-Q ultrapure water was purchased from Sartorius Stedim Biotech (Arium, Göttingen, Germany).


### 2.3. Breadfruit Drying Process

All the fruits were manually picked at the same maturity levels (full maturation stage: fruit suitable for fresh consumption by local population) from the plants based on selected qualitative parameters (firmness and total soluble solids), considering also literature and experience of the University researchers. Moreover, the appearance of the latex on the skin was considered. For each biological replication (n=3), 5 kg of fruits were considered. Samples were sorted, and damaged fruits were removed. The samples were then washed with bleach to avoid contamination. After removing the superficial green portion, the fruits were cut into halves. The heart was removed, and the remaining portion was peeled into pieces (each fruit piece is quite ellipsoidal, about 70 mm in length and 30 mm in width, with a weight of about 25 g) that were subsequently dried under the sun (Figure 2). Due to its cheapness, sun drying is a sustainable drying method traditionally used by local population in Comoros Islands in order to prepare breadfruit flour. Drying temperature ranges from 25°C to 35°C for about 3 days. However, drying time occasionally depends on weather conditions and the degree of sunshine, but in October, the sun is constant in the sky from morning until midafternoon (5.00 pm). Dried fruits were packaged into black plastic bags and preserved for chemical and nutritional characterisation.Figure 2
Different stages of breadfruit drying.


### 2.4. Preparation of Methanol Extracts

Pieces of 3-day sun-dried breadfruits were then divided into two portions: first one was grinded with a ceramic mortar and milled with an automatic grinder (sample name: powder/flour), while fruits of the other portion were reduced in size to 10 mm × 10 mm (sample name: small pieces). Two extraction methods were used to prepare methanol extracts. Extracts designated S and T were obtained from the methanolic solution of small pieces of dried breadfruit, and extracts designated S1 and T1 were obtained from the powder (flour) of dried breadfruit. The material for the extracts T and T1 came from site P1, while the extracts S and S1 came from site P2.For each sample, 10 g of powder (flour) and 10 g of small pieces of dried fruits were macerated in 50 mL of methanolic solution (methanol: double-distilled water, 95 : 5 v/v, pH adjusted with 1.5 mL of 37% HCl, pH = 1.2) for 72 h using a magnetic stirrer for 5–10 min per day. The mixture was then filtered using a Whatman™ filter paper (185 mm diameter), after which the filtrate was stored. A second extraction was then repeated from the recovered sample with the same extraction solvent. The filtrate was recovered, mixed with the first filtrate (for a total of 100 mL), and then stored at 4°C until analysis.


### 2.5. Spectrophotometric Analyses

#### 2.5.1. Antioxidant Power

The antioxidant capacity of breadfruit was assessed using a ferric reducing antioxidant power (FRAP) assay [33, 34]. This process is based on the reduction of the ferric (Fe3+) 2,4,6-tripyridyl-s-triazine (TPTZ) complex to its ferrous (Fe2+) form. The FRAP reagent was prepared daily by mixing TPTZ and FeCl3·6H2O solutions with acetate buffer (0.3 M). This mixture was then stirred and incubated at 37°C until analysis. Afterwards, 30 μL of the sample (15 μL of extract and 15 μL of extraction buffer, 1 : 2 dilution) were added to 90 μL of distilled water and to 900 μL of the FRAP reagent in a 2 mL microtube. The mixture was stirred and then incubated at 37°C for 30 min. The absorbance was read at 595 nm using a UV-Vis spectrophotometer (UV–1600PC, VWR, Milan, Italy). A standard curve was obtained using FeSO4·7H2O (concentration of 100–1000 mmol/L), and the results were expressed as millimoles Fe2+ equivalents per kilogramme (dry weight).


#### 2.5.2. Total Polyphenolic Content (TPC)

The Folin–Ciocalteu reagent consists of a mixture of phosphotungstic acid (H3PW12O40) and phosphomolybdic acid (H3PMo12O40) reduced by oxidising phenols in a mixture of blue oxides of tungsten and molybdenum [35]. A total of 0.5 g of fruit extract and 30 mL of double-distilled water were added to 2.5 mL of the Folin–Ciocalteu reagent and to 10 mL of 15% Na2CO3. After 2 h in darkness, absorbance was read at 765 nm. A standard calibration curve was plotted using gallic acid at concentrations of 0.02–0.1 mg/mL. The TPC was expressed as milligrammes of gallic acid equivalents (GAE) per 100 g of dry matter [36].


### 2.6. Chromatographic Analysis

#### 2.6.1. Analysis of Vitamin C and Other Compounds by HPLC

Each analysis was carried out in triplicate. Two millilitres of each sample were centrifuged for 5 min at 12000 rpm and 4°C (ALC Multispeed PK 121R refrigerated centrifuge, Milan, Italy). Samples were then filtered through a C 18 cartridge (Sep-Pak C 18, Waters Corporation, Milford, MA, USA) that retained the polyphenolic compounds. The first elution was the extract for analysing the vitamin C (AA plus DHAA) content. The vitamin C analysis was carried out by adding 250μL of OPDA (18.8 mM) to 750 μL of extract for the derivatisation of DHAA in the fluorophore 3-(1,2-dihydroxyethyl)furo[3,4-b]quinoxaline-1-one (DFQ). After 37 min in the dark, the samples were analysed via HPLC with a diode array detection system [37]. The extract obtained after elution of the solid phase with methanol was used for the HPLC analysis of polyphenols. AA and DHAA were evaluated using an Agilent 1100 HPLC system (Agilent 1200, Santa Clara, CA, USA) equipped with a G1311A quaternary pump, a manual injection valve, and a 20 μL sample loop coupled with an Agilent GI315D UV-Vis diode array detector. The separations of AA and DFQ were carried out on a Kinetex C18 column (Phenomenex, Bologna, Italy) (4.6 × 150 mm, 5 μm). The mobile phase was methanol : water (5 : 95, v/v) containing 5 mM cetrimide and 50 mM potassium dihydrogenphosphate (KH2PO4, pH = 2.5). The flow rate was 0.9 mL/min (isocratic analysis), and the wavelengths of detection were 348 nm for DHAA (DFQ) and 261 nm for AA. The vitamin C content was calculated by adding the contents of AA and DHAA, and the results were expressed in milligrammes per 100 g [37].


#### 2.6.2. Chromatographic Conditions for the Analysis of the Other Bioactive Compounds

Four different chromatographic methods were used to analyse samples: two for polyphenols, one for terpene compounds, and one for organic acids. In all the methods, the separation of bioactive compounds was obtained on a Phenomenex Kinetex C18 column (4.6 × 150 mm, 5μm). Different mobile phases were used: methanol with a 10 mM solution of potassium dihydrogenphosphate in water at a flow rate of 1.5 mL/min (method A, 20 min of gradient analysis of cinnamic acids and flavonols); a methanol : water : formic acid solution (5 : 95 : 0.1 v/v/v) and a methanol/formic acid mixture (100 : 0.1 v/v) at a flow rate of 0.6 mL/min (method B, 23 min of gradient analysis of benzoic acids, tannins, and catechins); water and acetonitrile at a flow rate of 1.0 mL/min (method C, 17 min of gradient analysis of monoterpenes); and 10 mM aqueous KH2PO4 solution (pH 2.8, adjusted with phosphoric acid) at a flow rate of 0.6 mL/min (method D, 13 min of isocratic analysis of organic acids) [38].UV spectra were recorded at 330 nm (for method A); 280 nm (for method B); 210, 220, 235, and 250 nm (for method C); and 214 nm (for method D).


### 2.7. Protein Analysis

The amount of nitrogen and crude protein was analysed using the Kjeldahl method (ISO 1871, 2009). Samples (1 g) and controls were mineralised at 420°C for 105 min. Distillation was performed using a Kjeltec™ 2200 system (Foss, Hillerød, Denmark) for 4 min. The protein content was calculated using a nitrogen-protein conversion factor of 6.25 [39].


### 2.8. Total Lipid Analysis

Determination of the total lipids of dried breadfruit samples was carried out according to the Soxhlet method using petroleum ether as the extraction solvent. The sample was continuously extracted with boiling petroleum ether, which gradually dissolved the fat. The solvent containing the fat was returned to the flask by successive pouring caused by the siphoning effect in the lateral bend. Because only the solvent could evaporate again, the fat accumulated in the flask until the extraction was complete. Upon completion, the petroleum ether was evaporated, typically using a rotary evaporator, and the fat was weighed [40].


### 2.9. Carbohydrate Analysis by HPLC

#### 2.9.1. Extraction of Carbohydrates

Ten grams of powder (flour) of dried breadfruit samples were suspended in 50 mL of 80% ethanolic solution followed by stirring for 15 min (at 30°C). The mixture was macerated overnight at room temperature. After 5 min of stirring, samples were filtered by Whatman 185 mm diameter paper, after which the filtrate was recovered in a test tube. A second extraction was repeated from the recovered samples. The mixture of the extractions constituted one sample for the analysis of sugars (100 mL).


#### 2.9.2. HPLC Chromatographic Conditions

HPLC analysis was carried out using a SphereClone NH2 column (4.6 × 250 mm, 5 μm), and the mobile phase was acetonitrile and water (85 : 15, v/v). The flow rate was 0.5 mL/min (12 min + 3 min conditioning time, isocratic analysis), and wavelengths were set at 200 and 286 nm (Agilent UV-Vis diode array detector).


### 2.10. Fibre Analysis

Dietary fibre referred to indigestible materials measured by a standard method, such as the enzymatic-gravimetric method [41]. The method was composed by an enzymatic digestion (α-amylase, protease, and amyloglucosidase) followed by a gravimetric measurement.


