The Moringa tree (Moringa spp.) is a multipurpose plant that has attracted considerable attention in agricultural, medical and industrial fields. This interest is attributed to its rich content of bioactive compounds that contribute to human nutrition and possess various therapeutic properties, in addition to its potential role in environmental sustainability and ecosystem conservation. The Moringa tree belongs to the genus Moringa, which represents the sole genus of the family Moringaceae (Fadala  et al., 2023).
       
Moringaceae comprises 13 species distributed across Africa, the Arabian Peninsula, Southeast Asia and South America. Among these, Moringa oleifera and Moringa peregrine are considered the most significant due to their high nutritional and medicinal value (Lamou, 2016). Moringa oleifera is the most widely cultivated species, characterized as a small to medium-sized tree, with a height ranging from 3 to 12 meters and a trunk diameter of 20 to 40 cm. Its leaves are pinnately compound, consisting of 23 pairs of leaflets, with the terminal leaflet being larger, obovate and pale green. The flowers are white, fragrant and consist of five petals. The fruit is a triangular pod, varying in length from 20 to 50 cm depending on the environmental conditions (Medina et al., 2007; Al-Rikabi  et al., 2025).
       
Moringa peregrine, also referred to as Moringa al-Shua, exhibits distinct morphological and physiological traits. Its leaves consist of three pairs of long, slender leaflets. The species demonstrates vigorous growth during the wet season, while its tuber enters dormancy during drought periods and resumes growth afterward. Its flowers are pink and fragrant and the fruits are pendulous in shape (Foidl et al., 2001; Omar and Fadala, 2025).
       
The leaves of both species are the most utilized plant part due to their high content of essential minerals, carbohydrates and proteins, making them a valuable source of nutrition and bioactive compounds. The leaves of Moringa species also contain a wide range of biologically active compounds, including vitamins, carotenoids, alkaloids and saponins (Anwar et al., 2007), as well as glycosides, flavonoids and plant sterols (Yadav et al., 2017). Several bioactive compounds have been isolated and identified from different parts of Moringa plant (leaves, seeds, bark, flowers, pods and root). It is expected to chart-out a new road map for drug discovery is the basic aspect sustainable exploitation of bioactive natural products (Fadala  et al., 2023; Abbas and Fadala, 2026). Among the flavonoids, quercetin and kaempferol are the most prominent. These bioactive compounds are distributed throughout various plant tissues and are produced as secondary metabolites during plant metabolism. Therefore, the study was aimed to identify and characterize the bioactive compounds in Moringa leaves using Gas Chromatography-Mass Spectrometry (GC-MS) technique.

Study location and duration
 
The experiment was conducted in a greenhouse covered with tarpaulin, located in an agricultural area in Al-Gharraf District, Dhi Qar Governorate, Iraq, during the 2023 growing season. In 2024, seedlings of two Moringa species were obtained from a private nursery in Baghdad Governorate for further analysis.
 
Sample collection and preparation
 
Fresh leaves from both Moringa species were collected, thoroughly washed under a continuous stream of tap water for 5 minutes and subsequently rinsed with distilled water to remove any remaining impurities. The leaves were then air-dried at room temperature and ground into a fine powder using a laboratory grinder. The powdered samples were stored in tightly sealed glass containers at 4^oC until further use.
 
Preparation of ethanol extracts from Moringa leaves
 
For extraction, 25 grams of the dried leaf powder were weighed and dissolved in 500 ml of ethanol (equivalent to 1 g of powder per 20 ml of ethanol), following the method described in the previous section. The mixture was allowed to macerate under continuous stirring to ensure efficient extraction of bioactive compounds. The resulting extract was filtered and concentrated for subsequent GC-MS analysis.
       
Following the method described by Pizzale et al., (2002), the extract was allowed to stand for 24 hours at room temperature to ensure effective extraction of bioactive compounds. Subsequently, the extract was filtered through Whatman No. 14 filter paper to remove insoluble residues. The filtrate was then concentrated using a rotary evaporator at 45^oC under reduced pressure. The concentrated extract was transferred into airtight containers and stored at 5^oC until further analysis.
 
GC-MS analysis
 
Gas chromatography-mass spectrometry (GC-MS) analysis was conducted at the laboratory of the Basra Oil Company to identify the chemical constituents present in the ethanol extracts of leaves from two Moringa species, including Moringa peregrine (G 7890). The analysis was carried out using an Agilent GC-MS system coupled with an MSD 5977 mass selective detector, equipped with an automated sampling device and electron impact (EI) ionization mode.
 
