Investigation of the Potential Use, Phytochemical and Element Contents of Acacia Plant Seeds Grown in Wild Form, Considered as Environmental Waste

. Recently, plant seeds food source, nutritional components supplements nutritional needs of people daily life their protein contents, carbohy-drates, a large number of secondary metabolites 7 − 9 ） . In ad-dition, the interest in oil is increasing due to the physiologi-cal effects of unsaturated fatty acids. Therefore, new plant seeds as an oil source should be investigated 10, 11 ） . It has been reported that similar plant seeds have been used for this purpose recently 12 ） . The characterisctics of oils change depending on the fatty acid composition, which is of great importance due to their beneficial effects 13 ） . Vegetable oils have been found to consist of a complex mixture contain-Abstract: In this study, the effect of altitude on oil amounts, antioxidant activity, polyphenol content and mineral contents of Acacia seeds collected from two different locations (up to 1100 m above sea level) was investigated. Total carotenoid and flavonoid contents of Acacia seeds were detected as 0.76 (Konya) and 1.06 µg/g (Taşucu-Mersin) to 1343.60 (Konya) and 184.53 mg/100 g (Taşucu-Mersin), respectively. Total phenol contents and antioxidant activity values of Acacia seeds were identified as 255.11 (Konya) and 190.00 mgGAE/Taşucu-Mersin) to 64.18% (Konya) and 75.21% (Taşucu-Mersin), respectively. The oils extracted from Acacia seeds in Konya and Mersin province contained 62.70% and 70.39% linoleic, 23.41% and 16.03% oleic, 6.45%and 6.04% palmitic and 2.93% and 4.94% stearic acids, respectively. While 3,4-dihydroxybenzoic acid amounts of seeds are determined as 3.89 (Konya) and 4.83 mg/100 g (Taşucu-Mersin), (+)-catechin contents of Acacia seeds were identified as 3.42 (Konya) and 9.51 mg/100 g (Taşucu-Mersin). Also, rutintrihydrate and ferulic contents of Acacia seeds were found as 23.37 (Konya) and 11.87 mg/100 g (Taşucu-Mersin) to 14.74 mg/100 g (Konya) and 1.12 mg/100 g (Taşucu-Mersin), respectively. Acacia seeds collected from Konya and Mersin contained 4003.75 and 3540.89 mg/kg P, 9819.12 and 16175.69 mg/kg K, 4347.47 and 5078.81 mg/kg P, 2195.77 and 2317.90 mg/kg Mg, 1015.75 and 2665.60 mg/ kg S and 187.53 and 905.52 mg/kg Na, respectively.

ing a wide variety of compounds 14 16 . Because, the composition of triacylglycerol is known as a measurement criterion for the quality of vegetable oils 17 19 . Recently, the continuous increase in the number of industries using vegetable oil as the raw material has led to a gradual increase in the demand for domestic vegetable oils 20,21 . As a result of various studies, it has been determined that Acacia species are rich in terpenes, flavonoids and condensed tannins 22 27 . Shrubs and legumes such as Acacia species are widely used as a feed source for livestock in tropical and subtropical regions of the World 28 . Plant seeds, especially underused plant seeds such as Acacia are now creating new oil sources. Limited information is known about the composition, quantitative values and quality characteristics of phenolic components in Acacia seeds. Also, it is not clear how the phenolic constituents, bioactive properties, fatty acids and mineral profiles of Acacia seeds may vary depending on locations. The objective of the present work was to investigate the effect of altitude on oil amounts, antioxidant activity, polyphenol content and mineral content of Acacia seeds collected from two different locations up to 1100 m above sea level .

Material
Acacia Robinia pseudoacacia and gladiçya Gleditsia triacanthos seeds were provided from Konya 1100 m high level and Taşucu-Mersin sea level provinces in Turkey. Seed samples were harvested in October 2020. During the day and night in Konya and Mersin, the temperatures varied between about 20-24 and 10-12 to 24-28 and 18-22 in October, respectively. Seeds brought to the laboratory in paper bags were immediately dried at normal room temperature for one week, and then they were stored upto analysis.

Moisture content
Moisture contents of Acacia seeds were detected at 105 using an oven Nüve FN055 Ankara, Turkey up to constant weight 29 .

Oil content
The oil yields of seeds belong to each color type were established according to AOAC 30 . About 10 g of seed powder was placed into a Soxhlet section and extracted using petroleum ether at 50 for 5 hours, and then it was evaporated by the evaporator at 50 .

