PHYTOCHEMICAL DIVERSITY AND NUTRITIONAL VALUE OF KENGER AT DIFFERENT LOCATIONS IN SULAIMANI REGION - IRAQ

Kenger ( Gundelia tournefortii L.) plants were collected based on the availability of these plants in the local market to determine the nutrition value, some phytochemical content, and element content during March 2023 at the vegetative stage at thirteen different locations in the Sulaimani region. Some vegetative traits and chemical contents were studied. The results indicated that the highest content of carbohydrates was (12.670%) at Mawat, the protein was (32.703%) at Hazarmerd, and fiber was (35.620%) at Penjwen locations respectively. The analysis of Kenger edible parts (stem and leaves) through GC-MS revealed the presence of a complex mixture of compounds varying from 13 to 19 compounds among the locations. Compounds such as carboxylic acids ranged from 25.217% at Penjwen to 77.043% at Temar, terpenes ranged from 15.912% in Temar to 57.749% in Penjwen, and sterols ranged from 5.351% at Qara Dagh to 24.688% at Hazarmerd. In other locations, these compounds were detected in between those ranges, with differences in their concentrations. According to the results obtained from ICP-OES analysis, macro-elements (Ca, K, P, S, Mg, and Na) contents between the studied locations, the highest concentration of 3.127% occurred at Temar location, the lowest value was obtained at Gulp location with 2.468%, with an average of 2.811% for all locations.

elements in medicinal plants is crucial to comprehending their pharmacological effects (48).The chemical constituents metal ions in plants, are particularly responsible for medicinal and nutritional properties and also for their toxicity.In the Kurdistan region, the Kenger plant is considered one of the delicious vegetables found in the local markets during spring, and people collect this plant in different locations which grown naturally.Because of the lack of research on this plant in this region, this research aims to provide a comprehensive understanding of the phytochemical constituents and nutritional values of Kenger and provide insights into the safety of consuming Kenger plants based on their elemental content, which may have a significant role and implications for its use in medicine and agriculture.

MATERIALS AND METHOD Plant collection and identification
Kenger plant material was collected during March 2023 in different locations at the Sulaimani region during the growth stage when the plants were at the edible stage, i.e.; before flowering (stem and leaves), (Table 1).The selection of the collected locations was based on the presence of this plant in the local market.Using the Global Positioning System (MGRS UTM GPS version 1.9.4) to recode the information about the locations.Sample specimens were identified and deposited in the University of Sulaimani, College of Agricultural Engineering Sciences.Some morphological traits of Kenger plant were studied at all of the locations, stem length (cm), number of leaves/plants, and dry stem weight/plant (g).Stem dry matter of the plant samples was determined by drying the plant material at 105 o C for 24 hours in an oven.Then, the oven dried samples were weighted and recorded.Chemical analysis: Analysis of Kenger samples for carbohydrate protein and fiber was carried out with the "Reflection Infrared Spectroscopy FT-IR Techniques IRA finitty-1 SHIMADZU-KYOTO JAPAN as an important technique in chemistry (Kurdistan, Sanandaj, Iran -Faculty of Basic Science -Chemistry Department), (51).