Identification of phytochemicals in leaf and seed extracts of two Moringa species
 
The bioactive compounds present in the ethanol extracts of leaves and seeds from two Moringa species were identified based on their retention times and corresponding mass spectral data obtained from GC-MS analysis. The mass spectra were interpreted by comparison with the National Institute of Standards and Technology (NIST) mass spectral database (Heavner et al., 2014). Further confirmation of compound identities was achieved using the Wiley Library 9 database. For each identified compound, the name, molecular weight, molecular formula and chemical structure were recorded and verified.
 
Identification of compounds
 
Interpretation on mass-spectrum GC-MS-MS was conducted using the database of National institute Standard and Technology (NIST) having more 62,000 patterns. The spectrum of the unknown components was compared with the spectrum of known components stored in the NIST library. The name, molecular weight and structure of the components of the test materials were ascertained.

Identification of medically active compounds in the leaves and seeds of two Moringa tree species
 
The results of the GC-MS analysis of the chemical constituents present in the leaves and seeds of two Moringa tree species, as presented in Tables (1-4), revealed the presence of numerous compounds with recognized medicinal and biological activities. Among the identified compounds were n-α-D-ribopyranoside, methyl, vitamin E and phytol. The most prominent bioactive compounds detected included 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-hexadecanoic acid and γ-sitosterol. In addition, fatty acid derivatives such as 9,12,15-octadecatrienoic acid (Z,Z,Z)-, were identified. Other compounds, including dibutyl phthalate, methyl ester (Z,Z,Z)-, were also detected in varying proportions.








       
GC-MS analysis of the second treatment, consisting of 1 g L^-1 NPK nanoparticles combined with Moringa oleifera (Table 1), revealed the presence of at least 41 distinct compounds. Among these, ethanol, 2,2-oxybis(s), exhibited the highest retention time, whereas several other compounds showed comparatively lower retention times. The diversity of the identified compounds reflects the rich phytochemical profile of Moringa species and supports their potential medicinal and pharmaceutical applications.
       
The GC-MS analysis results for the third treatment (2 g/L) - NPK nanoparticles + Moringa oleifera (Table 2) showed the presence of at least 53 compounds. Furfural-1H-Indole, 1-methyl-2-phenyl was the most active compound, while R.T. exhibited the shortest retention time and the highest retention time.
       
The results of the GC–MS analysis for the first treatment (0 + Peregrina), as presented in Table 3, revealed the presence of no fewer than 37 compounds. Among these, Isopropyl isothiocyanate exhibited the shortest retention time (R.T.), whereas Gamma-Sit sterol showed the longest retention time (R.T.).
      
The GC-MS analysis results for the third treatment (2 g/L - NPK nanoparticles + Peregrina) are shown in Table 4, revealing at least 37 compounds. Isopropyl-5-hydroxy-3 showed the lowest retention time, while R.T. had the lowest retention time. Methylacetophenone and TMS derivatives had the highest retention time.
 
Discussion of GC-MS analysis results
 
The GC-MS analysis results showed the appearance of several compounds with medicinal activity, the most important of which are shown in Table 5.
       
The compounds shown in Table 5 vary in their percentages from one cultivar to another and from one treatment to another. Fig 1 shows that the relative abundance of the compound Phytol varies according to the treatment; the second treatment (1 g L^-1 nano NPK + Licorice) achieved the highest relative abundance for this compound, reaching 37.91.




       
Phytol has significant medicinal importance, as studies have indicated that this compound is used as a precursor in the synthesis of vitamins E and K. It also possesses antioxidant, antimicrobial and anti-inflammatory properties, contributes to improving skin health and is widely used in cosmetic preparations. Fig 2 illustrates that the relative abundance of Vitamin E varies among different treatments. The first treatment (0 + olfiera) recorded the highest relative abundance of this compound, reaching 14.39%.


       
Vitamin E is of great medicinal importance, as studies have shown that it acts as a powerful antioxidant that protects cells from damage, enhances skin and eye health, supports the immune system and is sometimes used in the treatment of male infertility. Fig 3 indicates that the relative abundance of the compound methyl alpha-D-Ribopyranoside varies according to different treatments. The second treatment (1 g L^-1 nano-NPK + Olfiera) showed the highest relative abundance of this compound, reaching 8.04%.


