Turkish Journal of Biology Turk J Biol (2013) 37: 25-32 http://journals.tubitak.gov.tr/biology/ © TÜBİTAK Research Article doi:10.3906/biy-1204-38

Compositional studies and antioxidant potential of lebbeck (L.) Benth. pods and

1 2 3 4, Muhammad ZIA-UL-HAQ , Shakeel AHMAD , Mughal QAYUM , Sezai ERCİŞLİ * 1 Department of Pharmacognosy, University of Karachi, Karachi 75270, Pakistan 2 Department of Agronomy, Bahauddin Zakariya University, Multan 60800, Pakistan 3 Department of Pharmacy, Kohat University of Science and Technology, Kohat 2600, Pakistan 4 Department of Horticulture, Atatürk University, 25240 Erzurum, Turkey

Received: 20.04.2012 Accepted: 19.07.2012 Published Online: 10.01.2013 Printed: 01.02.2013

Abstract: (L.) Benth. is an important medicinal found in Pakistan. The present study evaluates the composition and antioxidant potential of various parts of the A. lebbeck . Compositional studies indicated carbohydrates as major components while saponin was found as a major antinutrient in both pods and seeds. Potassium was found in the highest amount and copper in the lowest. The amino acid profile indicated that arginine and lysine are present in excessive amounts in seeds while glutamic acid and aspartic acid are present in the highest concentrations in pods. While the linoleic acid was detected as the major fatty acid in pod and oil, α-tocopherol was determined as the major tocopherol component in oil. In vitro antioxidant assays such as ferric reducing antioxidant power, total radical-trapping antioxidant parameter, and Trolox equivalent antioxidant capacity showed that the examined extracts have potent antioxidant potential.

Key words: Albizia lebbeck (L.) Benth., amino acids, minerals, α-tocopherol, ferric reducing antioxidant power, digestibility

1. Introduction besides having anticancer and spermicidal properties (9). Albizia lebbeck (L.) Benth. (Mimosaceae), known locally The are aphrodisiac, emollient, and maturant, and as shirish, is an unarmed deciduous woody tree, 12–21 m their smell is useful in hemicranias (4). Flowers are used in height, having pale bark with glabrous young shoots. for the treatment of spermatorrhea. Its seeds are eaten It is cultivated in many parts of Pakistan in farmlands, after boiling by native people. The seeds are aphrodisiac, along roadsides, on irrigated plantations, along rivers, astringent, and used as brain tonic as well as for treating and as an ornamental plant in gardens due to its pleasant gonorrhea, while the seed oil is applied topically to cure appearance (1,2). The plant has a remarkable reputation leucoderma (10). Seeds are also used to cure piles, diarrhea, due to its food, feed, and medicinal value. It is considered and scrofulous swellings. The pod extract is believed to a potent alexipharmic, and every part of it is prescribed for possess antiprotozoal, hypoglycemic, antidiabetic, and the treatment of bites and stings from venomous animals. anticancer properties (6,11). Besides use as fuel wood, it is Its are reported to be good for ophthalmic diseases, grown as fodder for camels, goats, sheep, and other cattle night blindness, syphilis and ulcer (3,4), cold, cough, and in Pakistan, which eat its leaves, twigs, seeds, and pods. respiratory disorders (5,6). The leaves are also used as cattle fodder, mulch, and manure due to high nitrogen The plant is also used as a windbreak crop. contents (7). The bark is bitter, cooling, alexiteric, and Despite its versatile uses, there are limited data anthelmintic, and cures diseases of blood, leucoderma, on the composition of seeds and pods as well as the itching, skin disease, piles, excessive perspiration, antioxidant capacity of the stem, pods, and root of A. inflammation, bronchitis, and toothache and strengthens lebbeck. In continuation of our series of efforts to explore the gums and teeth; it is used for leprosy, deafness, boils, compositional studies and antioxidant potential of scabies, syphilis, paralysis, and weakness (8). After drying of Pakistan (12–20), we have analyzed the composition of and pounding, it is used as a soap substitute. Its roots seeds and pods and the antioxidant activity of the stem, alleviate spasms and stimulate the cardiovascular system, root, and pods. * Correspondence: [email protected] 25 ZIA-UL-HAQ et al. / Turk J Biol

