Phytochemical analysis of Clinopodium candidissimum (Munby) Kuntze growing in Algeria by an integrated HS-SPME-GC-MS, NMR and HPLC-DAD-MSn approach: valorisation of an endemic natural source of bioactive compounds

Abstract Clinopodium candidissimum (Munby) Kuntze (Lamiaceae) is used in traditional medicine and as a food condiment in Algeria, where it is known as Zaater cheleuh and Nabta elbida. Here, we report the comprehensive characterisation of non-volatile polar constituents extracted from C. candidissimum aerial parts (a mixture of inflorescences, stems and leaves), and their aroma profile. Qualitative 1H‐NMR and quali-quantitative HPLC-MSn analyses of fractions obtained with solvents at different polarity revealed the presence of aglyconic and glycosylated flavonoids (3.1%), phenylpropanoids (3.6%), gallic acid derivatives (0.76%), and triterpenoids (0.62%), among the others. On the other hand, HS‐SPME-GC-MS allowed to identify 38 volatile constituents, among which the oxygenated monoterpenes pulegone (44.8%), piperitenone (6.6%), isopulegone (5.8%) and neo-menthol (3.8%), and the sesquiterpene hydrocarbons germacrene D (16.2%) and bicyclogermacrene (3.0%) were the most abundant. Overall, results indicate that C. candidissimum represents an endemic natural source of antioxidants and bioactive compounds, and they will be useful for further studies on this species. Graphical Abstract


Introduction
Clinopodium candidissimum (Munby) Kuntze (Figure S1) is a strictly endemic species of Algeria (Qu ezel and Santa 1962;Melnikov 2017).This plant has been originally described in Algeria (Oran province) by Mynby as Melissa candidissima Munby, but later it has been reported with several synonyms, in particular Calamintha candidissima (Munby) Benth.and Satureja candidissima (Munby) Briq.(Engler et al. 1897;GBIF Secretariat 2021).Despite the fact that Dobignard and Chatelain retain Calamintha candidissima (Munby) Benth in their synonymic indexes of the flora of North Africa (Dobignard and Chatelain 2012), several works (Govaerts 1999;Govaerts 2003;Melnikov 2016;Melnikov 2017) retain rather Clinopodium candidissimum (Munby) Kuntze as an accepted name.This plant grows spontaneously between oleanders and rocky lawns in and around Oran, and it is known to the population as Zaater cheleuh and Nabta elbida (Qu ezel and Santa 1962).The richness of its essential oil confers to C. candidissimum several biological properties such as antimicrobial, insecticide, larvicide and herbicide, spasmolytic, anti-inflammatory and analgesic.This plant is effective in treating flu and infections, and as a dressing for the healing of burns and wounds.It has a warming effect and is recommended in case of respiratory tract infections (bronchitis) (Chevallier and Larousse 2001).It is used in cooking to flavour sauces or to prepare a traditional Algerian dish from the region of Sid safi, called 'Rfiss'.However, to the best of our knowledge, the chemical composition of C. candidissimum has been scarcely investigated up to now.In order to promote Algerian medicinal and edible plants, in this work we aimed at characterising the volatile composition and the non-volatile polar constituents of C. candidissimum aerial parts, using different extractive and analytical approaches.A headspace solid phase micro-extraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) method was used for the characterisation of volatiles, while integrated NMR and HPLC-DAD-MS n approaches allowed the identification and quantification of polar constituents.

Explorative NMR analysis
The NMR approach was used for a preliminary evaluation of the composition of C. candidissimum aerial parts fractions.To assess the presence of different classes of phytoconstituents, extraction of C. candidissimum aerial parts was performed using methanol, and this crude extract was then fractionated with solvents at different polarity, i.e., hexane, dichloromethane (DCM), ethyl acetate (EA) and butanol.The superimposition of the 1 H-NMR spectra obtained from the analyses of these fractions is reported in Figure S2.
Several signals in the spectrum of methanol crude extract are ascribable to fatty acids, sugars as well as phenolic constituents.A large number of signals in the more shielded part of the spectrum support the presence of aliphatic compounds, thus suggesting the presence of a hydrocarburic portion of constituents ascribable to terpenoids.Furthermore, several signals are clearly visible in the aromatic part of the spectrum, suggesting the presence of aromatic derivatives and phenolics.Among these, signals ascribable to rutin are observed: namely, the aromatic signals ascribable to the catechol ring and the H-6/H-8, as well as those of the anomeric proton of glucopyranosyl and rhamnopyranosyl units (Table S1).These are in good agreement with the literature, and with spectra of rutin acquired for comparison purposes (Napolitano et al. 2012).
Intense aliphatic signals supporting the presence of fatty acids as well as a series of sharp signals indicating the presence of quaternary methyl moieties of terpenoids are observed in the spectrum of the hexane fraction.These observations suggest a large presence of terpenoids.The spectrum of the DCM fraction presents a similar shape as hexane's one, but with a clearer aliphatic region (d 0.90-2.00).This latter shows several sharp signals suggesting the presence of quaternary methyl groups ascribable to terpenes, such as triterpenes (Sut et al. 2018).However, compared to hexane fraction, a lower amount of signals ascribable to fatty acid moieties is observed (Figure S2 and Table S1).The spectrum of the EA fraction showed the presence of triterpenoids that mostly can resume the structure of ursolic acid, due to the presence of six quaternary and one secondary methyl groups, the deshielded methylene linked to a hydroxyl group, and the olefinic signal.These assignments are in good agreement with 2ahydroxyursolic acid (Choi et al. 1991).The spectrum of the butanol fraction did not show significant signals.
On the basis of these data, we decided to proceed with HPLC-DAD-MS n for the analysis of phenolics and triterpenoids, and with HS-SPME-GC-MS for the analysis of lower molecular weight (i.e., volatile) terpenoids.