Carotenoid content
After the 2 g Acacia seed powder was mixed with 25 mL of acetone, it was vortexed for 10 min. Then, after it was filtrated by filter paper Whatman No. 1 , their phases were separated in a seperation funnel. The filtrate was mixed with 20 mL of petroleum ether and washed triplicate with 100 mL of distilled water. The absorbance value of seed extract dissolved in 25 mL petroleum ether was measured at 450 nm 31,32 .

Extraction procedure
For extraction of seed powder, after about 2.5 g ground Acacia seed was mixed with 25 mL methanol:water solution 80:20, v/v , it was shaken in rinsing water-bath for 72 h at 25 . Then, it was filtrated with Whatman No:1. The extract was evaporated at 40 in a rotary evaporator with vacuum, followed by dissolved in 10 mL methanol: water mixture 80/20, v/v . Before analyses, the extract was filtered through a 0.45 µm filter 33 .

Total phenolic content
The Folin-Ciocalteu was used for analysis of total phenol contents of Acacia seed extracts 32 . After 1 mL Folin-Ciocalteu and 10 mL 7.5 Na 2 CO 3 were added on seed extract, it was stirred by a vortex. After the final volume of the sample solution was completed to 25 mL with deionized water, the mixture was left in the dark for 1 hour. The absorbances of seed extracts were recorded at 750 nm in a spectrophotome. The results obtained were shown as mg gallic acid equivalent/100 g fw . 2.2.6 Total flavonoid content After 0.3 mL 7.5 NaNO 2 , 0.3 mL of AlCl 3 and 2 mL of NaOH, respectively, were added onto 1 mL extract, it was was mixed by vortex for 1 min 38 . Then, it was left in dark for 15 min. The absorbance of sample was determined at 510 nm. The results are given as mg catechin CA /100 g of fresh weight 34,35 .

Antioxidant activity
The DPPH 1.1-diphenyl-2-picrylhydrazyl was applied for free radical scavenging activity of Acacia seed extracts 36 . After 2 mL of DPPH in methanol was added onto 0.2 mL extract, it was stirred by vortex. Then, it was stored 30 min incubation in the dark at room. Later, the absorbances of extract samples were recorded at 517 nm. Following relation was used for calculation of antioxidant activity samples: Inhibition 100

Fatty acid composition
The fatty acid methyl esters FAMEs of Acacia seed oils 37 were determined using a gas chromatography Shimadzu GC-2010 consisting of flame-ionization detector FID and capillary column. The nitrogen with 1.51 mL/min flow rate and 80 mL/min were applied for the carrier gas and total flow rate, respectively Aljuhaimi et al., 2019 . The temperature of injection block and detector was 260 and column temperature was programmed 120 for 5 minutes and increased 240 at 4 /min and held 25 minutes at 240 30,38 .

Determination of phenolic compounds
A Shimadzu-HPLC equipped with a PDA detector and an Inertsil ODS-3 column was applid for the qualification and quantification of phenolic compounds were performed. 20 µl of the extract was injected and run at 1 mL/min at 30 for a total running time of 60 min. The peaks were measured at 280 and 330 nm using a PDA detector 38,39 . The total running time per sample was 60 min. 2.2.10 Determination of mineral Acacia seeds were dried in a drying cabinet at 70 up to constant weight. Then, 0.5 g Acacia seed powder digested by using 5 mL of 65 HNO 3 and 2 mL of 35 H 2 O 2 in a closed microwave system Cem-MARS Xpress were brought to 20 mL with ultra-deionized water till the volumes. ICP OES Varian-Vista, Australia was applied for mineral contents of Acacia seed sample 40 .

Statistical Analyses
All analysis were done three times and data of triplicate analyses were averaged and subjected to analysis of student t-test. Results were described as mean standard deviation of two different Acacia seeds 41 .

