Iraqi J Pharm Sci, Vol.31( 2 ) 2022 Comparison between black and white desert truffles DOI: https://doi.org/10.31351/vol31iss2pp184-192 184 Comparison of Phenolic Contents and Antioxidant Activities for Black and White Desert Truffles Spread in Syria Abdullah Zuhair AL-ATASSI*,1, Racha Aboualkher AL-KHATIB** and Mohammad Abdulraouf OTHMAN*  Department of Pharmaceutics and Pharmaceutical Technology, Faculty of Pharmacy, Damascus University, Damascus, Syria.  Department of Pharmacognosy, Faculty of Pharmacy, Damascus University, Damascus, Syria. Abstract Desert truffle is considered as a type of Syrian wild fungi that spreads heavily, and it occupies important rank in folk medicine, where its aqueous extract is used for the treatment of some eye and skin illnesses, and people prefer the use of black truffle. This work interested in studying of the most available species; Terfezia claveryi (black) and Tirmania pinoyi (white). The extracts of the two species of truffle were prepared by maceration with water, methanol, and ethanol 70%. Their total phenolic contents (TPC) and total flavonoid contents (TFC) were analyzed using Folin-ciocalteu and Aluminum chloride methods respectively, and their antioxidant activities was tested using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Ferric Reducing Antioxidant Power (FRAP) methods, after microscopic examination and detection of phytochemical components. Then, phenolic profile of ethanolic 70% extract of black truffle T.claveryi was studied by using LC-MS/MS. The values of TPC were between 25.3-43.6 mg GAE/g dry extract and TFC were between 2.5-6.8 mg QE/g dry extract. The values of DPPH (IC50) and FRAP were between 5.6-9.0 mg/ml and 90.1-153.4 µmol AAE/g dry extract respectively. There is a great similarity in content and activity of two species, also the aqueous extract is similar to other extracts in content and activity, and this means that the method of extract preparation in traditional medicine is reliable. It has been predicted about 14 phenolic compounds in the extract; as p-Hydroxy benzoic acid, Syringic acid and trans-Cinnamic acid. As a result, both truffle species are a new rich resource of antioxidant compounds which are usable in nutritional, cosmetic, and therapeutic applications. Keywords: DPPH, Flavonoids, FRAP, LC-MS/MS, Polyphenols, Truffle. السوداء والبيضاء المنتشرة الصحراوية كمأةل المحتوى الفينولي والفعالية المضادة لألكسدة لمقارنة في سوريا * ومحمد عبد الرؤوف عثمان و **ورشا أبو الخير الخطيب 1،*األتاسيعبد هللا زهير . سوريادمشق، كلية الصيدلة، جامعة دمشق، ، قسم الصيدالنيات والتكنولوجيا الصيدلية * . سوريادمشق، كلية الصيدلة، جامعة دمشق، عقاقير والنباتات الطبية، قسم ال ** ةالخالص ، حيث تُستخدم في الطب الشعبي وهي تشغل منزلة قيّمةالسورية، في البادية بكثرةالفطور المنتشرة نواعالصحراوية أحد أتعد الكمأة السوداء الكمأة استخدام الناس ويفضل والجلدية، العينية األمراض بعض عالج في المائية توافراً . خالصتها األكثر النوعين بدراسة البحث اهتم Terfezia claveryi (و )السوداء Tirmania pinoyi ،)رت خالصات نوعْي الكمأة إذ )البيضاء ، %70نقيع بالماء والميتانول واإليتانول بالت ُحّضِّ سودُ فعاليتها اختبار تم، وعلى الترتيب سيوكالتو وكلوريد األلمنيوم-بطريقتي فولين TFC الكلي والفالفونوئيدي TPCالكلي الفينولي محتواها رِّ دراسة الكشف عن المجموعات الكيميائية. ثم تم مجهري وال الفحصبعد ، FRAPوالمرجعة للحديد DPPHالمضادة لألكسدة الكابحة للجذر الحر 43.6-25.3بين TPC. تراوحت قيم LC-MS/MSباستخدام تقنية T.claveryiلنوع الكمأة السوداء %70التركيب الفينولي للخالصة اإليتانولية تراوحت ملغ مكافئ من الكيرسيتين لكل غرام خالصة جافة. 6.8-2.5بين TFCمن حمض العفص )الغاليك( لكل غرام خالصة جافة، وملغ مكافئ لكل غرام خالصة جافة على ميكرومول مكافئ من حمض األسكوربيك 153.4-90.1ملغ/مل، و 9.0-5.6بين FRAPو 50IC (DPPH)قيم صات، وهذا الترتيب. ُوجد تشابه كبير بين النوعين