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ABSTRACT

Background: Freesia (Freesia hybrida L.) is an economically valuable fragrant and decorative plant; nonetheless, research on its chemical composition remains scarce. The objective of this research was to discover the chemical constituents of freesia leaf extract by gas chromatography-mass spectrometry (GC-MS), emphasizing bioactive substances and their biological relevance.

Methods: Freesia leaves were procured from nurseries, extracted with methanol and then analyzed using GC-MS, with the chemical spectra being compared against verified reference libraries (NIST, Wiley).

Result: The study identified the existence of twenty-three chemical constituents, including oligosaccharides, fatty acids, aromatic compounds and heterocyclic compounds. The most notable compounds include hexadecenoic acid, recognized for its antioxidant and cholesterol-lowering effects; octadecinamide (Z-), exhibiting antibacterial and anti-inflammatory properties; oligosaccharides with antimicrobial capabilities and aromatic compounds like 2-methoxy-4-vinylphenol, known for its anti-inflammatory activity. These findings underscore the prospective significance of Freesia chemicals in medical and pharmacological contexts, establishing a foundational knowledge base for future investigations designed to validate biological activities using specialized bioassays.

INTRODUCTION

Freesia is from the Iridaceae family, which originated in South Africa (Mahmood et al., 2024). Flowers like freesia are commonly gathered, sold and bought. Freesia bulbs develop into perennial herbaceous monocots that bloom in winter and spring. Due to its export from its native environment, hybridization has produced a broad variety of commercial Freesia (F. hybrida). Freesia’s decorative value depends on its tuber quality and plant health (Ma et al., 2021). Its vibrant colors and elegant look make it perfect for flower arrangements. It is planted for its cut flowers as well as a blooming plant (Mahmood et al., 2023). White, purple, red and yellow flowers decorate its original flower stand (Al-Zurfy et al., 2018). This variety was produced from hybridization between several species, the most important of which are Freesia aurea, Freesia refracta and Freesia odorata (Huang et al., 2018).
       
Freesia is a popular cut-flower ornamental for its fragrant flowers, which are one of the top ten cut flowers on the global floricultural market and its demand has risen (Huang et al., 2018; Ahmad et al., 2019). According to study conducted by Li et al. (2020) and Shan et al. (2020) found flavonols, anthocyanins and proanthocyanidins in cultivar flowers. Most volatile organic compounds (VOCs) in 26 grown and 8 hybrid Freesia hybrida samples were terpenoids, mainly monoterpenoids. The most abundant chemicals were linalool and d-limonene (Weng et al., 2021). Because of volatile terpenes such as linalool, α-terpineol, β-ionone and copaene dominate the scent (Santilli et al., 2023; Gao et al., 2018). Freesia extracts are used in cosmetics, detergents and candles because of their pleasant scent. The protease from freesia flowers (Freesia refracta) was successfully used in cosmetic compositions by (Weng et al., 2021; Demir et al., 2023). In addition, essential oils are the main chemical components of Freesia, which is used to make medications and nutritional supplements which may boost immunity and health. Also, because of the economic importance of these flowers, many studies have appeared, including a study of the effect of spraying organic fertilizer extract and vitamin E on the growth and flowering characteristics of Freesia plants (Al-Zurfi et al., 2023). Delphinidin, petunidin, malvinidin, peonidin and cyanidin are anthocyanin aglycons discovered in F.Hybrida, together with kaempferol and quercetin (Al-Zurfi et al., 2023 ; Li et al., 2016). Six structural genes and two regulatory genes have been found to perform this role (Sun et al., 2016; Sun et al., 2017; Li et al., 2017; Ju et al., 2018). Al-Jaafari and Al-Zurfi (2024) investigated the effects of different concentrations of jasmonic acid and nano magnesium on the growth and blooming of freesia plants to increase bulb production, in response to consumer demand.
       
One reliable method for determining whether plant chemicals are volatile or semi-volatile is gas chromatography-mass spectrometry (GC-MS). Studying the chemical composition of freesia, which is still restricted, is a good fit for its high detection accuracy and speedy analysis. Therefore, the purpose of this work is to address a knowledge gap on this plant and draw attention to its potential for future pharmaceutical applications by conducting a thorough analysis of freesia leaf extract using GC-MS and identifying secondary chemicals of biological value.

MATERIALS AND METHODS

The research analyzed the chemicals in the freesia plant using the GC-MS technique, following the steps shown in Fig 1 and took place at the Ibn Al-Bitar Research Centre/Iraqi Ministry of Industry.
 

Fig 1: Diagram of the experimental methodology used for GC-MS analysis of Freesia hybrida leaves.