2. Materials and methods solution, the pH of the hydrolysate was measured and the 2.1. Sample collection percentage of in vitro protein digestibility was calculated A. lebbeck (L.) Benth. pods and seeds were sampled in a from the formula given below (28). collection from the Department of Agronomy, Bahauddin Y = 210.464 – 18.103X, Zakariya University, Multan, Pakistan. The plant materials where X = pH of protein suspension after digestion (10 were air-dried in shadow and laboratory conditions. After min) with multienzyme solution and Y = percentage of drying, the pods and seeds were crushed to coarse powder digestibility. by mortar and pestle. The powdered materials were used 2.7. In vitro starch digestibility for compositional analyses and extract preparations. Ten milliliters of HCl–KCl buffer, with a pH of 1.5, was 2.2. Proximate analysis added to 50 mg of flour sample, and then 0.2 mL of a solution Crude fiber, lipids, ash, protein, and carbohydrates were containing 1 g of pepsin in 10 mL of HCl–KCl buffer was determined according to AOAC methods (21). added to each sample. The samples were incubated at 40 °C for 1 h in a shaking water bath. Volume was completed 2.3. Antinutrient determination to 25 mL with Tris-maleate buffer, pH 6.9. Five milliliters The determination of the cyanide, oxalate, and saponin of α-amylase solution (2.6 UI) in Tris-maleate buffer was contents was carried out using previously reported added to each sample. Samples were then incubated at 37 methods (21–23). Trypsin inhibitor activity was estimated °C in a shaking water bath for 3 h. From this, a 1-mL aliquot using benzoyl-DL-arginine-p-nitroanilide hydrochloric as was taken and placed in a tube that was shaken vigorously substrate (24). at 100 °C for 5 min to inactivate the enzyme. Next, 3 mL 2.4. Mineral content of 0.4 M sodium acetate buffer, pH 4.75, was added to The samples after acid digestion were used for elemental the aliquot, and 60 µL of amyloglucosidase was used to analysis. Sodium and potassium were estimated by flame hydrolyze the digested starch to glucose over 45 min at 60 photometer (Flame Photometer Model-EEL). An atomic °C in a shaking water bath. Volume was adjusted from 10 absorption spectrophotometer (PerkinElmer Model 5000) to 100 mL with distilled water. Triplicate aliquots of 0.5 mL was used to measure calcium, manganese, magnesium, were incubated with glucose oxidase/peroxidase reagent. zinc, copper, and iron, while phosphorus was determined The glucose was converted into starch by multiplying by by the phosphovanado–molybdate method (10). The 0.9 (29). Percentage of starch digestibility was calculated samples were quantified against standard solutions of as percent starch hydrolyzed from the total starch content known concentration that were analyzed concurrently. of the sample. 2.5. Amino acid analysis 2.8. Fatty acid composition Samples (300 mg) were hydrolyzed with 6 M HCl in an The fatty acid composition of the extracted oils was evacuated test tube for 24 h at 105 °C. The dried residue was determined following the ISO draft standard ISO/FIDS dissolved in citrate buffer (pH 2.2) after flash evaporation. 5509 (30). One drop of oil was dissolved in 1 mL of Aliquots were analyzed in an automatic amino acid n-heptane, 50 µL of 2 M sodium methanolate in methanol analyzer (Hitachi PerkinElmer Model KLA 3B), using the was added, and the closed tube was agitated vigorously buffer system described earlier. Methionine and cystine for 1 min. After addition of 100 µL of water, the tube were analyzed separately after performic acid treatment was centrifuged at 4500 × g for 10 min and the lower and subsequent hydrolysis with HC (25). Tryptophan aqueous phase was removed. After 50 µL of 1 M HCl was was determined after alkali (NaOH) hydrolysis by the added to the n-heptane phase, the 2 phases were briefly colorimetric method (26). mixed and the lower aqueous phase was rejected. About 2.6. In vitro protein digestibility 20 mg of sodium hydrogen sulfate was added, and after A multienzyme technique was used to measure the in vitro centrifugation at 4500 × g for 10 min the top n-heptane protein digestibility (27). Fifty milliliters of glass-distilled phase was transferred to a vial and injected into a Varian water was added to the sample (the amount of the sample 5890 gas chromatograph with a capillary column, CP-Sil88 was adjusted so as to contain 6.25 mg/mL) and kept for 1 (100 m long, 0.25 mm i.d., film thickness 0.2 µm). The h at 5 °C to hydrate. The sample suspension was adjusted temperature program was: from 155 °C heated to 220 °C to pH 8.0 with 0.1 N HCl and/or 0.1 N NaOH while (1.5 °C/min), 10 min isotherm; injector 250 °C, detector stirring in a water bath maintained at 37 °C for 15 min. 250 °C; carrier gas 1.07 mL/min H2; split ratio 1:50; detector

The multienzyme solution (1.6 mg/mL trypsin, 3.1 mg/ gas 30 mL/min H2; 300 mL/min air and 30 mL/min N2; mL chymotrypsin, and 1.3 mg/mL peptidase maintained manual injection volume less than 1 µL. The integration in an ice bath at pH 8.0) was added (5 mL) to the protein software computed the peak areas, and percentages of suspension while stirring at a constant temperature of 37 fatty acid methyl esters (FAMEs) were obtained as weight °C. Exactly 10 min after the addition of the multienzyme percent by direct internal normalization.