من حيث المحتوى والفعالية، إضافةً إلى تشابه محتوى الخالصة المائية وفعاليتها مع بقية الخال الفينولية التي تم التحّري عنها في مركباً من المركبات 14تبيّن وجود يعني أن طريقة تحضير الخالصة في الطب الشعبي يمكن االعتماد عليها. أهمها من غنيّاً . trans-Cinnamic acidو Syringic acidو p-Hydroxy benzoic acidالخالصة، جديداً مصدراً بنوعيها الكمأة تشكل بالمركبات المضادة لألكسدة والتي يمكن االستفادة منها بالتطبيقات العالجية والتجميلية والغذائية. . DPPH ،FRAP ،LC-MS/MSالكمأة، عديدات الفينول، الفالفونوئيدات، المفتاحية: الكلمات 1Corresponding author E-mail: a111atassi@gmail.com Received: 30/10 /2021 Accepted:19 /1 /2022 Iraqi Journal of Pharmaceutical Science https://doi.org/10.31351/vol31iss2pp184-192 Iraqi J Pharm Sci, Vol.31(2) 2022 Comparison between black and white desert truffles 185 Introduction Truffle is one of the wild edible hypogeous macrofungi, and it belongs to the phylum Ascomycota that do not have cap, gills and stalk unlike other fungi such Basidiomycetes (1). Truffle has hundreds of species. The species that grow in arid and semi-arid areas in Mediterranean basin and middle east are called “desert truffle”, and are classified under genus of “Tirmania” and “Terfezia” that belong to Pezizaceae family (2). Some species were identified in Syria as Terfezia claveryi, Terfezia boudieri, Tirmania pinoyi and Tirmania nivea (3), where they grow naturally in the desert plains such as the region between Homs and Deir ezzor. The season of gathering is between March and May. The quantity of truffles is usually varied from season to season, and it is dependent on rainfall and temperature, so truffles absent some years when weather conditions are not suitable (4,5). Truffles have high values of proteins, minerals, unsaturated fatty acids, and antioxidant compounds (6,7), that candidates it to be as complements and complete food. Bedouins use boiled truffle extract in treatment of trachoma and illnesses of eyes and skin, and they consume truffle as meat alternative (8). The application of truffle extract on infected eyes was advised by Prophet Mohammad (Peace be upon him). Species of truffle have wide biological activities such as antioxidant (9), antimicrobial (10), anti-inflammatory (11), anticancer (12), and antidepressant (13). Types of wild and cultivated mushrooms are well known to contain various polyphenolic compounds which are considered as an excellent antioxidants (14,15). Some articles studied the antioxidant activity and the antioxidant compounds of the two species of desert truffle (Terfezia calveryi and Tirmania pinoyi). The same species of desert truffles from different geographical regions may not exhibit the same chemical composition, because many environmental factors such as amount of rain, soil types and climatic changes have influence on the chemical profile (16). Polyphenols are considered as secondary metabolites, and they are wide group of compounds that differ in structure and resources. They help the organism to defend against oxidant stress and free radicals which damage the cells (17). The use of modern technics in extraction of polyphenols increases efficacy, save time, and decrease amounts of solvents, furthermore they are convenient for thermo-sensitive compounds (18). DPPH method is considered as one of the most common methods to determine the antioxidant activity according to hydrogen atom or single electron transfer, and it depends on a stable organic radical (2,2-diphenyl-1- picrylhydrazyl) which changes from purple to yellow color when interacts with antioxidants (19). Ferric reducing antioxidant power (FRAP) is another method to determine the antioxidant activity according to single electron transfer, and it measures reducing of Fe (III) to Fe (II) by antioxidants (20). Desert truffle is famous fungi that has crucial value in traditional medicine, and there is a lack of information about chemical components and the importance of antioxidant activity of truffles that are harvested in Syria. This study aims to compare between the most two abundance species according to their phenolic contents and antioxidant activities. Materials and methods Chemicals 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) and 2,4,6-tri-2-pyridinyl-1,3,5-triazine (TPTZ) were from (Cayman chemical, USA). Folin-ciocalteu's phenol reagent (FC) was from (Sigma-Aldrich, Switzerland). L(+)-Ascorbic acid (AA), Ferric chloride hexahydrate, Sodium acetate anhydrous, and Sodium carbonate anhydrous were from (Panreac, Spain). gallic acid (GA) was from (Titan biotech, India). Quercetin (Q) was from (Sigma- Aldrich, USA). Aluminium chloride hexahydrate was from (Riedel-de Haen, Germany). All other chemicals were of analytical grades. Collection and preparation of fungi Ascocarps were collected from desert region in the east of Homs (Palmyra and Al-Sukhna) during April 2020. They were intact and showed no sign of spoilage. The identification of two species was made advising with Dr. Fawaz Al-Azmah and Dr. Hijazi Mando from faculty of agricultural engineering, Damascus university, by macroscopic and microscopic (OLYMPUS BX41, Japan) observations for fresh ascocarps and asci as described in the literature (21,22). Ascocarps were cleaned, peeled, and sliced. The slices were dried in an oven at (40°C) and the size was reduced mechanically, then stored at room temperature in dry place away from light until use. Determination of chemical composition These reactions were conducted according to standard methods (23–25). Two species of truffle were evaluated before extraction to determine the presence of alkaloids, anthraquinones, saponins, coumarins, flavonoids, tannins, steroid and triterpenoid glycosides. The resulting solutions were observed for color change and/or precipitate formation to indicate positive results. Preparation of extracts Three extracts of each type were prepared. Namely, powder of dried truffle was extracted by maceration with water, methanol and Ethanol 70% separately (10 g of dried truffle/100 ml of solvent) at room temperature, and the extracts were exposed to ultrasonication (PHYLO USH-10D, Italy) for 15 min at the beginning of extraction. After 24 h, the extracts were centrifugated and filtrated, then the solvent was evaporated by the rotary evaporator (Great Wall, Rotary evaporator R-1001-VN, China) Iraqi J Pharm Sci, Vol.31(2) 2022 Comparison between black and white desert truffles 186 at (45°C). Finally, the dried extracts were stored in a refrigerator. Determination of total phenolic content (TPC) Total phenolic content was estimated by Folin–Ciocalteu method adapted from (Al-laith, 2010) (9) with a slight modification. Briefly, 20 µl of the extract, standard and the blank were added to 1.58 ml H2O, followed by the addition of 100 µl concentrated FC reagent. After 3 min standing at room temperature, 300 µl of sodium carbonate buffer (200 g/l) was added. The mixture was incubated at (40°C) for 30 min. The absorbance was measured at 736 nm (pg instruments UV-VIS Specrtophotometer T80+, UK). The phenolics concentration was determined by comparing with the standard calibration curve of gallic acid (y=0.0009x+0.00177, R2=0.9961), and results were reported as mg of gallic acid equivalent (GAE)/ g of dry extract. Determination of total flavonoid content (TFC) Total flavonoid content was estimated by aluminium chloride colorimetric assay adapted from (Sembiring et al., 2018) (26) with a slight modification. Briefly, 0.5 