Sample collection and preparation of alcoholic extract
 
From June to October 2024, the leaves of the Freesia plant were gathered from agricultural nurseries in the Babylon Governorate (Fig 2). They were then washed under running water to remove dust and other contaminants, excluding any infected ones. After that, they were dried at room temperature to reduce the moisture content. The goal was to grind the dried leaves in to a fine powder that would increase the reaction surface.

Fig 2: Freesia plant, which gathered from babylonian agricultural nurseries.


       
20 grams of dried leaf powder was extracted using 100 ml of methanol (99%) under continuous stirring for 24 hours at room temperature (25±2°C). The extract was then filtered and concentrated using a rotary evaporator under a reduced pressure of 80-100 mbar at 40°C to obtain a concentrated extract. This was done to gently evaporate the methanolic solvent and prevent the degradation of heat-sensitive bioactive compounds during the concentration process (Al-Jayid et al., 2025). Note that the extraction and analysis method was carried out in three replicates and a control sample of methanol only (Blank sample) was used, free of the plant extract, to verify the cleanliness of the device and the absence of any possible interference resulting from the solvent or the tools used.
 
Preparation of the GC-MS device
 
After ensuring the readiness of the GC-MS device, including the ion source, mass analyzer and detector, the temperature and pressure must be adjusted according to the recommended specifications. A suitable gas chromatography column was selected to separate the compounds to be analyzed, which is Fused silica capillary column Elite-1 (30 × 0.25 mm × ID1EM of dimethylpolysiloxane), ensuring that the flow of the carrier helium gas flowed at a rate of 1.0 mL.min-1
       
In addition, the GC device can be programmed to gradually increase the column temperature according to a specific program: 110°C for 2 min; going up at 5°C min to 200°C and held for 9 min; rising at 5°C min to 280°C and held for 9 min. The program depends on the nature of the compounds to be separated. The prepared sample was then injected into the injection port at the beginning of the chromatographic column at a volume of 0.1ìl and a split ratio of 1:10 (Kavitha et al., 2023).
 
GC-MS analysis
 
Compounds are separated using a gas chromatograph according to their spectral characteristics. A Turbo-Mass Gold-Perkin Elmer mass spectrometry detector is used to identify and quantify the compounds in the methanolic extract. The results are compared with mass spectra reference libraries, such as the Wiley Spectral Library and the National Institute of Standards and Technology. The current study was limited to the initial qualitative characterization of the bioactive compounds. Quantitative analysis and statistical validation will be completed in future studies after the compounds recorded in the current qualitative study are separated (Kavitha et al., 2024).

RESULTS AND DISCUSSION

GC-MS chromatographic profile
 
In this investigation, 23 phytochemical substances were identified by GC-MS analysis of Freesia hybrid leaf samples (Fig 3). Oligosaccharides, carboxylic acids, aromatic hydrocarbons, ketene, unsaturated fatty acids and organic heterocyclic compounds were among them. Table 1 shows that there were some active chemicals found in the plant extracts studied and the mass spectrographs for the components found may be seen in Fig 4-26. Relative quantities of the different components were calculated using GC peak areas.

Table 1: Principal phytochemical constituents found in the methanolic extract of Freesia hybrida.



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Fig 3: Chromatogram profile for GC-MS in methanolic extract of Freesia hyprida.



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Fig 4: Mass spectrum of octanamide, N-(2-mercaptoethyl).



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Fig 5: Mass spectrum of D-glucose,6-O-á-galactopyranosyl.



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Fig 6: Mass spectrum of 3-hydroxydodecanoic acid.



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Fig 7: Mass spectrum of HEPES.



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Fig 8: Mass spectrum of n-Glycyl-DL-leucine.



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Fig 9: Mass spectrum of á-D-glucopyranoside, O-á-D-glucopyranosyl-(1. Fw).



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Fig 10: Mass spectrum of 2-propyl-tetrahydropyran-3-ol.



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Fig 11: Mass spectrum of methyl-6-oxoheptanoate.



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Fig 12: Mass spectrum of 6-acetyl-â-d-mannose.



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Fig 13: Mass spectrum of 9-octadecenamide, (Z).



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Fig 14: Mass spectrum of muramic acid.



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Fig 15: Mass spectrum of formamide, N-methyl-N-4-[1-(pyrrolidinyl)-2-butynyl].



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Fig 16: Mass spectrum of dithiocarbamate, S-methyl-, N-(2-methyl -3-oxobutyl).



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Fig 17: Mass spectrum of 2-methoxy-4-vinylphenol.



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Fig 18: Mass spectrum of 1,2-cyclopentanedicarboxylic acid ,4-(1,1-dimethyl).



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Fig 19: Mass spectrum of 9-hexadecenoic acid.