Plant Extract Preparation:
The 50g of the fine Kenger plant powder were dissolved in to 500 ml hexane (99%) in an 1L Erlenmeyer flask.The Kenger samples solution was sonicated for 2 h at 50 °C, to ensure complete extraction of the compounds.Then, the flask covered with aluminums foil and kept in a dark place for 24 hrs., the mixture was filtered using 0.4µm filter.The hexane solution was preserved in a sealed glass container and kept at -20 °C until use.GC-MS analysis: Agilent Technologies 7890A gas chromatograph with a mass selective detector and Agilent Technologies 5975C inert XL MSD mass spectrometer was used to analyze the aerial part of Kenger plant samples at University of Kurdistan, Faculty of Basic Science, Chemistry Department, Sanandaj, Iran.The Agilent 190915-433: 325°C (30 m × 250 µm × 0.25 µm) GC-MS column was designed to start at 40°C and increased by 10°C per minute to a maximum temperature of 280°C.The temperature of the heater front inlet was 250°C, the injector port was set to 290 °C, and helium was utilized as the carrier gas at a flow rate of 1 mL/min.The injection mode was split at a ratio of 5:1.At 70 eV, mass spectrometry was used in the electron impact mode (EI).Using the normalization of the GC peak areas, the percentages of the phytochemical components were calculated.The software automatically normalized the peaks by dividing each peak area by the total area and then multiplying by 100.Using the Wiley library, the relative proportions of the constituents were calculated (19).(29), at a high altitudes plant cell size did not reduce, instead, their organs become smaller due to a decreases in cell production.Esbati et al., (16), reported that during the vegetative stage, the mean dry matter increases.Data in Table 2 shows the mean carbohydrate percentage was higher in the Mawat location at 12.670% compared with the other locations, which was statistically significant (p<0.01).The location shows a variation in the amount of protein percentage content which was statically significant (p≤0.01),ranging from 17.427 % in the Kani Tu location and 32.703% in the Hazarmerd location.Hoy et al., (23) concluded that in the mature stage, the content of crude protein decreased.A study by Fang et al., (18) emphasized that carbohydrate and protein content could be increased at the maturity stage, meanwhile, carbohydrate contents decreased.In terms of Fiber content, Kenger samples from Hazarmerd location displayed the lowest value 20.560%, with the highest value 35.620% for the Penjwen location which was statistically significant (p≤0.01).The amount of solid tissues (sclerenchyma tissue) and preservatives increases when plant age is increased (31), which leads to an increase in the amount of crude fiber at the maturity stage.
The results obtained in this study were found to be partially in line with the study conducted by Tuncturk et al., (54), which reported that Kenger had abundant active compounds among the plant species studied.The amount of crude protein and crud fiber, were higher than other species.The dry matter content of Kenger plant was 17.46%, the total crude protein was 21.00% and the crude fiber was 36.44%.(7) revealed that stigmasterol acts as a tumorpromotion inhibitor in mice at two different stages of carcinogenesis.Topical application of a mixture of sitosterol and stigmasterol was observed to have anti-inflammatory action.They conclude that the anticancer properties of Kenger may be influenced by the presence of these sterols.A number of investigations conducted on Kenger plant revealed that it contains a high amount of α-amyrin, lupeol, βsitosterol, and 3-acetyl-lupeol (1,26) 5avenasterol, 7-stigmasterol, campesterol, 7avenasterol, and stigmasterol (45).α-amyrin composed 13.121% in the Mawat location and 1.483% in the Ahmad Awa location, while it was not detected in each of the Temar and Gulp locations.This compound was previously identified as having significant anticancer activity on four cancer cell lines (30).Hop-22(29)-en-3β-ol triterpenes detected in four locations, the highest abundance was detected in Bezhawa location 12.715% and the lowest was 3.124% in Hazarmerd location.This substance has been described as natural product with antiparasitic properties (32), in prokaryotes, it is widely distributed, while in plants, it is relatively rare.Lupeol, an additional phytochemical, was detected in some locations; the sample weighed between 7.25% in Mawat and 1.631% in Qara Dagh.It has been discovered to function as a new androgen receptor that targets β-catenin signaling during carcinogenesis to prevent human prostate cancer cells from proliferating (46).Olean-12-en-3-yl acetate is one of the derivatives of oleanolic acid, a naturally occurring triterpenoid compound found in various plants.Their major abundance in the Kenger plant is 6.871% in Bnary Qandil.