ml of extract or standard were added to 100 μl of the 10% aluminium chloride solution and followed by 1.5 ml of 96% ethanol. Finally, 100 μl of (1 M) sodium acetate was added to the mixture. Ethanol 96% was used as blank. The mixture was incubated at room temperature for 40 min protected from light. The absorbance was measured at 415 nm. Total flavonoid content was determined by comparing with the standard calibration curve of quercetin (y=0.0106x+0.077, R2=0.9986), and results were reported as mg of quercetin equivalent (QE)/ g of dry extract. DPPH radical scavenging activity DPPH˙ scavenging activity of the extracts was estimated according to (Gouzi et al., 2013) (27) with a slight modification. Various concentrations of extracts or positive control (ascorbic acid) (0.1 ml) were added to 2.9 ml of methanolic solution con- taining DPPH radical (0.1 mM). The absorbance was measured at 515 nm after the mixture was left to stand at room temperature for 30 min in a dark place. The methanol was used as a blank. DPPH free radical scavenging activity was calculated according to the following equation: DPPH radical scavenging activity (%) = [(Abs0 – Abs1)/Abs0] × 100 where Abs0 was the absorbance of the negative control (methanol), and Abs1 was the absorbance of the extract or the positive control. The extract concentration that scavenges 50% of DPPH radical (IC50) was calculated from the graph that plots the percentage of radical scavenging activity against the extract concentration, Also IC50 was calculated for the positive control (Ascorbic acid). Ferric Reducing Antioxidant Power (FRAP) assay The reducing power of extracts was estimated according to (Benzie et al., 1996) (28) with a slight modification. The FRAP reagent contained 2,4,6-tripyridyl-s-triazine (TPTZ) solution (10 mM) in HCl (40 mM), ferric chloride solution (20 mM), and acetate buffer (0.3 M) at pH 3.6 in the ratio 1:1:10 respectively. 100 μl of the extract, or standard, or blank was mixed with freshly prepared FRAP reagent (3 ml). The mixture was incubated at (37°C) for 30 min. The absorbance was measured at 595 nm. The results of FRAP assay were determined by comparing with the standard calibration curve of ascorbic acid (y=0.002x+0.0067, R2=0.9991), and were reported as micromoles of ascorbic acid equivalent (AAE)/ g of dry extract. LC-MS/MS analysis of selected phenolics Ethanolic 70% extract of T.claveryi was selected to qualitative detection of some phenolic compounds in it. This detection was carried out using liquid chromatography-mass spectroscopy (Agilent technologies LC-MS/MS, USA) according to procedure of (Orcˇic et al., 2014) (29) and (Kıvrak, 2015) (6). Data were acquired in Multiple Reaction Monitoring (MRM) mode, using the optimized compound specific parameters (precursor ion, product ion, fragmentor voltage, collision voltage). Nineteen phenolic compounds which were detected are the following: (Apigenin, Catechin, Chlorogenic acid, trans-Cinnamic acid, p-Coumaric acid, 3,4- Dihydroxybenzaldehyde, Eugenol, Ferulic acid, Gallic acid, Gentisic acid, Hesperidin, Homogentisic acid, p-Hydroxy benzoic acid, Protocatechuic acid, Pyrocatechol, Rutin, Syringic acid, Vanillic acid, and Vanillin). Statistical analysis The results were expressed as a mean of three frequencies followed by standard deviation. Statistical analysis was performed using SPSS software (version 25). One-way ANOVA (Tukey’s HSD) was used for comparison between means. Values were considered significantly different when P-value<0.05. Results and Discussion Macroscopic and microscopic identification of truffles, based on morphological characteristics of ascocarps and asci, reveals two species: Terfezia claveryi (black