### 2.11. Mineral Analysis

The contents of calcium, magnesium, potassium, and sodium were determined by atomic absorption spectrophotometry in an acetylene-air flame using a flame and graphite furnace atomic absorption spectrometer (PerkinElmer, PinAAcle 900T, Waltham, MA, USA). Reference solutions for each atom were prepared in standard solutions as a reference system in order to determine the quantity of each atom in samples previously mineralised by dry incineration (for Ca2+, Mg2+, K+, and Na+) or wet mineralisation (for Fe3+) at room pressure. In dry incineration, 3 g of the sample were homogenized and charred on a heating plate; the sample was then transferred to a stove and incinerated for 8 hours at a temperature of 380°C. The resulting ash was then solubilised in 65% HNO3 solution. In wet mineralisation, 2 g of the homogenized sample were added to a solution of 65% HNO3 : 64% HClO4 : 96% H2SO4 (24 : 24 : 1, v/v/v) and gradually warmed up (max 150–200°C) continuing until clarification. The wavelengths and relative widths were 422.7 nm and 0.7 nm for calcium, 285.2 nm and 0.7 nm for magnesium, 766.5 nm and 2.0 nm for potassium, and 589.6 nm and 0.7 nm for sodium, respectively. For phosphorus, the colorimetric method after dry mineralisation of the sample followed by solubilisation in HCl (6 N) and treatment with the molybdovanadate reagent was used. The optical density of the coloured solution was spectrophotometrically measured at 430 nm [40]. The iron content was determined by graphite furnace atomic absorption after wet mineralisation by the US EPA 200.0 method. The chloride was identified as sodium chloride.


### 2.12. Statistical Analysis

Student’st-test and ANOVA of independent samples were used to detect significant differences in the chemical and nutritional composition between breadfruit samples. Differences with P<0.05 were considered statistically significant. The results were expressed as mean values and their relative standard deviations (SD).


## 2.1. Material and Harvest Site

The study material consisted of two fruit samples ofArtocarpus altilis (Parkinson) that were harvested in October 2016 in the same region but at two different sites (P1—A and P2—B). The samples were collected in the southern region of Grande Comore (or Ngazidja in Shikomor, the Comorian language), near the city of Ouziouani (Figure 1). The geographical coordinates are 11°53′37″S and 43°25′24″E for site P1—A (250 m above the sea level) and 11°53′36″S and 43°25′27″E for site P1—B (254 m above the sea level) (in degrees, minutes, and seconds, resp.). Selected sampling sites are the most accessible areas in the southern region of Ngazidja where breadfruit plants grow, thanks to specific pedoclimatic conditions.Figure 1
Aerial view of sampling sites (village of Ouziouani in Grande Comore). A: sample P1; B: sample P2.


## 2.2. Solvents and Chemicals

Sodium carbonate, Folin–Ciocalteu phenol reagent, sodium acetate, citric acid, potassium chloride, hydrochloric acid, iron(III) chloride hexahydrate, 2,4,6-tripyridyl-S-triazine, and 1,2-dihydrochloride-phenylenediamine (OPDA) were used for analyses. All polyphenolic and terpene standards, potassium dihydrogenphosphate, phosphoric acid, methanol, and HPLC-grade acetonitrile were purchased from Sigma-Aldrich (St. Louis, MO, USA). Acetic acid, ethanol, organic acids, and HPLC-grade formic acid were purchased from Fluka BioChemika (Buchs, Switzerland). The disodium salt of ethylenediaminetetraacetic acid was purchased from AMRESCO (Solon, OH, USA). Sodium fluoride was purchased from Riedel-de Haen (Seelze, Germany). Cetyltrimethylammonium bromide (cetrimide), ascorbic acid (AA), and dehydroascorbic acid (DHAA) were purchased from Extrasynthese (Genay, France). Milli-Q ultrapure water was purchased from Sartorius Stedim Biotech (Arium, Göttingen, Germany).


## 2.3. Breadfruit Drying Process

All the fruits were manually picked at the same maturity levels (full maturation stage: fruit suitable for fresh consumption by local population) from the plants based on selected qualitative parameters (firmness and total soluble solids), considering also literature and experience of the University researchers. Moreover, the appearance of the latex on the skin was considered. For each biological replication (n=3), 5 kg of fruits were considered. Samples were sorted, and damaged fruits were removed. The samples were then washed with bleach to avoid contamination. After removing the superficial green portion, the fruits were cut into halves. The heart was removed, and the remaining portion was peeled into pieces (each fruit piece is quite ellipsoidal, about 70 mm in length and 30 mm in width, with a weight of about 25 g) that were subsequently dried under the sun (Figure 2). Due to its cheapness, sun drying is a sustainable drying method traditionally used by local population in Comoros Islands in order to prepare breadfruit flour. Drying temperature ranges from 25°C to 35°C for about 3 days. However, drying time occasionally depends on weather conditions and the degree of sunshine, but in October, the sun is constant in the sky from morning until midafternoon (5.00 pm). Dried fruits were packaged into black plastic bags and preserved for chemical and nutritional characterisation.Figure 2
Different stages of breadfruit drying.


## 2.4. Preparation of Methanol Extracts

Pieces of 3-day sun-dried breadfruits were then divided into two portions: first one was grinded with a ceramic mortar and milled with an automatic grinder (sample name: powder/flour), while fruits of the other portion were reduced in size to 10 mm × 10 mm (sample name: small pieces). Two extraction methods were used to prepare methanol extracts. Extracts designated S and T were obtained from the methanolic solution of small pieces of dried breadfruit, and extracts designated S1 and T1 were obtained from the powder (flour) of dried breadfruit. The material for the extracts T and T1 came from site P1, while the extracts S and S1 came from site P2.For each sample, 10 g of powder (flour) and 10 g of small pieces of dried fruits were macerated in 50 mL of methanolic solution (methanol: double-distilled water, 95 : 5 v/v, pH adjusted with 1.5 mL of 37% HCl, pH = 1.2) for 72 h using a magnetic stirrer for 5–10 min per day. The mixture was then filtered using a Whatman™ filter paper (185 mm diameter), after which the filtrate was stored. A second extraction was then repeated from the recovered sample with the same extraction solvent. The filtrate was recovered, mixed with the first filtrate (for a total of 100 mL), and then stored at 4°C until analysis.


## 2.5. Spectrophotometric Analyses

### 2.5.1. Antioxidant Power

The antioxidant capacity of breadfruit was assessed using a ferric reducing antioxidant power (FRAP) assay [33, 34]. This process is based on the reduction of the ferric (Fe3+) 2,4,6-tripyridyl-s-triazine (TPTZ) complex to its ferrous (Fe2+) form. The FRAP reagent was prepared daily by mixing TPTZ and FeCl3·6H2O solutions with acetate buffer (0.3 M). This mixture was then stirred and incubated at 37°C until analysis. Afterwards, 30 μL of the sample (15 μL of extract and 15 μL of extraction buffer, 1 : 2 dilution) were added to 90 μL of distilled water and to 900 μL of the FRAP reagent in a 2 mL microtube. The mixture was stirred and then incubated at 37°C for 30 min. The absorbance was read at 595 nm using a UV-Vis spectrophotometer (UV–1600PC, VWR, Milan, Italy). A standard curve was obtained using FeSO4·7H2O (concentration of 100–1000 mmol/L), and the results were expressed as millimoles Fe2+ equivalents per kilogramme (dry weight).


### 2.5.2. Total Polyphenolic Content (TPC)

The Folin–Ciocalteu reagent consists of a mixture of phosphotungstic acid (H3PW12O40) and phosphomolybdic acid (H3PMo12O40) reduced by oxidising phenols in a mixture of blue oxides of tungsten and molybdenum [35]. A total of 0.5 g of fruit extract and 30 mL of double-distilled water were added to 2.5 mL of the Folin–Ciocalteu reagent and to 10 mL of 15% Na2CO3. After 2 h in darkness, absorbance was read at 765 nm. A standard calibration curve was plotted using gallic acid at concentrations of 0.02–0.1 mg/mL. The TPC was expressed as milligrammes of gallic acid equivalents (GAE) per 100 g of dry matter [36].


## 2.5.1. Antioxidant Power

The antioxidant capacity of breadfruit was assessed using a ferric reducing antioxidant power (FRAP) assay [33, 34]. This process is based on the reduction of the ferric (Fe3+) 2,4,6-tripyridyl-s-triazine (TPTZ) complex to its ferrous (Fe2+) form. The FRAP reagent was prepared daily by mixing TPTZ and FeCl3·6H2O solutions with acetate buffer (0.3 M). This mixture was then stirred and incubated at 37°C until analysis. Afterwards, 30 μL of the sample (15 μL of extract and 15 μL of extraction buffer, 1 : 2 dilution) were added to 90 μL of distilled water and to 900 μL of the FRAP reagent in a 2 mL microtube. The mixture was stirred and then incubated at 37°C for 30 min. The absorbance was read at 595 nm using a UV-Vis spectrophotometer (UV–1600PC, VWR, Milan, Italy). A standard curve was obtained using FeSO4·7H2O (concentration of 100–1000 mmol/L), and the results were expressed as millimoles Fe2+ equivalents per kilogramme (dry weight).


## 2.5.2. Total Polyphenolic Content (TPC)

The Folin–Ciocalteu reagent consists of a mixture of phosphotungstic acid (H3PW12O40) and phosphomolybdic acid (H3PMo12O40) reduced by oxidising phenols in a mixture of blue oxides of tungsten and molybdenum [35]. A total of 0.5 g of fruit extract and 30 mL of double-distilled water were added to 2.5 mL of the Folin–Ciocalteu reagent and to 10 mL of 15% Na2CO3. After 2 h in darkness, absorbance was read at 765 nm. A standard calibration curve was plotted using gallic acid at concentrations of 0.02–0.1 mg/mL. The TPC was expressed as milligrammes of gallic acid equivalents (GAE) per 100 g of dry matter [36].


## 2.6. Chromatographic Analysis

### 2.6.1. Analysis of Vitamin C and Other Compounds by HPLC

Each analysis was carried out in triplicate. Two millilitres of each sample were centrifuged for 5 min at 12000 rpm and 4°C (ALC Multispeed PK 121R refrigerated centrifuge, Milan, Italy). Samples were then filtered through a C 18 cartridge (Sep-Pak C 18, Waters Corporation, Milford, MA, USA) that retained the polyphenolic compounds. The first elution was the extract for analysing the vitamin C (AA plus DHAA) content. The vitamin C analysis was carried out by adding 250μL of OPDA (18.8 mM) to 750 μL of extract for the derivatisation of DHAA in the fluorophore 3-(1,2-dihydroxyethyl)furo[3,4-b]quinoxaline-1-one (DFQ). After 37 min in the dark, the samples were analysed via HPLC with a diode array detection system [37]. The extract obtained after elution of the solid phase with methanol was used for the HPLC analysis of polyphenols. AA and DHAA were evaluated using an Agilent 1100 HPLC system (Agilent 1200, Santa Clara, CA, USA) equipped with a G1311A quaternary pump, a manual injection valve, and a 20 μL sample loop coupled with an Agilent GI315D UV-Vis diode array detector. The separations of AA and DFQ were carried out on a Kinetex C18 column (Phenomenex, Bologna, Italy) (4.6 × 150 mm, 5 μm). The mobile phase was methanol : water (5 : 95, v/v) containing 5 mM cetrimide and 50 mM potassium dihydrogenphosphate (KH2PO4, pH = 2.5). The flow rate was 0.9 mL/min (isocratic analysis), and the wavelengths of detection were 348 nm for DHAA (DFQ) and 261 nm for AA. The vitamin C content was calculated by adding the contents of AA and DHAA, and the results were expressed in milligrammes per 100 g [37].