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2.9. Tocopherol content extracts on hemolysis was calculated and expressed as For determination of tocopherols, a solution of 250 mg of percent inhibition. For comparison, α-tocopherol and studied samples oil in 25 mL of n-heptane was directly used BHA were used. for high-performance liquid chromatography (HPLC). 2.13. Total antioxidant capacity determination The HPLC analysis was conducted using a Merck/Hitachi Plant extracts were analyzed for their antioxidant capacity low-pressure gradient system fitted with an L-6000 pump, by 3 different antioxidant assays: Trolox equivalent a Merck-Hitachi F-1000 fluorescence spectrophotometer antioxidant capacity (TEAC) (35), ferric reducing (detector wavelengths for excitation 295 nm, for emission antioxidant power (FRAP) (36), and total radical-trapping 330 nm), and a D-2500 integration system; 20 µL of the antioxidant parameter (TRAP) (37). The TEAC and TRAP samples was injected by a Merck 655-A40 autosampler values were expressed as micromoles of Trolox per gram onto a diol-phase HPLC column 25 cm × 4.6 mm i.d. of plant extract, and FRAP values were expressed as (Merck) using a flow rate of 1.3 mL/min. The mobile phase micromoles of Fe2+ equivalents per gram of plant extract. used was n-heptane/tert-butyl methyl ether (99 + 1, v/v) 2.14. Statistical analysis (31). All of the analyses were performed in triplicate to minimize 2.10. Preparation of extracts for total phenolic, total the incidence of error. The MSTAT-C statistical computer flavonoid content, and antioxidant assays package was used during the analysis of variance and least A. lebbeck (L.) pods, stems, and roots were collected significant difference calculations. from the Department of Agronomy, Bahauddin Zakariya University, Multan, Pakistan. Plant material (0.5 kg each) 3. Results and discussion was macerated with aqueous methanolic mixture (80:20; Proximate composition (Table 1) indicated presence of v/v, 1 L) at room temperature for 15 days with occasional higher contents of carbohydrates (49.07 ± 0.19% to 57.89 shaking. The extracts obtained were filtered through filter ± 1.03%), crude fiber (5.62 ± 0.41% to 7.71 ± 0.49%), and paper under vacuum and concentrated under reduced proteins (34.17 ± 0.20% to 23.63 ± 0.77%) for seed and pod, pressure in a rotary evaporator (model Q-344B, Quimis) respectively. Our results for crude protein, crude fiber, and using a warm water bath (model Q-214M2, Quimis) to carbohydrate contents for seeds differ from those reported obtain a thick gummy mass, which was further dried in a earlier (38) as 19.40 ± 0.00%, 38.50 ± 0.01%, and 27.30 desiccator and stored in a vial in a refrigerator at 4 °C until ± 0.001% for carbohydrates, crude fiber, and proteins, further use. respectively. Similarly, carbohydrate (32.2 ± 0.03%) and 2.11. Determination of total phenolic and flavonoid crude fiber (11.63 ± 0.21%) values reported earlier (39) contents are very different from our findings. However, our results Extracts were assayed for total phenolic and flavonoid are in partial agreement with some previous findings contents by following the previously reported methods (40) of carbohydrates (43.19 ± 1.20%), crude fiber (2.11 of Heimler et al. (32) and Jia et al. (33), respectively, and ± 0.20%), and proteins (38.60 ± 0.40%). The differences the results were expressed as gallic acid equivalents mg observed may be due to agrogeoclimatic conditions that (GAE)/g and as mg catechin equivalents (CAE)/g. affect the composition of seeds. There is only one report 2.12. Antihemolytic assay on the proximate composition of pods (41). The study of Antihemolytic activity of extracts was studied according to the method of Naim et al. (34), whereby red blood cells Table 1. Proximate chemical composition (%) of Albizia lebbeck obtained from cow blood were diluted with phosphate seed and pod. Data are expressed as the mean ± standard buffered saline to prepare a 4% suspension. The solution deviation; values having different letters differ significantly (P < of extract (500 µg) in 1 mL of saline buffer was added to 0.05). 2 mL of this suspension, and the volume was made up to 5 mL with saline buffer. After incubating the mixture Component Seed Pod at ambient conditions for 5 min, 0.5 mL of 0.3% H2O2 solution in saline buffer was added to the mixture to induce Crude protein 34.17b ± 0.20 23.63b ± 0.77 oxidative degradation in the lipoprotein membrane. Total lipids 6.11c ± 1.40 7.08c ± 0.51 The concentration of H2O2 in the reaction mixture was adjusted to bring approximately 90% hemolysis in Total carbohydrates 49.07a ± 0.19 57.89a ± 1.03 blood cells. The reaction mixture was then centrifuged at 3000 rpm for 10 min and the extent of hemolysis was Crude fiber 5.62c ± 0.41 7.71c ± 0.49 determined by measuring the absorbance at 540 nm to Ash 5.03c ± 0.12 3.69d ± 0.80 measure hemoglobin liberation. Inhibitory activity of