truffle) locally called “Obaidi”, and Tirmania pinoyi (white truffle) locally called “Zubaidi” (Figures 1-4). Iraqi J Pharm Sci, Vol.31(2) 2022 Comparison between black and white desert truffles 187 According to Alsheikh (21,22), the ascocarp of two studied species have lobed shape and diameters of 4 to 10 cm. The studied species T.claveryi and T.pinoyi present different characteristics. T.claveryi has a brownish-yellow peridium and a gleba with fleshy appearance and yellow-pinkish color. Asci are subglobose and contain 8 globose spores ornamented with warts. T.pinoyi has a white peridium, and a gleba with fleshy appearance and white color. Asci are pear- shaped and contain 8 smooth globose spores which have double outer layer. Figure 1. An ascocarp of Terfezia claveryi and a cross section of it. Figure 2.An ascocarp of Tirmania pinoyi and a cross section of it. Figure 3. An ascus of Terfezia claveryi which contains 8 globose spores ornamented with warts (colored with alcoholic iodine, magnification ×40). Figure 4.An ascus of Tirmania pinoyi which contains 8 smooth globose spores (colored with alcoholic iodine, magnification ×40). The results of detection about phytochemical components for dried T.claveryi and T.pinoyi indicate the probability of containing alkaloids, coumarins, flavonoids, phenols, and steroid and triterpenoid glycosides. T.claveryi also may contain little of saponins (Table 1). This is the first time that determination of chemical composition to truffle was published. Table 1. Detection of phytochemical components for two species of truffle: Terfezia claveryi and Tirmania pinoyi. Note: - = absent, + = present Phytochemical Constituents Tests Results Terfezia claveryi Tirmania pinoyi Alkaloids Dragendorff + + Mayer + + Hager - - Anthraquinones Borntrager - - Saponins Foam + - Coumarins Alcoholic FeCl3 + + Fluoresence + + Flavonoids Shinoda + + NaOH + + Ammonia + - Tannins/phenols Ferric chloride + + Lead acetate + + Gelatin deposition - - Matchstick + - Steroid and triterpenoid glycosides Salkowski + + Iraqi J Pharm Sci, Vol.31(2) 2022 Comparison between black and white desert truffles 188 The extraction’s yields of for both studied species of truffle were in the following arrangement (aqueous> ethanolic 70%> methanolic). The aqueous extracts had the highest yield (44.2%, 43.5%) for T.claveryi and T.pinoyi respectively. When we compare the yields of two species for each solvent, we observe high similarity (Table 2). Table 2. Yields of extraction, phenolic and flavonoid contents, and antioxidant activities of Terfezia claveryi and Tirmania pinoyi extracts. Type of extract Yield of extraction (%) Total phenolic content (mg GAE/g dry extract) Total flavonoid content (mg QE/g dry extract) DPPH IC50 (mg/ml) FRAP (µmol AAE/g dry extract) AC 44.2±9.4 a 43.6±9.6 a 3.2 ± 0.4 a,b 9.0 ± 1.8 a 126.1 ± 25.2 a,b MC 16.7±7.3 b 28.7±4.4 a,b 2.5 ± 0.4 a 8.6 ± 0.6 a 136.3 ± 14.8 a,b EC 21.9±3.0 b 32.7±0.4 a,b 3.7 ± 0.1 b 6.1 ± 0.7 a,b 153.4 ± 12.4 a AP 43.5±1.4 a 25.3±4.6 b 3.9 ± 0.3 b 8.8 ± 1.4 a 90.1 ± 26.6 b MP 11.5±0.9 b 30.4±7.1 a,b 5.0 ± 0.6 c 8.5 ± 0.4 a,b 134.8 ± 20.0 a,b EP 23.8±0.7 b 33.3±2.8 a,b 6.8 ± 0.2 d 5.6 ± 0.8 b 135.1 ± 14.7 a,b Where AC, MC, and EC: Aqueous, Methanolic, and Ethanolic 70% extracts of Terfezia claveryi respectively. AP, MP, and EP: Aqueous, Methanolic, and Ethanolic 70% extracts of Tirmania pinoyi respectively. IC50: inhibitory concentration to 50% of substrate. Values are means ± SD of three measurements. Means within each column with same letters don’t differ significantly (P-value>0.05) according to Tukey’s test. To evaluate the phenolic profile of the truffle’s extracts, the total phenolic and flavonoid contents were determined. The