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Fig 20: Mass spectrum of 1-(3,6,6-trimethyl-1,6,7,7a-tetrahydrocyclopenta[c].



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Fig 21: Mass spectrum of 5,6,6-trimethyl-5-(3-oxobut-1-enyl)-1-oxaspiro [2.5].



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Fig 22: Mass spectrum of 2H-oxecin-2-one,3,4,7,8,9,10-hexahydro-4-hydro.



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Fig 23: Mass spectrum of propiolic acid, 3-(1-hydroxy-2-isopropyl-5-methyl.



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Fig 24: Mass spectrum of Z-(13,14-epoxy) tetradic-11-en-1-ol acetate.



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Fig 25: Mass spectrum of picrotoxin.



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Fig 26: Mass spectrum of 1-hexadecanol, 2-methyl.


 
Identification of phytochemical compounds
 
The methanolic extract of F. hybrida revealed a prominent peak during chromatogram GC-MS analysis and the constituents matching the peak were identified as follows. Octanamide, N-(2-mercapto ethyl)-, was identified as the first set-up peak with a retention duration of 3.150 min (Fig 4). The second peak was indicated to be D-Glucose, 6-O-α-galactopyranosyl, in 3.298 min (Fig 5).
       
The next peaks are considered to be 3-Hydroxydodecanoic acid, HEPES (4-(2-Hydroxyethyl) piperazine-1-ethane sulfonic acid), n-glycyl-DL-leucine; α- D- Glucopyranoside, O- α- D-glucopyranosyl (1. fwdarw. 3)- β- D-fructofuranosyl; 2-Propyl- tetrahydropyran -3-ol;  Methyl-6-oxoheptanoate; 6-Acetyl-β-d-mannose; 9-Octadecenamide,(Z)-; Muramic acid; Formamide,N-methyl-N-4-[1-(pyrrolidinyl)-2-butynyl]; Dithiocarbamate,S-methyl-, N-(2-methyl-3-oxobutyl); 2-Methoxy-4 vinylphenol; 1,2-Cyclopentanedicarboxylic acid,dimethyl ester; 9-Hexadecenoic acid;1-(3, 6, 6-Trimethyl-1,6,7,7 tetra hydro cyclopenta [c] pyran-1-y l) ethanone; 5,6,6-Trimethyl-5-(3-oxobut-1-enyl)-1-oxaspiro [2.5]; 2H- Oxecin- 2-one, 3, 4,7,8,9,10-hexahydro- 4-hydroxy-10-methyl-; Propiolic acid,3-(1-hydroxy- 2-isopropyl- 5-methylcyclohexyl); Z-(13,14-Epoxy) tetradec-11-en-1-olacetate; Picrotoxin;1-Hexadecanol,2-methyl- from the retention time 3.447 to 11.195 (Fig 6 to 26). To learn about the biological impacts and chemical makeup of Freesia chemical compounds, GC-MS analysis is a viable option. The chemical components of a plant sample may be separated and analyzed using GC-MS. Secondary metabolites include all of these substances. These secondary metabolites serve as a defense against microbial infections or animal predation, distinguishing them from the components of primary metabolism. They are not engaged in general metabolism, however. To activate or inhibit signal transduction pathways in live cells, chemicals derived from similar plant species may be necessary (Kuruppu et al., 2019; Muteab and AL-Abedy, 2025).
 
Biological activities of identified compounds
 
The current results show similarity in chemical composition to some other aromatic species of the Iridaceae family, particularly in their content of unsaturated fatty acids and aromatic compounds. This suggests that Freesia hybrida may share some of the metabolic pathways responsible for the production of volatile compounds with biological activity, these compounds may have anti-mutagenic, anti-cancer and other properties. Table 2 shows the most important compounds identified in freesia leaf extract and their biological activities as reported in previous studies, which enhances the possibility of using these compounds in future medical and therapeutic applications such as D-Glucose, 6-O-α-galactopyranosyl- and α-D-Glucopyranoside, O-α-D-glucopyranosyl (1.fwdarw.3)-ß-D-fructofuranosyl have antibacterial and antifungal activity (Kamal et al., 2015) and anti-diabetic, anti-hyperlipidemic, anti-oxidant activity (Kumar et al., 2015); Fatty acid metabolic problems are linked to 3-hydroxy dodecanoic acid, a medium-chain fatty acid (Chickos et al., 2002); Also, 2-Propyl-tetra hydropyran-3-ol has anti-angiogenic effect (Hussein et al., 2016); and anticancer activity as the compounds Methyl 6-oxoheptanoate (Al Tameme et al., 2015; Mokni et al., 2016), These compounds represent the chemical basis that may explain the traditional use of freesia as a fragrance and antimicrobial in some popular applications.

