First phytochemical and antioxidant potential of essential oil and hydrolate of Asperula hirsuta Desf.ABSTRACT
This study presents the first chemical and biological investigation of the essential oil and hydrolate of Asperula hirsuta Desf. (Rubiaceae) collected from Batna, Algeria. The essential oil and hydrolate were analyzed using GC-MS and HPLC techniques, respectively, and their phenolic and flavonoid contents as well as antioxidant activities were evaluated. A total of 45 components were identified in the essential oil, which was found to be rich in terpenes (27.26%) and saturated hydrocarbons (25.21%). The hydrolate was dominated by caffeic acid, representing 34.71% of its phenolic content. Regarding phytochemical content, the hydrolate exhibited the highest levels, with a total phenolic content (TPC) of 61.98±1.91mg GAE/g and a total flavonoid content (TFC) of 120.22±7.92mg RE/g. The hydrolate also showed higher antioxidant activity than the essential oil, as assessed by DPPH and CUPRAC tests. Notably, this study reports the first evaluation of FRAP and CUPRAC tests for Asperula species. In conclusion, the richness in bioactive compounds of A. hirsuta, combined with its remarkable antioxidant activity, could make it a natural antioxidant agent with considerable potential in various applications.
Keywords:
Asperula hirsuta; essential oil; hydrosol; GC-MS; antioxidant capacity
RESUMO
Este estudo apresenta a primeira investigação química e biológica do óleo essencial e do hidrolato de Asperula hirsuta Desf. (Rubiaceae) coletada em Batna, Argélia. O óleo essencial e o hidrolato foram analisados por GC-MS e HPLC, respectivamente, e seus teores de fenólicos e flavonoides, bem como suas atividades antioxidantes, foram avaliados. Um total de 45 componentes foram identificados no óleo essencial, que se mostrou rico em terpenos (27,26%) e hidrocarbonetos saturados (25,21%). O hidrolato foi dominado pelo ácido cafeico, representando 34,71% do seu teor de fenólicos. Em relação ao conteúdo fitoquímico, o hidrolato apresentou os níveis mais elevados, com um teor total de fenólicos (TPC) de 61,98±1,91mg GAE/g e um teor total de flavonoides (TFC) de 120,22±7,92mg RE/g. O hidrolato também apresentou maior atividade antioxidante do que o óleo essencial, conforme avaliado pelos testes DPPH e CUPRAC. Notavelmente, este estudo relata a primeira avaliação dos testes FRAP e ABTS para espécies de Asperula. Em conclusão, a riqueza em compostos bioativos da A. hirsuta, combinada com sua notável atividade antioxidante, pode torná-la um agente antioxidante natural com considerável potencial em diversas aplicações.
Palavras-chave:
Asperula hirsuta; óleo essencial; hidrossol; GC-MS; capacidade antioxidante
INTRODUCTION
In recent years, interest in medicinal plants as part of alternative therapeutic approaches has grown considerably, owing to their broad range of potential applications and therapeutic properties (Latif and Nawaz, 2025). These benefits are largely attributed to the high content of bioactive natural compounds in their extracts, particularly antioxidant molecules (Jain et al., 2025).
Essential oils and plant hydrolats also known as hydrolates or hydrosols or floral waters, are natural extracts rich in biologically active molecules (Al-Mansour, 2021). These compounds confer diverse medicinal properties, including skincare benefits as well as sedative, antimicrobial, anti-depressive and antioxidant activities (Vuko et al., 2021; Almeida et al., 2024). Such activities offer promising avenues for the development of new therapeutic strategies aimed at preventing chronic diseases and aging in which oxidative stress is frequently identified as a key factor (Al-Mansour, 2021), thereby explaining the growing scientific interest in these natural fractions. While essential oils have been extensively studied, mainly due to their richness in water insoluble volatile compounds, hydrolats, which are by-products of essential oil extraction, have received comparatively less attention despite containing unique water-soluble and polar metabolites (Aćimović et al., 2020).