total phenolic contents (TPC) for the analyzed truffle extracts which were evaluated by the FC method are shown in (Table 2). TPC values were between 25.3-43.6 mg GAE/g dry extract. Aqueous extract of T.claveryi (AC) was found to have the highest phenolic con- tent, on the other hand, the lowest phenolic content was found in aqueous extract of T.pinoyi (AP). Statistical study demonstrated that there were no significant differences between three extracts of the same species in TPC values. Boufeldja et al. (30) found that TPC for methanolic 80% extract of T.claveryi was 15.5 mg GAE/g dry weight, and this value resembles to present finding for aqueous extract. Regarding to T.pinoyi, TPC for methanolic extract (30.4 mg GAE/g dry extract) has been obviously above the values of (Stojkovic et al., 2013) (31) and (Gouzi et al., 2013) (27) (13.19 and 2.1 mg GAE/g dry extract) respectively. Depending on previous results, the TPC values for the two studied species of truffle were higher than TPC values for foods which are classified as rich in polyphenols such strawberry and cherries. This high level of polyphenols assists truffle to overcome severe stress condition in deserts (17,32). Values of total flavonoid contents (TFC) for the analyzed truffle extracts were between 2.5- 6.8 mg QE/g dry extract [methanolic extract of T.claveryi (MC) and ethanolic 70% extract of T.pinoyi (EP) respectively] (Table 2). The flavonoid contents for ethanolic 70% extracts were the highest among other extracts for both species of truffle. Values of TFC for T.pinoyi were approximately two fold the values of T.claveryi. Kivrak (6) found that TFC for ethyl acetate extract of T.claveryi was 4.71 mg QE/g dry extract, and this value is slightly higher than the value of ethanolic 70% extract in this study (3.74 mg QE/g dry extract). TFC of T.pinoyi had not been studied previously. To extensively characterize the antioxidant potential of truffle extracts, two antioxidant assays were applied. The combination of more than one method helps in understanding the mechanism of action for antioxidants. With regard to IC50 values of DPPH (Table 2), they were between 5.6-9.0 mg/ml [ethanolic 70% extract of T.pinoyi (EP) and aqueous extract of T.claveryi (AC) respectively]. There were no significant differences between two species of truffle in IC50 values of DPPH. Ethanolic 70% extract of T.pinoyi (EP) exhibited the highest scavenging activity with the lowest IC50 (5.64 mg/ml). The reported scavenging activity for all extracts is too lower than the one showed by ascorbic acid (positive control, IC50= 0.11 mg/ml), but the comparison between pure compounds and extracts should be avoided, because they are individual/purified compounds and not mixtures, where the concentration of each individual compound is actually too lower. It seems that the IC50 value for methanolic extract of T.claveryi (8.6 mg/ml) was comparable to that was reported by (Neggaz et al., 2015) (2) (8.6 mg/ml) for an extract which was prepared using Soxhlet, by applicating same protocol, but the value of maceration extract was (22.16 mg/ml). Gouzi et al. (27) and Stojkovic et Iraqi J Pharm Sci, Vol.31(2) 2022 Comparison between black and white desert truffles 189 al. (31) found that IC50 for methanolic extracts of T.pinoyi were (2.51 and 6.41 mg/ml respectively), and these values are lower than IC50 in this study (8.5 mg/ml), by applicating same protocol. The values of FRAP assay for investigated extracts were between 90.1-153.4 µmol AAE/g dry extract [aqueous extract of T.pinoyi (AP) and ethanolic 70% extract of T.claveryi (EC) respectively] (Table 2). There were no significant differences between the extracts of both truffle’s species, also