Antioxidant Activity, Total Phenolic Content and Selected Physicochemical Properties of White Mulberry (Morus Alba L.) Fruits

Abstract

In this study, total phenolic content, antioxidant activity, mineral content and selected physicochemical properties of three white mulberry phenotypes (Morus alba L.) grown in Turkey were measured. Moisture, total soluble solids, total sugar, sucrose, reducing sugar, protein, ash, pH, titratable acidity, color (L, a, b), vitamin C, total phenolic, antioxidant activity of fruits were determined between 72.85–79.75 g/100 g fresh fruit, 21.25–28.50 g/100 g fresh fruit, 12.18–17.02 g/100 g fresh fruit, 1.57–4.36 g/100 g fresh fruit, 9.42–15.46 g/100 g fresh fruit, 0.82–0.89 g/100 g fresh fruit, 2.20–2.65 g/100 g fresh fruit, 5.70–5.86, 0.25–0.28 g/100 g fresh fruit, 31.24–68.69, −2.46 to 15.68, 4.58–21.74, 10.15–21.50 mg/100 g fresh fruit, 18.16–19.24 μg gallic acid equivalent/mg of sample, and 33.96–38.96%, of sample respectively. Fruits were generally found high in P and low in Ca.

Keywords:

• Mulberry
• Morus alba L.
• Antioxidant activity
• Total phenolic
• Mineral content

INTRODUCTION

Mulberry is a fast-growing deciduous plant and it grows under different climatic conditions such as tropical, subtropical and temperate throughout world.[1] It has been cultivated in the Northern hemisphere for centuries. There are three types of mulberry fruits: red mulberry (Morus rubra L.), black mulberry (M. nigra L.) and white mulberry (M. alba L.). White mulberry originated in Western Asia, including Turkey, red mulberry in North and South America, and black mulberry in Southern Russia.[2] In Turkey, mulberry fruits are as important as the other temperate fruit species such as apricot, walnut, sour cherry, etc. Mulberry trees are extensively grown in southern Europe, India, China for its foliage as foods for silkworms. However, in Turkey and Greece mulberries are grown for fruit production rather than foliage.[3] In Turkey, growing conditions in most agriecological region is very suitable for cultivating high quality mulberry fruits, mainly from Morus alba, Morus nigra and Morus rubra.[4] Mulberry fruit can be eaten fresh or dried. It is also processed into mulberry juices, paste, marmalade, jam, pulp or jelly.[5] In Turkey, several traditional products such as pekmez,[6,7] pestil, and kome are made with the mulberry fruits as well.[3]

Mulberry fruits has been used medicinally in Turkey as a worming agent, as a remedy for dysentery, and as a laxative, hypoglycaemic, expectorant, anthelmintic, odontalgic, and emetic.[8] Local people traditionally believe that deep-coloured fruits, especially red and black mulberry fruits, are healthier for the human body.[3] Mulberry fruit is also a traditional Chinese edible fruit that is used effectively in folk medicines to treat fever, strengthen the joints, protect liver from damage, lower blood pressure, and facilitate discharge of urine. Recently, it has gained an important position in the local soft drink market, although its biological and pharmacological effects are still poorly defined.[9,10]

Epidemiological and experimental studies reveal a negative correlation between the consumption of diets rich in fruits, and vegetables and the risks for chronic angiogenic diseases, such as cardiovascular diseases, arthritis, chronic inflammation and cancers, diabets, stroke, neurodegenerative diseases, including Parkinson's and Alzheimer's diseases as well as inflammation and problems caused by cell and cutaneous aging.[11,12,13,14,15] These physiological functions of fruits and vegetables may be partly attributed by their abundance of phenolics. Deep-colored vegetables and fruits are good sources of phenolics, including flavonoids and anthocyanins, and carotenoids.[13] Fruits and vegetables contain many antioxidant compounds, including carotenoids, thiols, vitamins such as ascorbic acid and tocopherols, and phenolics.[16] Antioxidant components can delay or inhibit lipid oxidation, by inhibiting the intiation or propagation of oxidizing chain reactions, and are also involved in scavenging free radicals.[14]

Among berries, mulberry fruits are rich in phenolics and anthocyanins.[3,9,10,17] Phenolics possess a wide spectrum of biochemical activities such as antioxidant, antimutagenic and anticarcinogenic properties, as well as the ability to modify gene expression.[17] There are some studies about physical and chemical properties of fruits of different mulberry species.[18,19] In these studies, little information is reported on the phenolic content and antioxidant activity of mulberry fruits. Morever, the diversity on physical and chemical parameters of mulberry fruits within same species was not studied earlier. For these reasons, the present studies were carried out to investigate antioxidant activity, total phenolics, mineral composition, physicochemical properties of fruits of three white mulberry (Morus alba) phenotypes.

