Antioxidant components and antioxidant/antiradical ...
Journal of Food Composition and Analysis 23 (2010) 15–22
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Original Article
Antioxidant components and antioxidant/antiradical activities of desert truffle (Tirmania nivea) from various Middle Eastern origins Abdul Ameer Ahmed Al-Laith * Department of Biology, College of Science, University of Bahrain, P.O. Box 32038, Sakhir Campus, Building S41, Sakhir, Bahrain
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 November 2008 Received in revised form 2 July 2009 Accepted 6 July 2009
Dried desert truffles from Bahraini, Iranian, Moroccan and Saudi origins were examined for their antioxidant and antiradical activities using four analytical methods: ferric reducing ability (FRAP), DPPH, deoxyribose, and nitric oxide (NO). Chemical constituents contributing towards these activities were also investigated. Generally, these truffles possessed varying concentration of antioxidant chemicals averaging 9.6 0.15, 12.0 8.34, 1860 361, 1328 167 and 293 32 mg/100 g dw, for ascorbic, anthocyanins, total esterified phenolics, total free phenolics and total flavonoids, respectively; total carotenoids averaged 681 245 g/100 g dw. Dried truffles also varied with regard to their antioxidant and antiradical activities. The FRAP value averaged 15.41 3.51 mmol/100 g dw. Antiradical activity measured as percent inhibition of DPPH quenching averaged 30.6 12.97% and EC50 of 0.55 0.38 mg. The average EC50 of NO scavenging activity was 159.4 69.3 mg, whereas the average percent inhibition of deoxyribose degradation was 55.9 30.1%. The Iranian truffles yielded the highest in several variables, whereas the Moroccan truffles possessed the lowest values of many variables among the four tested samples. Significant correlation was established between total and free phenolics and FRAP values, and between flavonoids and percent radical inhibition using DPPH, NO and deoxyribose assays. ß 2009 Elsevier Inc. All rights reserved.
Keywords: Antioxidant activity Antiradical activity DPPH Flavonoids FRA Phenolics Truffle Fungi Wild food Middle East Bahrain Traditional food Food composition Food analysis
1. Introduction Desert truffles are a type of an obligate hypogeous ascomycetes ectomycorrhizal fungi formed in association with host roots of Helianthemum spp. and the soil inhabiting fungi Terfezia or Tirmania spp. (Mandeel and Al-Laith, 2007). The type of mycorrhizal fungus ramifies through the soil, absorbing nitrogen and other minerals, which is transported back to the host plant (Ewaze and Al-Naama, 1989). Desert truffles are seasonal and socio-economically important fungi. These truffles are edible and grow wild in the central-southern part of Bahrain. The truffles usually appear in the deserts following the rainy season between February and April in Bahrain as well as in many Gulf states (Al-Ruqaie, 2002; Moubasher, 1993). They are distributed naturally from North Africa (Morocco, Tunisia, Algeria and Egypt) to the Middle East (Saudi Arabia, Kuwait, Iraq, Iran, Lebanon, Syria and Jordon). The people in the Middle East region are
* Tel.: +973 17 437477; fax: +973 17 449158. E-mail addresses: [email protected], [email protected]. 0889-1575/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2009.07.005
considered the largest truffle consumers (Al-Rahmah, 2001), and the truffle commodity is regarded as a costly delicacy. In these countries, no data is available on total production and consumption. Among the various known edible desert truffle varieties, only two species of the dark brown color truffles belonging to the genus Terfezia, locally called Ikhlasi (Terfezia claveryi and Terfezia boudieri) and one species of the white color truffles belonging to the genus Tirmania, locally called Zubaidi (Tirmania nivea) are found on the Arabian Peninsula (Al-Rahmah, 2001). Ikhlasi is ovoid with a black skin and a pinkish-ivory interior with a nut-like flavor. Zubaidi is cream-colored with a more delicate flavor and is usually more expensive. The truffles of the desert are not so strongly flavoured, compared with the European truffles (Al-Sheik and Trappe, 1983). Nevertheless, the truffle is a type of tuber highly prized for its unique musky aroma and flavour (Omer et al., 1994). The popularity of truffles is believed to be due to their nutritional value and delicious taste. Wild edible fungi not only add flavor to bland staple foods but they are also valuable foods in their own right. As such, desert truffles are a rich source of protein, amino acids, fatty acids, minerals and carbohydrates (Al-Naama et al., 1988; Bokhary et al., 1987, 1989).
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Similar to other fungi, desert truffles comprise a vast and yet largely unexploited source of new pharmaceutical products. In search for new therapeutic alternatives, and most importantly for modern medicine, truffles represent an unlimited source of therapeutic compounds with anti-inflammatory, immunosuppressor, antimutagenic and anticarcinogenic properties (Hannan et al., 1989), antioxidant properties (Murcia et al., 2002), antimicrobial (Janakat et al., 2004, 2005) and steroidal glucoside with polyhydroxy ergosterol nucleus (tuberoside) (Gao et al., 2001). Specific and peculiar attributes of desert truffles like hypogeous, mycorrhizal and microaerophilic make their metabolism interesting model to study, especially, under stressful desert arid environment conditions. Truffles that live under microaerobic habitats are expected to show morphological, physiological and biochemical adaptations to an environment poor in oxygen as found under hypogeous conditions. As mycorrhizal symbiotic fungi of several species of plants, they exchange metabolites, mineral salts and ions. These adaptations are largely controlled by several environmental factors like the rainy season with its amount and timing, soil types and characteristics, water availability and climatic conditions (Bokhary et al., 1987; Bokhary and Parvez, 1993). Very little information is available relating to truffle metabolism and, in particular, oxidative metabolism (Pacioni et al., 1995). Oxidation is necessary to many organisms for the production of energy to support metabolism of aerobic cell. However, in some instances, the production of oxygen through uncontrolled metabolic pathways results in free radicals. This imbalance can result in damage to molecules, including lipids, DNA, carbohydrates and proteins (Dubost et al., 2007). Moreover, these free radicals are potentially involved in the onset of many diseases such as cancer, rheumatoid arthritis and atherosclerosis as well as in degenerative processes associated with aging (Halliwell and Gutteridge, 1984). Exogenous chemical and endogenous metabolic processes in the human body or in the food system might produce highly reactive free radicals, especially reactive oxygen species (ROS) capable of oxidizing biomolecules, resulting in cell death and tissue damage (Halliwell and Gutteridge, 2003). On a cellular basis, damage provoked by these free radical formations is usually protected by oxidative enzymes as well as compounds such ascorbic acid, tocopherols and phenolics. When the mechanism of antioxidant protection becomes unbalanced by factors such as aging, deterioration of physiological functions may occur resulting in diseases and accelerated aging. However, the antioxidant components present in truffles are of great interest as possible protective agents to help reduce oxidative damage. Previous reports had shown that the 120 expressions of some antioxidant enzymes were correlated with the microaerobic metabolism, growth rate and mycorrhizal symbiosis of truffles (Amicarelli et al., 1999). Wild and cultivated mushrooms are well known to contain various polyphenolic compounds which are recognized as an excellent antioxidant due to their ability to scavenge free radicals by acting as reducing agents, hydrogen donating antioxidants and singlet oxygen quenchers (Barros et al., 2007; Dubost et al., 2007; Elmastas et al., 2007). Although some common edible mushrooms have been found to possess antioxidant activity which is well correlated with their total phenolic contents (Lo and Cheung, 2005), data about the antioxidant/antiradical activity and the antioxidant compounds of desert truffles are lacking. Thus, there is an increasing demand to assess these attributes on truffles and other wild fungi. The importance of truffles as traditional food and medicine was recently surveyed by Mandeel and Al-Laith (2007). The aim of this study was to investigate the antioxidant and antiradical activities as well as the chemical components with antioxidant/antiradical properties of the desert truffle Tirmania nivea obtained from four Middle Eastern countries, namely Bahrain, Iran, Moroccan and Saudi Arabia.
2. Materials and methods 2.1. Chemicals L-Ascorbic acid, deoxyribose, 2,4-dinitrophenylhydrazine (DNPH) and sodium nitroprusside (SNP), were obtained from BDH (England, UK). 2,4,6-Tripyridyl-s-triazine (TPTZ), gallic acid, N-(1-naphthyl)ethylenediamine di-hydrochloride (NEDD) and sulfanilic acid 2-thiobarbituric acid were from Fluka Chemicals AG (Deisenhofen, Switzerland). 2,2-Diphenyl-1-picrylhydrazyl (DPPH), Folin–Ciocalteu (FC) reagent were from Sigma (St. Louis, MO, USA). All other chemicals were of analytical grade.
2.2. Equipment All spectrophotometric measurements were performed using Spectronic Genesys 20 (Spectronic Genesys, USA). Beckman benchtype and Beckman high speed equipped with J-21 rotor (Beckman, USA) were used for centrifugation. Air-circulated oven (LKB, UK) was used for drying samples. 2.3. Desert truffle source and description Samples of Bahraini desert truffles were a kind gift collected from the southern area of Bahrain during the month of April 2007. Iranian truffles were also a kind gift collected from the western part of Iran. Moroccan and Saudi truffles were commercially imported to Bahrain and purchased from local markets during May 2007. When received, all truffle samples were unwashed, unpacked, and not stored at room temperature. They were intact and showed no sign of spoilage. The Bahraini, Moroccan and Saudi samples were of similar irregular spherical shape and size with an average weight of 33.0, 30.4 and 30.3 g, respectively, whereas the Iranian samples were larger with an average weight of 64.3 g. The color of flesh of first three was similar, dark creamy, whereas the flesh color of the Iranian was light creamy. Superficially, the Bahraini, Moroccan and Saudi samples cannot be distinguished from each other. All truffle samples were identified as white variety Zubaidi (Tirmania nivea). They were characterized by earthy musky aroma and light flavor. 2.4. Truffle preparation A total of 3–5 ascocarps (tubers) composite samples of each truffle source were cleaned from soil, peeled, sliced and dried at 65 8C in an air-circulated oven until constant weight (3 days). Dried slices were subjected to milling for 2 min using Micro-mill (TechniLab Instruments, NJ, USA). The resulting fine pulverized powder was capped and stored at room temperature (23 2 8C) in the dark. Moisture content was performed in duplicate. 2.5. Antioxidant activity by ferric reducing ability (FRAP) assay For the ferric reducing ability (FRAP) assay, one gram of fine truffle powder was extracted with 10 mL of phosphate buffer (75 mM, pH 7.4) for 30 min with occasional shaking. The suspension was centrifuged at 2000 g for 15 min. The clear extract was used to measure the antioxidant activity by FRAP assay according to Benzie and Strain (1996). The reaction mixture was freshly prepared by mixing acetate buffer (300 mM, pH 3.6), FeCl36H2O (20 mM) and TPTZ (10 mM) in a ratio of 10:1:1. The clear extract (0.1 mL) was added to 3 mL reaction mixture, mixed and kept at room temperature. Absorbance was conducted at 593 nm at 0 and 6 min. Ascorbic acid was used as a positive control to generate the standard curve and FRAP value was calculated relevant to the activity of ascorbic acid and expressed as AA equivalent (AAE). Under these
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conditions, ascorbic acid has a relative activity of 2. Each sample was analyzed in triplicate and the FRAP values were expressed as mmol/ 100 g on dry-weight basis. For dose-response experiment, which was performed in duplicate, varying amounts of dried truffle (0.25–2 mg) were extracted and analyzed similarly.