HPLC-DAD-MS n analysis of secondary metabolites in the crude methanol extract
HPLC-DAD-MS n analysis of C. candidissimum aerial parts (a mixture of inflorescences, stems and leaves) was performed on the methanol crude extract, since it was the one containing the broadest range of phytochemicals, from phenolics to terpenes.HPLC-DAD-MS n allowed to identify 30 secondary metabolites, whose details are reported in Table S2.By using the HPLC-DAD technique, total contents of flavonoids, phenolic acids and gallic acid derivatives were assessed at 3.1%, 3.6% and 0.76% of whole extract, respectively.The total phenolic content was comparable to that already reported for C. vulgaris (Khan et al. 2018).Rutin resulted as the most abundant phenolic compound among those identified, followed by several caffeic acid oligomers, caffeoylquinic acid isomers and the gallic acid derivative hexa-O-galloyl-glucoside.Several phenolic constituents identified in C. candidissimum have been already reported by other authors in other Calamintha spp.For instance, the glycosylated flavonoids hyperoside and 6,8-C-dihexosylapigenin, and the phenolic acids chlorogenic acid, rosmarinic acid and salvianolic acid B have been detected in the leaves of C. nepeta from Italy (Pacifico et al. 2015).
Regarding triterpenes, ursolic acids and its hydroxylated derivative were identified, and their amounts reached the 0.62% of the whole crude extract.Previously published phytochemical data on Calamintha genus have shown that ursolic acid and its derivatives are common constituents within this genus (Khodja et al. 2018).Conversely, our LC-MS results show that triterpenes are only minor constituents of C. candidissimum crude extract, although abundant signals attributable to the same class of phytochemicals were observed in the NMR data.These contrasting results suggest that further in depth studies are required for a more comprehensive elucidation of the triterpenes content of this plant.Chemical structures of the most abundant polar constituents identified in C. candidissimum are reported in Figure S3.

HS-SPME-GC-MS analysis
The analysis on the mixture of ground stems, leaves and flowers allowed to characterise 98.7% of the total volatiles emission and to identify 38 constituents (Table S3).The composition of the volatile bouquet was dominated by oxygenated monoterpenes (66.2%), which included pulegone (44.8%), piperitenone (6.6%), isopulegone (5.8%) and neo-menthol (3.8%).Sesquiterpene hydrocarbons were found as the second major group of chemicals (27.4%), including germacrene D (16.2%) and bicyclogermacrene (3.0%).Chemical structures of the main volatiles identified in C. candidissimum are reported in Figure S4.No previous studies are present in the literature about the spontaneous volatile emission of this species.Even if the SPME results cannot be directly compared with essential oil composition, these results partially reflect already published data regarding the chemical composition of essential oils from the aerial parts of C. candidissimum, since several studies have indicated pulegone and menthone as the two major components.For instance, the constituents identified in C. candidissimum essential oil by Benyoucef et al. (Benyoucef et al. 2020) were principally oxygenated monoterpenes, represented by pulegone (70.4%), menthol (5.2%) and isomenthone (4.5%).Satureja calamintha subsp.nepeta from Portugal has isomenthone, 1,8-cineole and trans-isopulegone as dominant components (Marongiu et al. 2010).Finally, in a study involving wild C. nepeta from Corsica, Baldovini et al. identified three chemotypes, whose essential oils were characterised by menthone/pulegone, pulegone, and piperitone and piperitenone oxides as main components, respectively (Baldovini et al. 2000).
Within the Satureja genus, a great variability of the volatile profile has been reported, although carvacrol, thymol, p-cymene, borneol, b-caryophyllene, germacrene D, and caryophyllene oxide have been indicated as major compounds (Azaz et al. 2002).Differences between our results and those previously published may be due to the different analytical techniques used, the part of the plant studied, the age of the plant, the period of the vegetative cycle, or even to ecological and genetic factors.

Experimental
Included in supplemental file.

Conclusion
Our findings show that endemic species Clinopodium candidissimum (Munby) Kuntze from Algeria contains valuable components endowed with possible biological activities, including antioxidant and volatile compounds, which could find wide practical application in various areas, such as formulation and production of food additives and healthy supplements.Overall, the results of this investigation represent a starting point for further research on this plant species.These findings could be useful for other research: in particular, the combination of traditional knowledge with currently available research tools may open up new perspectives for drug discovery from natural sources.