Chemical characterization of plant extracts is essential to identify their constituents, elucidate their mechanisms of action, and evaluate their potential toxicity. Although plant families such as Lamiaceae and Rutaceae have been extensively studied (Popa et al., 2021; Slougui et al., 2023), research on the cosmopolitan and highly diverse Rubiaceae family, which comprises approximately 13,000 species across 630 genera (Karou et al., 2011) remains scarce, highlighting the need for further phytochemical and biological investigations. Medicinal plants of the Rubiaceae family are reported in the literature to possess strong antioxidant properties, which supports their traditional therapeutic use (Heitzman et al., 2005; Lakić et al., 2010; Martins and Nunez, 2015; Suksungworn and Duangsrisai, 2021).
Within this family, the genus Asperula has been traditionally used in Turkish and Ukrainian medicine for its antidiarrheal, tonic, diuretic, antihypoxic, and sedative properties (Iurchenko et al., 2015; Özgen et al., 2018). In Algeria, however, this genus remains largely unexplored, particularly from a phytochemical and biological perspective. Previous studies have reported the presence of iridoids, flavonoids, anthraquinones, and phenolic acids in Asperula species (Park et al., 2002; Özgen et al., 2006; Tzakou et al., 2011; Kırmızıbekmez et al., 2014, 2017).
Asperula hirsuta Desf., widely distributed across North Africa (except Egypt) and common in the Algerian Tell and mountainous regions, is locally known as ‘Fouaou’. Owing to its broad distribution and adaptation to mountainous environments, this species is one of the five Asperula taxa recorded in the Algerian flora (Quezel and Santa, 1963), it represents a valuable source of natural bioactive compounds. Its medicinal and pharmacological properties, however, remain poorly studied. Therefore, the present study aimed to characterize, for the first time, the chemical profile of its essential oil (AHEO) using gas chromatography-mass spectrometry (GC-MS) and that of its hydrolate (AHHD) using high-performance liquid chromatography (HPLC), as well as to evaluate the antioxidant potential of both fractions through various in vitro assays.
ETHICAL ASPECTS
Not applicable. This research was not submitted to the Ethics Committee on Animal Use.
MATERIALS AND METHODS
Asperula hirsuta leaves, stems, and flowers were collected in Marouana region, wilaya of Batna in eastern Algeria in May 2024, and identified by Dr. Bachir Oudjehih from Batna university.
The fresh aerial parts (554g) were used to extract essential oils by steam distillation for three hours using a Kaiser Lang apparatus, with the circulating steam maintained at 100 °C. The oil was recovered by decantation using n-hexane, then transferred into an amber vial and stored at 4 °C until further use.
The hydrolate (AHHD) is collected in a distilled water fraction obtained at the same time as the essential oil extraction from the plant by steam distillation. Then it was stored at 4 °C until use.
The AHHD (2 ml) was then poured into a petri dish wrapped in cling film and frozen at (-60 °C) before being concentrated in a freeze-dryer for approximately 18h in order to prepare it for subsequent analyses.
The AHEO dissolved in n-hexane (500µL) was used for analysis; it was previously dried over anhydrous sodium sulfate and filtered. The volatile compounds were analyzed using a Gas Chromatography Mass Spectrometry system (GC-MS, Shimadzu QP-2020) equipped with an Rxi-5MS capillary column (30m×0.25 mm, 0.25μm film thickness). The oven temperature was initially set at 60°C for 5min, then increased to 150°C at a rate of 10°C/min, held for 3min, and finally raised to 300°C at 10°C/min and maintained for 5min. The injector and interface temperatures were 280°C, and the ion source temperature was 250°C. Helium was used as the carrier gas at a constant flow rate of 1.0mL/min with a split ratio of 1:10. Data were acquired in full-scan mode over a mass range of 30-800 Da.