Table 2: Summary of major identified compounds and their reported biological activities.


       
The alcoholic extract of Freesia also contained the compound 6-Acetyl-ß-d-mannose which has anti-inflammatory and anti-oxidant effects (Sosa et al., 2016); and 9-Octadecenamide, (Z) which has Anti-inflammatory activity and antibacterial activity (Hadi et al., 2016); Further bioactivities of hexadecanoic acid include nematicide, insecticide, hypocholesterolemic, antioxidant and larvicide for mosquitoes (Rajalakshmi and Mohan, 2016; Kumar et al., 2017). Because of its antioxidant and anti-inflammatory characteristics, freesia may have therapeutic uses due to the presence of chemicals like 9-Hexadecenoic acid and 2-Methoxy-4-vinylphenol. Because of these qualities, plant extracts may find application in the production of natural medicinal and cosmetic goods. Al-Marzoqi et al. (2015) indicated that Dithiocarbanate, S-methyl-, N-(2-methyl-3-oxobutyl) has anti-cancer agents and anti-inflammatory effects (Kadhim et al., 2016) and anti-bacterial activity (Al-Khafaji, 2018). Beyond its dual roles as a pediculicide and pesticide, picrotoxin stimulates the central nervous system and the respiratory system (Böttger et al., 2018) and Propiolic acid, 3-(1-hydroxy-2-isopropyl-5- methylcyclohexyl) has anti-cancer activity (Rajalakshmi and Mohan, 2016; Hepokur et al., 2020) indicated that 5,6,6-Trimethyl-5-(3-oxobut-1-enyl)-1-oxaspiro [2.5] is one chemical compound of Thymbra capitata ethanolic extract which have antimicrobial, antioxidant, cytotoxic activities. Also, (Singh et al., 2021) mentioned that 1-(3, 6, 6-Trimethyl-1, 6, 7, 7a-tetrahydro cyclopenta [c]pyran-1-yl) ethanone compound has antihistamine activity and the anti-germination, anti-inflammatory, analgesic and antibacterial properties of 2-methyl-4-vinyphenol are well-documented (Ibibia et al., 2016).
       
Freesia hybrida and other Iridaceae species shared metabolic traits, according to Weng et al., (2021). Previous research has shown that phenolic compounds, fatty acids and volatile organic compounds from this family have antioxidant, antibacterial and plant defense properties. Li et al. (2024) studied saffron (Caffron crocus), a member of the Iridaceae family, to identify secondary metabolites. This finding suggests that similar environmental conditions or genetic routes may affect metabolite synthesis, which could lead to similar health benefits across species. They found 88 favorable metabolites, including lipids, alkaloids, amino acids, terpenoids, organic acids and flavonoids. These metabolites primarily participate in metabolic pathways, nucleotide metabolism, purine metabolism and the production of flavonoids. Many plant species have well-studied flavonoid biosynthesis pathways (Gao et al., 2020; Wu et al., 2022). Due to the fact that chemical identification depends on comparing spectra with reference libraries and GC-MS can only identify volatile compounds (no heavy or non-volatile compounds are detected), there may be some restrictions to the accuracy of identification. Hence, these findings should be seen as first clues for understanding plant chemical composition; more studies using liquid chromatography-mass spectrometry (LC-MS) or Nuclear Magnetic Resonance spectroscopy (NMR) to confirm the existence of non-volatile chemicals are suggested.
       
The discovery of these chemicals’ biological activity in freesia is a significant step towards comprehending the chemical variety of this plant genus, even if some of these activities have been seen in other plants.  Not only do these findings corroborate earlier efforts, but they also point to uncharted territory with promise in areas like aromatics and pharmaceuticals. The limitations of the current study, including the lack of quantitative quantification of the identified compounds, are a major limitation that should be taken into account. However, this work constitutes a preliminary step toward identifying key chemical patterns that can be quantitatively analyzed in future research.

CONCLUSION

Based on the information provided, it is clear that GC-MS technology is capable of identifying heterocyclic organic compounds, oligosaccharides, carboxylic acids, aromatic hydrocarbons, chitin, unsaturated fatty acids and other bioactive compounds in freesia extracts. Their biological activities can be validated in the future using specialized bioassays. By separating these compounds, the purified extract could contribute to the development of new drugs or the improvement of existing treatments.

ACKNOWLEDGEMENT

The Biology Department at Babylon University College of Science for Women supported the present study and provided invaluable assistance with this project.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

CONFLICT OF INTEREST

The authors assert that no conflicts of interest exist.