### 2.6.2. Chromatographic Conditions for the Analysis of the Other Bioactive Compounds

Four different chromatographic methods were used to analyse samples: two for polyphenols, one for terpene compounds, and one for organic acids. In all the methods, the separation of bioactive compounds was obtained on a Phenomenex Kinetex C18 column (4.6 × 150 mm, 5μm). Different mobile phases were used: methanol with a 10 mM solution of potassium dihydrogenphosphate in water at a flow rate of 1.5 mL/min (method A, 20 min of gradient analysis of cinnamic acids and flavonols); a methanol : water : formic acid solution (5 : 95 : 0.1 v/v/v) and a methanol/formic acid mixture (100 : 0.1 v/v) at a flow rate of 0.6 mL/min (method B, 23 min of gradient analysis of benzoic acids, tannins, and catechins); water and acetonitrile at a flow rate of 1.0 mL/min (method C, 17 min of gradient analysis of monoterpenes); and 10 mM aqueous KH2PO4 solution (pH 2.8, adjusted with phosphoric acid) at a flow rate of 0.6 mL/min (method D, 13 min of isocratic analysis of organic acids) [38].UV spectra were recorded at 330 nm (for method A); 280 nm (for method B); 210, 220, 235, and 250 nm (for method C); and 214 nm (for method D).


## 2.6.1. Analysis of Vitamin C and Other Compounds by HPLC

Each analysis was carried out in triplicate. Two millilitres of each sample were centrifuged for 5 min at 12000 rpm and 4°C (ALC Multispeed PK 121R refrigerated centrifuge, Milan, Italy). Samples were then filtered through a C 18 cartridge (Sep-Pak C 18, Waters Corporation, Milford, MA, USA) that retained the polyphenolic compounds. The first elution was the extract for analysing the vitamin C (AA plus DHAA) content. The vitamin C analysis was carried out by adding 250μL of OPDA (18.8 mM) to 750 μL of extract for the derivatisation of DHAA in the fluorophore 3-(1,2-dihydroxyethyl)furo[3,4-b]quinoxaline-1-one (DFQ). After 37 min in the dark, the samples were analysed via HPLC with a diode array detection system [37]. The extract obtained after elution of the solid phase with methanol was used for the HPLC analysis of polyphenols. AA and DHAA were evaluated using an Agilent 1100 HPLC system (Agilent 1200, Santa Clara, CA, USA) equipped with a G1311A quaternary pump, a manual injection valve, and a 20 μL sample loop coupled with an Agilent GI315D UV-Vis diode array detector. The separations of AA and DFQ were carried out on a Kinetex C18 column (Phenomenex, Bologna, Italy) (4.6 × 150 mm, 5 μm). The mobile phase was methanol : water (5 : 95, v/v) containing 5 mM cetrimide and 50 mM potassium dihydrogenphosphate (KH2PO4, pH = 2.5). The flow rate was 0.9 mL/min (isocratic analysis), and the wavelengths of detection were 348 nm for DHAA (DFQ) and 261 nm for AA. The vitamin C content was calculated by adding the contents of AA and DHAA, and the results were expressed in milligrammes per 100 g [37].


## 2.6.2. Chromatographic Conditions for the Analysis of the Other Bioactive Compounds

Four different chromatographic methods were used to analyse samples: two for polyphenols, one for terpene compounds, and one for organic acids. In all the methods, the separation of bioactive compounds was obtained on a Phenomenex Kinetex C18 column (4.6 × 150 mm, 5μm). Different mobile phases were used: methanol with a 10 mM solution of potassium dihydrogenphosphate in water at a flow rate of 1.5 mL/min (method A, 20 min of gradient analysis of cinnamic acids and flavonols); a methanol : water : formic acid solution (5 : 95 : 0.1 v/v/v) and a methanol/formic acid mixture (100 : 0.1 v/v) at a flow rate of 0.6 mL/min (method B, 23 min of gradient analysis of benzoic acids, tannins, and catechins); water and acetonitrile at a flow rate of 1.0 mL/min (method C, 17 min of gradient analysis of monoterpenes); and 10 mM aqueous KH2PO4 solution (pH 2.8, adjusted with phosphoric acid) at a flow rate of 0.6 mL/min (method D, 13 min of isocratic analysis of organic acids) [38].UV spectra were recorded at 330 nm (for method A); 280 nm (for method B); 210, 220, 235, and 250 nm (for method C); and 214 nm (for method D).


## 2.7. Protein Analysis

The amount of nitrogen and crude protein was analysed using the Kjeldahl method (ISO 1871, 2009). Samples (1 g) and controls were mineralised at 420°C for 105 min. Distillation was performed using a Kjeltec™ 2200 system (Foss, Hillerød, Denmark) for 4 min. The protein content was calculated using a nitrogen-protein conversion factor of 6.25 [39].


## 2.8. Total Lipid Analysis

Determination of the total lipids of dried breadfruit samples was carried out according to the Soxhlet method using petroleum ether as the extraction solvent. The sample was continuously extracted with boiling petroleum ether, which gradually dissolved the fat. The solvent containing the fat was returned to the flask by successive pouring caused by the siphoning effect in the lateral bend. Because only the solvent could evaporate again, the fat accumulated in the flask until the extraction was complete. Upon completion, the petroleum ether was evaporated, typically using a rotary evaporator, and the fat was weighed [40].


## 2.9. Carbohydrate Analysis by HPLC

### 2.9.1. Extraction of Carbohydrates

Ten grams of powder (flour) of dried breadfruit samples were suspended in 50 mL of 80% ethanolic solution followed by stirring for 15 min (at 30°C). The mixture was macerated overnight at room temperature. After 5 min of stirring, samples were filtered by Whatman 185 mm diameter paper, after which the filtrate was recovered in a test tube. A second extraction was repeated from the recovered samples. The mixture of the extractions constituted one sample for the analysis of sugars (100 mL).


### 2.9.2. HPLC Chromatographic Conditions

HPLC analysis was carried out using a SphereClone NH2 column (4.6 × 250 mm, 5 μm), and the mobile phase was acetonitrile and water (85 : 15, v/v). The flow rate was 0.5 mL/min (12 min + 3 min conditioning time, isocratic analysis), and wavelengths were set at 200 and 286 nm (Agilent UV-Vis diode array detector).


## 2.9.1. Extraction of Carbohydrates

Ten grams of powder (flour) of dried breadfruit samples were suspended in 50 mL of 80% ethanolic solution followed by stirring for 15 min (at 30°C). The mixture was macerated overnight at room temperature. After 5 min of stirring, samples were filtered by Whatman 185 mm diameter paper, after which the filtrate was recovered in a test tube. A second extraction was repeated from the recovered samples. The mixture of the extractions constituted one sample for the analysis of sugars (100 mL).


## 2.9.2. HPLC Chromatographic Conditions

HPLC analysis was carried out using a SphereClone NH2 column (4.6 × 250 mm, 5 μm), and the mobile phase was acetonitrile and water (85 : 15, v/v). The flow rate was 0.5 mL/min (12 min + 3 min conditioning time, isocratic analysis), and wavelengths were set at 200 and 286 nm (Agilent UV-Vis diode array detector).


## 2.10. Fibre Analysis

Dietary fibre referred to indigestible materials measured by a standard method, such as the enzymatic-gravimetric method [41]. The method was composed by an enzymatic digestion (α-amylase, protease, and amyloglucosidase) followed by a gravimetric measurement.


## 2.11. Mineral Analysis

The contents of calcium, magnesium, potassium, and sodium were determined by atomic absorption spectrophotometry in an acetylene-air flame using a flame and graphite furnace atomic absorption spectrometer (PerkinElmer, PinAAcle 900T, Waltham, MA, USA). Reference solutions for each atom were prepared in standard solutions as a reference system in order to determine the quantity of each atom in samples previously mineralised by dry incineration (for Ca2+, Mg2+, K+, and Na+) or wet mineralisation (for Fe3+) at room pressure. In dry incineration, 3 g of the sample were homogenized and charred on a heating plate; the sample was then transferred to a stove and incinerated for 8 hours at a temperature of 380°C. The resulting ash was then solubilised in 65% HNO3 solution. In wet mineralisation, 2 g of the homogenized sample were added to a solution of 65% HNO3 : 64% HClO4 : 96% H2SO4 (24 : 24 : 1, v/v/v) and gradually warmed up (max 150–200°C) continuing until clarification. The wavelengths and relative widths were 422.7 nm and 0.7 nm for calcium, 285.2 nm and 0.7 nm for magnesium, 766.5 nm and 2.0 nm for potassium, and 589.6 nm and 0.7 nm for sodium, respectively. For phosphorus, the colorimetric method after dry mineralisation of the sample followed by solubilisation in HCl (6 N) and treatment with the molybdovanadate reagent was used. The optical density of the coloured solution was spectrophotometrically measured at 430 nm [40]. The iron content was determined by graphite furnace atomic absorption after wet mineralisation by the US EPA 200.0 method. The chloride was identified as sodium chloride.


## 2.12. Statistical Analysis

Student’st-test and ANOVA of independent samples were used to detect significant differences in the chemical and nutritional composition between breadfruit samples. Differences with P<0.05 were considered statistically significant. The results were expressed as mean values and their relative standard deviations (SD).