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2.9. Determination of free, bound phenolics and flavonoids
where AC was the absorbance of the control (blank) and AE was the absorbance of the extract. For the calculation of the amount that resulted in 50% inhibition of DPPH quenching, varying amounts of dried truffle (0.25–2 mg) were extracted as described above. Each sample was analyzed in triplicate.
Free, bound phenolics and flavonoids were determined according to Vinson et al. (2001). For free phenolics, 0.5 g dried truffle was extracted with 5 mL of 80% methanol. The suspension was incubated in a water bath at 90 8C for 3 h, centrifuged and the clear extract was collected and used for free phenolics as described below. Bound phenolics were similarly extracted using acidified methanolic solution (1.2 M HCl in 80% methanol). Total flavonoids were estimated after formaldehyde precipitation. Briefly, 2.5 mL of the clear methanolic extract, prepared for free phenolics and was combined with 2.5 mL acidified methanolic solution and 1.25 mL of 0.8% formaldehyde solution. The mixture was left to stand overnight at room temperature and later subjected to centrifugation at 2000 g for 15 min. The clear solution was collected. Flavonoids contents were estimated by the difference between free phenolics and free phenolics after formaldehyde precipitation.1 A modified Folin–Ciocalteu method reported by Waterhouse (2001) was used to estimate phenolics. Briefly, 20 mL of the sample, standard and the blank were added to 1.58 mL H2O, followed by the addition of 100 mL concentrated FC reagent. After 3 min standing at room temperature, 300 mL of sodium carbonate buffer (200 g/L) was added. The mixture was incubated at 40 8C for 30 min. Absorbance was measured at 765 nm. Gallic acid was used as a standard. All phenolics are reported as gallic acid equivalent (GAE)/mg 100 g dw).
2.7. Nitric oxide (NO) radical scavenging activity assay
2.10. Determination of total anthocyanins by pH differential assay
This assay is based on the spontaneous disintegration of sodium nitroprusside (SNP) at physiological pH yielding NO radical which reacts with oxygen to produce nitrite ions that can be measured by Griess reagent (Sreejayan and Rao, 1997). Ground (0.1 g) truffle samples were extracted using 10 mL using 75 mM phosphate buffer (pH 7.5). Extraction was continued for 1 h with occasional vortexing, followed by centrifugation as mentioned above. An aliquot (100 mL) of the clear supernatant was mixed with 400 mL of the same extraction buffer and 500 mL of SNP solution (10 mM final concentration). The reaction mixture was incubated at room temperature for 30 min, followed by the addition of one mL of Griess reagent (prepared by mixing equal volumes of 1% NEDD and 0.1% sulfanilic acid), standing at room temperature for 10 min and reading the absorbance at 542 nm. A standard curve of nitrite was prepared. The NO radical scavenging activity was calculated as follows: AC AE % inhibition ¼ 100 AC
Total anthocyanins were estimated by (Rodriguez-Saona and Wrolstad, 2001). A fine powder of the dried truffle samples (50 mg) was mixed with 4 mL H2O, sonicated in a water bath for 3 min, and kept at room temperature for 15 min with occasional vortexing followed by centrifugation for 10 min at 2000 g. Next, 1 mL of the clear supernatant was mixed with 24 mL of the buffer of pH 1.0 (made of 25 parts of 1.49 M KCl/100 mL and 67 parts of 0.2 N HCl). Additional one mL of the extract was mixed with 24 mL of the sodium acetate buffer of pH 4.5 (1.64 g/100 mL). The absorbance was measured at 510 and 700 nm and used to calculate the absorbance of anthocyanins (Ab) as:
2.6. DPPH radical scavenging activity DPPH radical assay was performed according to BrandWilliams et al. (1995) with slight modifications. Ground truffle samples (0.1 g each) were extracted using 10 mL of 80% methanolic solution. Extraction was continued for 1 h with occasional vortexing, followed by centrifugation as mentioned above. A standard DPPH assay consisted of an addition of 100 mL of the extract to 2.9 mL of 80% methanolic solution containing 56 mg/mL DPPH while mixing and incubating at room temperature for 15 min. Absorbance was measured at 517 nm. Radical scavenging activity is expressed as percent inhibition: AC AE DPPH% inhibition ¼ 100 AC
where AC was the absorbance of the control (blank) and AE was the absorbance of the extract. 2.8. Hydroxyl radical scavenging activity by deoxyribose assay The deoxyribose method employed was essentially as described by Halliwell et al. (1987). Samples were prepared as described for NO radical scavenging activity. An aliquot (100 mL) was added to a reaction mixture prepared by mixing 120 mL of 20 mM deoxyribose, 500 mL of 75 mM phosphate buffer (pH 7.4), 40 mL of 20 mM H2O2 and 40 mL of 500 mM FeSO4. The mixture was incubated at 37 8C for 30 min, then the reaction was stopped by the addition of 0.5 mL of 2.8% TCA and 0.4 mL of 0.6% TBA. The tubes were subsequently incubated in a water bath (100 8C) for 15 min and then cooled to room temperature; the absorbance was measured at 532 nm. The experiment was carried out in triplicate. Hydroxyl radical scavenging activity (% inhibition) was calculated as for NO radical described above.
Ab ¼ ðA510 A700 ÞpH 1 ðA510 A700 ÞpH 4:5 and Total anthocyanins ðmg=100 gÞ ¼
Ab
e
MW
V 100 G
where e is the molar extinction coefficient taken as 26,900, MW is the molecular weight of a typical anthocyanin taken as 449.2, DF is the dilution factor, V is the final volume and G is the sample weight in grams. Each sample was analyzed in triplicate. 2.11. Estimation of total carotenoids Total carotenoids were estimated according to Lichtenthaler and Wellburn (1985). A fine powder of dried truffles (100 mg) was extracted for 2 min using 3 mL acetone:ethanol (1:1) containing 20 mg BHT per 100 mL in a capped tube protected from light with aluminum foil. The clear extract was aspirated and the truffle residue was further re-extracted twice. The combined extract was brought to 10 mL, centrifuged at 2000 g for 10 min and the
1 The total free phenolic content was first estimated (A), then the same clear solution was treated with formaldehyde to precipitate flavonoids and the remaining phenolic content was again estimated (B). B was subtracted from A to give total flavonoids content.