Thunbergol is a monocyclic diterpene alcohol (17).Its abundance was 13.393% in the Ahmad Awa location.Gitoxigenin, which weighed 17.017% of the Kenger plant sample in Hazarmerd.It has anti-cancer properties, especially for renal adenocarcinoma (33).Aneogammacer-22(29)-en-3-ol, acetate (3.β.,21β.)detected in some sample locations, the highest peak area was detected at Penjwen location with 18.95%.The compound is a triterpenoid found in medicinal plants (283).The results obtained above were in agreement with the finding by Abu-Lafi et al., (1), the GC-MS analysis detected 27 phytochemical constituents, such as gitoxigenin, α-amyrin, artemisinin, sitosterol, stigmasterol, and lupeol, which have been reported to have anticancer properties.As it was noticed from Tables (4a, b, and c) the detected phytoconstituents varied among the locations, resulting in discrepancies in the total number of phytoconstituents.Kani Tu and Penjwen locations gave the maximum phytochemical compounds in comparison with the other locations; this is due to specific biotic and abiotic biological conditions present in their location.Despite their varying proportions and quantities across all locations, certain of the chemicals discovered in the current investigation were common (42).Such variations in the concentration of common plant secondary metabolites in plants could be the consequence of particular biosynthetic variations that are either up-or down-regulated (36).Geographical location is one of the most important factors that causes variations in phytoconstituents.Studies have revealed that the concentration of a specific secondary metabolite varies among plants of the same species that grow in various environments.This is because, to combat environmental stress, the plant must produce secondary metabolites in a specific quantity and quality.As a result, research on each environmental element is crucial to understanding plant availability and adaptability in a given area (39).The average temperature is lower at higher elevations than at the lowest points of the mountains.An important factor in the growth and development of plants is elevation.Sunlight exposure, water absorption, and nutrient availability vary with altitude (27).
Other research has found that altitude influences the chemical composition of various plants (22).Ali-Shtayeh et al., (6), discovered that a number of both internal and external factors, including plant genetic structure, growing conditions, soil characteristics, and used plant parts, have an impact on the chemical composition of plant.The analysis of macro and micro elements The results obtained from the ICP-OES analysis, macro-elements (Ca, K, P, S, Mg, and Na) content varied from 3.127%% in Temar to 2.468 in Gulp, with an average of % for all locations, while micro-elements (Fe, Cu, Zn, Mn, Se, Cr, and Mo) were relatively rare in the Kenger plant samples from all of the locations (Table 3).Among the determined elements, calcium (Ca) has the highest concentration in the whole Kenger plant sample.The calcium contents ranged from 7843 mg/kg in Gulp to 11968 mg/kg in Temar and accounted for 0.946% as an average of all locations.High dietary calcium consumption, particularly from plant sources, lowers the chance of osteoporosis and increases bone mineral density.In addition to being a potential source of calcium, vegetables may also include vitamins and minerals that have extra positive effects on bone health (40).Potassium (K) was the second most prevalent element found in plant samples, with concentrations ranging from 5864 mg/kg in the Gulp location to 9974 mg/kg in the Ahmad Awa location.Potassium is found in both plant and animal tissues since it is a necessary element for all living cells to operate properly.It is particularly found in fruits and plants (8).Phosphor (P) is the third most abundant macro element detected in Kenger plant samples.Phosphor is an important macro element in plants; the content varied from 1442 mg/kg in the Azmar location to 4919 mg/kg in the Bnary Qandil location.Sulfur (S) is a macro element.It is one of the most abundant minerals in the human body.It is the third most important mineral in the body, after calcium and phosphorus (37).The average in plant samples was 3270 mg/kg, and it ranged from 2342 mg/kg in Bezhawa to 4716 mg/kg in Temar.Magnesium (Mg) is a necessary element, and deficiencies in it are recognized to have typical symptoms in people, animals, and plants.Magnesium is a prevalent intracellular element found in all living cells, second only to potassium.Plants contain 1 to 8 g/kg (52).In the present study, Mg contents ranged from 1547 mg/kg in Mawat to 2455 mg/kg in Qaramani and were within the reference value of plant content.Sodium (Na) is an important element for the maintenance of acid-base equilibrium and the osmotic pressure of body liquids (10).The sodium contents