## 3. Results and Discussion

### 3.1. Nutraceutical Properties

#### 3.1.1. Total Polyphenolic Content

All the methanolic extracts showed similar TPC values, ranging from 28.30 ± 3.71 to 29.69 ± 1.40 mgGAE/100 g of dried weight (DW) (Table 1). The T1 extract showed the highest TPC value, followed by S1, S, and T, but there were very few differences. The type of the material (small pieces or powder) had no effect on the extraction of polyphenols.Table 1
Total polyphenol content, antioxidant activity, and vitamin C of the different breadfruit extracts.
Material
Extract
TPC (mgGAE/100 gDW)
Antioxidant activity (mmol·Fe2+/kgDW)
Vitamin C (mg/100 gDW)
Small pieces
S
28.90 ± 4.68
5.44 ± 0.35
6.32 ± 0.12
T
28.30 ± 3.71
6.40 ± 1.02
6.25 ± 0.16
Flour
S1
29.26 ± 2.78
2.29 ± 0.32
35.30 ± 1.48
T1
29.69 ± 1.40
1.99 ± 0.33
35.40 ± 1.46
Samples T and T1 are from site P1, and samples S and S1 are from site P2. Values represent the mean of three measurements ± SD. SD = standard deviation. DW = dry weight of the plant material.The ANOVA and Student’st-test showed no significant differences (P<0.05) between the extracts. The TPC for dried breadfruit samples could not be compared with previous results because no studies have reported these data. However, many studies have reported the TPC of fresh breadfruit. Breadfruit TPC was different from that of a study conducted in Malaysia by Nur Arina and Azrina [42]. Their study reported that breadfruit samples contained approximately 54.042 ± 0.596 mgGAE/100 g of fresh weight (FW) of TPC, which was almost double the results of the present study (29.69 ± 1.40 mgGAE/100 gDW). Despite the low TPC content observed in the present study compared with the results of Nur Arina and Azrina [42], the TPC values in the study by Lee et al. [43] did not show large differences (38.446 mgGAE/100 gFW). This difference between the results could be due to the different genotypes of the breadfruit samples. In addition, this variability could be due to geographical origin, processing stage (fresh or dried fruits), and postharvest storage conditions of the fruits [44].


#### 3.1.2. Antioxidant Activity

Thein vitro antioxidant activity of the extracts was evaluated using the FRAP method, which consists of an iron reduction technique. The results obtained in the present study are reported in Table 1. Antioxidant activity did not vary widely among the samples. The highest value was 6.40 ± 1.02 mmol Fe2+/kgDW, and the lowest value was 1.99 ± 0.33 mmol·Fe2+/kgDW. Student’s t-test and ANOVA were used to compare the values of antioxidant activity among the four extracts. There were no significant differences (P<0.05) between the small-piece extracts (T and S) and the flour extracts (T1 and S1), whereas the extracts from two different appearances (T versus T1 and S versus S1) presented significant differences (P<0.05). The value of 0.15 ± 0.07 mmol Fe2+/100 gFW reported by Stangeland et al. [45] on the fresh pulp of jackfruit was much lower than values observed for the four breadfruit extracts. On the contrary, the work of Nur Arina and Azrina [42] on breadfruit reported an antioxidant activity of 2.210 ± 0.085 mmol·Fe2+/100 gFW, which is similar to the values obtained in the present study.


### 3.2. HPLC Fingerprint

#### 3.2.1. Vitamin C

The vitamin C values of the different samples were reported in Table1. These results showed that extraction from the small pieces of dried breadfruit was not a good extraction method for vitamin C because of the size of pieces that constitutes samples and their physical interaction with extraction solvent: indeed, the flour consists of a powder that is most in contact with the extraction solvent, while it is more difficult to solvate larger pieces of dried fruits. The extracts obtained from the powder showed good vitamin C values between 35.30 ± 1.48 and 35.40 ± 1.46 mg/100 gDW. The mixture of the solvent and breadfruit powder (flour) produced a good extraction yield compared with that of the small pieces of dried breadfruit. After analysing these data using ANOVA and Student’s t-tests, no significant differences were observed between the two breadfruit sampling sites. A study on dried breadfruit flour by Christina et al. [46] reported a maximum vitamin C content of 22.7 mg/100 gDW.


#### 3.2.2. Polyphenols

The HPLC analysis of the different dried breadfruit samples showed that breadfruit may be a good source of phenolic constituents. The main identified phenolic groups were cinnamic acids, with a maximum of 51.88 ± 2.63 mg/100 gDW for chlorogenic acid and 3.21 ± 0.09 mg/100 gDW for caffeic acid; tannins, with 26.59 ±5.38 mg/100 gDW for castalagin and 15.99 ± 5.65 mg/100 gDW for vescalagin; benzoic acid, with 5.69 ± 0.08 mg/100 gDW for ellagic acid and 5.56 ± 0.04 mg/100 gDW for gallic acid; and catechins, with a maximum of 8.04 ± 0.44 mg/100 gDW for epicatechin (Tables 2 and 3). Using Student’s t-test and ANOVA to compare the polyphenolic composition of the different samples, no significant differences were found (P<0.05). In general, the analysis of the polyphenolic composition of dried breadfruit samples could not be compared with previous results because no studies of these parameters exist. However, the major polyphenols in breadfruit have pharmacological activities. For example, chlorogenic acid delays the intestinal reabsorption of glucose and its passage through the blood [47]. Chlorogenic acid inhibits the hydrolysis of potato starch in vitro [48]. Another in vitro study reported that chlorogenic acid protects against the oxidation of low density lipoprotein, which is the first step in the formation of atheroma deposits [49]. The breadfruit tree is used in traditional medicine in the Pacific region to treat specific diseases such as diabetes and hypertension [11]. The high amount of chlorogenic acid in breadfruit could play an important role against these diseases. Because of its inhibitory effect on the conversion of histidine to histamine, catechin can aid histamine-mediated immune disorders in numerous pathologies, including gastric ulcers [50]. Catechins play an important role in antioxidant activity and also in the prevention of cardiovascular disease [51]. Gallic acid has antioxidant and cytotoxic activity against cancer cells, including those of leukaemia and prostate cancer [52]. Gallic acid also exhibits antiviral activity against the HSV-2 herpes simplex virus [53].Table 2
Cinnamic acid composition of the different breadfruit samples.
Materials
Extracts
Caffeic acid (mg/100 gDW)
Chlorogenic acid (mg/100 gDW)
Coumaric acid (mg/100 gDW)
Ferulic acid (mg/100 gDW)
Small pieces
S
2.31 ± 1.26
30.27 ± 35.03
n.q.
n.q.
T
2.49 ± 1.21
43.30 ± 3.93
n.q.
n.d.
Flour
S1
3.21 ± 0.09
51.88 ± 2.63
n.d.
n.q.
T1
2.65 ± 1.00
43.80 ± 5.43
n.q.
n.d.
Samples T and T1 are from site P1, and samples S and S1 are from site P2. Values represent the mean of three measurements ± SD. SD = standard deviation. DW = dry weight of the plant material. n.d. = not detected. n.q. = not quantified.Table 3
Benzoic acid, catechin, and tannin composition of breadfruit extracts.
Materials
Extracts
Ellagic acid (mg/100 gDW)
Gallic acid (mg/100 gDW)
Catechin (mg/100 gDW)
Epicatechin (mg/100 gDW)
Castalagin (mg/100 gDW)
Vescalagin (mg/100 gDW)
Piece
S
5.25 ± 0.18
5.56 ± 0.04
n.d.
8.04 ± 0.44
26.59 ± 5.38
15.99 ± 5.65
T
5.17 ± 0.36
5.46 ± 0.12
n.q.
7.76 ± 0.31
15.66 ± 5.42
13.53 ± 4.94
Flour
S1
5.43 ± 0.12
5.32 ± 0.42
n.d.
7.31 ± 1.38
26.33 ± 2.36
12.03 ± 4.29
T1
5.69 ± 0.08
5.23 ± 0.14
n.q.
6.96 ± 0.52
13.37 ± 5.17
5.79 ± 1.27
Samples T and T1 are from site P1, and samples S and S1 are from site P2. Values represent the mean of three measurements ± SD. SD = standard deviation. DW = dry weight of the plant material. n.d. = not detected. n.q. = not quantified.


#### 3.2.3. Organic Acids

Among the identified organic acids, quinic acid was the predominant acid among all dried breadfruit extracts in this study; quinic acid was followed by citric acid and traces of tartaric acid. Quinic acid levels in the samples ranged from 77.25 ± 6.04 (S1) to 32.55 ± 0.35 mg/100 g (T, P1) (Table4). The obtained results showed no significant differences (P<0.05) between the two breadfruit sampling sites, which suggest that the breadfruit samples could belong to the same genotype. Organic acids are one of the most important factors influencing fruit flavour, and they are very important to human health. Studies have shown the importance of organic acids such as malic acid, citric acid, and tartaric acid in the prevention and elimination of kidney stones [54].Table 4
Organic acid composition of breadfruit samples.
Materials
Extracts
Citric acid (mg/100 gDW)
Malic acid (mg/100 gDW)
Oxalic acid (mg/100 gDW)
Quinic acid (mg/100 gDW)
Succinic acid (mg/100 gDW)
Tartaric acid (mg/100 gDW)
Small pieces
S
3.41 ± 0.87
n.d.
n.d.
45.66 ± 6.09
n.d.
2.61 ± 1.75
T
2.49 ± 1.31
n.d.
n.q.
32.55 ± 0.35
n.d.
1.77 ± 0.28
Flour
S1
6.53 ± 0.90
n.d.
n.d.
77.25 ± 6.04
n.d.
n.d.
T1
5.07 ± 0.77
n.d.
n.q.
48.86 ± 4.56
n.d.
n.q.
Samples T and T1 are from site P1, and samples S and S1 are from site P2. Values represent the mean of three measurements ± SD. SD = standard deviation. DW = dry weight of the plant material. n.d. = not detected. n.q. = not quantified.