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absorbance was measured at 470 nm. Total carotenoids were estimated using the following formula: Total carotenoids ðmg=100 gÞ ¼
Ab final volume 106 A1% 100 G
tested dried truffle obtained from Bahrain, Iran, Morocco and Saudi Arabia. 3.3. Ascorbic acid and total carotenoids
where A1% equals 2500, and G is sample weight in g. Samples were analyzed in triplicate. 2.12. Determination of ascorbic acid using 2,4dinitrophenylhydrazine (DNPH) Ascorbic acid content was determined according to Roe and Osterling (1943). A fine powder of dried truffle samples (100 mg) was extracted 3 times with meta-phosphoric acid (4.5%), each 2.5 mL, using vigorous vortexing. The three extracts were combined, centrifuged at 2000 g for 10 min. Then 1 ml of the clear supernatant was taken and 0.2 mL TCA 2.8% was added to precipitate proteins; it was kept in ice for 5 min and then centrifuged at 12,000 rpm for 15 min. 0.2 mL of the de-proteinated extract was mixed with 0.8 mL of 0.2 M acetic acid and 0.25 of 2% DNPH reagent (in 9 N H2SO4), incubated at 60 8 C for 60 min and transferred to an ice bath. After 5 min, 1.25 mL of 85% H2SO4 was added and kept at room temperature for 30 min. Absorbance was measured at 540 nm. The pure ascorbic acid was treated similarly and used as a standard. All samples were analyzed in triplicate. 2.13. Statistical analysis Excel (Microsoft Corporation, USA) and SPSS (version 12.1, SPSS, Chicago, IL, USA) were used for statistical analysis. Data are expressed as means SD. One-way analysis of variance was used for source (origin). Statistical significance between means was determined using Duncan’s multiple range test and set as p < 0.05. Pearson’s correlation coefficient (r) was used to evaluate the association between variables. 3. Results and discussion 3.1. Moisture content The average moisture content of fresh Bahraini, Iranian, Moroccan and Saudi truffles were 77.4 0.77, 75.0 0.26, 75.1 2.02 and 78.6 0.65%, respectively, with an overall average of 76.4 1.53%. Content values were not significantly different (p = 0.052). The moisture content values reported here are in close agreement with those reported by other researchers (Bokhary and Parvez, 1993). However, moisture content as high as 93% was reported for some American edible mushrooms (Dubost et al., 2007). 3.2. Antioxidant components Shown in Table 1 is the concentration of the components contributing towards the antioxidant/antiradical activities in the
Ascorbic acid content ranged between 5.9 and 11.4 mg/100 g, with an average of 9.6 0.15 mg/100 g. Truffles from Bahrain and Iran were not significantly different (p > 0.05) in their ascorbic acid content, but were both significantly different from the other two sources. Bahraini dried truffles had the highest level of ascorbic acid, followed by Iranian, Saudi and Moroccan truffles, in that order. The Moroccan truffles possessed only about 50% of ascorbic acid content compared to the others. Shamekh et al. (1985) reported a value of ascorbic acid content of Libyan dehydrated truffle (Terfezia boudieri) similar to those of Bahraini, Iranian and Saudi truffles, whereas Sawaya et al. (1985) reported values ranging from 0.71 to 5 mg/100 g for various Saudi fresh truffles. Barros et al. (2007) reported that wild edible Portuguese mushrooms contained between 13 and 35 mg/ 100 g dw of ascorbic acid, a value close to the ones reported in this study. Total carotenoids ranged between 405 and 1051 mg/100 g and averaged 681 245 mg/100 g. Bahraini and Moroccan truffles were not significantly different. These truffles significantly differed from the Iranian and Saudi truffles (p < 0.05). The Iranian truffles possessed the highest total carotenoids, which amounted to twice as much as what was found in Bahraini and Saudi truffles. Elmastas et al. (2007) reported that the average content of b-carotene of several edible wild mushrooms ranged from 700 to 3600 mg/100 g, whereas Barros et al. (2007) reported a range from 188 to 297 mg/ 100 g. 3.4. Phenolics Concentration of total free and esterified phenolics, flavonoids and anthocyanins of dried truffles from the four origins are also presented in Table 1. The overall mean of esterified phenolics, free- and non-flavonoid phenolics were 1860 361, 1328 167 and 1034 124 mg/100 g, respectively, whereas the average flavonoids content was 293 (32) mg/100 g. Saudi and Bahraini truffles showed the highest concentration of total free phenolics and were statistically similar. The Moroccan truffles possessed the lowest contents of all investigated phenolics and were significantly different from others. Values of total phenolics reported here are within the range reported in wild mushrooms by other investigators. Total polyphenols of several Turkish edible wild mushrooms ranged between 800 and 2600 mg/100 g (Elmastas et al. (2007). Dubost et al. (2007) reported total phenolic values ranging between 400 and 1065 mg/100 g. The highest amount of anthocyanins were found in Saudi truffles (23.6 mg/100 g) followed by Moroccan (15.1 mg/100 g), whereas the Bahraini and Iranian truffles possessed the lowest, 4.7 and 4.5 mg/100 g, respectively (Table 1). Truffles from the former two sources were not significantly different. Saudi truffles
Table 1 Mean (SD) of ascorbic acid, total carotenoids, anthocyanins, total esterified and free phenolics, total flavonoids and non-flavonoids of dried desert truffles of different origins. Origin
Ascorbic acid (mg/100 g)
Total carotenoids (mg/100 g)
Total anthocyanins (mg/100 g)
Total esterified phenolics (mg/100 g)
Total free phenolics (mg/100 g)
Total flavonoids (mg/100 g)
Non-flavonoids (mg/100 g)
Bahraini Iranian Moroccan Saudi
11.4 10.9 5.9 10.3
594 1051 675 405
4.7 4.5 15.1 23.6
2206 1600 1445 2188
1418 1328 1083 1484
290 326 257 306
1128 1002 826 1178
(0.50) (0.26) (0.25) (0.50)
a a b c
(35) a (127) b (21) a (42) c
(0.18) (0.23) (0.14) (1.52)
a a b c
(68) (82) (28) (52)
a b c a
(51) (37) (85) (81)
a,b a c b
(20) a (10) a (6) b (29) a
(42) a 36) b (26) b (49) a
Data are expressed as a dry-weight-basis and presented as means (SD) (n = 3). All values of phenolic compounds, except for anthocyanins, are expressed as mg gallic acid equivalent (GAE)/100 g. Non-flavonoids were calculated by difference between total free phenolics and total flavonoids. Mean values within each column followed by different letter differ significantly at p < 0.05.
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Table 2 DPPH% inhibition (SD), EC50 and antiradical efficiency of dried desert truffle of various origins. Origin
Bahraini Iranian Morocco Saudi
(% inhibition)
52.5 69.2 24.5 51.0
(2.5) (0.9) (2.8) (3.8)
a b c a
DPPH radical assay EC50a
AEb
0.5 0.3 1.1 0.5
2 3.33 0.91 2
a EC50 is the amount of truffle (mg/assay; 3 mL) required to decrease the initial concentration by 50%. b AE: antiradical efficiency and calculated as AE = 1/EC. The EC50 for the positive control ascorbic acid was 7.2 (0.06) mg/mL. Values within the same column followed by the same letter are not significantly different (p > 0.05).
contained 5 times the amount of anthocyanins found in Bahraini and Iranian truffles, but only 1.6 times of the Moroccan truffles. Seasonal variations, geographical origins and climatic conditions all have been reported to influence the antioxidant constituents of plant foods and natural products (Alasalvar et al., 2005; Dragland et al., 2003; Gil et al., 2002; Ou et al., 2002). Thus, it is expected that truffles originated from different geographical origins would also show some variations. Furthermore, it is also expected that the antioxidant attributes of truffles may be affected by the nature and the extent of association with its host root associate Helianthemum spp. 3.5. Free radical activity using DPPH Percent inhibition of DPPH quenching of the dried truffles ranged between 24.5 and 69.2% with an average of 30.6 13% (Table 2). Iranian truffles, which possessed the highest percent inhibition, were significantly different from others (p = 0.001). The dose-dependent percent inhibition of DPPH quenching of the truffles from the four origins is shown in Fig. 1. The pattern of DPPH inhibition was the same for all. However, the rate of inhibition varied and depended on origin. Percent of inhibition linearly increased at low concentrations, leveled out and then reversed at higher concentrations. DPPH was found to give a reversible reaction with some phenolics bearing o-methoxyphenol structure, which may lead to a false low reading of antioxidant capacity (Bondet et al., 1997). As shown in Fig. 1, the Iranian truffles showed the highest DPPH percent inhibition at all concentrations, whereas the Moroccan truffles possessed the lowest activity at all levels tested. Bahraini and Saudi truffles were similar in their antiradical activity, which was intermediate (Fig. 2). Under experimental conditions employed, 85–90% inhibition could be achieved at 0.9–1.0 mg/ assay with Bahraini, Iranian and Saudi truffles, whereas the Moroccan truffles exhibited only 70% inhibition at such a level.
Fig. 1. Percent inhibition of hydroxyl radical activity by dried truffle of various origins as measured by DPPH quenching assay.
Fig. 2. Antioxidant activity of different concentrations of dried truffle measured by FRAP assay and expressed as mM.
The EC50 of DPPH quenching calculated from these data were 0.3, 0.5, and 1.1 mg for Iranian, Bahraini = Saudi and Moroccan truffles, respectively. The EC50 for the positive control ascorbic acid was much lower (7.2 0.06 mg/mL). DPPH reacts with different antioxidants at different molar ratios (Brand-Williams et al., 1995). More quenching of DPPH, in molar basis, takes place with phenolics and flavonoids possessing hydroxyl groups in ortho positions in the aromatic rings or having higher numbers of hydroxyl groups. As a natural product, variation in chemical composition of truffles due to geographical and climatic variables is expected (Hussain and Al-Ruqaie, 1999). Such variation may be responsible for both the differences between truffle sources in antioxidant activity, as well as the different reaction behavior found in DPPH and FRAP assays. A strong correlation was found between the DPPP percent inhibition and flavonoids content of the truffle (r = 0.966, p = 0.000), but not with total and free phenolics. Similar degrees of correlation between DPPP% inhibition and flavonoids content in plant materials and model systems (Tsimogiannis and Oreopolou, 2004; Khlebnikova et al., 2007) have been previously reported. The antioxidant and antiradical properties of flavonoids are attributed to their ability to act as transient metals chelaters, radical scavengers, and their involvement in electron and hydrogen atom transfer. 3.6. Antioxidant activity by ferric reducing antioxidant power (FRAP) FRAP values as a measure of the antioxidant activity is shown in Table 3. Bahraini and Saudi truffles possessed the highest FRAP values (18.62 and 18.06 mmol/100 g, respectively), but were not significantly different. The Iranian truffles showed the lowest FRAP values (10.43 mmol/100 g), whereas the Moroccan truffles were 418 intermediate in FRAP values (14.62 mmol/100 g). Recently, Halverson et al. (2006) published a list of the antioxidant content of various foods commonly found in the American diet using the FRAP assay. They found that the higher antioxidant content was associated with plant food and the FRAP values of the 50 food commodities, with the highest antioxidant content ranging between 1.01 and 124.55 mmol/100 g. Thus, according to such criteria, dried truffles from different origins may be considered among those edible foods with high antioxidant activity. Though the effect of dehydration on antioxidant/antiradical activity of desert truffles was not thoroughly investigated, preliminary results indicate that the process of dehydration apparently did not adversely affect the antioxidant activity as measured by the FRAP assay; i.e. the average FRAP values corrected
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Table 3 FRAP values, nitric oxide (NO) radical effective concentration (EC50), and scavenging activity of hydroxyl radicals of dried desert truffle of various origins. Origin
Antioxidant activity (FRAP values) (mmol/100 g)
Bahraini Iranian Morocco Saudi
18.62 10.34 14.62 18.06
(0.50) (0.84) (0.54) (1.07)
ac b c a
Nitric oxide radical (NO) EC50 (mg/mL)a
Hydroxyl radical scavenging activity (% inhibition)b
115 102 250 170
65.7 91.0 11.3 55.7
(4.0) a (3.5) a (11.5) b (8.0) c
(2.5) (2.0) (2.5) (1.5)
a b c d
a EC50 (mg/mL) is the effective concentration at which 50% of the NO radical are inhibited. The EC50 for the positive control ascorbic acid was 15.8 (0.25) mg/mL. Mean values within each column followed by different letter differ significantly at p > 0.05. b Percent inhibition of hydroxyl radical activity (deoxyribose assay) is for 2.5 mg of dried truffles per assay. c Values within the same column followed by the same letter are not significantly different (p < 0.05).