27 ZIA-UL-HAQ et al. / Turk J Biol proteins from unconventional and wild sources is very Table 3. Mineral contents (mg/100 g) of Albizia lebbeck seed and useful to analyze the feasibility of their incorporation into pod. Data are expressed as the mean ± standard deviation; values the diet as a way to combat the nutritional deficiencies of having different letters differ significantly (P < 0.05). the poorest populations, resulting from the intake of foods that do not meet nutritional requirements. The high ash, Mineral Seed Pod crude fiber, and carbohydrate content indicate that pod and seeds should be suitable for use in animal feed as Calcium 391.38b ± 0.27 104.36c ± 1.42 supplementary material. Copper 0.56f ± 0.04 1.02f ± 051 As seen in Table 2, cyanide, oxalate, saponin, and trypsin inhibitory activity were determined as antinutrient Iron 92.34c ± 1.52 7.35e ± 0.73 compounds and the content of saponin was the highest in seeds and pods. The antinutrients observed were cyanide Magnesium 79.08c ± 1.18 159.18b ± 0.44 (0.21 ± 0.13 and 2.35 ± 1.01 mg/kg), oxalate (0.04 ± 0.45 Manganese 39. 51d ± 0.35 35.29d ± 0.65 and 1.53 ± 0.62 mg/kg), saponin (834.13 ± 0.69 and 1174.08 ± 3.05 mg/kg), and trypsin inhibitor activity (16.4 Phosphorus 31.45d ± 0.47 6.11e ± 0.89 ± 0.31 and 44.56 ± 1.88 µg/mg flour). Our results for seeds are in partial agreement with some previous findings Potassium 599.22a ± 1.66 178.63a ± 0.92 (38,39,42,43) due to different measuring units involved. Sodium 381.75b ± 1.73 106.32c ± 0.11 We encountered no research about antinutritional content of A. lebbeck pod. The antinutrients may reduce feed intake Zinc 18.07e ± 0.77 3.09.80f ± 1.04 and bioavailability of minerals; however, the content of Na:K 0.63 0.09 these antinutrients is very low and may not cause any undesirable effects. The relatively high saponin content Ca:P 12.44 0.21 of the pods may be responsible for spermicidal effects of pods. It may be suggested that the removal of the pod from the seed should increase the nutritional value of the agrogeoclimatological conditions, maturity, and collection material, because the pods include higher antinutritional time, as well as plant tissue, soil, water, and fertilizer components than the seed. composition, along with permissibility, selectivity, and Mineral contents indicated that potassium was in the absorbability of for the uptake of these metals (20). highest concentration (599.22 ± 1.66 and 178.63 ± 0.92 Amino acid profile generally indicates the nutritive mg/100 g), followed by sodium (381.75 ± 1.73 and 106.32 value of a protein. The data (Table 4) indicated that arginine ± 0.11 mg/100 g), while copper was found in the lowest (14.59 ± 0.16%), lysine (13.48 ± 0.07%), and histidines amount (0.56 ± 0.04 and 1.02 ± 051 mg/100 g) for seed and (11.36 ± 0.28%) are present in excessive amounts in seed, pod, respectively (Table 3). Our results are different from while glutamic acid (17.22 ± 0.42%) and aspartic acid (3.03 those reported earlier (38,39,41,42) for seed and pod. The ± 0.90%) are present in the highest concentrations in pod. results indicate the presence of all essential elements in These results are comparable to those of earlier studies (44) sufficient quantities. The seed could also be a good source for seed, despite differences due to different units involved. of magnesium, iron, and selenium in animal diets. The The in vitro protein and starch digestibility was in presence of metals in plants usually depends on cultivars, the range of 41.00 ± 0.13% to 35.81 ± 1.67% and 62.21 ± 0.62% to 47.53 ± 2.12% for seeds and pods, respectively Table 2. Antinutrient contents of Albizia lebbeck seed and pod. (Table 5). There is only one report on digestibility of the Data are expressed as the mean ± standard deviation; values carbohydrates and protein of pods (41). starches having different letters differ significantly (P < 0.05). are generally known to contain more amylose and are less digestible. Antinutrient Seed Pod Owing to their curative properties and health-boosting nutritional bioconstituents, the plant’s oils are used against Cyanide (mg/kg) 0.21c ± 0.13 2.35c ± 1.01 leprosy. Visual observation of oil revealed it to have a light brown color. Fatty acid composition (Table 6) indicated the Oxalate (mg/kg) 0.04c ± 0.45 1.53c ± 0.62 presence of linoleic acid in highest contents (47.13 ± 0.82% Saponin (mg/kg) 834.13a ± 1.69 1174.08a ± 3.05 and 52.39 ± 0.38%), while sufficient amounts of oleic acid (23.37 ± 0.62% and 32.00 ± 0.41%) and palmitic acid (17.02 Trypsin inhibitor activity 16.04b ± 1.31 44.56b ± 1.88 ± 0.24% and 8.83 ± 0.82%) are also present in the seed and (µg/mg flour) pod, respectively. Tocopherol contents (Table 7) are well

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Table 4. Amino acid composition (%) of Albizia lebbeck seed and Table 6. Fatty acid (%) profile ofAlbizia lebbeck seed and pod. pod. Data are expressed as the mean ± standard deviation; values Data are expressed as the mean ± standard deviation; values having different letters differ significantly (P < 0.05). having different letters differ significantly (P < 0.05).