#### 3.2.4. Monoterpenes

Monoterpenes were among the major molecules identified in dried breadfruit. Different monoterpene compounds were detected: limonene, with a maximum of 247.91 ± 29.29 mg/100 gDW; phellandrene, with 56.67 ± 57.77 mg/100 gDW; and sabinene, with a maximum of 52.98 ± 1.08 mg/100 gDW (Table 5). Student’s t-test and ANOVA did not show significant differences (P<0.05) between the two breadfruit sampling sites. No studies regarding the identification of monoterpenes in breadfruit have been carried out until now. However, studies have demonstrated the pharmacological potential of monoterpenes in the treatment of inflammatory diseases [55]. This study may justify the traditional use of breadfruit as a remedy for inflammatory diseases [56].Table 5
Monoterpene composition of different breadfruit extracts.
Materials
Extracts
Limonene (mg/100 gDW)
Phellandrene (mg/100 gDW)
Sabinene (mg/100 gDW)
γ-Terpinene (mg/100 gDW)
Terpinolene (mg/100 gDW)
Small pieces
S
247.91 ± 29.29
100.29 ± 5.87
41.33 ± 9.28
n.d.
n.d.
T
235.21 ± 52.29
56.67 ± 5.77
37.24 ± 3.50
n.q.
n.q
Flour
S1
140.15 ± 24.46
20.16 ± 1.10
41.70 ± 10.09
n.d.
n.d.
T1
145.64 ± 40.78
44.63 ± 4.27
52.98 ± 1.08
n.q.
n.q.
Samples T and T1 are from site P1, and samples S and S1 are from site P2. Values represent the mean of three measurements ± SD. SD = standard deviation. DW = dry weight of the plant material. n.d. = not detected. n.q. = not quantified.


### 3.3. Macro- and Micromolecules

#### 3.3.1. Proteins

The protein composition of breadfruit is shown in Table6. The protein content presented an average value of 4.44 g/100 gflour. This value is very similar to the value of 4.05 ± 0.01 g/100 gflour reported by Graham and De Bravo [27]. The protein content reported by Huang et al. [57] showed a clear difference, averaging 1.2 g/100 gflour. The work of Christina et al. [46] on breadfruit varieties showed minimum and maximum values of approximately 1.9 g/100 gflour and 18.7 g/100 gflour, respectively. The minimum value is significantly lower than the value obtained in this study. However, the maximum value is much greater than that obtained in this work.Table 6
Macronutrient content of dried breadfruit flour samples.
Breadfruit flour
Protein (g/100 gDW)
Lipid (g/100 gDW)
Fibre (g/100 gDW)
P1
4.46
0.66
18.86
P2
4.42
0.68
20.12
Mean value
4.44
0.77
19.49
DW = dry weight of the plant material.


#### 3.3.2. Lipids

The lipid content of breadfruit is summarised in Table6. No variation was observed between the two flour samples, which presented an average value of 0.77 g/100 g. This value is slightly higher than the minimum value found in the work of Christina et al. [46] (0.5 g/100 gflour). Graham and De Bravo [27] recorded a slight increase in the lipid content of 1.14 ± 0.07 g/100 g in breadfruit flour.


#### 3.3.3. Fibre

The values of raw fibre between the two flour samples are shown in Table6. In both samples of breadfruit flour, the amount of fibre varied slightly between the two samples, ranging from 18.86 to 20.12 g/100 g of dried flour. These values are considerably higher than those found in the work of Christina et al. [46] that reported values between 0.8 and 15.3 g/100 g of breadfruit flour.


#### 3.3.4. Sugars

Simple sugars identified in the flour of dried breadfruit in decreasing order included glucose, sucrose, and fructose, and their mean values were 2.80 ± 0.52, 1.51 ± 0.25, and 0.34 ± 0.06 g/100 g (dried breadfruit flour), respectively (Table7). Chromatograms are shown in Figures 3–6. The analysis of Student’s t-test and ANOVA results showed significant differences (P<0.05) in the glucose content compared with that of fructose and sucrose. These results showed lower values of simple sugars compared with those results published by Christina et al. [46]. In previous studies, glucose and sucrose were proven to be the dominant sugars in fully mature breadfruits [58, 59].Table 7
Sugar content of dried breadfruits.
Sample
Fructose (g/100 gDW)
Glucose (g/100 gDW)
Sucrose (g/100 gDW)
Total sugars (g/100 gDW)
P1
0.35 ± 0.09
2.83 ± 0.40
0.90 ± 0.22
4.08 ± 0.71
P2
0.33 ± 0.04
2.76 ± 0.65
2.11 ± 0.28
5.20 ± 0.97
Mean value
0.34 ± 0.06
2.80 ± 0.52
1.51 ± 0.25
4.64 ± 0.83
Values represent the mean of three measurements ± SD. SD = standard deviation. P1 = flour sample from site 1. P2 = flour sample from site 2. DW = dry weight of the plant material.Figure 3
Glucose peak of the P1 sample and relative retention time (TR = 4.790 min).Figure 4
Glucose peak of the P2 sample and relative retention time (TR = 4.801 min).Figure 5
Fructose and sucrose peaks of the P1 sample, with fructose (TR = 9.616 min) and sucrose (TR = 11.177 min) retention times.Figure 6
Fructose and sucrose peaks of the P2 sample, with fructose (TR = 9.507 min) and sucrose (TR = 11.199 min) retention times.


#### 3.3.5. Minerals

The results of the mineral composition of dried breadfruit flour are shown in Table8. Calcium was the most abundant mineral in fruits, with an overall mean of 779 mg/100 gDW, followed by other micronutrients, including phosphorus, magnesium, sodium, iron, potassium, and chloride (in the form of NaCl), whose means were 73.5, 42, 13.2, 1.25, 1.195, and 0.435 mg/100 gDW, respectively. Some elements, such as calcium, are markedly important for the growth and development of bones, in particular for those of human infants. Student’s t-test and ANOVA tests revealed slight significant differences (P<0.05) for calcium and magnesium between the samples of the two sites, whereas the others (sodium, phosphorus, potassium, and iron) did not differ between the two samples. These values were largely greater than the results of Graham and De Bravo [27] regarding breadfruit flour, although the reported value of potassium was close to the mean value of the present research. Other studies also reported similar results, despite some differences, as shown by Jones et al. [17] for iron and sodium. However, only the potassium value was truly lower if compared with that reported in the study by Christina et al. [46]. Calcium and magnesium are essential to many human functions, including preservation of the nervous system and muscular balance, and calcium has a protective effect against bone loss [60]. Other studies have highlighted the beneficial effects of calcium on colon cancer [61], cardiovascular events [62], and hypertension [63]. Magnesium deficiency can lead to neuromuscular hyperexcitability, which results in signs of latent tetany [64].Table 8
Mineral content in dried flour of breadfruit.
Flour
Calcium (mg/100 gDW)
Iron (mg/100 gDW)
Phosphorus (mg/100 gDW)
Magnesium (mg/100 gDW)
Chlorides (mg/100 gDW)
Potassium (mg/100 gDW)
Sodium (mg/100 gDW)
P1
678
1.05
76
32
0.46
1.174
12.7
P2
880
1.45
71
52
0.41
1.217
13.7
Mean value
779
1.25
73.5
42
0.435
1.195
13.2
P1 = flour sample from site 1. P2 = flour sample from site 2.


## 3.1. Nutraceutical Properties

### 3.1.1. Total Polyphenolic Content

All the methanolic extracts showed similar TPC values, ranging from 28.30 ± 3.71 to 29.69 ± 1.40 mgGAE/100 g of dried weight (DW) (Table 1). The T1 extract showed the highest TPC value, followed by S1, S, and T, but there were very few differences. The type of the material (small pieces or powder) had no effect on the extraction of polyphenols.Table 1
Total polyphenol content, antioxidant activity, and vitamin C of the different breadfruit extracts.
Material
Extract
TPC (mgGAE/100 gDW)
Antioxidant activity (mmol·Fe2+/kgDW)
Vitamin C (mg/100 gDW)
Small pieces
S
28.90 ± 4.68
5.44 ± 0.35
6.32 ± 0.12
T
28.30 ± 3.71
6.40 ± 1.02
6.25 ± 0.16
Flour
S1
29.26 ± 2.78
2.29 ± 0.32
35.30 ± 1.48
T1
29.69 ± 1.40
1.99 ± 0.33
35.40 ± 1.46
Samples T and T1 are from site P1, and samples S and S1 are from site P2. Values represent the mean of three measurements ± SD. SD = standard deviation. DW = dry weight of the plant material.The ANOVA and Student’st-test showed no significant differences (P<0.05) between the extracts. The TPC for dried breadfruit samples could not be compared with previous results because no studies have reported these data. However, many studies have reported the TPC of fresh breadfruit. Breadfruit TPC was different from that of a study conducted in Malaysia by Nur Arina and Azrina [42]. Their study reported that breadfruit samples contained approximately 54.042 ± 0.596 mgGAE/100 g of fresh weight (FW) of TPC, which was almost double the results of the present study (29.69 ± 1.40 mgGAE/100 gDW). Despite the low TPC content observed in the present study compared with the results of Nur Arina and Azrina [42], the TPC values in the study by Lee et al. [43] did not show large differences (38.446 mgGAE/100 gFW). This difference between the results could be due to the different genotypes of the breadfruit samples. In addition, this variability could be due to geographical origin, processing stage (fresh or dried fruits), and postharvest storage conditions of the fruits [44].


### 3.1.2. Antioxidant Activity

Thein vitro antioxidant activity of the extracts was evaluated using the FRAP method, which consists of an iron reduction technique. The results obtained in the present study are reported in Table 1. Antioxidant activity did not vary widely among the samples. The highest value was 6.40 ± 1.02 mmol Fe2+/kgDW, and the lowest value was 1.99 ± 0.33 mmol·Fe2+/kgDW. Student’s t-test and ANOVA were used to compare the values of antioxidant activity among the four extracts. There were no significant differences (P<0.05) between the small-piece extracts (T and S) and the flour extracts (T1 and S1), whereas the extracts from two different appearances (T versus T1 and S versus S1) presented significant differences (P<0.05). The value of 0.15 ± 0.07 mmol Fe2+/100 gFW reported by Stangeland et al. [45] on the fresh pulp of jackfruit was much lower than values observed for the four breadfruit extracts. On the contrary, the work of Nur Arina and Azrina [42] on breadfruit reported an antioxidant activity of 2.210 ± 0.085 mmol·Fe2+/100 gFW, which is similar to the values obtained in the present study.


