Introduction

Malvaviscusis a small genus of perennial herbs or shrubs in the Hibiscus or mallow family, Malvaceae. It includes about 11 species with a natural distribution in the tropics and subtropics [1, 2]. Despite their close resemblance to Hibiscus plants, members of the genus Malvaviscus are generally distinguished by having ten, rather than five, capitate stigmas overlying a central style, in addition to the production of schizocarpic fruits formed of five separate parts [1, 3]. Malvaviscus arboreus Cav. (syn. Malvaviscus mollis (Aiton) DC. and Hibiscus malvaviscusL.) is a perennial deciduous shrub natively found in Central and South America but was also cultivated and naturalized in some tropical and subtropical regions of Africa, Asia, and Australia [4-6]. It is commonly known as Wax mallow, Turk's cap, and Sleeping Hibiscus; the latter name comes from its tightly wrapped petals that are slightly expanding only at the top [1]. The fast-growing shrubs of Sleeping Hibiscus are up to 1 m in length carrying ovate to broadly cordate leaves and red solitary flowers [7]. Malvaviscus plants, including M. arboreus, are widely grown as both garden ornamentals and medicinal plants with varied ethnomedical uses, mostly for wounds, fever, hypertension, sore throat, bronchitis, gastritis, and liver problems [1, 8-11]. The flowers, fruits, and leaves of M. arboreus are also useful for preparation of jellies, salads, herbal dyes, and herbal teas [1]. Previous phytochemical work on M. arboreus leaves and flowers revealed the presence of a range of phenolic metabolites, e.g. flavonoids, anthocyanins, and phenolic acids, along with sterols, terpenoids, and fatty acids [10, 12, 13]. Moreover, different extracts of this plant species were shown to possess antioxidant, hepatoprotective, cytotoxic, and anti-infective properties [14, 15]. Yet no reports are available in the literature on the volatile composition of Malvaviscus plants except for that by Vazquez-Cahuich et al. [16], where solely two compounds, namely eugenol and 1,4-dichlorobenzene, were detected at very low concentrations among the hydrodistilled leaf volatiles of M. arboreus growing in Mexico. Hence, in continuation of our research on M. arboreus plants cultivated in Egypt [12, 14, 17‒19], the present work was designed to identify and compare the volatile constituents of its leaves and flowers using the headspace GC-MS technique.

Experimental

Plant material

The fresh leaves and flowers of M. arboreus were collected at the flowering stage from plants cultivated in the campus of Minia University, Minia, Egypt. The plant was identified by Prof. Mahmoud A. Hassan, Department of Horticulture, Faculty of Agriculture, Minia University. A voucher sample with the number Mn-Ph-Cog-027 was kept in the herbarium of Pharmacognosy Department, Faculty of Pharmacy, Minia University.

Headspace GC-MS analysis

Volatiles were extracted using a solid phase microextraction (SPME) fiber coated with polydimethylsiloxane-divinylbenzene (65 μm film thickness; Supelco, Bellefonte, PA, USA). For each sample, the SPME device was inserted into the sealed vial by penetrating the silicone septum, followed by exposing the fiber to the headspace of the plant material (10.0 g) after 30 min. The fiber was exposed to each sample at 40 °C for 30 min. After extraction, the fiber was immediately inserted into the heated GC injector port for 10 min for desorption in the splitless mode. Samples were investigated in duplicate. GC-MS analyses were carried out using a Shimadzu GC-MS (QP-2010 Ultra, Kyoto, Japan) equipped with a Rtx-5MS column (5% diphenyl/ 95% dimethyl polysiloxane; 30 m × 0.25 mm i.d., 0.25 μm film thickness; Agilent, Palo Alto, CA, USA) and a headspace AOC-5000 auto-injector. Injections were performed in the splitless mode at 210 °C. A flame ionization detector was used at 230 ºC, whereas the temperatures of the interface and ion-source were kept at 280 and 230 ºC, respectively. Helium was used as a carrier gas at a flow rate of 1 mL/min. The programmed temperature technique was started at 40 ºC (for 2 min), then increased at 5 ºC/min to 210 °C with a 5-min hold at that final temperature, with the total run time was 40 min. Electron impact mass spectra were obtained at 70 eV; scanning from 35 to 500 m/z. A blank system with no plant material was also run at the same conditions as a control. The percentage composition of the identified volatiles was calculated from the total ion chromatogram (TIC), while they were identified by comparing their retention indices (RI) and mass spectra with some databases, including Wiley library 9, the National Institute Standard and Technology (NIST), and The Pherobase (Database of insect pheromones and semiochemicals) in addition to the literature [20-23].