for the moisture content of fresh truffle of Bahraini, Iranian, and Saudi origins were 13.05 (0.55), 10.85 (0.71), and 14.58 (0.30) mmol/100 g, respectively. On the other hand, dried truffles from Bahrain possessed significantly higher antioxidant activity than the fresh truffles (p = 0.0003). Halverson et al. (2006) reported that several processes, such as microwave cooking, steaming or boiling, roasting and baking resulted in increasing the antioxidant content (FRAP values) of many foods of plant origins. When samples of dried truffles from the four origins were tested by FRAP assay in the range of 0–2 mg/assay, they all showed a similar pattern (Fig. 2). Clearly, the FRAP values increased nonlinearly in a dose-dependent relationship. The rate of increase, however, differed. At relatively high amount of truffles (between 1 and 2 mg) the ranking order of antioxidant activity was as follows: Bahraini > Moroccan > Saudi > Iranian. Bahraini truffles were about 1.8 times higher in antioxidant activity as compared with the Iranian truffles. At such a high level (i.e. 2 mg), a significant difference between the four sources was observed (p = 0.0034). No significant difference was found between Bahraini, Moroccan and Saudi truffles at relatively lower amount (i.e. 0.25 mg/assay). Several investigators have reported a linear relationship between total phenolics and FRAP values (Cai et al., 2004). However, other studies (Wu et al., 2004; Dubost et al., 2007) reported a nonlinear relationship with strong correlation, which may indicate that phenolics accept electrons at a greater rate as the total free phenolics increase. The chemistry of antioxidant capacity assays has recently been evaluated (Huang et al., 2005). FRAP is an electron-transferred-based assay, and uses Fe (III)(TPTZ) as an oxidant. However, it is subjected to interference by many metal chelators present in food capable of binding with to Fe(III), and forming complexes that could react with antioxidants (Huang et al., 2005). In our study, a strong correlation was found between FRAP values measured at a fixed concentration of dried truffle (2.5 mg/assay), total (esterified) (r = 0.801, p = 0.001) and free phenolics (r = 0.973, p = 0.000), but not with flavonoids (p = 0.287). 3.7. Nitric oxide radical scavenging activity Extracts of dried truffles exerted inhibition activity against the production of nitric oxide (NO) radical from its artificial source sodium nitroprusside (Table 3). Dried truffles from all the tested sources were indifferent (p > 0.05) and possessed relatively high NO antiradical activity (>93%), which hampered the direct comparison of NO percent inhibition between dried truffles of different origins. The scavenging of NO radicals of dried truffle (25–500 mg/mL) was concentration-dependent, exhibiting a hyperbolic manner for
Fig. 3. Percent inhibition of nitric oxide (NO) radical production by dried truffle of various origins as measured by deoxyribose assay.
all tested samples (Fig. 3). At 500 mg/mL the maximum inhibition of NO radical ranged between 68 and 88%. The effective concentration at which 50% of the NO radical are inhibited (EC50 in mg/mL is presented in Table 3. Iranian truffles possessed the strongest EC50 (102 mg/mL) and was not statistically different from the EC50 of the Bahraini truffles. The Moroccan truffles possessed the weakest EC50 (250 mg/mL). All the tested truffles exhibited low scavenging activity compared to the EC50 of the positive control, ascorbic acid, which measured 15.8 0.25 mg/mL. It is known that the mechanism of inhibition of the production of the short-lived NO radical from SNP involves either direct scavenging of NO by the antiradical or via the scavenging of the oxide (oxygen), which reacts with the formed NO radical leading to the formation the nitrite ions (Janzen et al., 1993). Phenolics, particularly flavonoids, have been suggested to scavenge NO either by H-atom abstraction or reduction of NO by single electron transfer (Janzen et al., 1993). Inhibition of NO production by plant polyphenols is well-documented (Van Acker et al., 1995; Klotz and Sies, 2003). Potency of NO radical inhibition has been correlated with the number of hydroxyl residues of polyphenolics, including flavonoids. In this study, a good correlation between percent inhibition of NO production and flavonoids content was observed (r = 0.735, p = 0.024), but not with total and free phenolics. Some other studies reported that phenolic groups were not essential for NO scavenging activity (Sreejayan and Rao, 1997). 3.8. Hydroxyl radical activity using deoxyribose assay Hydroxyl radicals degrade the sugar deoxyribose (2-deoxy-Dribose) via Fenton-type reactions leading to the production of complex products which can be estimated as malonaldehyde (MDA). The presence of an antiradical prevents or decreases MDA production. Extract from all tested dried truffles exhibited antiradical activity and decreased the degradation of deoxyribose as shown in Table 3. This activity was significantly different for all sources (p = 0.003). The Iranian truffles exhibited the highest percent of inhibition of deoxyribose breakage (91%), whereas the Moroccan dried truffles exhibited 501, considerably very low antiradical activity using the deoxyribose assay (11.3%). The total flavonoids and percent of inhibition of deoxyribose degradation of the studied truffles were highly correlated (r = 0.939, p = 0.000). However, no correlation was found with total and free phenolics. According to Eberhardt (2001), the deoxyribose assay does not measure hydroxyl radicals only, but other species which are kinetically indistinguishable from hydroxyl radicals, too.
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4. Conclusions The extract of dried desert truffles (Tirmania nivea) from four different Middle Eastern countries showed varying degree of antioxidant and antiradical activities based on four analytical methods. It appears that truffles from a single tested source did not yield the highest values either in all assays used, or in the contents of the relevant antioxidant constituents. However, it is evident that the dried Iranian truffles scored highest in most parameters tested. Furthermore, the high correlation observed between the various assays employed and phenolic contents is a strong indication that these phenolics (total, free and flavonoids) are among the predominant source of antioxidant activity in desert truffles. Acknowledgments This work was financially supported by the University of Bahrain. The author thanks Professor Ahmed Y. Ali and Dr. Q. Mandeel for their critical reading of the manuscript. I am most grateful to Dr. Mandeel for confirming the authenticity of the tested truffle. I also thank Mrs. Naima Fakhroo for providing the Iranian truffles. References Alasalvar, C., Al-Farsi, M., Quantick, P.C., Shahidi, F., Wiktorowicz, R., 2005. Effect of chill and modified atmosphere packaging (MAP) on antioxidant activity, anthocyanins, carotenoids, phenolics and sensory quality of ready-to-eat shredded orange and purple carrots. Food Chemistry 89, 69–76. Al-Naama, N.M., Ewaze, J.O., Nema, J.H., 1988. Chemical constituents of Iraqi truffles. Iraqi Journal of Agricultural Sciences 6, 51–56. Al-Rahmah, A.N., 2001. Truffle of Deserts and Jungles. King Saud University Publications, Riyadh, Saudi Arabia, p. 272 (in Arabic). Al-Ruqaie, I.M., 2002. Effect of different treatment processes and preservation methods on the quality of truffles. I. Conventional methods (drying/freezing). Pakistan Journal of Biological Sciences 5, 1088–1093. Al-Sheik, A.M., Trappe, J.M., 1983. Desert truffles: the genus Tirmania. Transactions of the British Mycological Society 81, 83–90. Amicarelli, F., Bonfigli, A., Colafafarina, S., Cimini, A.M., Prutti, B., Cessare, P., Ceru, M.P., Di Ilio, C., Pacioni, G., Miranda, M., 1999. Glutathione dependent enzymes and antioxidant defences in truffles: organisms living in microaerobic environments. Mycological Research 103, 1643–1648. Barros, L., Ferreira, M.-J., Queiro´s, B., Ferreira, I.C.F.R., Baptista, P., 2007. Total phenols, ascorbic acid, b-carotene and lycopene in Portuguese wild edible mushrooms and their antioxidant activities. Food Chemistry 103, 413–419. Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of ‘‘antioxidant power’’: the FRAP assay. Analytical Biochemistry 239, 70–76. Bokhary, H.A., Parvez, S., 1993. Chemical composition of desert truffles Terfezia claveryi. Journal of Food Composition and Analysis 6, 285–293. Bokhary, H.A., Suleiman, A.A., Basalah, M.O., 1989. The fatty acid components of the desert truffle ‘‘AlKamah’’ of Saudi Arabia. Journal of Food Protection 52, 668– 669. Bokhary, H.A., Suleiman, A.A., Basalah, M.O., Parvez, S., 1987. Chemical composition of desert truffles from Saudi Arabia. Canadian Institute of Food Technology Journal 20, 336–341. Bondet, V., Brand-Williams, W., Berset, C., 1997. Kinetics and mechanisms of antioxidant activity using DPPH free radical method. Food Science and Technology-Leb 30 (6), 609–615. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of free radical method to evaluate antioxidant activity. Lebensmittel Wissenschaft und Technologie (Food Science and Technology) 28, 25–30. Cai, Y., Luo, Q., Sun, M., Corke, H., 2004. Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sciences 74, 2157–2184. Dragland, S., Senoo, H., Waka, K., Holte, K., Blomhoff, R., 2003. Several cultinary and medicinal herbs are important sources of dietary antioxidant. Journal of Nutrition 133, 1286–1290. Dubost, N.J., Ou, B., Beelman, R.B., 2007. Quantification of polyphenols and ergothioneine in cultivated mushroom and correlation to total antioxidant capacity. Food Chemistry 105, 727–735. Eberhardt, M.K., 2001. Reactive oxygen metabolites: chemistry and medical consequences. CRC Press, pp. 70–71. Elmastas, M., Isildak, O., Turkekul, I., Temur, N., 2007. Determination of antioxidant activity and antioxidant compounds in wild edible mushrooms. Journal of Food Composition and Analysis 20, 337–345.