## 3.1.1. Total Polyphenolic Content

All the methanolic extracts showed similar TPC values, ranging from 28.30 ± 3.71 to 29.69 ± 1.40 mgGAE/100 g of dried weight (DW) (Table 1). The T1 extract showed the highest TPC value, followed by S1, S, and T, but there were very few differences. The type of the material (small pieces or powder) had no effect on the extraction of polyphenols.Table 1
Total polyphenol content, antioxidant activity, and vitamin C of the different breadfruit extracts.
Material
Extract
TPC (mgGAE/100 gDW)
Antioxidant activity (mmol·Fe2+/kgDW)
Vitamin C (mg/100 gDW)
Small pieces
S
28.90 ± 4.68
5.44 ± 0.35
6.32 ± 0.12
T
28.30 ± 3.71
6.40 ± 1.02
6.25 ± 0.16
Flour
S1
29.26 ± 2.78
2.29 ± 0.32
35.30 ± 1.48
T1
29.69 ± 1.40
1.99 ± 0.33
35.40 ± 1.46
Samples T and T1 are from site P1, and samples S and S1 are from site P2. Values represent the mean of three measurements ± SD. SD = standard deviation. DW = dry weight of the plant material.The ANOVA and Student’st-test showed no significant differences (P<0.05) between the extracts. The TPC for dried breadfruit samples could not be compared with previous results because no studies have reported these data. However, many studies have reported the TPC of fresh breadfruit. Breadfruit TPC was different from that of a study conducted in Malaysia by Nur Arina and Azrina [42]. Their study reported that breadfruit samples contained approximately 54.042 ± 0.596 mgGAE/100 g of fresh weight (FW) of TPC, which was almost double the results of the present study (29.69 ± 1.40 mgGAE/100 gDW). Despite the low TPC content observed in the present study compared with the results of Nur Arina and Azrina [42], the TPC values in the study by Lee et al. [43] did not show large differences (38.446 mgGAE/100 gFW). This difference between the results could be due to the different genotypes of the breadfruit samples. In addition, this variability could be due to geographical origin, processing stage (fresh or dried fruits), and postharvest storage conditions of the fruits [44].


## 3.1.2. Antioxidant Activity

Thein vitro antioxidant activity of the extracts was evaluated using the FRAP method, which consists of an iron reduction technique. The results obtained in the present study are reported in Table 1. Antioxidant activity did not vary widely among the samples. The highest value was 6.40 ± 1.02 mmol Fe2+/kgDW, and the lowest value was 1.99 ± 0.33 mmol·Fe2+/kgDW. Student’s t-test and ANOVA were used to compare the values of antioxidant activity among the four extracts. There were no significant differences (P<0.05) between the small-piece extracts (T and S) and the flour extracts (T1 and S1), whereas the extracts from two different appearances (T versus T1 and S versus S1) presented significant differences (P<0.05). The value of 0.15 ± 0.07 mmol Fe2+/100 gFW reported by Stangeland et al. [45] on the fresh pulp of jackfruit was much lower than values observed for the four breadfruit extracts. On the contrary, the work of Nur Arina and Azrina [42] on breadfruit reported an antioxidant activity of 2.210 ± 0.085 mmol·Fe2+/100 gFW, which is similar to the values obtained in the present study.


## 3.2. HPLC Fingerprint

### 3.2.1. Vitamin C

The vitamin C values of the different samples were reported in Table1. These results showed that extraction from the small pieces of dried breadfruit was not a good extraction method for vitamin C because of the size of pieces that constitutes samples and their physical interaction with extraction solvent: indeed, the flour consists of a powder that is most in contact with the extraction solvent, while it is more difficult to solvate larger pieces of dried fruits. The extracts obtained from the powder showed good vitamin C values between 35.30 ± 1.48 and 35.40 ± 1.46 mg/100 gDW. The mixture of the solvent and breadfruit powder (flour) produced a good extraction yield compared with that of the small pieces of dried breadfruit. After analysing these data using ANOVA and Student’s t-tests, no significant differences were observed between the two breadfruit sampling sites. A study on dried breadfruit flour by Christina et al. [46] reported a maximum vitamin C content of 22.7 mg/100 gDW.


### 3.2.2. Polyphenols

The HPLC analysis of the different dried breadfruit samples showed that breadfruit may be a good source of phenolic constituents. The main identified phenolic groups were cinnamic acids, with a maximum of 51.88 ± 2.63 mg/100 gDW for chlorogenic acid and 3.21 ± 0.09 mg/100 gDW for caffeic acid; tannins, with 26.59 ±5.38 mg/100 gDW for castalagin and 15.99 ± 5.65 mg/100 gDW for vescalagin; benzoic acid, with 5.69 ± 0.08 mg/100 gDW for ellagic acid and 5.56 ± 0.04 mg/100 gDW for gallic acid; and catechins, with a maximum of 8.04 ± 0.44 mg/100 gDW for epicatechin (Tables 2 and 3). Using Student’s t-test and ANOVA to compare the polyphenolic composition of the different samples, no significant differences were found (P<0.05). In general, the analysis of the polyphenolic composition of dried breadfruit samples could not be compared with previous results because no studies of these parameters exist. However, the major polyphenols in breadfruit have pharmacological activities. For example, chlorogenic acid delays the intestinal reabsorption of glucose and its passage through the blood [47]. Chlorogenic acid inhibits the hydrolysis of potato starch in vitro [48]. Another in vitro study reported that chlorogenic acid protects against the oxidation of low density lipoprotein, which is the first step in the formation of atheroma deposits [49]. The breadfruit tree is used in traditional medicine in the Pacific region to treat specific diseases such as diabetes and hypertension [11]. The high amount of chlorogenic acid in breadfruit could play an important role against these diseases. Because of its inhibitory effect on the conversion of histidine to histamine, catechin can aid histamine-mediated immune disorders in numerous pathologies, including gastric ulcers [50]. Catechins play an important role in antioxidant activity and also in the prevention of cardiovascular disease [51]. Gallic acid has antioxidant and cytotoxic activity against cancer cells, including those of leukaemia and prostate cancer [52]. Gallic acid also exhibits antiviral activity against the HSV-2 herpes simplex virus [53].Table 2
Cinnamic acid composition of the different breadfruit samples.
Materials
Extracts
Caffeic acid (mg/100 gDW)
Chlorogenic acid (mg/100 gDW)
Coumaric acid (mg/100 gDW)
Ferulic acid (mg/100 gDW)
Small pieces
S
2.31 ± 1.26
30.27 ± 35.03
n.q.
n.q.
T
2.49 ± 1.21
43.30 ± 3.93
n.q.
n.d.
Flour
S1
3.21 ± 0.09
51.88 ± 2.63
n.d.
n.q.
T1
2.65 ± 1.00
43.80 ± 5.43
n.q.
n.d.
Samples T and T1 are from site P1, and samples S and S1 are from site P2. Values represent the mean of three measurements ± SD. SD = standard deviation. DW = dry weight of the plant material. n.d. = not detected. n.q. = not quantified.Table 3
Benzoic acid, catechin, and tannin composition of breadfruit extracts.
Materials
Extracts
Ellagic acid (mg/100 gDW)
Gallic acid (mg/100 gDW)
Catechin (mg/100 gDW)
Epicatechin (mg/100 gDW)
Castalagin (mg/100 gDW)
Vescalagin (mg/100 gDW)
Piece
S
5.25 ± 0.18
5.56 ± 0.04
n.d.
8.04 ± 0.44
26.59 ± 5.38
15.99 ± 5.65
T
5.17 ± 0.36
5.46 ± 0.12
n.q.
7.76 ± 0.31
15.66 ± 5.42
13.53 ± 4.94
Flour
S1
5.43 ± 0.12
5.32 ± 0.42
n.d.
7.31 ± 1.38
26.33 ± 2.36
12.03 ± 4.29
T1
5.69 ± 0.08
5.23 ± 0.14
n.q.
6.96 ± 0.52
13.37 ± 5.17
5.79 ± 1.27
Samples T and T1 are from site P1, and samples S and S1 are from site P2. Values represent the mean of three measurements ± SD. SD = standard deviation. DW = dry weight of the plant material. n.d. = not detected. n.q. = not quantified.


### 3.2.3. Organic Acids

Among the identified organic acids, quinic acid was the predominant acid among all dried breadfruit extracts in this study; quinic acid was followed by citric acid and traces of tartaric acid. Quinic acid levels in the samples ranged from 77.25 ± 6.04 (S1) to 32.55 ± 0.35 mg/100 g (T, P1) (Table4). The obtained results showed no significant differences (P<0.05) between the two breadfruit sampling sites, which suggest that the breadfruit samples could belong to the same genotype. Organic acids are one of the most important factors influencing fruit flavour, and they are very important to human health. Studies have shown the importance of organic acids such as malic acid, citric acid, and tartaric acid in the prevention and elimination of kidney stones [54].Table 4
Organic acid composition of breadfruit samples.
Materials
Extracts
Citric acid (mg/100 gDW)
Malic acid (mg/100 gDW)
Oxalic acid (mg/100 gDW)
Quinic acid (mg/100 gDW)
Succinic acid (mg/100 gDW)
Tartaric acid (mg/100 gDW)
Small pieces
S
3.41 ± 0.87
n.d.
n.d.
45.66 ± 6.09
n.d.
2.61 ± 1.75
T
2.49 ± 1.31
n.d.
n.q.
32.55 ± 0.35
n.d.
1.77 ± 0.28
Flour
S1
6.53 ± 0.90
n.d.
n.d.
77.25 ± 6.04
n.d.
n.d.
T1
5.07 ± 0.77
n.d.
n.q.
48.86 ± 4.56
n.d.
n.q.
Samples T and T1 are from site P1, and samples S and S1 are from site P2. Values represent the mean of three measurements ± SD. SD = standard deviation. DW = dry weight of the plant material. n.d. = not detected. n.q. = not quantified.


### 3.2.4. Monoterpenes

Monoterpenes were among the major molecules identified in dried breadfruit. Different monoterpene compounds were detected: limonene, with a maximum of 247.91 ± 29.29 mg/100 gDW; phellandrene, with 56.67 ± 57.77 mg/100 gDW; and sabinene, with a maximum of 52.98 ± 1.08 mg/100 gDW (Table 5). Student’s t-test and ANOVA did not show significant differences (P<0.05) between the two breadfruit sampling sites. No studies regarding the identification of monoterpenes in breadfruit have been carried out until now. However, studies have demonstrated the pharmacological potential of monoterpenes in the treatment of inflammatory diseases [55]. This study may justify the traditional use of breadfruit as a remedy for inflammatory diseases [56].Table 5
Monoterpene composition of different breadfruit extracts.
Materials
Extracts
Limonene (mg/100 gDW)
Phellandrene (mg/100 gDW)
Sabinene (mg/100 gDW)
γ-Terpinene (mg/100 gDW)
Terpinolene (mg/100 gDW)
Small pieces
S
247.91 ± 29.29
100.29 ± 5.87
41.33 ± 9.28
n.d.
n.d.
T
235.21 ± 52.29
56.67 ± 5.77
37.24 ± 3.50
n.q.
n.q
Flour
S1
140.15 ± 24.46
20.16 ± 1.10
41.70 ± 10.09
n.d.
n.d.
T1
145.64 ± 40.78
44.63 ± 4.27
52.98 ± 1.08
n.q.
n.q.
Samples T and T1 are from site P1, and samples S and S1 are from site P2. Values represent the mean of three measurements ± SD. SD = standard deviation. DW = dry weight of the plant material. n.d. = not detected. n.q. = not quantified.