Results and discussion

Headspace GC-MS analysis of M. arboreus leaves and flowers revealed notable qualitative and quantitative variations of their volatile constituents. A total of 39 compounds were detected and identified, including 11 from the flowers and 36 from the leaves, which indicates the higher abundance of volatiles in the latter (Table 1; Supplementary information: Fig. S1-S4). As shown in Table 1, the volatile blends of both plant parts were found to comprise varying levels of isoprenoids, fatty acid derivatives, phenyl propanoids/benzenoids, in addition to a group of other miscellaneous compounds. The chief proportion of floral volatiles was occupied by phenyl propanoids (41.64%) owing to the noticeable content of anethole (32.32%), the major volatile constituent identified from the flowers, along with its related compound, methyl chavicol (estragole; 9.32%). Both compounds were also detected in the leaves, albeit at much lower levels (7.39 and 1.66%, respectively). In contrast, fatty acid-derived compounds were detected as the major contributors to the volatile composition of M. arboreus leaves; accounting for 54.30% of their total volatiles, of which acetic acid (18.16%) was the main identified component followed by 2-cyclohexenone (9.60%). Although M. arboreus leaves enjoyed a chemically varied group of fatty acid derivatives, its flowers displayed an evidently lower emission level (8.68%) of these volatiles, with acetic acid (5.06%) and n-hexanol (3.62%) were merely observed, but also at a lesser extent as compared with the leaves (Table 1).

Furthermore, the volatile profile of M. arboreus exhibited a significant range of terpenoids that amounted to 20.39% in the leaves and 32.22% in the flowers (Table 1). This considerable isoprenoid contents of both the leaves and flowers were largely dominated by irregular terpenoids that accounted for 14.51 and 19.18% of their total volatiles, respectively. Generally, irregular terpenoids are volatile carotenoid-derived compounds that essentially contribute to the odour profile of plants. They are produced through several fragmentation patterns of various carotenoids found in different plant parts, mostly by enzymatic oxidation and photo-oxidation [24]. As depicted in Table 1, a variety of irregular terpenoids was emitted by the leaves of M. arboreus, encompassing small acyclic C₆ and C₈ ketones (e.g. methyl isobutenyl ketone and 6-methyl-5-heptene-2-one), C₁₀ aldehydic derivatives (e.g. safranal and β-cyclocitral), in addition to some C₁₃-norisoprenoids (e.g. vitispirane and geranyl acetone), among which, methyl isobutenyl ketone (4.80%) was found to predominate. On the contrary, none of these volatile components was detected in the flowers except for methyl isobutenyl ketone, which was given off at a higher percentage (19.18%). In the same context, although M. arboreus flowers exhibited a relatively higher content of total monoterpenoids (7.42%) in comparison with its leaves (5.88%), only two monoterpenoids were identified from the flowers, namely limonene (5.03%) and fenchone (2.39%). The monoterpenoid fraction of the leaves, on the other hand, was dominated by limonene (2.34%), whereas β-fenchol (0.97%), rather than fenchone, was observed among their montoerpenoids. It is also worth noting that the volatile mixtures of both plant parts of M. arboreus have totally lacked sesquiterpenoids, while only one diterpene hydrocarbon, namely phytane, was exclusively emitted from the flowers at a considerable level (5.62%) (Table 1).