MATERIALS AND METHODS

Materials and Chemicals

In this study, fruits of three mulberry phenotypes belongs to Morus alba L. were used. Mulberry fruits were harvested in full mature stage (June 2006) from Coruh valley in Turkey. Fresh white mulberries harvested by hand and kept in a refrigerator at 4°C (∼24 h) until analyses and drying. Berries of uniform in size were used. Fruit width and length of samples were 18.90–20.60 mm and 21.30–27.45 mm, respectively. All reagents used were of analytical grade.

Preparation of Fruit for Antioxidant and Phenolic Analysis

The fruits were cleaned and cut into small pieces before being dried in a hot air-blowing oven at 50°C. All samples, after drying, had water contents below 10%. They were ground to a fine powder in a mechanical blender and kept at room temperature prior to extraction. The dried plants were used for the analysis of antioxidant activity and phenolic compounds. Ten mg ground sample was mixed with 10 ml water and stirred for 30 min on a magnetic stirrer. The suspension was filtered through Whatman No. l filter paper. Final solutions were used as stock solution for the analysis of antioxidant activity and phenolic compounds.

Chemical Analyses

Moisture, total soluble solid (TSS), protein, ash, pH and titratable acidity were determined according to standard AOAC method;[20] pH was determined with a ATI ORION 420A model pH meter; titratable acidity, expressed as percentage of citric acid, was determined with 0.1 N NaOH up to pH 8.1; solubles dry matter was determined with an Abbe-Zeis refractometer; protein content was determined by the Kjeldal method (N × 6.25). Total sugar, reducing sugar and sucrose contents were analysed by the Lane-Eynon method.[21] Reducing sugar concentration was measured before inversion whereas total sugar was determined after inversion. Sucrose was calculated by subtracting the reducing sugar concentration from the total invert sugar and multiplying the result by 0.95.

In order to determine the mineral composition (K, Ca, Mg, Fe, Mn, Zn, and Cu), fruit were burned with nitric acid and a percloric acid solution on a hot plate at 200°C. Then, the absorbance of the extract was measured with the Atomic Absorbance Spectrophotometer. The amounts of minerals were calculated with a standard curve. Phosphorus content of the extract was analysed by determining the absorbance of the colour yellow, obtained from the Barton reaction, at 680 nm wavelengths, and comparing the results to a standard curve.[22]

(1)

For color analysis, the instrument was calibrated with a white reference before measurements. Color of mulberry fruit was analysed by measuring Hunter L (brightness; 100: white, 0: black), a ( + : red; −: green) and b ( + : yellow; −: blue) parameters with a colorimeter (Model CR 200, Chromometer, Minolta, Japan).

Determination of Total Phenolic Content

The concentration of total phenolics in the water extract of mulbery fruit was determined by the Folin–Ciocalteau colorimetric method.[23] Briefly, one millilitre of the solution (contains 1 mg sample) extract in water was pipetted into a flask. Then 46 ml of distilled water and 1 ml of Folin and Ciocalteu's reagent was added and mixed throughly. The mixture was left to stand for 3 min and 3.0 ml of 2% sodium carbonate were added. After 120 min incubation at ambient temperature with shaking, the resulting absorbance was measured at 760 nm. Measurements were carried out in dublicate, the calibration curve was performed with gallic acid, and the results were expressed as μg of gallic acid equivalents per milligram of sample (μg GAE/ mg of sample).