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    ABSTRACT

    Background: Freesia (Freesia hybrida L.) is an economically valuable fragrant and decorative plant; nonetheless, research on its chemical composition remains scarce. The objective of this research was to discover the chemical constituents of freesia leaf extract by gas chromatography-mass spectrometry (GC-MS), emphasizing bioactive substances and their biological relevance.

    Methods: Freesia leaves were procured from nurseries, extracted with methanol and then analyzed using GC-MS, with the chemical spectra being compared against verified reference libraries (NIST, Wiley).

    Result: The study identified the existence of twenty-three chemical constituents, including oligosaccharides, fatty acids, aromatic compounds and heterocyclic compounds. The most notable compounds include hexadecenoic acid, recognized for its antioxidant and cholesterol-lowering effects; octadecinamide (Z-), exhibiting antibacterial and anti-inflammatory properties; oligosaccharides with antimicrobial capabilities and aromatic compounds like 2-methoxy-4-vinylphenol, known for its anti-inflammatory activity. These findings underscore the prospective significance of Freesia chemicals in medical and pharmacological contexts, establishing a foundational knowledge base for future investigations designed to validate biological activities using specialized bioassays.

    INTRODUCTION

    Freesia is from the Iridaceae family, which originated in South Africa (Mahmood et al., 2024). Flowers like freesia are commonly gathered, sold and bought. Freesia bulbs develop into perennial herbaceous monocots that bloom in winter and spring. Due to its export from its native environment, hybridization has produced a broad variety of commercial Freesia (F. hybrida). Freesia’s decorative value depends on its tuber quality and plant health (Ma et al., 2021). Its vibrant colors and elegant look make it perfect for flower arrangements. It is planted for its cut flowers as well as a blooming plant (Mahmood et al., 2023). White, purple, red and yellow flowers decorate its original flower stand (Al-Zurfy et al., 2018). This variety was produced from hybridization between several species, the most important of which are Freesia aurea, Freesia refracta and Freesia odorata (Huang et al., 2018).
           
    Freesia     is a popular cut-flower ornamental for its fragrant flowers, which are one of the top ten cut flowers on the global floricultural market and its demand has risen (Huang et al., 2018; Ahmad et al., 2019). According to study conducted by Li et al. (2020) and Shan et al. (2020) found flavonols, anthocyanins and proanthocyanidins in cultivar flowers. Most volatile organic compounds (VOCs) in 26 grown and 8 hybrid Freesia hybrida samples were terpenoids, mainly monoterpenoids. The most abundant chemicals were linalool and d-limonene (Weng et al., 2021). Because of volatile terpenes such as linalool, α-terpineol, β-ionone and copaene dominate the scent (Santilli et al., 2023; Gao et al., 2018). Freesia extracts are used in cosmetics, detergents and candles because of their pleasant scent. The protease from freesia flowers (Freesia refracta) was successfully used in cosmetic compositions by (Weng et al., 2021; Demir et al., 2023). In addition, essential oils are the main chemical components of Freesia, which is used to make medications and nutritional supplements which may boost immunity and health. Also, because of the economic importance of these flowers, many studies have appeared, including a study of the effect of spraying organic fertilizer extract and vitamin E on the growth and flowering characteristics of Freesia plants (Al-Zurfi et al., 2023). Delphinidin, petunidin, malvinidin, peonidin and cyanidin are anthocyanin aglycons discovered in F.Hybrida, together with kaempferol and quercetin (Al-Zurfi et al., 2023 ; Li et al., 2016). Six structural genes and two regulatory genes have been found to perform this role (Sun et al., 2016; Sun et al., 2017; Li et al., 2017; Ju et al., 2018). Al-Jaafari and Al-Zurfi (2024) investigated the effects of different concentrations of jasmonic acid and nano magnesium on the growth and blooming of freesia plants to increase bulb production, in response to consumer demand.
           
    One reliable method for determining whether plant chemicals are volatile or semi-volatile is gas chromatography-mass spectrometry (GC-MS). Studying the chemical composition of freesia, which is still restricted, is a good fit for its high detection accuracy and speedy analysis. Therefore, the purpose of this work is to address a knowledge gap on this plant and draw attention to its potential for future pharmaceutical applications by conducting a thorough analysis of freesia leaf extract using GC-MS and identifying secondary chemicals of biological value.

    MATERIALS AND METHODS

    The research analyzed the chemicals in the freesia plant using the GC-MS technique, following the steps shown in Fig 1 and took place at the Ibn Al-Bitar Research Centre/Iraqi Ministry of Industry.
     

    Fig 1: Diagram of the experimental methodology used for GC-MS analysis of Freesia hybrida leaves.