of all the samples were found to be about 957 mg/kg in Azmar and 1403 mg/kg in Mawat.Two minerals that are necessary for human life are sodium and potassium.These ions play a role in numerous physiological functions.Their equilibrium is crucial to the operation of numerous organs and/or physiological systems.The nerve impulse is one of the main functions of the body's potassium/sodium balance (41).Significant element amounts in plants fall within a limited range, and high concentrations of metals may be toxic to people.These results are consistent with those of the researchers when taking into account earlier research on the macroelement concentrations of a few edible wild and medicinal plants.It is commonly known that a variety of factors, such as soil properties and water availability, have an impact on plant composition and significantly alter the mineral content of plants (47).In this study, there was also a wide variation in a macroelements content among the studied location plant samples.Determining the microelements content of the Kenger plant for consumers is very important because it is used as a food and medicinal plant.The miner element Fe was the highest among microelements and ranged from 4 mg/kg in Bezhawa, Mawat and Azmar to 109 mg/kg in Qara Dagh, with an average of 47 mg/kg.The Fe content in some samples reached the highest reference values (17-50 mg/kg) (25).The cupper (Cu) element ranged from 27 mg/kg in Azmar to 56 mg/kg in Temar samples; it was the second highest among the microelements.According to a study by Akguc et al., (4) the typical range for copper in plants was between 4 and 15 mg/kg.When copper concentrations were higher than these ranges, they were found to have harmful impacts on human health.In this study, it was determined that the Cu content of the studied samples was observed differently and was below the specified limit values.In addition, the Cu amount of the studied species is higher than the limit value determined by FAO's standard, which is 30 mg/kg.The minimum and maximum levels of Zn measured were 26 in Mawat and 58 mg/kg in Temar, respectively.The amount of Mn varies in the range of 22 mg/kg in Azmar to 38 mg/kg in Temar.The findings showed that of all the plant samples, the Mn content was the lowest.The World Health Organization (WHO) states that there are set permitted levels for zinc and manganese, also known as micronutrients, that allow for sufficient dietary consumption.Adult Zn and Mn recommendations range from 5.0 to 22.0 mg and 2.0 to 20.0 mg, respectively (50).Both humans and animals require selenium as a trace element.It has immunological, anti-inflammatory, and antioxidant qualities in addition to defense against cancer, toxins, and cardiac issues (11).Its recommended daily uptake was 0.15-0.4mg/kg (13).In Kenger the Se concentrations of plant species diversified between 3 mg/kg in Bezhawa, Gulp, Ahmad Awa and, Qara Dagh and 23 mg/kg in Penjwen, with an average of 8 mg/kg.According to WHO data, there are no acceptable limit values for the Cr element.In the study Yener et al., (57), the amount of chromium varies in the range of 0.15-4.8mg/kg.In this study, the chrome contents of thirteen plant samples from different locations were measured.According to our findings, chrome content was determined to be lowest at 2 mg/kg in Hazarmerd and then highest at 11 mg/kg in Temar location.The molybdenum contents of samples varied from 2 mg/kg in Azmar, Ahmad Awa, and Nalparez to 5 mg/kg in Bnary Qandil.The minimum daily requirement for Mo by adults is estimated at about 0.02 mg/kg (55).The total element content ranges from 24854 mg/kg at Gulp to 31491 mg/kg at Temar.These components, together with additional chemical substances, are crucial pharmacodynamic building blocks for medicinal plants (43).(34 -47), while a range of (32 -48) was recorded for Zinc content with 40 ± 9 mg/kg.Manganese content was 30 ± 4 mg/kg with a range of (26 -33).Selenium recorded 7 ± 6 mg/kg with a wide interval of (2 -12).The means of Chromium and Molybdenum contents were 5 and 3 with a standard deviation of 2 and 0.9 mg/kg and the range of (3 -7) and (2 -4)

Table 2 . Means of the growth traits and chemical contents of Kenger at different locations Locations (MASL) Stem length (cm) No. of leaves/plant Stem dry weight/plant (g) Carbohydrate (%) Protein (%) Fiber (%) Qaramani (566) 9.833 7.333 2.087 8.267 30.320 32.093 Bezhawa (591) 14.500 6.667 3.853 7.380 20.237 33.547 Gulp
Phytochemical compounds were screened using GC-MS at all locations as it has been shown in (Table3a, b, c).Along with the total area for each detected chemical class in Kenger plants, each study location revealed the presence of a complex mixture of compounds ranged from 13 to 19 compounds with corresponding retention times, relative peak areas, and percentages of similarity relative to the reference library in the GC-MS.