Determination of Antioxidant Activity

The antioxidant activity of mulberry water extracts was determined according to the β-carotene bleaching method described by Kaur and Kapoor[24] with some modifications. Briefly, 4 ml of β-carotene solution (0.1 mg in 1ml chloroform), 40 mg of linoleic acid and 400 mg of Tween 40 were transferred to a round-bottom flask. The mixture was then evaporated at 50°C by means of a rotary evaporator to remove chloroform. Then, 100 ml of oxygenated distilled water were added slowly to the residue and vigorously agitated to give a stable emulsion. Then, 800 μl of extracts were added to 3 ml aliquots of β-carotene/linoleic acid emulsion. As soon as the emulsion was added to each tube, the zero time absorbance was measured at 470 nm using a spectrophotometer. The mixtures were incubated at 50°C for 100 min. The measurement was carried out at 10 min intervals for 100 min. Water instead of plant extract was used as control. A blank, devoid of β-carotene, was prepared for background subtraction. Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) were used as a standard. All samples were assayed in dublicate. Degradation rate (DR) was calculated according to first order kinetics, using the following equation based on:

(2)

where ln is natural log; a is the initial absorbance (470 nm) at time 0; b is the absorbance (470 nm) at 100 min; and t is time. Antioxidant activity (AA) was expressed as percent of inhibition relative to the control, using the following formula:

(3)

Statistical Analysis

Analysis of variance was performed by ANOVA procedures (SPSS 9.0 for Windows). Significant differences between means were determined by Duncan's Multiple Range tests. P values < 0.05 were regarded as significant.

RESULTS AND DISCUSSION

Physicochemical Properties of Mulberry Fruit

The physicochemical properties of mulberry fruits are given in Table 1. In this study, mulberry fruits at the full mature stage. Fruit width and length of samples were 18.90–20.60 mm and 21.30–27.45 mm, respectively. There were statistically differences among white mulberry phenotypes in terms of moisture, total soluble solids, total sugar, sucrose, reducing sugar, ash, and color (L, a, b) of fruits (p < 0.05). Moisture, total soluble solids, total sugar, sucrose, reducing sugar, protein, ash, pH, titratable acidity, and color (L, a, b), of fruits were established between 72.85–79.75 g/100 g fresh fruit, 21.25–28.50 g/100 g fresh fruit, 12.18–17.02 g/100 g fresh fruit, 1.57– 4.36 g/100 g fresh fruit, 9.42–15.46 g/100 g fresh fruit, 0.82–0.89 g/100 g fresh fruit, 2.20–2.65 g/100 g fresh fruit, 5.70–5.86, 0.25–0.28 (% citric acid), 31.24 to 68.69, −2.46 to 15.68 and 4.58–21.74, respectively. In a previous study, it was reported that white mulberry fruit at the full ripe stage. contained 71.5% moisture, 29.50% total dry matter, 20.4% total soluble solid, 0.25% total acidity (citric acid), pH 5.60. Fruit colour was determined as L value 78.4, a value −13.6 and b value 16.2.[3] Erdogan,[4] reported that total soluble solids of mulberry fruit at full maturity level varied between 14–25%. Güneş and Çekiç,[25] found that white mulberry fruit width and length were 15.32–21.28 mm and 25.75–34.85 mm at full maturity level, respectively. This mulberry fruits contained 12.37–18.50% total dry matter, 13.0–18.6% total soluble solid, pH 5.53–6.12, 0.10–0.26% total acidity. Our results were found similar by minor differences compared with above studies. Chemical and physical characteristics of fruits depend on the ripening stage, genotypes differences, soil, climatic conditions, post-harvest practices and handling procedure after harvesting. Mulberry fruits are sensitive to storage because they have a high level of moisture content. Hence, fresh mulberry fruits are preserved in some form. Unwashed berries can be kept several days in a refrigerator in a container or is drying.[2]

Table 1 Physicochemical properties of mulberry fruit

	Mulberry phenotypes
Parameters	1	2	3
Moisture g/100 g fresh fruit	74.64 ± 0.65^b	79.75 ± 0.35^a	72.85 ± 0.21c
TSS, g/100 g fresh fruit	26.25 ± 0.35^b	21.25 ± 0.35^c	28.50 ± 0.00^a
Total sugar g/100 g fresh fruit	16.82 ± 0.11^a	17.02 ± 0.34^a	12.18 ± 0.18b
Sucrose g/100 g fresh fruit	4.36 ± 0.25^a	1.57 ± 0.25^c	2.76 ± 0.15b
Reducing sugar g/100 g fresh fruit	12.46 ± 0.06^b	15.46 ± 0.09^a	9.42 ± 0.03c
Protein g/100 g fresh fruit	0.83 ± 0.02^a	0.82 ± 0.02^a	0.89 ± 0.05^a
Ash g/100 g fresh fruit	2.65 ± 0.07^a	2.20 ± 0.00^c	2.45 ± 0.07b
pH	5.86 ± 0.16^a	5.79 ± 0.04^a	5.70 ± 0.02^a
Titratable acidity g/100 g fresh fruit	0.26 ± 0.07^a	0.25 ± 0.00^a	0.28 ± 0.01^a
L	68.69 ± 0.51^a	55.69 ± 0.41b	31.24 ± 1.27c
a	−2.23 ± 0.43^b	−2.46 ± 0.64b	15.68 ± 0.65^a
b	21.74 ± 0.00^a	18.25 ± 0.61b	4.58 ± 0.48c
Means within the same line followed by same letter are not statistically significant (P < 0.05).