Amino acid Seed Pod Component Seed Pod

Alanine 4.06b ± 0.74 3.68 ± 0.35b 16:0 17.02c ± 0.24 8.83c ± 0.82

Arginine 14.59a ± 0.16 11.44 ± 0.57a 16:1 0.89d ± 0.89 0.17d ± 0.17

Aspartic acid 8.07a ± 0.39 13.03a ± 0. 90 18:0 4.76d ± 0.71 1.22d ± 0.26

Cystine 0.64b ± 0.26 1.20b ± 0.07 18:1 23.37b ± 0.62 32.00b ± 0.41

Glutamic acid 6.22b ± 0.71 17.22a ± 0.42 18:2 47.13a ± 0.82 52.39a ± 0.38

Glycine 3.06b ± 0.02 3. 63b ± 0.91 18:3 5.01d ± 0.56 2.45d ± 0. 80

Histidine 11.36a ± 0.28 2.01b ± 0.74 20:0 1.14d ± 0.43 0.88d ± 0.59

Isoleucine 4.21b ± 0.71 3.03b ± 0.36 20:1 0.68d ± 0.22 0.57d ± 0.08

Leucine 5.34b ± 0.49 6.84b ± 0.66

Lysine 13.48a ± 0.07 5.62b ± 0.54 Table 7. Tocopherol profile (mg/100 g) of oils of Albizia lebbeck seed and pod. Data are expressed as the mean ± standard Methionine 0.55b ± 0.52 1.70b ± 0.37 deviation; values having different letters differ significantly (P < 0.05). Phenylaniline 5.02b ± 0.18 6.30b ± 0.96

Proline 3.81b ± 0.18 4.03b ± 0.63 Tocopherol Seed Pod

Serine 4.01b ± 0.12 5.06b ± 0.29 α-Tocopherol 51.70a ± 0.26 35.99a ± 0.09 Threonine 3.23b ± 0.06 3.91b ± 0.39 β-Tocopherol 11.04c ± 0.39 2.04c ± 0.35 Tryptophan 4.61b ± 0.24 1.00b ± 0.61 γ-Tocopherol 20.66b ± 0.11 13.29b ± 1.17 Tyrosine 4.71b ± 0.41 2.30b ± 0.30 δ-Tocopherol 1.53d ± 0.74 0.04d ± 0.58 Valine 3.03b ± 0.04 3.10b ± 0.39 Total 84.93 ± 0.55 51.36 ± 2.19

Table 5. In vitro protein and starch digestibility of Albizia be interesting for the production of naturally occurring lebbeck seed and pod. Data are expressed as the mean ± standard tocopherols for the stabilization of fats and oils against deviation; values having different letters differ significantly (P < oxidative deterioration and for applications in dietary, 0.05). pharmaceutical, or biomedical products. Total phenolic and flavonoid content determination Parameter Seed Pod from herbal extracts is considered necessary to study In vitro protein digestibility 41.00b ± 1.55 35.81b ± 1.67 the potential of plants for disease prevention and is the first step in the determination of the antioxidant activity In vitro starch digestibility 62.21a ± 1.88 47.53a ± 2.12 of herbal extracts (45). The total phenolic and flavonoid contents of the pods (51.21 ± 0.25 and 39.07 ± 0.63 mg/g), stem (209.08 ± 1.03 and 371.27 ± 2.12 mg/g), and root in line with those reported earlier (44), and appreciable (102.01 ± 1.41 and 89.08 ± 1.02 mg/g) appear in Table 8. amounts of these constituents are present. α-Tocopherol Significant levels of polyphenolic compounds have also was found in the highest amount (51.7 ± 0.26 mg/100 g and been found in the extracts of various parts of A. lebbeck 35.99 ± 0.09 mg/100 g) in total tocopherol content while from Egypt (46), India (47), Brazil (48), and Cuba (49). δ-tocopherol was found in the lowest amount (1.53 ± 0.74 However, there are very few data on the total phenolic and mg/100 g and 0.04 ± 0.58 mg/100 g) for seeds and pods, total flavonoid contents of the root, stem, and pods of A. respectively (Table 7). High amounts of tocopherols can lebbeck.