Table 3 a. Phytochemicals of Kenger at extract verified by GC/MS from all locations
* %

of similarity relative to the reference library in the GC/MS Iraqi Journal of Agricultural Sciences -2024:55(1):479-493 Ahmed 486Table 3 b. Phytochemicals of Kenger at extract verified by GC/MS from all locations
* %

Table 4 . Macro and microelements (mg/kg) content in Kenger plant samples in all locations
Based on the obtained results, it is noteworthy that most of the Kenger plant samples exhibited concentrations of microelements exceeding the recommended daily intake, potentially reaching levels considered toxic for human consumption.However, it is crucial to underscore that the concentrations, as presented in the table, are expressed in milligrams per kilogram (mg/kg), and, therefore, the ratio itself may not be a sole determinant of safety.Instead, the overall quantity consumed by an individual holds paramount significance in assessing the potential health risks associated with these elevated microelement concentrations.CONCLUSIONS It can be concluded that the GC-MS analysis of phytochemical compounds in Kenger plant extract across various locations has revealed a diverse and rich source of chemical compounds such as fatty acids, sterols, and terpenoids with the potential for various medicinal and therapeutic applications.Macro elements contents across locations in Kenger shows that the grand mean of Calcium content was 9464 with a standard deviation of 1500 mg/kg and an interval of (8193 -10735).Potassium recorded 8520 ± 1239 mg/kg with a range of (7470 -9570).A range of (2593 -4478) was obtained for Phosphorus content with 3535 ± 1112 mg/kg, whilst Sulfur gave 3270 ± 707 with a range of (2671 -3869).The means of Magnesium and Sodium contents were 2107 and 1206 with a standard deviation of 248 and 136 mg/kg and the range of (1897 -2318) and (1090 -1321), respectively.Concerning the Micro elements contents, Ferus gave 47 ± 33 mg/kg with an interval of (18 -75) across locations.Cupper recorded 41 ± 7 mg/kg with a range of , respectively.and F. Ozgokce 1993.Chemical composition of some edible wild plants grown in Eastern Anatolia.Am.J. Essent.Oils Nat.Prod.2: 31-34.55.Turnlund, J. R., W. R. Keyes, and G. L. Peiffer 1993.A stable isotope study of the dietary molybdenum requirement of young men.Trace Elem.man Anim.8: 189-192.1993.56.Yasukawa, K., M. Takido, T. Matsumoto, M. Takeuchi, and S. Nakagawa 1991.Sterol and triterpene derivatives from plants inhibit the effects of a tumor promoter, and sitosterol and betulinic acid inhibit tumor formation in mouse skin two-stage carcinogenesis.Oncology 48(1): 72-76.57.Yener, İ 2019.Trace element analysis in some plants species by inductively coupled plasma optical emission spectrometry (ICP-OES).J. Inst.Sci.Technol.9(3): 1492-1502.58.Zangeneh, M. M., N. Goodarzi, A. Zangeneh, R. Tahvilian, and F. Najafi.2018.Amelioration of renal structural changes in STZ-induced diabetic mice with ethanolic extract of Allium saralicum RM Fritsch.Comp.Clin.Path.27: 861-867.