## 3.2.1. Vitamin C

The vitamin C values of the different samples were reported in Table1. These results showed that extraction from the small pieces of dried breadfruit was not a good extraction method for vitamin C because of the size of pieces that constitutes samples and their physical interaction with extraction solvent: indeed, the flour consists of a powder that is most in contact with the extraction solvent, while it is more difficult to solvate larger pieces of dried fruits. The extracts obtained from the powder showed good vitamin C values between 35.30 ± 1.48 and 35.40 ± 1.46 mg/100 gDW. The mixture of the solvent and breadfruit powder (flour) produced a good extraction yield compared with that of the small pieces of dried breadfruit. After analysing these data using ANOVA and Student’s t-tests, no significant differences were observed between the two breadfruit sampling sites. A study on dried breadfruit flour by Christina et al. [46] reported a maximum vitamin C content of 22.7 mg/100 gDW.


## 3.2.2. Polyphenols

The HPLC analysis of the different dried breadfruit samples showed that breadfruit may be a good source of phenolic constituents. The main identified phenolic groups were cinnamic acids, with a maximum of 51.88 ± 2.63 mg/100 gDW for chlorogenic acid and 3.21 ± 0.09 mg/100 gDW for caffeic acid; tannins, with 26.59 ±5.38 mg/100 gDW for castalagin and 15.99 ± 5.65 mg/100 gDW for vescalagin; benzoic acid, with 5.69 ± 0.08 mg/100 gDW for ellagic acid and 5.56 ± 0.04 mg/100 gDW for gallic acid; and catechins, with a maximum of 8.04 ± 0.44 mg/100 gDW for epicatechin (Tables 2 and 3). Using Student’s t-test and ANOVA to compare the polyphenolic composition of the different samples, no significant differences were found (P<0.05). In general, the analysis of the polyphenolic composition of dried breadfruit samples could not be compared with previous results because no studies of these parameters exist. However, the major polyphenols in breadfruit have pharmacological activities. For example, chlorogenic acid delays the intestinal reabsorption of glucose and its passage through the blood [47]. Chlorogenic acid inhibits the hydrolysis of potato starch in vitro [48]. Another in vitro study reported that chlorogenic acid protects against the oxidation of low density lipoprotein, which is the first step in the formation of atheroma deposits [49]. The breadfruit tree is used in traditional medicine in the Pacific region to treat specific diseases such as diabetes and hypertension [11]. The high amount of chlorogenic acid in breadfruit could play an important role against these diseases. Because of its inhibitory effect on the conversion of histidine to histamine, catechin can aid histamine-mediated immune disorders in numerous pathologies, including gastric ulcers [50]. Catechins play an important role in antioxidant activity and also in the prevention of cardiovascular disease [51]. Gallic acid has antioxidant and cytotoxic activity against cancer cells, including those of leukaemia and prostate cancer [52]. Gallic acid also exhibits antiviral activity against the HSV-2 herpes simplex virus [53].Table 2
Cinnamic acid composition of the different breadfruit samples.
Materials
Extracts
Caffeic acid (mg/100 gDW)
Chlorogenic acid (mg/100 gDW)
Coumaric acid (mg/100 gDW)
Ferulic acid (mg/100 gDW)
Small pieces
S
2.31 ± 1.26
30.27 ± 35.03
n.q.
n.q.
T
2.49 ± 1.21
43.30 ± 3.93
n.q.
n.d.
Flour
S1
3.21 ± 0.09
51.88 ± 2.63
n.d.
n.q.
T1
2.65 ± 1.00
43.80 ± 5.43
n.q.
n.d.
Samples T and T1 are from site P1, and samples S and S1 are from site P2. Values represent the mean of three measurements ± SD. SD = standard deviation. DW = dry weight of the plant material. n.d. = not detected. n.q. = not quantified.Table 3
Benzoic acid, catechin, and tannin composition of breadfruit extracts.
Materials
Extracts
Ellagic acid (mg/100 gDW)
Gallic acid (mg/100 gDW)
Catechin (mg/100 gDW)
Epicatechin (mg/100 gDW)
Castalagin (mg/100 gDW)
Vescalagin (mg/100 gDW)
Piece
S
5.25 ± 0.18
5.56 ± 0.04
n.d.
8.04 ± 0.44
26.59 ± 5.38
15.99 ± 5.65
T
5.17 ± 0.36
5.46 ± 0.12
n.q.
7.76 ± 0.31
15.66 ± 5.42
13.53 ± 4.94
Flour
S1
5.43 ± 0.12
5.32 ± 0.42
n.d.
7.31 ± 1.38
26.33 ± 2.36
12.03 ± 4.29
T1
5.69 ± 0.08
5.23 ± 0.14
n.q.
6.96 ± 0.52
13.37 ± 5.17
5.79 ± 1.27
Samples T and T1 are from site P1, and samples S and S1 are from site P2. Values represent the mean of three measurements ± SD. SD = standard deviation. DW = dry weight of the plant material. n.d. = not detected. n.q. = not quantified.


## 3.2.3. Organic Acids

Among the identified organic acids, quinic acid was the predominant acid among all dried breadfruit extracts in this study; quinic acid was followed by citric acid and traces of tartaric acid. Quinic acid levels in the samples ranged from 77.25 ± 6.04 (S1) to 32.55 ± 0.35 mg/100 g (T, P1) (Table4). The obtained results showed no significant differences (P<0.05) between the two breadfruit sampling sites, which suggest that the breadfruit samples could belong to the same genotype. Organic acids are one of the most important factors influencing fruit flavour, and they are very important to human health. Studies have shown the importance of organic acids such as malic acid, citric acid, and tartaric acid in the prevention and elimination of kidney stones [54].Table 4
Organic acid composition of breadfruit samples.
Materials
Extracts
Citric acid (mg/100 gDW)
Malic acid (mg/100 gDW)
Oxalic acid (mg/100 gDW)
Quinic acid (mg/100 gDW)
Succinic acid (mg/100 gDW)
Tartaric acid (mg/100 gDW)
Small pieces
S
3.41 ± 0.87
n.d.
n.d.
45.66 ± 6.09
n.d.
2.61 ± 1.75
T
2.49 ± 1.31
n.d.
n.q.
32.55 ± 0.35
n.d.
1.77 ± 0.28
Flour
S1
6.53 ± 0.90
n.d.
n.d.
77.25 ± 6.04
n.d.
n.d.
T1
5.07 ± 0.77
n.d.
n.q.
48.86 ± 4.56
n.d.
n.q.
Samples T and T1 are from site P1, and samples S and S1 are from site P2. Values represent the mean of three measurements ± SD. SD = standard deviation. DW = dry weight of the plant material. n.d. = not detected. n.q. = not quantified.


## 3.2.4. Monoterpenes

Monoterpenes were among the major molecules identified in dried breadfruit. Different monoterpene compounds were detected: limonene, with a maximum of 247.91 ± 29.29 mg/100 gDW; phellandrene, with 56.67 ± 57.77 mg/100 gDW; and sabinene, with a maximum of 52.98 ± 1.08 mg/100 gDW (Table 5). Student’s t-test and ANOVA did not show significant differences (P<0.05) between the two breadfruit sampling sites. No studies regarding the identification of monoterpenes in breadfruit have been carried out until now. However, studies have demonstrated the pharmacological potential of monoterpenes in the treatment of inflammatory diseases [55]. This study may justify the traditional use of breadfruit as a remedy for inflammatory diseases [56].Table 5
Monoterpene composition of different breadfruit extracts.
Materials
Extracts
Limonene (mg/100 gDW)
Phellandrene (mg/100 gDW)
Sabinene (mg/100 gDW)
γ-Terpinene (mg/100 gDW)
Terpinolene (mg/100 gDW)
Small pieces
S
247.91 ± 29.29
100.29 ± 5.87
41.33 ± 9.28
n.d.
n.d.
T
235.21 ± 52.29
56.67 ± 5.77
37.24 ± 3.50
n.q.
n.q
Flour
S1
140.15 ± 24.46
20.16 ± 1.10
41.70 ± 10.09
n.d.
n.d.
T1
145.64 ± 40.78
44.63 ± 4.27
52.98 ± 1.08
n.q.
n.q.
Samples T and T1 are from site P1, and samples S and S1 are from site P2. Values represent the mean of three measurements ± SD. SD = standard deviation. DW = dry weight of the plant material. n.d. = not detected. n.q. = not quantified.


## 3.3. Macro- and Micromolecules

### 3.3.1. Proteins

The protein composition of breadfruit is shown in Table6. The protein content presented an average value of 4.44 g/100 gflour. This value is very similar to the value of 4.05 ± 0.01 g/100 gflour reported by Graham and De Bravo [27]. The protein content reported by Huang et al. [57] showed a clear difference, averaging 1.2 g/100 gflour. The work of Christina et al. [46] on breadfruit varieties showed minimum and maximum values of approximately 1.9 g/100 gflour and 18.7 g/100 gflour, respectively. The minimum value is significantly lower than the value obtained in this study. However, the maximum value is much greater than that obtained in this work.Table 6
Macronutrient content of dried breadfruit flour samples.
Breadfruit flour
Protein (g/100 gDW)
Lipid (g/100 gDW)
Fibre (g/100 gDW)
P1
4.46
0.66
18.86
P2
4.42
0.68
20.12
Mean value
4.44
0.77
19.49
DW = dry weight of the plant material.