The AHHD phenolic composition was determined using high-performance liquid chromatography (HPLC) equipped with a photodiode array (PDA) detector (SPD M20A, Shimadzu, Kyoto, Japan). Chromatographic separation of phenolics was carried out on an ACE C18 column (250mm x 4.6mm, 3µm particle size) paired with a guard column (4.0 x 10mm, 2µm particle size) from Advanced Chromatography Technologies Ltd (Aberdeen, UK). A gradient elution system was employed using mobile phase A consisting of a Milli-Q 0.75% formic acid in water, and mobile phase B consisting of 0.75% formic acid in Methanol. The injection volume was 10µL. The column was held at 40°C, and the sampler was kept at room temperature (25°C), with a flow rate of 0.5mL/min. The gradient was 10% B for the first 5 minutes, followed by 55% B for the next 40 minutes, then 90% B for 4 minutes, followed by a return to 10% B for the 8 minutes (of column re-equilibration)
The total phenolic content (TPC) of the essential oil and hydrosol was determined using the Folin-Ciocalteu method, as described by (Aydar et al., 2023). The results are expressed as milligrams of gallic acid equivalents (mg GAE/g extract), based on the gallic acid calibration curve.
The total flavonoid content (TFC) of the hydrosol was evaluated using a colorimetric method involving complex formation with aluminum chloride (AlCl₃), according to the reported procedures (Jia et al., 1999) with slight modifications. The quantification was performed based on a rutin calibration curve, and the results were expressed as milligrams of rutin equivalents per gram of extract (mg RE/g extract).
The essential oil antioxidant potential was estimated by DPPH and CUPRAC tests; and for the hydrolat by DPPH, CUPRAC, ABTS and FRAP methods. According to reported procedures (Kumaran and Karunakaran, 2006), the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging activity of both the essential oil and the hydrolate was evaluated. Serial dilutions ranging from 1/2 to 1/20 were prepared. The results were expressed as IC₅₀ values (mg/mL), in mg Trolox equivalents (TE) per g of extract, and as the percentage of DPPH radical scavenging activity (%).
Based on the protocol used by Deighton et al. (2000) where 100µl of hydrolate were mixed with 900µl of the FRAP (Ferric Reducing Antioxidant Power) reagent (obtained by mixing 300mM sodium acetate solution at Ph = 3.6, 10mM 2,4,6-Tris (2-pyridyl)-s-triazine (TPTZ) solution prepared with 40mM HCl, and 20mM FeCl3 solution at a ratio of 10:1:1). The absorbance was measured 4 minutes later, at 593nm. The results were expressed in mg Trolox equivalent (TE)/g extract.
The ABTS•⁺ (2,2’-azinobis 3-ethyl-benzothyazoline 6-sulphonate diammonium salt) assay was carried out according to the method followed by Kamiloglu et al, (2015). This test assesses the antioxidant capacity of the extracts to scavenge ABTS•⁺ free radicals. The antioxidant activity was expressed as the percentage of inhibition (I%) and as Trolox equivalents (mg TE/g extract). The percentage of inhibition, which reflects the ability of the sample to neutralize free radicals, was calculated based on the decrease in absorbance measured at 734nm. The activity was then converted to Trolox equivalents using a calibration curve established with different concentrations of Trolox.
According to (Apak et al., 2005), the antioxidant activity was determined by measuring the reducing power using the CUPRAC (Cupric Ion Reducing Antioxidant Capacity) assay. The results were expressed as milligrams of Trolox equivalents per gram of extract (mg TE/g extract) and as percentage inhibition (I%), reflecting the ability of the sample to reduce Cu²⁺ to Cu⁺ ions in the reaction medium.
RESULTS
GC-MS analysis of AHEO allowed to the identification of forty-five compounds (Table 1, Figure 1), belonging to different chemical classes (Figure3). Terpenes (27.26%) and saturated hydrocarbons (25.21%) represented the major classes of the total composition. The main volatile constituents were hexadecanoic acid (palmitic acid) (12.41%), octadecane (11.86%), 6,10,14-trimethyl-2-pentadecanone (10.38%), phytol (7.86%), and heneicosane (5.49%).