Aside from the aforementioned groups of biosynthetically related volatiles, a number of miscellaneous compounds have also contributed to the headspace composition of M. arboreus leaves and flowers, constituting 13.91 and 17.46% of their total volatile blends, respectively (Table 1). Among such varied volatiles, a considerable concentration of furanoids was noticed, including furfural that was emitted at a marked level from the flowers (7.59%) followed by 2,5-dimethylfuran (7.08%). Conversely, only negligible amount of furfural (0.84%) was recorded from the leaves, while 2,5-dimethylfuran was totally absent. Some other furan derivatives, e.g. 2-pentylfuran, 2-ethylfuran, and 2-(methoxymethyl)-furan were also characterized at different quantitative ratios, particularly from the leaves. Nevertheless, the assortment of miscellaneous compounds identified from the leaves was predominated by 2-ethyl-5,5-dimethyl-1,3-cyclopentadiene (5.29%) that, on the other hand, was not detected in the flowers (Table 1).

Finally, it is also worth mentioning that the volatile mixtures of the two investigated plant parts of M. arboreus displayed comparable amounts of hydrocarbons (10.46% in leaves and 10.65% in flowers) and oxygenated compounds (89.54% in leaves and 89.35% in flowers), with a noticeable greater contribution of the latter to their total volatiles. Among the family of oxygenated volatiles described from M. arboreus, a diversity of chemical classes was recorded (Table 2; Fig. 1). In this regard, phenolic ethers (41.64%) have dominated the oxygenated compounds emitted from the flowers followed by ketones (21.57%), with anethole (32.32%) being the major identified component. In contrast, the main proportion of the volatile principles of the leaves was primarily represented by ketones (27.40%), although acetic acid (18.16%) was the chief constituent given off by the leaves. Moreover, while methyl isobutenyl ketone (19.18%) has dominated the ketone content of the flowers, 2-cyclohexenone (9.60%) was the principal ketone detected from the leaves. Another important difference between the volatile compositions of the studied plant parts from M. arboreus is the marked prevalence of alcohols in the leaves (14.88%) as compared with the flowers (3.62%) (Table 2; Fig. 1). Of these, cis-3-hexenol (5.85%) and n-hexanol (3.62%) were the major alcohols identified from the leaves and flowers, respectively. On the other hand, both the leaves and flowers of M. arboreus demonstrated relatively comparable levels of aldehydes, accounting for 7.99 and 7.59% of their headspace volatiles, respectively, with furfural (7.59%) being the major aldehyde observed in the flowers, whereas 2-methylbutanal (5.28%) was uniquely characterized from the leaves as the chief component of their aldehyde fraction. Moreover, minor concentrations of esters (2.67%), phenols (0.84%), and oxides (0.74%) were found to contribute to the volatile emission of M. arboreus, but only from the leaves (Table 2; Fig. 1).

Conclusion

Comparative headspace GC-MS investigation of M. arboreus leaves and flowers has revealed different qualitative and quantitative patterns of their volatile organic compounds, with a marked abundance of volatiles in the leaves. Volatile emissions from both plant parts were dominated by a structurally varied pool of oxygenated compounds, of which ketones and phenolic ethers prevailed in the leaves and flowers, respectively. The volatile profile of the leaves was also characterized by the prevalence of acetic acid, while anethole was the most abundant compound in the flowers. Besides, the volatile composition of M. arboreus showed considerable amounts of irregular terpenoids and furanoids, particularly in the flowers, whereas no sesquiterpenoids were detected in both organs. As the first comprehensive report on the volatile metabolites of M. arboreus, this work would help expand both the current phytochemical knowledge and future research on plants belonging to the genus Malvaviscus.

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Author notes

*Corresponding author: John Refaat Fahim, email: johnrefaat82@yahoo.com; john.michael@mu.edu.eg Tel.: +2-086-2369075; Fax: +2-086-2369075.

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