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Halverson, B.L., Carlsen, M.H., Phillips, K.M., Bøhn, S.K., Holte, K., Jacobs Jr., D.R., Blomhoff, R., 2006. Content of redox-active compounds (i.e. antioxidants) in foods consumed in the United States. American Journal of Clinical Nutrition 84, 95–135. Hannan, M.A., Al-Dakan, A.A., Aboul-Enein, H.Y., Al-Othaimeen, A.A., 1989. Mutagenic and antimutagenic factor(s) extracted from desert mushroom using different solvents. Mutagenesis 4, 111–114. Huang, D., Ou, B., Prior, R.L., 2005. The chemistry behind antioxidant capacity assays. Journal of Agricultural & Food Chemistry 53, 1841–1856. Hussain, G., Al-Ruqaie, I.M., 1999. Occurrence, chemical composition, and nutritional value of truffle: an overview. Pakistan Journal of Biological Sciences 2, 510–514. Janakat, S., Al-Fakhiri, S., Sallal, A.K., 2004. A promising peptide antibiotic from Terfezia claveryi aqueous extract against Staphylococcus aureus in vitro. Phototherapy Research 18, 810–813. 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Journal of Food Composition and Analysis journal homepage: www.elsevier.com/locate/jfca
Original Article
Antioxidant components and antioxidant/antiradical activities of desert truffle (Tirmania nivea) from various Middle Eastern origins Abdul Ameer Ahmed Al-Laith * Department of Biology, College of Science, University of Bahrain, P.O. Box 32038, Sakhir Campus, Building S41, Sakhir, Bahrain
A R T I C L E I N F O
A B S T R A C T
Article history: Received 10 November 2008 Received in revised form 2 July 2009 Accepted 6 July 2009
Dried desert truffles from Bahraini, Iranian, Moroccan and Saudi origins were examined for their antioxidant and antiradical activities using four analytical methods: ferric reducing ability (FRAP), DPPH, deoxyribose, and nitric oxide (NO). Chemical constituents contributing towards these activities were also investigated. Generally, these truffles possessed varying concentration of antioxidant chemicals averaging 9.6 0.15, 12.0 8.34, 1860 361, 1328 167 and 293 32 mg/100 g dw, for ascorbic, anthocyanins, total esterified phenolics, total free phenolics and total flavonoids, respectively; total carotenoids averaged 681 245 g/100 g dw. Dried truffles also varied with regard to their antioxidant and antiradical activities. The FRAP value averaged 15.41 3.51 mmol/100 g dw. Antiradical activity measured as percent inhibition of DPPH quenching averaged 30.6 12.97% and EC50 of 0.55 0.38 mg. The average EC50 of NO scavenging activity was 159.4 69.3 mg, whereas the average percent inhibition of deoxyribose degradation was 55.9 30.1%. The Iranian truffles yielded the highest in several variables, whereas the Moroccan truffles possessed the lowest values of many variables among the four tested samples. Significant correlation was established between total and free phenolics and FRAP values, and between flavonoids and percent radical inhibition using DPPH, NO and deoxyribose assays. ß 2009 Elsevier Inc. All rights reserved.
Keywords: Antioxidant activity Antiradical activity DPPH Flavonoids FRA Phenolics Truffle Fungi Wild food Middle East Bahrain Traditional food Food composition Food analysis
1. Introduction Desert truffles are a type of an obligate hypogeous ascomycetes ectomycorrhizal fungi formed in association with host roots of Helianthemum spp. and the soil inhabiting fungi Terfezia or Tirmania spp. (Mandeel and Al-Laith, 2007). The type of mycorrhizal fungus ramifies through the soil, absorbing nitrogen and other minerals, which is transported back to the host plant (Ewaze and Al-Naama, 1989). Desert truffles are seasonal and socio-economically important fungi. These truffles are edible and grow wild in the central-southern part of Bahrain. The truffles usually appear in the deserts following the rainy season between February and April in Bahrain as well as in many Gulf states (Al-Ruqaie, 2002; Moubasher, 1993). They are distributed naturally from North Africa (Morocco, Tunisia, Algeria and Egypt) to the Middle East (Saudi Arabia, Kuwait, Iraq, Iran, Lebanon, Syria and Jordon). The people in the Middle East region are
* Tel.: +973 17 437477; fax: +973 17 449158. E-mail addresses: [email protected], [email protected]. 0889-1575/$ – see front matter ß 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jfca.2009.07.005
considered the largest truffle consumers (Al-Rahmah, 2001), and the truffle commodity is regarded as a costly delicacy. In these countries, no data is available on total production and consumption. Among the various known edible desert truffle varieties, only two species of the dark brown color truffles belonging to the genus Terfezia, locally called Ikhlasi (Terfezia claveryi and Terfezia boudieri) and one species of the white color truffles belonging to the genus Tirmania, locally called Zubaidi (Tirmania nivea) are found on the Arabian Peninsula (Al-Rahmah, 2001). Ikhlasi is ovoid with a black skin and a pinkish-ivory interior with a nut-like flavor. Zubaidi is cream-colored with a more delicate flavor and is usually more expensive. The truffles of the desert are not so strongly flavoured, compared with the European truffles (Al-Sheik and Trappe, 1983). Nevertheless, the truffle is a type of tuber highly prized for its unique musky aroma and flavour (Omer et al., 1994). The popularity of truffles is believed to be due to their nutritional value and delicious taste. Wild edible fungi not only add flavor to bland staple foods but they are also valuable foods in their own right. As such, desert truffles are a rich source of protein, amino acids, fatty acids, minerals and carbohydrates (Al-Naama et al., 1988; Bokhary et al., 1987, 1989).
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Similar to other fungi, desert truffles comprise a vast and yet largely unexploited source of new pharmaceutical products. In search for new therapeutic alternatives, and most importantly for modern medicine, truffles represent an unlimited source of therapeutic compounds with anti-inflammatory, immunosuppressor, antimutagenic and anticarcinogenic properties (Hannan et al., 1989), antioxidant properties (Murcia et al., 2002), antimicrobial (Janakat et al., 2004, 2005) and steroidal glucoside with polyhydroxy ergosterol nucleus (tuberoside) (Gao et al., 2001). Specific and peculiar attributes of desert truffles like hypogeous, mycorrhizal and microaerophilic make their metabolism interesting model to study, especially, under stressful desert arid environment conditions. Truffles that live under microaerobic habitats are expected to show morphological, physiological and biochemical adaptations to an environment poor in oxygen as found under hypogeous conditions. As mycorrhizal symbiotic fungi of several species of plants, they exchange metabolites, mineral salts and ions. These adaptations are largely controlled by several environmental factors like the rainy season with its amount and timing, soil types and characteristics, water availability and climatic conditions (Bokhary et al., 1987; Bokhary and Parvez, 1993). Very little information is available relating to truffle metabolism and, in particular, oxidative metabolism (Pacioni et al., 1995). Oxidation is necessary to many organisms for the production of energy to support metabolism of aerobic cell. However, in some instances, the production of oxygen through uncontrolled metabolic pathways results in free radicals. This imbalance can result in damage to molecules, including lipids, DNA, carbohydrates and proteins (Dubost et al., 2007). Moreover, these free radicals are potentially involved in the onset of many diseases such as cancer, rheumatoid arthritis and atherosclerosis as well as in degenerative processes associated with aging (Halliwell and Gutteridge, 1984). Exogenous chemical and endogenous metabolic processes in the human body or in the food system might produce highly reactive free radicals, especially reactive oxygen species (ROS) capable of oxidizing biomolecules, resulting in cell death and tissue damage (Halliwell and Gutteridge, 2003). On a cellular basis, damage provoked by these free radical formations is usually protected by oxidative enzymes as well as compounds such ascorbic acid, tocopherols and phenolics. When the mechanism of antioxidant protection becomes unbalanced by factors such as aging, deterioration of physiological functions may occur resulting in diseases and accelerated aging. However, the antioxidant components present in truffles are of great interest as possible protective agents to help reduce oxidative damage. Previous reports had shown that the 120 expressions of some antioxidant enzymes were correlated with the microaerobic metabolism, growth rate and mycorrhizal symbiosis of truffles (Amicarelli et al., 1999). Wild and cultivated mushrooms are well known to contain various polyphenolic compounds which are recognized as an excellent antioxidant due to their ability to scavenge free radicals by acting as reducing agents, hydrogen donating antioxidants and singlet oxygen quenchers (Barros et al., 2007; Dubost et al., 2007; Elmastas et al., 2007). Although some common edible mushrooms have been found to possess antioxidant activity which is well correlated with their total phenolic contents (Lo and Cheung, 2005), data about the antioxidant/antiradical activity and the antioxidant compounds of desert truffles are lacking. Thus, there is an increasing demand to assess these attributes on truffles and other wild fungi. The importance of truffles as traditional food and medicine was recently surveyed by Mandeel and Al-Laith (2007). The aim of this study was to investigate the antioxidant and antiradical activities as well as the chemical components with antioxidant/antiradical properties of the desert truffle Tirmania nivea obtained from four Middle Eastern countries, namely Bahrain, Iran, Moroccan and Saudi Arabia.
2. Materials and methods 2.1. Chemicals L-Ascorbic acid, deoxyribose, 2,4-dinitrophenylhydrazine (DNPH) and sodium nitroprusside (SNP), were obtained from BDH (England, UK). 2,4,6-Tripyridyl-s-triazine (TPTZ), gallic acid, N-(1-naphthyl)ethylenediamine di-hydrochloride (NEDD) and sulfanilic acid 2-thiobarbituric acid were from Fluka Chemicals AG (Deisenhofen, Switzerland). 2,2-Diphenyl-1-picrylhydrazyl (DPPH), Folin–Ciocalteu (FC) reagent were from Sigma (St. Louis, MO, USA). All other chemicals were of analytical grade.