The HPLC analysis of AHHD obtained from the aerial parts was identified for the first time based on their retention times, shows the presence of four majority peaks classified into two categories of polyphenol compounds: phenolic acids and flavonoids (Fig. 2, Table2).
Two phenolic acids were detected in AHHD; caffeic acid was the predominant compound (34.71%), reaching a concentration of 5.24±0.025 ppm, followed by chlorogenic acid (17.6%) with 7.12±0.21 ppm. In addition, two flavonols were identified: rutin (quercetin-3-O-rutinoside) and rhamnetin, with rutin constituting the major flavonoid (21.25%) of the total peak area) with 22.80±0.41 ppm.
The TPC and the TFC of AHEO and AHHD aerial parts are reported in Table 3.
In this study, the TPC of the investigated AHEO extract (12.76±3.79mg GAE/g extract) was found to be markedly lower than that of the corresponding hydrosol (61.98mg GAE/g extract). The TFC of AHHD was estimated at 120.22±7.92mg RE/g sample.
The in vitro antioxidant activity evaluation of AHEO and AHHD was determined, for the first time, by DPPH, ABTS, CUPRAC and FRAP assays. The results that were obtained are given in Table 4 and Table 5. To date, no published studies have reported the antioxidant activity of essential oils or hydrolates from Asperula species (Rubiaceae) and A. hirsuta. The available works mainly concern polar or semi-polar extracts (methanolic, aqueous, etc.) of different Asperula species. This gap in the literature highlights the originality and relevance of investigating the antioxidant activity of Asperula essential oils and hydrolates.
The essential oil of A. hirsuta exhibited moderate DPPH radical scavenging activity with an IC₅₀ value of 21.70±0.70mg/mL, compared to Trolox, the reference compound, which showed much stronger activity (IC₅₀= 0.129mg/mL). In contrast, the hydrosol demonstrated a lower IC₅₀ value (1.57±0.14mg/mL), indicating a better capacity to scavenge free radicals.
Regarding the cupric reducing antioxidant capacity of A. hirsuta, it revealed a clear difference between the essential oil and the hydrosol. The hydrosol exhibited a high reducing capacity (330.83±38.33 mg TE/g extract), while the essential oil displayed a much lower value (14.00±1.89 mg TE/g extract). This study is the first to report the CUPRAC reducing capacity of Asperula extracts, including A. hirsuta. Additionally, the hydrosol demonstrated significant ABTS radical scavenging activity (101.49±6.4mg TE/g extract).
The ferric reducing antioxidant power assay also indicated significant antioxidant activity in the hydrosol, with a value of 58.61±4.94mg TE/g extract.
DISCUSSION
To the best of our knowledge, only one study on the essential oils of the Asperula genus has been published. In that work, terpenes accounted for only 5.96% of the essential oil of A. oppositifolia, whereas hydrocarbons were the predominant class at 39.85%, followed by alcohols at 28.70% (Halimi and Nasrabadi, 2015). The oil was mainly composed of 2-(6,6-dimethylbicyclo [3.1.1] hept-2-en-2-yl) ethanol (17.16%), decane (8.47%), dibutyl phthalate (5.59%), and 1-bromonaphthalene (4%). These profiles differ markedly from those of A. hirsuta, in which terpenes represented 27.26%, hydrocarbons 25.21%, and alcohols only 4.54%, with terpenes being the predominant class and alcohols the least abundant.
Palmitic acid (Hexadecanoic acid), the major constituent of the studied oil, is a saturated fatty acid recognized for its broad bioactive potential. It is widely used in the pharmaceutical industry as a key component in the formulation of antioxidant drugs and has been associated with beneficial effects in the management of several metabolic disorders, including obesity, type 2 diabetes, cardiovascular diseases, and certain cancers (Carta et al., 2017).
In addition to palmitic acid, the oil also contains other biologically active constituents. Among them, octadecane has been reported to possess significant antibacterial, antifungal, antioxidant, and anti-inflammatory activities ( Al-Marzoqi et al., 2015).