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Polyunsaturated fatty acids are major constituents of same trend was observed for TEAC and TRAP assays, and erythrocyte membranes and are vulnerable to free radical the order of antioxidant potential observed was stem > root attack leading to erythrocyte membrane damage and > pod. Usually the stem has a higher antioxidant potential hemolysis. Phenolic compounds present in crude extracts than other plant parts, as per our previous findings for may have the ability to quench hydroxyl radicals before the other (19). The distinct scavenging activities of radicals attack biomolecules of the erythrocyte membrane methanolic extracts from different parts may be due to to cause oxidative hemolysis. The percent inhibition the diverse chemical nature of various phytochemicals. observed for pods, stem, root, BHA, and α-tocopherol All parts of A. lebbeck may be good candidates for further was 41.02 ± 0.29, 48.21 ± 0.76, 56.11 ± 0.12, 69.43 ± 0.34, development as antioxidant remedies. The presented results and 63.14 ± 0.19 in 500 µg/mL, respectively (Table 9). Like are in the agreement with those published earlier (47). They the other parameters, no previous study exists on stem, suggest that the pods, root, and stem may act as free radical pods, and root. The results from the antioxidant assays scavengers, which are capable of transforming reactive showed that examined extracts showed potent antioxidant species into stable nonradical products. activity (Table 10), indicating their ability to act as radical A simple linear regression analysis was used to analyze scavengers to various extents. Antioxidant capacity assays the correlation between the TEAC values and total phenolic mainly focus on thermodynamic conversion and measure contents (Figure 1a). A positive linear correlation was the number of electrons donated or radicals quenched by a given antioxidant molecule. In the FRAP assay, antioxidant 1200 components present in crude extract donate a single a 1000 electron to the ferric-tripyridyltriazine (Fe(II)-TPTZ) complex, reducing it to blue ferrous TPTZ (Fe(II)-TPTZ) 800 complex, which absorbs strongly at 593 nm. The highest FRAP values were observed for stem extracts (514.23 ± 2.02 600 µmol/g), indicating greater antioxidant capacity than in root TEAC (254.90 ± 1.50 µmol/g) and pod (137.22 ± 1.24 µmol/g). The 400 TPC vs TEAC (µmol/g) 200 Table 8. Total phenolic content (mg/g dry basis) and total TPC vs FRAP (µmol/g) flavonoid content (mg/g dry basis) of Albizia lebbeck root, stem, TPC vs TRAP (µmol/g) 0 and pods. Data are expressed as the mean ± standard deviation; values having different letters differ significantly (P < 0.05). b 500 Total Total Sample phenolic content flavonoid content 400

Pod 51.21c ± 0.25 39.07c ± 0.63 FRAP 300 Root 102.01b ± 1.41 89.08b ± 1.02 200 Stem 209.08a ± 1.03 371.27a ± 2.12

100 c Table 9. In vitro antihemolytic activity of Albizia lebbeck pods 180 and leaves of root, stem, and pods. Data are expressed as the mean ± standard deviation; values having different letters differ 160 significantly (P < 0.05). 140 Sample Antihemolytic activity (%) TRAP 120 Pod 41.02d ± 0.29 100 Root 48.21c ± 0.76

Stem 56.11b ± 0.12 80 40 60 80 100 120 140 160 180 200 220 BHA 69.43a ± 0.34 Total phenol c contents Figure 1. Correlation between the total phenolic contents (TPC) α-Tocopherol 63.14a ± 0.19 and a) TEAC, b) FRAP, and c) TRAP assays.

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Table 10. Antioxidant activity of Albizia lebbeck pods and leaves of root, stem, and pods. Data are expressed as the mean ± standard deviation; values having different letters differ significantly (P < 0.05).

Sample TEAC (µmol/g) FRAP (µmol/g) TRAP (µmol/g)

Pod 207.50c ± 2.05 137.22c ± 1.24 98.39b ± 1.12

Root 483.23b ± 1.42 254.90b ± 1.50 188.07a ± 0.88

Stem 981.33a ± 3.01 514.23a ± 2.02 98.21b ± 1.74 observed. The regression equation Y = –29.95 + 4.86 was This study indicates A. lebbeck to be a cheap, reliable, statistically significant (F = 3.22, P < 0.0245), and the and safe plant-based resource to meet the demand for coefficient of determination was2 R = 0.99. The results suggest protein-rich foods. It may provide healthy and useful food that the 2 methods are generally consistent for evaluating by providing many of the nutrients the human body needs, antioxidant capacities. Similarly for the correlation between as it is high in protein, cholesterol-free, high in dietary FRAP values and total phenolic contents (Figure 1b), the fibers, and low in saturated fat. On the basis of the results regression equation Y = 13.08.2 + 2.39x was statistically of this study, it is concluded that extracts of A. lebbeck significant (F = 11.29, P < 0.0067), and the coefficient of have significant antioxidant activity along with nutritional determination (R2) was 0.92. For the correlation between TRAP values and total phenolic contents, the regression value, as compared to other well-characterized, standard equation Y = 144.201 – 0.13x was also statistically significant antioxidant systems in vitro. This study highlights the need (F = 0.547, P < 0.8696), and the coefficient of determination for further research on the isolation and characterization was R2 = 0.041 (Figure 1c). The results indicate a positive of the constituents from extracts in order to decode the linear correlation, and the total antioxidant capacities of A. specific phytochemical constituent(s) responsible for the lebbeck can be attributed to their phenolic contents. antioxidant activity of the plant.