between three extracts of the same species in FRAP values. Our results were in agreement with those reported by (Dahham et al., 2018) (33) where the values of reducing power for examined extracts of T.calveryi were also in this arrangement (ethanolic 70%> methanolic> aqueous). The value of FRAP for methanolic extract of T.pinoyi in this study (134.8 µmol AAE/g extract) was higher than that in the work of (Gouzi et al., 2013) (27) (126.8 µmol AAE/g extract). These results demonstrate that the reducing power of truffle was high, and it was among the best foods in this field. The results of LC-MS/MS revealed the presence of 14 from 19 phenolic compounds which were detected in ethanolic 70% extract of T.claveryi, and they are listed in (Table 3) starting by the most intensity. Figure 5 presents the chromatograms of some founded phenolics in the extract. Table 3.Phenolic compounds that are founded in ethanolic 70% extract of T.claveryi (starting by the most intensity). Where MW: Molecular Weight, Rt: Retention time. Rt (min) Product ion [M- H]- (m/z) Precursor ion [M- H]- (m/z) Total MW Compound name 6.457 93 137 138.12 p-Hydroxy benzoic acid 1 8.015 155.1 199 198.17 Syringic acid 2 5.598 103 147 148.16 trans-Cinnamic acid 3 15.844 119 163 164.16 p-Coumaric acid 4 7.275 125 169.1 170.12 Gallic acid 5 3.955 123 167 168.15 Homogentisic acid 6 2.543 109 153 154.12 Protocatechuic acid 7 6.542 92 151.1 152.15 Vanillin 8 3.079 134 193 194.18 Ferulic acid 9 6.376 301 609 610.5 Rutin 10 5.099 107.9 166.9 168.15 Vanillic acid 11 2.115 151 269 270.24 Apigenin 12 8.002 245 289 290.27 Catechin 13 15.209 303 611 610.6 Hesperidin 14 Iraqi J Pharm Sci, Vol.31(2) 2022 Comparison between black and white desert truffles 190 Figure 5.Chromatograms of some phenolics in ethanolic 70% extract of T.claveryi by LC-MS/MS. Where (a): Hydroxy benzoic acid, (b): Trans-cinnamic acid, (c): Vanillin, (d): Vanillic acid Kıvrak (6) found that p-Hydroxy benzoic acid was the most abundance in the same studied truffle, and this resemble present results, But there is no existence of three compounds (Gentisic acid, Pyrocatechol, and 3,4-Dihydroxybenzaldehyde) as a result of this study, also there are no existence of two phenolics (Chlorogenic acid and Eugenol) which are founded by Vahdani et al (34). This variation in phenolic profile for studied samples of the same species may be due to variation of synthesis of these compounds as a result to differences in climate conditions, soil and geographical regions, or the difference in the method of extraction and the differences in the parameters of analytical methods. The prediction of the presence of these phenolic compounds supports medical, cosmetic and nutritional applications of truffle. Conclusion The results of this work reveal that two studied species of desert truffle are rich in phenolic compounds, and all of their extracts showed appreciable DPPH free radical scavenging activity and reducing power with FRAP assay, so they can act as antioxidant by donating a hydrogen or an electron. There is great similarity in content and activity between two studied species. The water can be use continually for extracting the phenolic compounds as it is used in traditional medicine, instead of other non-safe polar solvents, because the values of TPC are similar in the two cases. More profound studies of truffle and fungi generally which exist heavily in Syrian environment should be performed, that may provide a great economic and medical benefits. (d) (a) (b) (c) Iraqi J Pharm Sci, Vol.31(2) 2022 Comparison between black and white desert truffles 191 References 1. Enshasy H El, Elsayed EA, Aziz R, Wadaan MA. 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