    Sample collection and preparation of alcoholic extract
     
    From June to October 2024, the leaves of the Freesia plant were gathered from agricultural nurseries in the Babylon Governorate (Fig 2). They were then washed under running water to remove dust and other contaminants, excluding any infected ones. After that, they were dried at room temperature to reduce the moisture content. The goal was to grind the dried leaves in to a fine powder that would increase the reaction surface.

    Fig 2: Freesia plant, which gathered from babylonian agricultural nurseries.


           
    20 grams of dried leaf powder was extracted using 100 ml of methanol (99%) under continuous stirring for 24 hours at room temperature (25±2°C). The extract was then filtered and concentrated using a rotary evaporator under a reduced pressure of 80-100 mbar at 40°C to obtain a concentrated extract. This was done to gently evaporate the methanolic solvent and prevent the degradation of heat-sensitive bioactive compounds during the concentration process (Al-Jayid et al., 2025). Note that the extraction and analysis method was carried out in three replicates and a control sample of methanol only (Blank sample) was used, free of the plant extract, to verify the cleanliness of the device and the absence of any possible interference resulting from the solvent or the tools used.
     
    Preparation of the GC-MS device
     
    After ensuring the readiness of the GC-MS device, including the ion source, mass analyzer and detector, the temperature and pressure must be adjusted according to the recommended specifications. A suitable gas chromatography column was selected to separate the compounds to be analyzed, which is Fused silica capillary column Elite-1 (30 × 0.25 mm × ID1EM of dimethylpolysiloxane), ensuring that the flow of the carrier helium gas flowed at a rate of 1.0 mL.min-1
           
    In addition, the GC device can be programmed to gradually increase the column temperature according to a specific program: 110°C for 2 min; going up at 5°C min to 200°C and held for 9 min; rising at 5°C min to 280°C and held for 9 min. The program depends on the nature of the compounds to be separated. The prepared sample was then injected into the injection port at the beginning of the chromatographic column at a volume of 0.1ìl and a split ratio of 1:10 (Kavitha et al., 2023).
     
    GC-MS analysis
     
    Compounds are separated using a gas chromatograph according to their spectral characteristics. A Turbo-Mass Gold-Perkin Elmer mass spectrometry detector is used to identify and quantify the compounds in the methanolic extract. The results are compared with mass spectra reference libraries, such as the Wiley Spectral Library and the National Institute of Standards and Technology. The current study was limited to the initial qualitative characterization of the bioactive compounds. Quantitative analysis and statistical validation will be completed in future studies after the compounds recorded in the current qualitative study are separated (Kavitha et al., 2024).

    RESULTS AND DISCUSSION

    GC-MS chromatographic profile
     
    In this investigation, 23 phytochemical substances were identified by GC-MS analysis of Freesia hybrid leaf samples (Fig 3). Oligosaccharides, carboxylic acids, aromatic hydrocarbons, ketene, unsaturated fatty acids and organic heterocyclic compounds were among them. Table 1 shows that there were some active chemicals found in the plant extracts studied and the mass spectrographs for the components found may be seen in Fig 4-26. Relative quantities of the different components were calculated using GC peak areas.

    Table 1: Principal phytochemical constituents found in the methanolic extract of Freesia hybrida.



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    Fig 3: Chromatogram profile for GC-MS in methanolic extract of Freesia hyprida.



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    Fig 4: Mass spectrum of octanamide, N-(2-mercaptoethyl).



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    Fig 5: Mass spectrum of D-glucose,6-O-á-galactopyranosyl.



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    Fig 6: Mass spectrum of 3-hydroxydodecanoic acid.



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    Fig 7: Mass spectrum of HEPES.



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    Fig 8: Mass spectrum of n-Glycyl-DL-leucine.



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    Fig 9: Mass spectrum of á-D-glucopyranoside, O-á-D-glucopyranosyl-(1. Fw).



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    Fig 10: Mass spectrum of 2-propyl-tetrahydropyran-3-ol.



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    Fig 11: Mass spectrum of methyl-6-oxoheptanoate.



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    Fig 12: Mass spectrum of 6-acetyl-â-d-mannose.



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    Fig 13: Mass spectrum of 9-octadecenamide, (Z).



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    Fig 14: Mass spectrum of muramic acid.



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    Fig 15: Mass spectrum of formamide, N-methyl-N-4-[1-(pyrrolidinyl)-2-butynyl].



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    Fig 16: Mass spectrum of dithiocarbamate, S-methyl-, N-(2-methyl -3-oxobutyl).



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    Fig 17: Mass spectrum of 2-methoxy-4-vinylphenol.



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    Fig 18: Mass spectrum of 1,2-cyclopentanedicarboxylic acid ,4-(1,1-dimethyl).



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    Fig 19: Mass spectrum of 9-hexadecenoic acid.