2.2. Equipment All spectrophotometric measurements were performed using Spectronic Genesys 20 (Spectronic Genesys, USA). Beckman benchtype and Beckman high speed equipped with J-21 rotor (Beckman, USA) were used for centrifugation. Air-circulated oven (LKB, UK) was used for drying samples. 2.3. Desert truffle source and description Samples of Bahraini desert truffles were a kind gift collected from the southern area of Bahrain during the month of April 2007. Iranian truffles were also a kind gift collected from the western part of Iran. Moroccan and Saudi truffles were commercially imported to Bahrain and purchased from local markets during May 2007. When received, all truffle samples were unwashed, unpacked, and not stored at room temperature. They were intact and showed no sign of spoilage. The Bahraini, Moroccan and Saudi samples were of similar irregular spherical shape and size with an average weight of 33.0, 30.4 and 30.3 g, respectively, whereas the Iranian samples were larger with an average weight of 64.3 g. The color of flesh of first three was similar, dark creamy, whereas the flesh color of the Iranian was light creamy. Superficially, the Bahraini, Moroccan and Saudi samples cannot be distinguished from each other. All truffle samples were identified as white variety Zubaidi (Tirmania nivea). They were characterized by earthy musky aroma and light flavor. 2.4. Truffle preparation A total of 3–5 ascocarps (tubers) composite samples of each truffle source were cleaned from soil, peeled, sliced and dried at 65 8C in an air-circulated oven until constant weight (3 days). Dried slices were subjected to milling for 2 min using Micro-mill (TechniLab Instruments, NJ, USA). The resulting fine pulverized powder was capped and stored at room temperature (23 2 8C) in the dark. Moisture content was performed in duplicate. 2.5. Antioxidant activity by ferric reducing ability (FRAP) assay For the ferric reducing ability (FRAP) assay, one gram of fine truffle powder was extracted with 10 mL of phosphate buffer (75 mM, pH 7.4) for 30 min with occasional shaking. The suspension was centrifuged at 2000 g for 15 min. The clear extract was used to measure the antioxidant activity by FRAP assay according to Benzie and Strain (1996). The reaction mixture was freshly prepared by mixing acetate buffer (300 mM, pH 3.6), FeCl36H2O (20 mM) and TPTZ (10 mM) in a ratio of 10:1:1. The clear extract (0.1 mL) was added to 3 mL reaction mixture, mixed and kept at room temperature. Absorbance was conducted at 593 nm at 0 and 6 min. Ascorbic acid was used as a positive control to generate the standard curve and FRAP value was calculated relevant to the activity of ascorbic acid and expressed as AA equivalent (AAE). Under these
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conditions, ascorbic acid has a relative activity of 2. Each sample was analyzed in triplicate and the FRAP values were expressed as mmol/ 100 g on dry-weight basis. For dose-response experiment, which was performed in duplicate, varying amounts of dried truffle (0.25–2 mg) were extracted and analyzed similarly.
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2.9. Determination of free, bound phenolics and flavonoids
where AC was the absorbance of the control (blank) and AE was the absorbance of the extract. For the calculation of the amount that resulted in 50% inhibition of DPPH quenching, varying amounts of dried truffle (0.25–2 mg) were extracted as described above. Each sample was analyzed in triplicate.
Free, bound phenolics and flavonoids were determined according to Vinson et al. (2001). For free phenolics, 0.5 g dried truffle was extracted with 5 mL of 80% methanol. The suspension was incubated in a water bath at 90 8C for 3 h, centrifuged and the clear extract was collected and used for free phenolics as described below. Bound phenolics were similarly extracted using acidified methanolic solution (1.2 M HCl in 80% methanol). Total flavonoids were estimated after formaldehyde precipitation. Briefly, 2.5 mL of the clear methanolic extract, prepared for free phenolics and was combined with 2.5 mL acidified methanolic solution and 1.25 mL of 0.8% formaldehyde solution. The mixture was left to stand overnight at room temperature and later subjected to centrifugation at 2000 g for 15 min. The clear solution was collected. Flavonoids contents were estimated by the difference between free phenolics and free phenolics after formaldehyde precipitation.1 A modified Folin–Ciocalteu method reported by Waterhouse (2001) was used to estimate phenolics. Briefly, 20 mL of the sample, standard and the blank were added to 1.58 mL H2O, followed by the addition of 100 mL concentrated FC reagent. After 3 min standing at room temperature, 300 mL of sodium carbonate buffer (200 g/L) was added. The mixture was incubated at 40 8C for 30 min. Absorbance was measured at 765 nm. Gallic acid was used as a standard. All phenolics are reported as gallic acid equivalent (GAE)/mg 100 g dw).
2.7. Nitric oxide (NO) radical scavenging activity assay
2.10. Determination of total anthocyanins by pH differential assay
This assay is based on the spontaneous disintegration of sodium nitroprusside (SNP) at physiological pH yielding NO radical which reacts with oxygen to produce nitrite ions that can be measured by Griess reagent (Sreejayan and Rao, 1997). Ground (0.1 g) truffle samples were extracted using 10 mL using 75 mM phosphate buffer (pH 7.5). Extraction was continued for 1 h with occasional vortexing, followed by centrifugation as mentioned above. An aliquot (100 mL) of the clear supernatant was mixed with 400 mL of the same extraction buffer and 500 mL of SNP solution (10 mM final concentration). The reaction mixture was incubated at room temperature for 30 min, followed by the addition of one mL of Griess reagent (prepared by mixing equal volumes of 1% NEDD and 0.1% sulfanilic acid), standing at room temperature for 10 min and reading the absorbance at 542 nm. A standard curve of nitrite was prepared. The NO radical scavenging activity was calculated as follows: AC AE % inhibition ¼ 100 AC
Total anthocyanins were estimated by (Rodriguez-Saona and Wrolstad, 2001). A fine powder of the dried truffle samples (50 mg) was mixed with 4 mL H2O, sonicated in a water bath for 3 min, and kept at room temperature for 15 min with occasional vortexing followed by centrifugation for 10 min at 2000 g. Next, 1 mL of the clear supernatant was mixed with 24 mL of the buffer of pH 1.0 (made of 25 parts of 1.49 M KCl/100 mL and 67 parts of 0.2 N HCl). Additional one mL of the extract was mixed with 24 mL of the sodium acetate buffer of pH 4.5 (1.64 g/100 mL). The absorbance was measured at 510 and 700 nm and used to calculate the absorbance of anthocyanins (Ab) as:
2.6. DPPH radical scavenging activity DPPH radical assay was performed according to BrandWilliams et al. (1995) with slight modifications. Ground truffle samples (0.1 g each) were extracted using 10 mL of 80% methanolic solution. Extraction was continued for 1 h with occasional vortexing, followed by centrifugation as mentioned above. A standard DPPH assay consisted of an addition of 100 mL of the extract to 2.9 mL of 80% methanolic solution containing 56 mg/mL DPPH while mixing and incubating at room temperature for 15 min. Absorbance was measured at 517 nm. Radical scavenging activity is expressed as percent inhibition: AC AE DPPH% inhibition ¼ 100 AC
where AC was the absorbance of the control (blank) and AE was the absorbance of the extract. 2.8. Hydroxyl radical scavenging activity by deoxyribose assay The deoxyribose method employed was essentially as described by Halliwell et al. (1987). Samples were prepared as described for NO radical scavenging activity. An aliquot (100 mL) was added to a reaction mixture prepared by mixing 120 mL of 20 mM deoxyribose, 500 mL of 75 mM phosphate buffer (pH 7.4), 40 mL of 20 mM H2O2 and 40 mL of 500 mM FeSO4. The mixture was incubated at 37 8C for 30 min, then the reaction was stopped by the addition of 0.5 mL of 2.8% TCA and 0.4 mL of 0.6% TBA. The tubes were subsequently incubated in a water bath (100 8C) for 15 min and then cooled to room temperature; the absorbance was measured at 532 nm. The experiment was carried out in triplicate. Hydroxyl radical scavenging activity (% inhibition) was calculated as for NO radical described above.
Ab ¼ ðA510 A700 ÞpH 1 ðA510 A700 ÞpH 4:5 and Total anthocyanins ðmg=100 gÞ ¼
Ab
e
MW
V 100 G
where e is the molar extinction coefficient taken as 26,900, MW is the molecular weight of a typical anthocyanin taken as 449.2, DF is the dilution factor, V is the final volume and G is the sample weight in grams. Each sample was analyzed in triplicate. 2.11. Estimation of total carotenoids Total carotenoids were estimated according to Lichtenthaler and Wellburn (1985). A fine powder of dried truffles (100 mg) was extracted for 2 min using 3 mL acetone:ethanol (1:1) containing 20 mg BHT per 100 mL in a capped tube protected from light with aluminum foil. The clear extract was aspirated and the truffle residue was further re-extracted twice. The combined extract was brought to 10 mL, centrifuged at 2000 g for 10 min and the
1 The total free phenolic content was first estimated (A), then the same clear solution was treated with formaldehyde to precipitate flavonoids and the remaining phenolic content was again estimated (B). B was subtracted from A to give total flavonoids content.