Ascorbic Acid, Total Phenolics and Antioxidant Activity of Mulberry Fruit Extracts

Table 2 shows the contents of ascorbic acid, total phenolics and antioxidant activity of mulberry fruit extracts. There were statistically differences among phenotypes in terms of Vitamin C (p < 0.05). The content of vitamin C of the fruit varied from 10.15–21.50 mg/100 g fresh sample with phenotype 3 showed the highest vitamin C content. Lale,[26] reported that the vitamin C found in fruits of white mulberry cultivars was 17.81 mg/100 ml of fresh fruit juice. Wills et al.[19] found that mulberry fruits contains 10 mg/100 g fresh fruit vitamin C. Our results are similar with Lale,[26] and Wills et al.[19] Vitamin C have a high antioxidant capacity and it is highly bioavailable and is therefore the most important water-soluble antioxidant in cells and an efficient scavenger of reactive oxygen species.[27]

Table 2 Vitamin C, phenolic compounds, antioxidant activity of mulberry fruits

Mulberry phenotypes	Vitamin C (mg/100 gof fresh fruit	Phenolic compounds (μg GAE/ mg of sample)	Antioxidant activity(%) of sample
1	12.73 ± 0.14^b	18.15 ± 0.50a	33.95 ± 0.10d
2	10.15 ± 0.17^c	19.11 ± 0.16a	38.92 ± 0.53c
3	21.50 ± 0.21^a	19.23 ± 0.33a	37.87 ± 0.31c
BHA (100 mg/L)	—	—	61.46 ± 0.11b
BHA(200 mg/L)	—	—	80.12 ± 0.12a
Means within the same lines followed by same letter are not statistically significant (P < 0.05).

The total phenolic content of mulberry fruit ranged from 18.16 to 19.24 μg GAE /mg of sample (Table 2). Phenotype 3 had the highest total phenolic content, followed by phenotype 2 (19.12 μg/mg GAE), and phenotype 1. Earlier, total phenolic content in mulberry fruit of three different species was reported which ranged from 181 (Morus alba)-1422 (Morus nigra) mgGAE/100g fresh weight.[3] Lin and Tang[13] reported that mulberry fruit (Morus alba) contained 1515 mgGAE/100 g fresh matter of total phenolic. Bae and Suh[9] determined that the mulberry fruit contained phenolic substances 959.9–2570.4 μg/g fresh sample. The variation of phenolic compounds in the fruits depends on many factors, such as degree of maturity at harvest, genetic differences, and environmental conditions during fruit development. Mulberry fruit has many physiological functions. Many phenolic compounds are antioxidants. They are may contribute to reducing human diseases such as cancer, arteriosclerosis, brain disorders and hearth diseases.[28]

The antioxidant activity of plants is mainly contributed by the active compounds present in them. There are several methods for determination of antioxidant activities. In this study, β-carotene bleaching method were used due to β-carotene shows strong biological activity and is physiologically important compound.[15] In the β-carotene bleaching assay, linoleic acid produces hydroperoxides as free radicals during incubation at 50°C. The presence of antioxidants in the extract will minimise the oxidation of β-carotene by hydroperoxides. Hydroperoxides formed in this system will be neutralized by the antioxidants from the extracts. Thus, the degradation rate of β-carotene depends on the antioxidant activity of the extracts.[14] The results are summarized in Table 2. It was found that the water extracts of mulberry fruits showed antioxidant activity. There were statistically differences among phenotypes in terms of antioxidant activity (p < 0.05). All types showed lower antioxidant activity than BHA and BHT. The antioxidant activity of phenotype 2 and 3 had higher antioxidant activity, compared to phenotype 1.