Furthermore,6,10,14-trimethyl-2-pentadecanone, another major component of A. hirsuta oil, is a sesquiterpenoid known for its antibacterial and anti-inflammatory properties, suggesting potential applications in pain management (Wei et al., 2016; Avoseh et al., 2021). Phytol is a diterpene classified as long-chain, unsaturated acyclic alcohol. It demonstrates a remarkably broad spectrum of biological activities, including antianxiety, cytotoxic, metabolism-modulating, antioxidant, apoptosis-inducing, antinociceptive, anti-inflammatory, immunomodulatory, and antimicrobial effects. Its widespread occurrence in nature, combined with this diverse pharmacological profile, highlights its significance as a compound of considerable commercial value (Islam et al., 2018). Antioxidant activity is regarded as one of the most crucial properties of bioactive compounds, since oxidative stress is implicated in the development or progression of many diseases (Besednova et al., 2017).
To the best of our knowledge, no previous studies have reported the identification of phenolic compounds in the hydrolate of the Asperula genus, highlighting a significant divergence from the profiles observed in other extracts of Asperula species described in the literature. The relative percentages of phenolic compounds were calculated based on the peak area values reported by Özgen et al., (2018). They identified p-hydroxy benzoic acid (90.83%) as the major one in the methanolic extract of A. taurina subsp. caucasica, along with benzoic acid (3.45%) and protocatechuic acid (5.73%). Iurchenko et al., (2015) analyzed the hydromethanolic extract of A. odorata L. (syn. Galium odoratum), a species widely used in Ukrainian traditional medicine, and reported chlorogenic acid (16.75mg/g) as one of the main compounds, with p-coumaric acid (0.13mg/g) and 4,5-dicaffeoylquinic acid (1.86mg/g). However, according to the literature, caffeic acid has not been previously reported in the genus Asperula and was detected for the first time in A.hirsuta in this study.
Previous phytochemical investigations on the genus Asperula have consistently reported the presence of flavonoids, most of which belong to the flavonol glycoside group, particularly quercetin derivatives and flavone glycosides (Özgen et al., 2018). The aglycone rhamnetin has not been reported in Asperula species; however, in our study on A. hirsuta, this flavonol was detected in the hydrolate. Rhamnetin can be found in various natural sources, including fruits, vegetables, and herbs, as well as in their derived products. It is a secondary metabolite with several pharmacological properties, including antioxidant, anticancer, anti-inflammatory, antiviral, and antibacterial activities (Medeiros et al., 2022).
The AHHD contains polyphenols, particularly flavonoids and phenolic acids, which are well known for their antioxidant activity and associated health benefits. As noted in the previous sections, rutin, quercetin and their derivatives are recognized for their beneficial in humans including the prevention of various diseases such as cancer, cardiovascular disorders, diabetes, obesity, liver dysfunction and infectious diseases (Rasouli et al., 2017).
The results suggest that the differences observed in the phytochemical profiles of Asperula species could be attributed to the extraction method and the type of extract (dry extract, essential oil or hydrolate).
However, when compared with literature data on A. hirsuta, the phenolic content of the hydrolate was lower than that reported for the methanolic extract (144.68 ± 10.87 mg GAE/g extract) and the aqueous extract (109.36 ± 19.41 mg GAE/g extract) obtained from aerial parts of A. hirsuta collected in Sétif (Algeria) (Kherbache et al., 2024). Nevertheless, the TFC results indicate that the hydrolate possesses a higher concentration of flavonoids than other Asperula extracts, highlighting its potential as a valuable source of bioactive compounds.
These differences can be attributed to the solvents nature and the extraction methods employed, which strongly influence both the chemical composition and the concentration of phenolic metabolites.