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absorbance was measured at 470 nm. Total carotenoids were estimated using the following formula: Total carotenoids ðmg=100 gÞ ¼
Ab final volume 106 A1% 100 G
tested dried truffle obtained from Bahrain, Iran, Morocco and Saudi Arabia. 3.3. Ascorbic acid and total carotenoids
where A1% equals 2500, and G is sample weight in g. Samples were analyzed in triplicate. 2.12. Determination of ascorbic acid using 2,4dinitrophenylhydrazine (DNPH) Ascorbic acid content was determined according to Roe and Osterling (1943). A fine powder of dried truffle samples (100 mg) was extracted 3 times with meta-phosphoric acid (4.5%), each 2.5 mL, using vigorous vortexing. The three extracts were combined, centrifuged at 2000 g for 10 min. Then 1 ml of the clear supernatant was taken and 0.2 mL TCA 2.8% was added to precipitate proteins; it was kept in ice for 5 min and then centrifuged at 12,000 rpm for 15 min. 0.2 mL of the de-proteinated extract was mixed with 0.8 mL of 0.2 M acetic acid and 0.25 of 2% DNPH reagent (in 9 N H2SO4), incubated at 60 8 C for 60 min and transferred to an ice bath. After 5 min, 1.25 mL of 85% H2SO4 was added and kept at room temperature for 30 min. Absorbance was measured at 540 nm. The pure ascorbic acid was treated similarly and used as a standard. All samples were analyzed in triplicate. 2.13. Statistical analysis Excel (Microsoft Corporation, USA) and SPSS (version 12.1, SPSS, Chicago, IL, USA) were used for statistical analysis. Data are expressed as means SD. One-way analysis of variance was used for source (origin). Statistical significance between means was determined using Duncan’s multiple range test and set as p < 0.05. Pearson’s correlation coefficient (r) was used to evaluate the association between variables. 3. Results and discussion 3.1. Moisture content The average moisture content of fresh Bahraini, Iranian, Moroccan and Saudi truffles were 77.4 0.77, 75.0 0.26, 75.1 2.02 and 78.6 0.65%, respectively, with an overall average of 76.4 1.53%. Content values were not significantly different (p = 0.052). The moisture content values reported here are in close agreement with those reported by other researchers (Bokhary and Parvez, 1993). However, moisture content as high as 93% was reported for some American edible mushrooms (Dubost et al., 2007). 3.2. Antioxidant components Shown in Table 1 is the concentration of the components contributing towards the antioxidant/antiradical activities in the
Ascorbic acid content ranged between 5.9 and 11.4 mg/100 g, with an average of 9.6 0.15 mg/100 g. Truffles from Bahrain and Iran were not significantly different (p > 0.05) in their ascorbic acid content, but were both significantly different from the other two sources. Bahraini dried truffles had the highest level of ascorbic acid, followed by Iranian, Saudi and Moroccan truffles, in that order. The Moroccan truffles possessed only about 50% of ascorbic acid content compared to the others. Shamekh et al. (1985) reported a value of ascorbic acid content of Libyan dehydrated truffle (Terfezia boudieri) similar to those of Bahraini, Iranian and Saudi truffles, whereas Sawaya et al. (1985) reported values ranging from 0.71 to 5 mg/100 g for various Saudi fresh truffles. Barros et al. (2007) reported that wild edible Portuguese mushrooms contained between 13 and 35 mg/ 100 g dw of ascorbic acid, a value close to the ones reported in this study. Total carotenoids ranged between 405 and 1051 mg/100 g and averaged 681 245 mg/100 g. Bahraini and Moroccan truffles were not significantly different. These truffles significantly differed from the Iranian and Saudi truffles (p < 0.05). The Iranian truffles possessed the highest total carotenoids, which amounted to twice as much as what was found in Bahraini and Saudi truffles. Elmastas et al. (2007) reported that the average content of b-carotene of several edible wild mushrooms ranged from 700 to 3600 mg/100 g, whereas Barros et al. (2007) reported a range from 188 to 297 mg/ 100 g. 3.4. Phenolics Concentration of total free and esterified phenolics, flavonoids and anthocyanins of dried truffles from the four origins are also presented in Table 1. The overall mean of esterified phenolics, free- and non-flavonoid phenolics were 1860 361, 1328 167 and 1034 124 mg/100 g, respectively, whereas the average flavonoids content was 293 (32) mg/100 g. Saudi and Bahraini truffles showed the highest concentration of total free phenolics and were statistically similar. The Moroccan truffles possessed the lowest contents of all investigated phenolics and were significantly different from others. Values of total phenolics reported here are within the range reported in wild mushrooms by other investigators. Total polyphenols of several Turkish edible wild mushrooms ranged between 800 and 2600 mg/100 g (Elmastas et al. (2007). Dubost et al. (2007) reported total phenolic values ranging between 400 and 1065 mg/100 g. The highest amount of anthocyanins were found in Saudi truffles (23.6 mg/100 g) followed by Moroccan (15.1 mg/100 g), whereas the Bahraini and Iranian truffles possessed the lowest, 4.7 and 4.5 mg/100 g, respectively (Table 1). Truffles from the former two sources were not significantly different. Saudi truffles
Table 1 Mean (SD) of ascorbic acid, total carotenoids, anthocyanins, total esterified and free phenolics, total flavonoids and non-flavonoids of dried desert truffles of different origins. Origin
Ascorbic acid (mg/100 g)
Total carotenoids (mg/100 g)
Total anthocyanins (mg/100 g)
Total esterified phenolics (mg/100 g)
Total free phenolics (mg/100 g)
Total flavonoids (mg/100 g)
Non-flavonoids (mg/100 g)
Bahraini Iranian Moroccan Saudi
11.4 10.9 5.9 10.3
594 1051 675 405
4.7 4.5 15.1 23.6
2206 1600 1445 2188
1418 1328 1083 1484
290 326 257 306
1128 1002 826 1178
(0.50) (0.26) (0.25) (0.50)
a a b c
(35) a (127) b (21) a (42) c
(0.18) (0.23) (0.14) (1.52)
a a b c
(68) (82) (28) (52)
a b c a
(51) (37) (85) (81)
a,b a c b
(20) a (10) a (6) b (29) a
(42) a 36) b (26) b (49) a
Data are expressed as a dry-weight-basis and presented as means (SD) (n = 3). All values of phenolic compounds, except for anthocyanins, are expressed as mg gallic acid equivalent (GAE)/100 g. Non-flavonoids were calculated by difference between total free phenolics and total flavonoids. Mean values within each column followed by different letter differ significantly at p < 0.05.
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Table 2 DPPH% inhibition (SD), EC50 and antiradical efficiency of dried desert truffle of various origins. Origin
Bahraini Iranian Morocco Saudi
(% inhibition)
52.5 69.2 24.5 51.0
(2.5) (0.9) (2.8) (3.8)
a b c a
DPPH radical assay EC50a
AEb
0.5 0.3 1.1 0.5
2 3.33 0.91 2
a EC50 is the amount of truffle (mg/assay; 3 mL) required to decrease the initial concentration by 50%. b AE: antiradical efficiency and calculated as AE = 1/EC. The EC50 for the positive control ascorbic acid was 7.2 (0.06) mg/mL. Values within the same column followed by the same letter are not significantly different (p > 0.05).
contained 5 times the amount of anthocyanins found in Bahraini and Iranian truffles, but only 1.6 times of the Moroccan truffles. Seasonal variations, geographical origins and climatic conditions all have been reported to influence the antioxidant constituents of plant foods and natural products (Alasalvar et al., 2005; Dragland et al., 2003; Gil et al., 2002; Ou et al., 2002). Thus, it is expected that truffles originated from different geographical origins would also show some variations. Furthermore, it is also expected that the antioxidant attributes of truffles may be affected by the nature and the extent of association with its host root associate Helianthemum spp. 3.5. Free radical activity using DPPH Percent inhibition of DPPH quenching of the dried truffles ranged between 24.5 and 69.2% with an average of 30.6 13% (Table 2). Iranian truffles, which possessed the highest percent inhibition, were significantly different from others (p = 0.001). The dose-dependent percent inhibition of DPPH quenching of the truffles from the four origins is shown in Fig. 1. The pattern of DPPH inhibition was the same for all. However, the rate of inhibition varied and depended on origin. Percent of inhibition linearly increased at low concentrations, leveled out and then reversed at higher concentrations. DPPH was found to give a reversible reaction with some phenolics bearing o-methoxyphenol structure, which may lead to a false low reading of antioxidant capacity (Bondet et al., 1997). As shown in Fig. 1, the Iranian truffles showed the highest DPPH percent inhibition at all concentrations, whereas the Moroccan truffles possessed the lowest activity at all levels tested. Bahraini and Saudi truffles were similar in their antiradical activity, which was intermediate (Fig. 2). Under experimental conditions employed, 85–90% inhibition could be achieved at 0.9–1.0 mg/ assay with Bahraini, Iranian and Saudi truffles, whereas the Moroccan truffles exhibited only 70% inhibition at such a level.
Fig. 1. Percent inhibition of hydroxyl radical activity by dried truffle of various origins as measured by DPPH quenching assay.
Fig. 2. Antioxidant activity of different concentrations of dried truffle measured by FRAP assay and expressed as mM.
The EC50 of DPPH quenching calculated from these data were 0.3, 0.5, and 1.1 mg for Iranian, Bahraini = Saudi and Moroccan truffles, respectively. The EC50 for the positive control ascorbic acid was much lower (7.2 0.06 mg/mL). DPPH reacts with different antioxidants at different molar ratios (Brand-Williams et al., 1995). More quenching of DPPH, in molar basis, takes place with phenolics and flavonoids possessing hydroxyl groups in ortho positions in the aromatic rings or having higher numbers of hydroxyl groups. As a natural product, variation in chemical composition of truffles due to geographical and climatic variables is expected (Hussain and Al-Ruqaie, 1999). Such variation may be responsible for both the differences between truffle sources in antioxidant activity, as well as the different reaction behavior found in DPPH and FRAP assays. A strong correlation was found between the DPPP percent inhibition and flavonoids content of the truffle (r = 0.966, p = 0.000), but not with total and free phenolics. Similar degrees of correlation between DPPP% inhibition and flavonoids content in plant materials and model systems (Tsimogiannis and Oreopolou, 2004; Khlebnikova et al., 2007) have been previously reported. The antioxidant and antiradical properties of flavonoids are attributed to their ability to act as transient metals chelaters, radical scavengers, and their involvement in electron and hydrogen atom transfer. 3.6. Antioxidant activity by ferric reducing antioxidant power (FRAP) FRAP values as a measure of the antioxidant activity is shown in Table 3. Bahraini and Saudi truffles possessed the highest FRAP values (18.62 and 18.06 mmol/100 g, respectively), but were not significantly different. The Iranian truffles showed the lowest FRAP values (10.43 mmol/100 g), whereas the Moroccan truffles were 418 intermediate in FRAP values (14.62 mmol/100 g). Recently, Halverson et al. (2006) published a list of the antioxidant content of various foods commonly found in the American diet using the FRAP assay. They found that the higher antioxidant content was associated with plant food and the FRAP values of the 50 food commodities, with the highest antioxidant content ranging between 1.01 and 124.55 mmol/100 g. Thus, according to such criteria, dried truffles from different origins may be considered among those edible foods with high antioxidant activity. Though the effect of dehydration on antioxidant/antiradical activity of desert truffles was not thoroughly investigated, preliminary results indicate that the process of dehydration apparently did not adversely affect the antioxidant activity as measured by the FRAP assay; i.e. the average FRAP values corrected
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Table 3 FRAP values, nitric oxide (NO) radical effective concentration (EC50), and scavenging activity of hydroxyl radicals of dried desert truffle of various origins. Origin
Antioxidant activity (FRAP values) (mmol/100 g)
Bahraini Iranian Morocco Saudi
18.62 10.34 14.62 18.06
(0.50) (0.84) (0.54) (1.07)
ac b c a
Nitric oxide radical (NO) EC50 (mg/mL)a
Hydroxyl radical scavenging activity (% inhibition)b
115 102 250 170
65.7 91.0 11.3 55.7
(4.0) a (3.5) a (11.5) b (8.0) c
(2.5) (2.0) (2.5) (1.5)
a b c d
a EC50 (mg/mL) is the effective concentration at which 50% of the NO radical are inhibited. The EC50 for the positive control ascorbic acid was 15.8 (0.25) mg/mL. Mean values within each column followed by different letter differ significantly at p > 0.05. b Percent inhibition of hydroxyl radical activity (deoxyribose assay) is for 2.5 mg of dried truffles per assay. c Values within the same column followed by the same letter are not significantly different (p < 0.05).