Mineral Contents of Mulberry Fruit

Table 3 shows concentration of minerals in the mulberry fruit. The mineral content of mulberry fruit samples ranged from 0.3 to 0.7 mg/100 g fresh fruit for Fe, 19 to 20 mg/100 g fresh fruit for Mg, 159 to 510 mg/100 g fresh fruit for Ca, 45 to 49 mg/100 g fresh fruit for K, 0.2 to 0.4 mg/100 g fresh fruit for Cu, 0.4 to 2 mg/100 g fresh fruit for Zn, 2 mg/100 g fresh fruit for Mn, 3 to 4 mg/100 g fresh fruit for Na and 4101 to 7483 mg/100 g fresh fruit for P. The highest minerals were P and followed by Ca, K, Mg Na, Mn, Zn, Fe and Cu. Significant differences (p < 0.05) were found among the phenotypes in P, Cu, Fe, Zn, Ca content of mulberry fruits whereas significant differences (p < 0.05) were detected in Mn. No significant differences were detected in Mg, Na and K. The mineral composition of mulberry fruit (100 g edible portion) was 310 mg K, 6 g Na, 20 g Ca, 12 g Mg, 0.3 g Fe, 0.2 g Zn as reported by Wills et al.[19] Ercisli and Orhan,[3] determined that the mulberry fruit contained N 0.75%, P 247 mg/100 g, K 1668 mg/100 g, Ca 152 mg/100 g, Mg 106 mg/100 g, Na 60 mg/100 g, Fe 4.2 mg/100 g, Cu 0.5 mg/100 g, Mn 3.8 mg/100 g and Zn 2.8 mg/100 g. The results of this study show differences when compared with the result of Ercisli and Orhan[3] and Wills et al.[19] Differences in mineral contents are due to species, varieties, maturity, agricultural practices, growing conditions, such as soil and geographical conditions, climate.[29] Minerals have important and vital role in metabolism, health and disease.[30] They are required for normal cellular function, and are critical for enzyme activation, bone formation, hemoglobin composition, gene expression, and amino acid, lipid and carbohydrate metabolism.[29] Micronutrients are involved in numerous biochemical processes and an adequate intake of certain micronutrients relates to the prevention of deficiency diseases. Iron deficiency anaemia, for example, affects one third of the world population. Fruits and vegetables are valuable sources of minerals.[31]

Table 3 Mineral content of mulberry fruit (mg/100 g of fresh fruit)

	Minerals
Phenotypes	P	K	Ca	Mg	Na	Fe	Mn	Zn	Cu
1	7483 ± 0.07^a	45 ± 3.21a	510 ± 51.52a	20 ± 0.00a	4 ± 0.53a	0.7 ± 0.01a	2 ± 0.04a	2 ± 0.10a	0.4 ± 0.01a
2	2029 ± 0.03^c	48 ± 4.28a	159 ± 4.59b	19 ± 1.55a	3 ± 0.53b	0.3 ± 0.01b	2 ± 0.06a,b	0.4 ± 0.02c	0.2 ± 0.01b
3	4101 ± 0.30^b	49 ± 2.67a	220 ± 9.19b	20 ± 0.00a	4 ± 0.17b	0.3 ± 0.04b	2 ± 0.06c	0.8 ± 0.12b	0.2 ± 0.03b
Means within the same line followed by same letter are not statistically significant (P < 0.05).

CONCLUSIONS

According to these results, the highest moisture (79.75 g/ 100g fresh fruit) and the lowest TSS (21.25 g/100 g fresh fruit) were found for phenotype 2. The sucrose and reducing sugar were the lowest for phenotype 3. When comparing the three phenotype pH and titratable acidity were similar. Mulberry fruits contained 10.150–21.500 mg vitamin C/100 g of fresh weight. This study confirmed that mulberry contain significant amounts of phenolic substance and antioxidant activity. The total phenolic content of mulberry fruit ranged from 18.16 to 19.24 μg GAE/mg of sample. Antioxidant activity of mulberry fruit samples ranged from 33.955 to 38.925% of sample. Mulberry fruit is a good source of antioxidant and minerals. Mulberry fruits are appear as outstanding mineral sources, especially in phosphorus and calcium, followed by potassium, magnesium and sodium. These parameters varied between phenotypes used significantly suggesting that these phenotypes could be preserved as mulberry genetic resources for fruit properties.

ACKNOWLEDGMENT

This research was financially supported by the Research Fund of Erzurum Ataturk University, Turkey (BAP- 2005/56).

Notes

8. Baytop, T. Türkiye'de Bitkiler Ile Tedavi. I.U. Yayinlari No:3255, Eczacilik Fak., 40. Istanbul, Turkey, 1996; 444 pp.