       
The medical importance of α-D-Ribopyranoside, methyl has been highlighted in several studies, indicating that this compound is involved in the synthesis of nucleosides and vitamins and plays an essential role in supporting vital cellular functions. Fig 4 illustrates that the relative abundance of n-Hexadecanoic acid varies among the different treatments. The second treatment (1 g/L NPK nanoparticles + riboflavin) exhibited the highest relative abundance of this compound, reaching 8.12%.


       
The compound 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl is of considerable medical importance. Previous studies have indicated its potential applications in skin lightening and the treatment of hyperpigmentation, in addition to its antioxidant and antibacterial properties, which enhance its value in pharmaceutical and biomedical applications. 9,12,15-Octadecatrienoic acid (Z,Z,Z), the results illustrated in Fig 5 show that its relative abundance varies depending on the applied treatments. The first treatment (0 + Z) exhibited the highest relative abundance of this compound, reaching 7.56%, indicating a pronounced effect of the treatment on its concentration.


       
Octadecatrienoic acid (Z,Z,Z) exhibits significant medical relevance, as previous studies have demonstrated its anti-inflammatory properties, its role in supporting brain and skin functions and its importance as an essential fatty acid for cardiovascular health. As illustrated in Fig 6, the relative abundance of γ-Sitosterol varies among the applied treatments. Notably, the first treatment (0) + (S) resulted in the highest relative abundance of this compound, reaching a value of 4.42.


       
γ-Sitosterol is a bioactive compound of medical significance. Previous studies have reported that it reduces LDL (low-density lipoprotein) cholesterol, exhibits anti-inflammatory effects, supports prostate health and inhibits the proliferation of cancerous cells (Mohammed et al., 2018). The relative abundance of Methyl-β-D-thiogalactoside was found to vary among the applied treatments) Hussien et al., 2025). As illustrated in Fig 7, the third treatment (2 g/L NPK nanoparticles + sulfate) resulted in the highest relative abundance of this compound, reaching a value of 45.54.


 
Medical significance
 
Methyl-β-D-thiogalactoside has been reported in studies as a valuable compound in biological research, serving as a substrate for enzymes and being utilized in the analysis of lactase activity and glycolytic enzymes.
 
9,12,15-Octadecatrienoic acid, methyl ester
 
The relative abundance of this compound varied among the applied treatments, as shown in Fig 8. The second treatment (1 g/L NPK nanoparticles + Moringa oleifera) resulted in the highest relative abundance, reaching a value of 1.69.


 
9,12,15-Octadecatrienoic acid, methyl ester (Z,Z,Z)
 
This compound is of medical significance, as studies have demonstrated its role as a dietary supplement and its anti-inflammatory and antioxidant properties, contributing to the support of brain and skin functions.
 
Dibutyl phthalate
 
The relative abundance of this compound varied among the applied treatments, as illustrated in Fig 9. The second treatment (1 g/L NPK nanoparticles + sulfate) resulted in the highest relative abundance.


       
Dibutyl phthalate is an industrially significant compound commonly used as a plasticizer in the production of plastics. However, chronic exposure to this compound has toxic effects and it is recognized as an environmental pollutant and endocrine disruptor. The therapeutic effects of medicinal plants are often attributed not to a single active constituent but to the synergistic interaction of multiple compounds. Several studies have reported that plant extracts and oils containing 9,12-octadecadienoic acid (linoleic acid; O-9,12,15) exhibit notable antioxidant activity and antimicrobial efficacy. Moreover, highlighted that the high concentration of n-hexadecanoic acid (Palmitic Acid) in Moringa oleifera leaf extracts contributes to anti-inflammatory and anticancer effects (Beniwal et al., 2025; Kikraliya et al., 2024; Moond et al., 2023).

The study concludes that GC-MS analysis of the chemical constituents present in the leaves and seeds of two Moringa tree species, revealed the presence of numerous compounds with recognized medicinal and biological activities. Among the identified compounds were n-α-D-ribopyranoside, methyl, vitamin E and phytol. The most prominent bioactive compounds detected included 4H-pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-hexadecanoic acid and γ-sitosterol. In addition, fatty acid derivatives such as 9,12,15-octadecatrienoic acid (Z,Z,Z)-, methyl-9,12,15-octadecatrienoic acid and methyl-β-D-thiogalactoside were identified. Other compounds, including dibutyl phthalate, methyl ester (Z,Z,Z)-, were also detected in varying proportions.

The authors declare there is no conflict of interest.