for the moisture content of fresh truffle of Bahraini, Iranian, and Saudi origins were 13.05 (0.55), 10.85 (0.71), and 14.58 (0.30) mmol/100 g, respectively. On the other hand, dried truffles from Bahrain possessed significantly higher antioxidant activity than the fresh truffles (p = 0.0003). Halverson et al. (2006) reported that several processes, such as microwave cooking, steaming or boiling, roasting and baking resulted in increasing the antioxidant content (FRAP values) of many foods of plant origins. When samples of dried truffles from the four origins were tested by FRAP assay in the range of 0–2 mg/assay, they all showed a similar pattern (Fig. 2). Clearly, the FRAP values increased nonlinearly in a dose-dependent relationship. The rate of increase, however, differed. At relatively high amount of truffles (between 1 and 2 mg) the ranking order of antioxidant activity was as follows: Bahraini > Moroccan > Saudi > Iranian. Bahraini truffles were about 1.8 times higher in antioxidant activity as compared with the Iranian truffles. At such a high level (i.e. 2 mg), a significant difference between the four sources was observed (p = 0.0034). No significant difference was found between Bahraini, Moroccan and Saudi truffles at relatively lower amount (i.e. 0.25 mg/assay). Several investigators have reported a linear relationship between total phenolics and FRAP values (Cai et al., 2004). However, other studies (Wu et al., 2004; Dubost et al., 2007) reported a nonlinear relationship with strong correlation, which may indicate that phenolics accept electrons at a greater rate as the total free phenolics increase. The chemistry of antioxidant capacity assays has recently been evaluated (Huang et al., 2005). FRAP is an electron-transferred-based assay, and uses Fe (III)(TPTZ) as an oxidant. However, it is subjected to interference by many metal chelators present in food capable of binding with to Fe(III), and forming complexes that could react with antioxidants (Huang et al., 2005). In our study, a strong correlation was found between FRAP values measured at a fixed concentration of dried truffle (2.5 mg/assay), total (esterified) (r = 0.801, p = 0.001) and free phenolics (r = 0.973, p = 0.000), but not with flavonoids (p = 0.287). 3.7. Nitric oxide radical scavenging activity Extracts of dried truffles exerted inhibition activity against the production of nitric oxide (NO) radical from its artificial source sodium nitroprusside (Table 3). Dried truffles from all the tested sources were indifferent (p > 0.05) and possessed relatively high NO antiradical activity (>93%), which hampered the direct comparison of NO percent inhibition between dried truffles of different origins. The scavenging of NO radicals of dried truffle (25–500 mg/mL) was concentration-dependent, exhibiting a hyperbolic manner for
Fig. 3. Percent inhibition of nitric oxide (NO) radical production by dried truffle of various origins as measured by deoxyribose assay.
all tested samples (Fig. 3). At 500 mg/mL the maximum inhibition of NO radical ranged between 68 and 88%. The effective concentration at which 50% of the NO radical are inhibited (EC50 in mg/mL is presented in Table 3. Iranian truffles possessed the strongest EC50 (102 mg/mL) and was not statistically different from the EC50 of the Bahraini truffles. The Moroccan truffles possessed the weakest EC50 (250 mg/mL). All the tested truffles exhibited low scavenging activity compared to the EC50 of the positive control, ascorbic acid, which measured 15.8 0.25 mg/mL. It is known that the mechanism of inhibition of the production of the short-lived NO radical from SNP involves either direct scavenging of NO by the antiradical or via the scavenging of the oxide (oxygen), which reacts with the formed NO radical leading to the formation the nitrite ions (Janzen et al., 1993). Phenolics, particularly flavonoids, have been suggested to scavenge NO either by H-atom abstraction or reduction of NO by single electron transfer (Janzen et al., 1993). Inhibition of NO production by plant polyphenols is well-documented (Van Acker et al., 1995; Klotz and Sies, 2003). Potency of NO radical inhibition has been correlated with the number of hydroxyl residues of polyphenolics, including flavonoids. In this study, a good correlation between percent inhibition of NO production and flavonoids content was observed (r = 0.735, p = 0.024), but not with total and free phenolics. Some other studies reported that phenolic groups were not essential for NO scavenging activity (Sreejayan and Rao, 1997). 3.8. Hydroxyl radical activity using deoxyribose assay Hydroxyl radicals degrade the sugar deoxyribose (2-deoxy-Dribose) via Fenton-type reactions leading to the production of complex products which can be estimated as malonaldehyde (MDA). The presence of an antiradical prevents or decreases MDA production. Extract from all tested dried truffles exhibited antiradical activity and decreased the degradation of deoxyribose as shown in Table 3. This activity was significantly different for all sources (p = 0.003). The Iranian truffles exhibited the highest percent of inhibition of deoxyribose breakage (91%), whereas the Moroccan dried truffles exhibited 501, considerably very low antiradical activity using the deoxyribose assay (11.3%). The total flavonoids and percent of inhibition of deoxyribose degradation of the studied truffles were highly correlated (r = 0.939, p = 0.000). However, no correlation was found with total and free phenolics. According to Eberhardt (2001), the deoxyribose assay does not measure hydroxyl radicals only, but other species which are kinetically indistinguishable from hydroxyl radicals, too.
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4. Conclusions The extract of dried desert truffles (Tirmania nivea) from four different Middle Eastern countries showed varying degree of antioxidant and antiradical activities based on four analytical methods. It appears that truffles from a single tested source did not yield the highest values either in all assays used, or in the contents of the relevant antioxidant constituents. However, it is evident that the dried Iranian truffles scored highest in most parameters tested. Furthermore, the high correlation observed between the various assays employed and phenolic contents is a strong indication that these phenolics (total, free and flavonoids) are among the predominant source of antioxidant activity in desert truffles. Acknowledgments This work was financially supported by the University of Bahrain. The author thanks Professor Ahmed Y. Ali and Dr. Q. Mandeel for their critical reading of the manuscript. I am most grateful to Dr. Mandeel for confirming the authenticity of the tested truffle. I also thank Mrs. Naima Fakhroo for providing the Iranian truffles. References Alasalvar, C., Al-Farsi, M., Quantick, P.C., Shahidi, F., Wiktorowicz, R., 2005. Effect of chill and modified atmosphere packaging (MAP) on antioxidant activity, anthocyanins, carotenoids, phenolics and sensory quality of ready-to-eat shredded orange and purple carrots. Food Chemistry 89, 69–76. Al-Naama, N.M., Ewaze, J.O., Nema, J.H., 1988. Chemical constituents of Iraqi truffles. Iraqi Journal of Agricultural Sciences 6, 51–56. Al-Rahmah, A.N., 2001. Truffle of Deserts and Jungles. King Saud University Publications, Riyadh, Saudi Arabia, p. 272 (in Arabic). Al-Ruqaie, I.M., 2002. Effect of different treatment processes and preservation methods on the quality of truffles. I. Conventional methods (drying/freezing). Pakistan Journal of Biological Sciences 5, 1088–1093. Al-Sheik, A.M., Trappe, J.M., 1983. Desert truffles: the genus Tirmania. Transactions of the British Mycological Society 81, 83–90. Amicarelli, F., Bonfigli, A., Colafafarina, S., Cimini, A.M., Prutti, B., Cessare, P., Ceru, M.P., Di Ilio, C., Pacioni, G., Miranda, M., 1999. Glutathione dependent enzymes and antioxidant defences in truffles: organisms living in microaerobic environments. Mycological Research 103, 1643–1648. Barros, L., Ferreira, M.-J., Queiro´s, B., Ferreira, I.C.F.R., Baptista, P., 2007. Total phenols, ascorbic acid, b-carotene and lycopene in Portuguese wild edible mushrooms and their antioxidant activities. Food Chemistry 103, 413–419. Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of ‘‘antioxidant power’’: the FRAP assay. Analytical Biochemistry 239, 70–76. Bokhary, H.A., Parvez, S., 1993. Chemical composition of desert truffles Terfezia claveryi. Journal of Food Composition and Analysis 6, 285–293. Bokhary, H.A., Suleiman, A.A., Basalah, M.O., 1989. The fatty acid components of the desert truffle ‘‘AlKamah’’ of Saudi Arabia. Journal of Food Protection 52, 668– 669. Bokhary, H.A., Suleiman, A.A., Basalah, M.O., Parvez, S., 1987. Chemical composition of desert truffles from Saudi Arabia. Canadian Institute of Food Technology Journal 20, 336–341. Bondet, V., Brand-Williams, W., Berset, C., 1997. Kinetics and mechanisms of antioxidant activity using DPPH free radical method. Food Science and Technology-Leb 30 (6), 609–615. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of free radical method to evaluate antioxidant activity. Lebensmittel Wissenschaft und Technologie (Food Science and Technology) 28, 25–30. Cai, Y., Luo, Q., Sun, M., Corke, H., 2004. Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sciences 74, 2157–2184. Dragland, S., Senoo, H., Waka, K., Holte, K., Blomhoff, R., 2003. Several cultinary and medicinal herbs are important sources of dietary antioxidant. Journal of Nutrition 133, 1286–1290. Dubost, N.J., Ou, B., Beelman, R.B., 2007. Quantification of polyphenols and ergothioneine in cultivated mushroom and correlation to total antioxidant capacity. Food Chemistry 105, 727–735. Eberhardt, M.K., 2001. Reactive oxygen metabolites: chemistry and medical consequences. CRC Press, pp. 70–71. Elmastas, M., Isildak, O., Turkekul, I., Temur, N., 2007. Determination of antioxidant activity and antioxidant compounds in wild edible mushrooms. Journal of Food Composition and Analysis 20, 337–345.
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