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    Fig 20: Mass spectrum of 1-(3,6,6-trimethyl-1,6,7,7a-tetrahydrocyclopenta[c].



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    Fig 21: Mass spectrum of 5,6,6-trimethyl-5-(3-oxobut-1-enyl)-1-oxaspiro [2.5].



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    Fig 22: Mass spectrum of 2H-oxecin-2-one,3,4,7,8,9,10-hexahydro-4-hydro.



    ​

    Fig 23: Mass spectrum of propiolic acid, 3-(1-hydroxy-2-isopropyl-5-methyl.



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    Fig 24: Mass spectrum of Z-(13,14-epoxy) tetradic-11-en-1-ol acetate.



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    Fig 25: Mass spectrum of picrotoxin.



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    Fig 26: Mass spectrum of 1-hexadecanol, 2-methyl.


     
    Identification of phytochemical compounds
     
    The methanolic extract of F. hybrida revealed a prominent peak during chromatogram GC-MS analysis and the constituents matching the peak were identified as follows. Octanamide, N-(2-mercapto ethyl)-, was identified as the first set-up peak with a retention duration of 3.150 min (Fig 4). The second peak was indicated to be D-Glucose, 6-O-α-galactopyranosyl, in 3.298 min (Fig 5).
           
    The next peaks are considered to be 3-Hydroxydodecanoic acid, HEPES (4-(2-Hydroxyethyl) piperazine-1-ethane sulfonic acid), n-glycyl-DL-leucine; α- D- Glucopyranoside, O- α- D-glucopyranosyl (1. fwdarw. 3)- β- D-fructofuranosyl; 2-Propyl- tetrahydropyran -3-ol;  Methyl-6-oxoheptanoate; 6-Acetyl-β-d-mannose; 9-Octadecenamide,(Z)-; Muramic acid; Formamide,N-methyl-N-4-[1-(pyrrolidinyl)-2-butynyl]; Dithiocarbamate,S-methyl-, N-(2-methyl-3-oxobutyl); 2-Methoxy-4 vinylphenol; 1,2-Cyclopentanedicarboxylic acid,dimethyl ester; 9-Hexadecenoic acid;1-(3, 6, 6-Trimethyl-1,6,7,7 tetra hydro cyclopenta [c] pyran-1-y l) ethanone; 5,6,6-Trimethyl-5-(3-oxobut-1-enyl)-1-oxaspiro [2.5]; 2H- Oxecin- 2-one, 3, 4,7,8,9,10-hexahydro- 4-hydroxy-10-methyl-; Propiolic acid,3-(1-hydroxy- 2-isopropyl- 5-methylcyclohexyl); Z-(13,14-Epoxy) tetradec-11-en-1-olacetate; Picrotoxin;1-Hexadecanol,2-methyl- from the retention time 3.447 to 11.195 (Fig 6 to 26). To learn about the biological impacts and chemical makeup of Freesia chemical compounds, GC-MS analysis is a viable option. The chemical components of a plant sample may be separated and analyzed using GC-MS. Secondary metabolites include all of these substances. These secondary metabolites serve as a defense against microbial infections or animal predation, distinguishing them from the components of primary metabolism. They are not engaged in general metabolism, however. To activate or inhibit signal transduction pathways in live cells, chemicals derived from similar plant species may be necessary (Kuruppu et al., 2019; Muteab and AL-Abedy, 2025).
     
    Biological activities of identified compounds
     
    The current results show similarity in chemical composition to some other aromatic species of the Iridaceae family, particularly in their content of unsaturated fatty acids and aromatic compounds. This suggests that Freesia hybrida may share some of the metabolic pathways responsible for the production of volatile compounds with biological activity, these compounds may have anti-mutagenic, anti-cancer and other properties. Table 2 shows the most important compounds identified in freesia leaf extract and their biological activities as reported in previous studies, which enhances the possibility of using these compounds in future medical and therapeutic applications such as D-Glucose, 6-O-α-galactopyranosyl- and α-D-Glucopyranoside, O-α-D-glucopyranosyl (1.fwdarw.3)-ß-D-fructofuranosyl have antibacterial and antifungal activity (Kamal et al., 2015) and anti-diabetic, anti-hyperlipidemic, anti-oxidant activity (Kumar et al., 2015); Fatty acid metabolic problems are linked to 3-hydroxy dodecanoic acid, a medium-chain fatty acid (Chickos et al., 2002); Also, 2-Propyl-tetra hydropyran-3-ol has anti-angiogenic effect (Hussein et al., 2016); and anticancer activity as the compounds Methyl 6-oxoheptanoate (Al Tameme et al., 2015; Mokni et al., 2016), These compounds represent the chemical basis that may explain the traditional use of freesia as a fragrance and antimicrobial in some popular applications.