The physico-chemical and bioactive properties of
Acacia seeds The physico-chemical and bioactive properties of Acacia seeds provided from two different locations are shown in Table 1. Results showed differences depending on altitude and locations. While moisture contents of seeds are measured as 7.41 Konya and 7.50 Taşucu-Mersin , the oil contents of Acacia seeds were determined as 4.30 Konya and 3.00 Taşucu-Mersin . Also, while total carotenoid amounts of Acacia seeds are detected as 0.76 Konya and 1.06 µg/g Taşucu-Mersin , total flavonoid contents of seed samples were recorded as 1343.60 mg/100 g Konya and 184.53 mg/100 g Taşucu-Mersin . Total phenol amounts of Acacia seeds were identified as 255.11 mgGAE/100 g Konya and 190.00 mgGAE/100 g Taşucu-Mersin while antioxidant activity values of Acacia seeds are found as 64.18 Konya and 75.21 Taşucu-Mersin . In general, oil contents, bioactive compounds of Acacia seeds provided from Konya distinct were determined higher than those of values of Acacia seeds growing in Mersin province. The seeds of 12 species of Acacia contained 2.5-10.2 oil 42 . The oil contents of Acacia cyclops, Acacia cyanophylla and Acacia mollissima seeds were 8.85 , 11.13 and 7.16 , respectively 43 . Acacia arabica and A. raddiana seeds contained 10.06 and 5.14 oils, respectively 3 . The oil contents of Acacia species changed between 6.83 A. ligulata and 12.18 A. salicina 18 . Youzbachi et al. 27 determined that A. cyanophylla seed in Tunisia contained about 10 oil. Dry matter, total phenolic, and total flavonoid contents of Acacia victoriae seeds roasted at different times changed between 13.96 and 15.49 , 1.19 and 12.19 mg GAE/g and 0.23 and 1.93 mgCE/g, respectively 25 . The total phenol, total flavonoid and total carotenoid contents of Acacia seed extracts changed between 17.5 and 18.2 mgGAE/g, 4.9 and 6.3 mgCAE/g and 14.2 and 15.5 mg/g, respectively. Wild Acacia seeds contained 10.2 oil and 180.44 mgGAE/100 g total phenol 44 . The antioxidant activity values of the methanol extracts of Acacia nilotica seeds varied between 73 and 78 45 . Some variations in this study were observed compared to previous results. These differences among results can be probably due to climatic conditions, altitude, some analytical conditions and genetic factors. There was established statistically significant variations among physico-chemical and bioactive properties of Acacia seeds p 0.05 . The content of total phenolic substances constantly changes depending on various factors such as plant species, genetics, location, and climate 46,47 .

The fatty acid compositions of Acacia seed oils
The fatty acids of Acacia seed oils are presented in Table  2. Acacia seed oil extracted from Acacia seeds grown in Konya province contained 62.70 linoleic, 23.41 oleic, 6.45 palmitic and 2.93 stearic acids. The oil obtained In general, stearic, oleic, arachidic, linolenic, behenic and arachidonic acid contents of Acacia seed oil provided from Mersin province were higher compared to results of Acacia seed oil provided from Konya distinct. The fatty acid profiles of both Acacia samples were different as quantity. Acacia seed oil contained more than 70 of unsaturated fatty acids and Acacia seed oil provided collected from Konya showed the highest level with about 86.00 Table 2 . While palmitic acid was high in Acacia seed oil from myrtle, oleic acid was high in Acacia seed oil from Konya. The fact that Mersin location is warmer than Konya may have caused the palmitic acid content to be high. Adewusia et al. 48 reported that Acacia colei and Acacia tumida seed oils contained 9.4-14.6 palmitic, 3.5-7.0 stearic, 18.0-23.5 oleic, 31.7-55.9 linoleic, 1.2-4.1 arachidonic and 1.0-1.8 lignoceric acids. The oil of Acacia species grown in Tunisia contained 9.63-15.10 palmitic, 0.8-2.10 stearic, 15.06-27.13 oleic, 52.39-68.1 linoleic, 0.47-1.08 arachidic acids 18 . The oil of wild Acacia seeds contained 0.14 myristic, 9.97 palmitic, 0.39 palmitoleic, 1.82 stearic, 22.71 oleic, 61.29 linoleic and 0.52 arachidic acids 44 . Mukhanwati 45 reported that Acacia nilotica seed oils contained 13.4-13.7 palmitic, 4.4-4.5 stearic, 30.6-31.8 oleic, 39.6-40.5 linoleic and 2.6-2.8 linolenic and 1.4-1.6 behenic acids. Indeed, previous studies on a large number of plant seeds have shown that environmental conditions have a significant effect on fatty acids. Results exhibited some fluctuations depending on location and altitude. These differences can be probably due to climatic factors of locations, altitude, soil structure, enetic position and harvest time.