References

1. Ali SI. Flora of West Pakistan, Mimosaceae. Shamim Printing 10. Kirtikar KR, Basu BD. Indian Medicinal Plants. Lalit Mohan Press. Karachi, Pakistan; 1973: pp. 1–41. Publication. Allahabad, India; 2000. 2. Burkil HM. The Useful Plants of West Tropical Africa. Royal 11. Uma B, Prabhakar K, Rajendran S et al. Antimicrobial activity Botanic Gardens, Kew. London; 2000: pp. 11–13. of Albizzia lebbeck Benth against infectious diarrhoea. The 3. Hussain MM, Rahman MS, Jabbar A et al. Phytochemical and Internet J Micro 7: 1–6, 2009. biological investigations of Albizzia lebbeck Benth. Bol Latin 12. Zia-Ul-Haq M, Iqbal S, Ahmad S et al. Nutritional and Car Plant Med Aromat 7: 273–278, 2008. compositional study of desi chickpea (Cicer arietinum L.) 4. Pathak N, Gohil P, Patel NB et al. Curative effect of Albizia cultivars grown in Punjab, Pakistan. Food Chem 105: 1357– lebbeck methanolic extract against adjuvant arthritis-with 1363, 2007. special reference to bone erosion. Int J Pharm Sci Drug Res 1: 13. Zia-Ul-Haq M, Ahmad M, Iqbal S et al. Characterization and 183–187, 2009. compositional studies of oil from seeds of desi chickpea (Cicer 5. Pratibha N, Saxena VS, Amit A et al. Anti-inflammatory arietinum L.) cultivars grown in Pakistan. J Am Oil Chem Soc activities of Aller-7, a novel polyherbal formulation for allergic 84: 1143–1148, 2007. rhinitis. Int J Tissue Res 26: 43–51, 2004. 14. Zia-Ul-Haq M, Iqbal S, Ahmad M. Characteristics of oil from 6. Saha A, Ahmed M. The analgesic and anti-inflammatory seeds of 4 mungbean [Vigna radiata (L.) Wilczek] cultivars activities of the extract of Albizia lebbeck in animal model. Pak grown in Pakistan. J Am Oil Chem Soc 85: 851–856, 2008. J Pharm Sci 22: 74–77, 2009. 15. Zia-Ul-Haq M, Iqbal S, Ahmad S et al. Antioxidant potential 7. Benthal AP. The of Calcutta and its Neighborhood. of desi chickpea varieties commonly consumed in Pakistan. J Thacker-Spink and Co. Ltd. London; 1933: pp. 140–225. Food Lipid 15: 26–342, 2008. 8. Verma N, Srivastav RK. Analgesic, antipyretic and antiinflammatory activities of Albizia lebbeck Benth. seeds. 16. Zia-Ul-Haq M, Ahmad S, Ahmad M et al. Effects of cultivar Pharma 3: 1209–1216, 2011. and row spacing on tocopherol and sterol composition of chickpea (Cicer arietinum L) seed oil. Tarim Bilim Der 15: 9. Kumar A, Saluja AK, Shah UD et al. Pharmacological potential 25–30, 2009. of Albizzia lebbeck: a review. Pharm Rev 1: 171–174, 2007.