### 3.3.2. Lipids

The lipid content of breadfruit is summarised in Table6. No variation was observed between the two flour samples, which presented an average value of 0.77 g/100 g. This value is slightly higher than the minimum value found in the work of Christina et al. [46] (0.5 g/100 gflour). Graham and De Bravo [27] recorded a slight increase in the lipid content of 1.14 ± 0.07 g/100 g in breadfruit flour.


### 3.3.3. Fibre

The values of raw fibre between the two flour samples are shown in Table6. In both samples of breadfruit flour, the amount of fibre varied slightly between the two samples, ranging from 18.86 to 20.12 g/100 g of dried flour. These values are considerably higher than those found in the work of Christina et al. [46] that reported values between 0.8 and 15.3 g/100 g of breadfruit flour.


### 3.3.4. Sugars

Simple sugars identified in the flour of dried breadfruit in decreasing order included glucose, sucrose, and fructose, and their mean values were 2.80 ± 0.52, 1.51 ± 0.25, and 0.34 ± 0.06 g/100 g (dried breadfruit flour), respectively (Table7). Chromatograms are shown in Figures 3–6. The analysis of Student’s t-test and ANOVA results showed significant differences (P<0.05) in the glucose content compared with that of fructose and sucrose. These results showed lower values of simple sugars compared with those results published by Christina et al. [46]. In previous studies, glucose and sucrose were proven to be the dominant sugars in fully mature breadfruits [58, 59].Table 7
Sugar content of dried breadfruits.
Sample
Fructose (g/100 gDW)
Glucose (g/100 gDW)
Sucrose (g/100 gDW)
Total sugars (g/100 gDW)
P1
0.35 ± 0.09
2.83 ± 0.40
0.90 ± 0.22
4.08 ± 0.71
P2
0.33 ± 0.04
2.76 ± 0.65
2.11 ± 0.28
5.20 ± 0.97
Mean value
0.34 ± 0.06
2.80 ± 0.52
1.51 ± 0.25
4.64 ± 0.83
Values represent the mean of three measurements ± SD. SD = standard deviation. P1 = flour sample from site 1. P2 = flour sample from site 2. DW = dry weight of the plant material.Figure 3
Glucose peak of the P1 sample and relative retention time (TR = 4.790 min).Figure 4
Glucose peak of the P2 sample and relative retention time (TR = 4.801 min).Figure 5
Fructose and sucrose peaks of the P1 sample, with fructose (TR = 9.616 min) and sucrose (TR = 11.177 min) retention times.Figure 6
Fructose and sucrose peaks of the P2 sample, with fructose (TR = 9.507 min) and sucrose (TR = 11.199 min) retention times.


### 3.3.5. Minerals

The results of the mineral composition of dried breadfruit flour are shown in Table8. Calcium was the most abundant mineral in fruits, with an overall mean of 779 mg/100 gDW, followed by other micronutrients, including phosphorus, magnesium, sodium, iron, potassium, and chloride (in the form of NaCl), whose means were 73.5, 42, 13.2, 1.25, 1.195, and 0.435 mg/100 gDW, respectively. Some elements, such as calcium, are markedly important for the growth and development of bones, in particular for those of human infants. Student’s t-test and ANOVA tests revealed slight significant differences (P<0.05) for calcium and magnesium between the samples of the two sites, whereas the others (sodium, phosphorus, potassium, and iron) did not differ between the two samples. These values were largely greater than the results of Graham and De Bravo [27] regarding breadfruit flour, although the reported value of potassium was close to the mean value of the present research. Other studies also reported similar results, despite some differences, as shown by Jones et al. [17] for iron and sodium. However, only the potassium value was truly lower if compared with that reported in the study by Christina et al. [46]. Calcium and magnesium are essential to many human functions, including preservation of the nervous system and muscular balance, and calcium has a protective effect against bone loss [60]. Other studies have highlighted the beneficial effects of calcium on colon cancer [61], cardiovascular events [62], and hypertension [63]. Magnesium deficiency can lead to neuromuscular hyperexcitability, which results in signs of latent tetany [64].Table 8
Mineral content in dried flour of breadfruit.
Flour
Calcium (mg/100 gDW)
Iron (mg/100 gDW)
Phosphorus (mg/100 gDW)
Magnesium (mg/100 gDW)
Chlorides (mg/100 gDW)
Potassium (mg/100 gDW)
Sodium (mg/100 gDW)
P1
678
1.05
76
32
0.46
1.174
12.7
P2
880
1.45
71
52
0.41
1.217
13.7
Mean value
779
1.25
73.5
42
0.435
1.195
13.2
P1 = flour sample from site 1. P2 = flour sample from site 2.


## 3.3.1. Proteins

The protein composition of breadfruit is shown in Table6. The protein content presented an average value of 4.44 g/100 gflour. This value is very similar to the value of 4.05 ± 0.01 g/100 gflour reported by Graham and De Bravo [27]. The protein content reported by Huang et al. [57] showed a clear difference, averaging 1.2 g/100 gflour. The work of Christina et al. [46] on breadfruit varieties showed minimum and maximum values of approximately 1.9 g/100 gflour and 18.7 g/100 gflour, respectively. The minimum value is significantly lower than the value obtained in this study. However, the maximum value is much greater than that obtained in this work.Table 6
Macronutrient content of dried breadfruit flour samples.
Breadfruit flour
Protein (g/100 gDW)
Lipid (g/100 gDW)
Fibre (g/100 gDW)
P1
4.46
0.66
18.86
P2
4.42
0.68
20.12
Mean value
4.44
0.77
19.49
DW = dry weight of the plant material.


## 3.3.2. Lipids

The lipid content of breadfruit is summarised in Table6. No variation was observed between the two flour samples, which presented an average value of 0.77 g/100 g. This value is slightly higher than the minimum value found in the work of Christina et al. [46] (0.5 g/100 gflour). Graham and De Bravo [27] recorded a slight increase in the lipid content of 1.14 ± 0.07 g/100 g in breadfruit flour.


## 3.3.3. Fibre

The values of raw fibre between the two flour samples are shown in Table6. In both samples of breadfruit flour, the amount of fibre varied slightly between the two samples, ranging from 18.86 to 20.12 g/100 g of dried flour. These values are considerably higher than those found in the work of Christina et al. [46] that reported values between 0.8 and 15.3 g/100 g of breadfruit flour.


## 3.3.4. Sugars

Simple sugars identified in the flour of dried breadfruit in decreasing order included glucose, sucrose, and fructose, and their mean values were 2.80 ± 0.52, 1.51 ± 0.25, and 0.34 ± 0.06 g/100 g (dried breadfruit flour), respectively (Table7). Chromatograms are shown in Figures 3–6. The analysis of Student’s t-test and ANOVA results showed significant differences (P<0.05) in the glucose content compared with that of fructose and sucrose. These results showed lower values of simple sugars compared with those results published by Christina et al. [46]. In previous studies, glucose and sucrose were proven to be the dominant sugars in fully mature breadfruits [58, 59].Table 7
Sugar content of dried breadfruits.
Sample
Fructose (g/100 gDW)
Glucose (g/100 gDW)
Sucrose (g/100 gDW)
Total sugars (g/100 gDW)
P1
0.35 ± 0.09
2.83 ± 0.40
0.90 ± 0.22
4.08 ± 0.71
P2
0.33 ± 0.04
2.76 ± 0.65
2.11 ± 0.28
5.20 ± 0.97
Mean value
0.34 ± 0.06
2.80 ± 0.52
1.51 ± 0.25
4.64 ± 0.83
Values represent the mean of three measurements ± SD. SD = standard deviation. P1 = flour sample from site 1. P2 = flour sample from site 2. DW = dry weight of the plant material.Figure 3
Glucose peak of the P1 sample and relative retention time (TR = 4.790 min).Figure 4
Glucose peak of the P2 sample and relative retention time (TR = 4.801 min).Figure 5
Fructose and sucrose peaks of the P1 sample, with fructose (TR = 9.616 min) and sucrose (TR = 11.177 min) retention times.Figure 6
Fructose and sucrose peaks of the P2 sample, with fructose (TR = 9.507 min) and sucrose (TR = 11.199 min) retention times.


## 3.3.5. Minerals

The results of the mineral composition of dried breadfruit flour are shown in Table8. Calcium was the most abundant mineral in fruits, with an overall mean of 779 mg/100 gDW, followed by other micronutrients, including phosphorus, magnesium, sodium, iron, potassium, and chloride (in the form of NaCl), whose means were 73.5, 42, 13.2, 1.25, 1.195, and 0.435 mg/100 gDW, respectively. Some elements, such as calcium, are markedly important for the growth and development of bones, in particular for those of human infants. Student’s t-test and ANOVA tests revealed slight significant differences (P<0.05) for calcium and magnesium between the samples of the two sites, whereas the others (sodium, phosphorus, potassium, and iron) did not differ between the two samples. These values were largely greater than the results of Graham and De Bravo [27] regarding breadfruit flour, although the reported value of potassium was close to the mean value of the present research. Other studies also reported similar results, despite some differences, as shown by Jones et al. [17] for iron and sodium. However, only the potassium value was truly lower if compared with that reported in the study by Christina et al. [46]. Calcium and magnesium are essential to many human functions, including preservation of the nervous system and muscular balance, and calcium has a protective effect against bone loss [60]. Other studies have highlighted the beneficial effects of calcium on colon cancer [61], cardiovascular events [62], and hypertension [63]. Magnesium deficiency can lead to neuromuscular hyperexcitability, which results in signs of latent tetany [64].Table 8
Mineral content in dried flour of breadfruit.
Flour
Calcium (mg/100 gDW)
Iron (mg/100 gDW)
Phosphorus (mg/100 gDW)
Magnesium (mg/100 gDW)
Chlorides (mg/100 gDW)
Potassium (mg/100 gDW)
Sodium (mg/100 gDW)
P1
678
1.05
76
32
0.46
1.174
12.7
P2
880
1.45
71
52
0.41
1.217
13.7
Mean value
779
1.25
73.5
42
0.435
1.195
13.2
P1 = flour sample from site 1. P2 = flour sample from site 2.


## 4. Conclusion

The results of this study are important because they are derived from the first analytical study on breadfruit consumed by the Comorian population. The presented data provide information on the nutrition and health properties of breadfruit by the identification of the main biologically active molecules in breadfruit. Nutritionists and epidemiologists could use these data for the evaluation of food-health relationships among the Comorian population.



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*Source: 5697928-2018-06-24.xml*