The phenolic compounds and their quantity values of
Acacia seeds collected from two different locations The phenolic compounds and their quantity values of Acacia seeds provided from both locations are illustrated in Table 3. The major phenolic constituents of Acacia seed samples were 3,4-dihydroxybenzoic acid, -catechin, caffeic acid, syringic acid, rutintrihydrate, p-coumaric acid and ferulic acid. While 3,4-dihydroxybenzoic acid amounts of seed samples are determined as 3.89 mg/100 g Konya  and 4.83 mg/100 g Taşucu-Mersin , -catechin contents of Acacia seeds were identified as 3.42 mg/100 g Konya and 9.51 mg/100 g Taşucu-Mersin . Also, rutintrihydrate contents of Acacia seeds were found as 23.37 mg/100 g Konya and 11.87 mg/100 g Taşucu-Mersin while ferulic acid amounts of Acacia seed samples are detected as 14.74 mg/100 g Konya and 1.12 mg/100 g Taşucu-Mersin . In addition, quercetin amounts of seed samples were measured as 3.64 mg/100 g Konya and 4.60 mg/100 g Taşucu-Mersin . Also, Acacia seeds provided from Konya province contained 2.91 gallic acid, 5.29 syringic acid, 2.44 p-coumaric acid and 2.76 mg/100 g kaempferol. In general, the amounts of phenolic constituents of Acacia seeds provided from Konya were higher except 3,4-dihydroxybenzoic acid, caffeic acid, and quercetin compared to results of Acacia seeds provided from Mersin distinct. There were established statistically significant variations among the amounts of phenolics of both Acacia seeds p 0.05 . Climatic and soil factors had affected on phenolic constituents of Acacia seeds provided from Konya and Mersin provinces. In addition, it is thought that phenolic components in Konya Acacia seeds are partially high due to the fact that the temperature is partially lower than Mersin during the growing period of the Acacia plant in Konya location and the weather is cool. Jelessi et al. 43 25 reported that succinic and gallic acid contents of acetone extract of wattle seeds Acacia victoriae Bentham changed between 0.06 and 0.32 mg/mL to 0.01 and 0.11 mg/mL, respectively. These values are similar or high to those obtained with the seeds of other plants commonly used, such as peanut, corn and soybeen. The differences in the results may have resulted from the genetic structure of the plant, environmental factors such as temperature, rainy, location and altitude.
3.4 The mineral contents of Acacia seeds collected from two different locations The mineral contents of Acacia seeds provided from Konya and Mersin Taşucu-Silifke locations are presented in Table 4. Locations had affected on mineral profiles of Acacia seeds. In general, mineral contents of Acacia seeds collected from Mersin district were found higher than that of collected from Konya province. Acacia seeds collected from Konya  and 100 mg/100 g K, 51.4 and 50.7 mg/100 g P, 25 and 27 mg/100 g Na, 2.5 and 2.4 mg/100 g Mg, 18 and 20 mg/100 g Fe, 2.4 and 2.5 mg/100 g Zn, 1.9 and 1.4 mg/100 g Co, 3.0 and 3.2 mg/100 g Mn, and 0.3 and 0.2 mg/100 g Cu, respectively 45 . Gebeyew et al. 48 determined 850-936 g kg Ca, 41-64 g/kg Mg, 131-141 g/kg P, 195-481 g/kg S, 169-372 g/ kg Mn, 42-121 g/kg Mo, 2.6-4.7 g/kg Zn, 1.6-2.4 g/kg Co, 30.0-88.4 g/kg Cu, 24.7-74.4 g/kg Fe and 2.4-11.2 g /kg Se in some selected Acacia species seeds. Acacia turnda, A. colei seed oils contained 308 and 597 mg/kg Mg, 1424 and 2146 mg/kg Ca, 17 and 48 mg/kg Zn, 194 and 91 mg/kg Fe and 12 and 11 mg/kg Cu, respectively 21 . When the results were compared with results of previous studies, it was observed that some minerals increase and decrease. It has been observed that Acacia seeds obtained from both locations are rich in minerals. Therefore, it is thought that these seeds can be added to feeds as mineral additives. There were observed statistically significant differences among mineral contents of both Acacia seeds depending on locations p 0.05 . These differences in the results obtained may be due to altitude, maturation, climatic factors, precipitation, genetic, locations, and soil structure.

Conclusion
In general, oil contents, total phenolic and total flavonoid amounts of Acacia seeds provided from Konya distinct were determined higher than those of values of Acacia seeds growing in Mersin province. As a result of this study, Acacia seed oil can be a new source of oil due to its high linoleic-oleic acid contents and has economic importance that can meet the edible oil needs of the population living in the areas where it is grown. In addition, oleic, arachidic, linolenic, and behenic acid contents of Acacia seed oil provided from Mersin province were higher compared to results of Acacia seed oil provided from Konya distinct. The amounts of phenolic constituents of Acacia seeds provided from Konya were higher except 3,4-dihydroxybenzoic acid, caffeic acid, and quercetin compared to results of Acacia seeds provided from Mersin distinct. Locations had affected on mineral profiles of Acacia seeds. But, it has been observed that Acacia seeds obtained from both locations are rich in minerals.