31 ZIA-UL-HAQ et al. / Turk J Biol

17. Zia-Ul-Haq M, Ahmad S, Chiavaro E et al. Studies of oil from 35. Pellegrini N, Serafini M, Colombi B et al. Total antioxidant cowpea (Vigna unguiculata (L.) Walp.) cultivars commonly capacity of plant foods, beverages and oils consumed in Italy grown in Pakistan. Pak J Bot 42: 1333–1341, 2010. assessed by 3 different in vitro assays. J Nut 133: 2812–2819, 2003. 18. Zia-Ul-Haq M, Ahmad S, Shad MA et al. Compositional studies of some of lentil cultivars commonly consumed in 36. Benzie IFF, Strain JJ. The ferric reducing ability of plasma Pakistan. Pak J Bot 43: 1563–1567, 2011. (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal Biol 239: 70–76, 1996. 19. Zia-Ul-Haq M, Ahmad S, Iqbal S et al. Antioxidant potential of lentil cultivars commonly consumed in Pakistan. Oxid Comm 37. Ghiselli A, Serafini M, Maiani G et al. A fluorescence-based 34: 819–831, 2011. method for measuring total plasma antioxidant capability. Free Rad Bio Med 18: 29–36, 1995. 20. Zia-Ul-Haq M, Shahid SA, Ahmad S. Mineral contents and antioxidant potential of selected legumes of Pakistan.J Med 38. Muhammad NO, Jimoh FO, Nafiu MO et al. Nutrients and Plants Res 6: 2256–2260, 2012. antinutrients analysis of Albizia lebbeck seed. Biores Bull 4: 161–165, 2010. 21. AOAC. Official Methods of Analysis of the Association of Official Analytical Chemists. Association of Official Analytical 39. Mishra SS, Gothecha VK, Sharma A. Albizia lebbeck: a short Chemists. Washington, DC; 1990. review. J Herbal Med Toxic 4: 9–15, 2010. 22. Iwuoha CI, Kalu FA. Calcium oxalate and physico-chemical 40. Oderinde RA, Adewuyi A, Ajayi IA. Determination of the properties of coco yam (Colocasia esculenta and Xanthoso mineral contents, characterization and analysis of the fat- maagittifolium) tuber flours as affected by processing. Food soluble vitamins of Caesalpinia pulcherrima and Albizia lebbeck Chem 54: 61–66, 1995. seeds and seed oils. Seed Sci Biotech 2: 74–78, 2008. 23. Joslyn MA. Methods in Food Analysis. Academic Press. 41. Hassan LG, Umar KJ, Atiku I. Nutritional evaluation of Albizia London; 1970. lebbeck (L.) pods as source of feeds for livestock. Amer J Food Tech 2: 435–439, 2007. 24. Kakade ML, Simons N, Liener IE. An evaluation of natural vs. synthetic substrates for measuring the antitryptic activity of 42. Adubiaro HO, Olaofe O, Akintayo ET. Chemical composition, soybean samples. Cereal Chem 46: 518–528, 1969. calcium, zinc and phytate interrelationships in Albizia lebbeck and Daniellia oliveri seeds. Elect J Envir Agric Food Chem 10: 25. Khalil LA, Durani FR. Haulm and hull of peas as a protein 2523–2530, 2011. source in animal feed. Sarhad J Agri 6: 219–225, 1990. 43. Carvalho AFU, Farias DF, Bezerra LCBR et al. Preliminary 26. Freidman M, Finely JW. Methods of tryptophan analysis. J Agri assessment of the nutritional composition of underexploited Food Chem 19: 626–631, 1971. wild legumes from semi-arid Caatinga and moist forest 27. Ekpenyong TE, Borchers RL. Digestibility of proteins of environments of northeastern Brazil J Food Comp Anal 24: winged bean seed. J Food Sci Tech 16: 92–95, 1979. 487–493, 2011. 28. Hsu HW, Vavak DL, Satterlee LD et al. A multienzyme 44. Mariod A, Matthäus B. Physico-chemical properties, fatty acid technique for estimating protein digestibility. J Food Sci 42: and tocopherol composition of oils from some Sudanese oil 1269–1271, 1977. bearing sources. Grasasy Aceit 59: 321–326, 2008. 29. Goni I, Garcia-Alonso A, Saura-Calixto FA. A starch hydrolysis 45. Tosun M, Ercisli S, Sengul M et al. Antioxidant properties and procedure to estimate glycemic index. Nut Res 17: 427–437, total phenolic content of eight Salvia species from Turkey. Biol 1997. Res 42: 175–181, 2009. 30. International Organization for Standardization. ISO/FIDS 46. El-Hawary S, El-Fouly K, Sokkar NM et al. A phytochemical 5509. International Standards, 1st ed. Geneva; 1997. profile ofAlbizia lebbeck (L.) Benth. cultivated in Egypt. Asian 31. Balz M, Shulte E, Their HP. Trennung von Tocopherol und J Biochem 6: 122–141, 2011. Tocotrienolendurch HPLC. Fat Sci Tech 94: 209–213, 1992. 47. Anusuya N, Anusuya S, Manian R et al. Antioxidant and free 32. Heimler D, Vignolini P, Dini MG et al. Rapid tests to assess the radical scavenging activity of certain dietary and medicinal antioxidant activity of Phaseolus vulgaris L. dry bean. J Agri plant extracts. Food 3: 47–52, 2011. Food Chem 53: 3053–3056, 2005. 48. Miranda CG, Arantes MDCB, Rezende MH et al. 33. Jia Z, Tang M, Wu J. The determination of flavonoid contents in Pharmacognostic characterization of leaves and seeds of mulberry and their scavenging effects on superoxide radicals. Albizia lebbeck (L.) Benth. (). Rev Braz Pharm 19: Food Chem 64: 555–559, 1999. 536–544, 2009. 34. Naim M, Gestener B, Bondi A et al. Antioxidant and 49. Rodríguez Y, Chongo B, La O et al. Chemical characteristics antihemolytic activities of soybean isoflavones. J Agri Food of Albizia lebbeck and determination of its nutritive potential Chem 24: 1174–1177, 1976. through the technique of gas in vitro production. Cuban J Agri Sci 39: 305–310, 2005.

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