The hydrolate of A. hirsuta exhibited stronger radical scavenging activity (DPPH) than its essential oil as well as a greater activity than that reported for the methanol extract of G. rotundifolium(IC₅₀= 126.11 mg/mL) studied by Kumar et al. (2025), given the taxonomic relationship between Asperula and Galium genera. However, this value was lower than those reported for the endemic A. lilaciflora subsp. phrygia (IC₅₀= 0.38 mg/mL) and A. pestalozzae (IC₅₀= 0.134±0.017 mg/mL), as reported by (Kayiş and Kaya, 2023, 2024) .
The CUPRAC capacity is consistent with the nature of the hydrosol extract, which mainly contains phenolic compounds, as presented in the current study. These compounds are considered excellent electron donors due to their high content of hydroxyl groups (Gutiérrez-Del-Río et al., 2021). The radical scavenging capacity was also evaluated using the ABTS•⁺ assay. The results for the AHHD further corroborate the antioxidant potential previously reported for other Asperula species, although direct comparisons are limited due to differences in measurement units. The ferric reducing antioxidant power of AHHD is higher than those reported by (Mocan et al., 2019) for different Galium species (Rubiaceae) extracts. Reported FRAP values for Galium species were 45.2±1.1 mg TE/g (G. purpureum), 19.4±0.9 mg TE/g (G. pseudoaristatum), 12.6±0.2 mg TE/g (G. rivale), 17.19±0.04 mg TE/g (G. album), and 21.9±0.9 mg TE/g (G. verum). Strong correlations have been reported between DPPH and FRAP antioxidant activities and the total flavonoid and phenolic contents in methanolic extracts of Rubiaceae plants (Suksungworn and Duangsrisai, 2021), consistent with the findings of the present study on AHHD. Overall, it is worth mentioning that the Rubiaceae species exhibit potential as a promising source of natural antioxidants. In the other hand, in most studies, there is an association between TPC, TFC and antioxidant ability (Liaquat et al., 2023; Esguerra et al., 2024) according to the fact that phenolic substances are the major plant antioxidants. They act as chelators of metal ions, and as scavengers of free radicals that catalyze ROS (reactive oxygen species) formation implicated in cell damage (Rudrapal et al., 2022; Zhou et al., 2024). Terpenoids and polyphenols (like quercetin and other flavonoids) are important phytochemicals with a broad range of antioxidant effects (Gutiérrez-del-Río et al., 2021). Although these compounds constitute the major constituents of the hydrolate and essential oil, the minor components also contribute significantly, acting synergistically to enhance the plant’s overall antioxidant potential.
CONCLUSION
The present study highlighted the importance of chromatographic techniques in elucidating the chemical composition of largely uninvestigated Rubiaceae species, particularly those of the Asperula genus, thereby contributing to a better understanding of their potential medicinal value. This work provides the first comprehensive characterization of both the essential oil and the hydrosol of the Algerian A. hirsuta, a member of the Rubiaceae family whose medicinal and economic relevance remains underexplored.
Despite the absence of directly comparable literature, the results clearly demonstrate the ability of A. hirsuta to neutralize free radicals, an activity attributable to the presence of antioxidant phytochemicals namely phenolic acids and flavonoids identified through HPLC analysis of the hydrosol. In addition, GC-MS investigation revealed that the essential oil of A. hirsuta is particularly rich in biologically active molecules, with terpenes exhibiting metal-reducing properties, a feature that is not commonly reported within the Rubiaceae family.
Overall, the antioxidant potential of A. hirsuta essential oil and hydrosol appears closely linked to their richness in bioactive constituents. These findings establish a foundational reference for this species and open new avenues for further research into its pharmacological and therapeutic applications.
ACKNOWLEDGEMENT
The authors would like to thank MESRS Algeria (Ministère de l’Enseignement Supérieur et de la Recherche Scientifique), and the Department of Food Engineering, Faculty of Chemical and Metallurgical Engineering of Istanbul Technical University, Türkiye for their provision of laboratories. They would also like to express their gratitude to Dr. HAZMOUNE Hichem for performing the essential oils extraction from the plant material.
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