    Table 2: Summary of major identified compounds and their reported biological activities.


           
    The alcoholic extract of Freesia also contained the compound 6-Acetyl-ß-d-mannose which has anti-inflammatory and anti-oxidant effects (Sosa et al., 2016); and 9-Octadecenamide, (Z) which has Anti-inflammatory activity and antibacterial activity (Hadi et al., 2016); Further bioactivities of hexadecanoic acid include nematicide, insecticide, hypocholesterolemic, antioxidant and larvicide for mosquitoes (Rajalakshmi and Mohan, 2016; Kumar et al., 2017). Because of its antioxidant and anti-inflammatory characteristics, freesia may have therapeutic uses due to the presence of chemicals like 9-Hexadecenoic acid and 2-Methoxy-4-vinylphenol. Because of these qualities, plant extracts may find application in the production of natural medicinal and cosmetic goods. Al-Marzoqi et al. (2015) indicated that Dithiocarbanate, S-methyl-, N-(2-methyl-3-oxobutyl) has anti-cancer agents and anti-inflammatory effects (Kadhim et al., 2016) and anti-bacterial activity (Al-Khafaji, 2018). Beyond its dual roles as a pediculicide and pesticide, picrotoxin stimulates the central nervous system and the respiratory system (Böttger et al., 2018) and Propiolic acid, 3-(1-hydroxy-2-isopropyl-5- methylcyclohexyl) has anti-cancer activity (Rajalakshmi and Mohan, 2016; Hepokur et al., 2020) indicated that 5,6,6-Trimethyl-5-(3-oxobut-1-enyl)-1-oxaspiro [2.5] is one chemical compound of Thymbra capitata ethanolic extract which have antimicrobial, antioxidant, cytotoxic activities. Also, (Singh et al., 2021) mentioned that 1-(3, 6, 6-Trimethyl-1, 6, 7, 7a-tetrahydro cyclopenta [c]pyran-1-yl) ethanone compound has antihistamine activity and the anti-germination, anti-inflammatory, analgesic and antibacterial properties of 2-methyl-4-vinyphenol are well-documented (Ibibia et al., 2016).
           
    Freesia hybrida     and other Iridaceae species shared metabolic traits, according to Weng et al., (2021). Previous research has shown that phenolic compounds, fatty acids and volatile organic compounds from this family have antioxidant, antibacterial and plant defense properties. Li et al. (2024) studied saffron (Caffron crocus), a member of the Iridaceae family, to identify secondary metabolites. This finding suggests that similar environmental conditions or genetic routes may affect metabolite synthesis, which could lead to similar health benefits across species. They found 88 favorable metabolites, including lipids, alkaloids, amino acids, terpenoids, organic acids and flavonoids. These metabolites primarily participate in metabolic pathways, nucleotide metabolism, purine metabolism and the production of flavonoids. Many plant species have well-studied flavonoid biosynthesis pathways (Gao et al., 2020; Wu et al., 2022). Due to the fact that chemical identification depends on comparing spectra with reference libraries and GC-MS can only identify volatile compounds (no heavy or non-volatile compounds are detected), there may be some restrictions to the accuracy of identification. Hence, these findings should be seen as first clues for understanding plant chemical composition; more studies using liquid chromatography-mass spectrometry (LC-MS) or Nuclear Magnetic Resonance spectroscopy (NMR) to confirm the existence of non-volatile chemicals are suggested.
           
    The discovery of these chemicals’ biological activity in freesia is a significant step towards comprehending the chemical variety of this plant genus, even if some of these activities have been seen in other plants.  Not only do these findings corroborate earlier efforts, but they also point to uncharted territory with promise in areas like aromatics and pharmaceuticals. The limitations of the current study, including the lack of quantitative quantification of the identified compounds, are a major limitation that should be taken into account. However, this work constitutes a preliminary step toward identifying key chemical patterns that can be quantitatively analyzed in future research.

    CONCLUSION

    Based on the information provided, it is clear that GC-MS technology is capable of identifying heterocyclic organic compounds, oligosaccharides, carboxylic acids, aromatic hydrocarbons, chitin, unsaturated fatty acids and other bioactive compounds in freesia extracts. Their biological activities can be validated in the future using specialized bioassays. By separating these compounds, the purified extract could contribute to the development of new drugs or the improvement of existing treatments.

    ACKNOWLEDGEMENT

    The Biology Department at Babylon University College of Science for Women supported the present study and provided invaluable assistance with this project.
     
    Disclaimers
     
    The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.

    CONFLICT OF INTEREST

    The authors assert that no conflicts of interest exist.

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