HPLC and GC/MS analysis of Prunus ulmifolia Franch. (syn. Aflatunia ulmifolia (Franch.) Vassilcz.) leaves growing in South-Eastern Kazakhstan

Abstract Prunus ulmifolia Franch. (Rosaceae) was investigated for its phytochemical composition from South-Eastern Kazakhstan for the first time. HPLC analysis confirmed rutin (0.88%) in ethanol extract, and the extract also exhibited antioxidant activity. The GC/MS analysis identified total 44 components from main groups e.g. oxygenated monoterpenes (51.06%), oxygenated sesquiterpenes (20.33%), non-terpene derivatives (18.71%), and sesquiterpene hydrocarbons (7.89%), and the maximum content was of acyclic alcoholic monoterpenoid citronellol (36.58%). The hierarchical cluster analysis (HCA) from previous reports and present study was used to demonstrate the variations between essential oil compositions in different Prunus species. It formed three main clusters, cluster I consisted of species with benzaldehyde as dominant component. Cluster II included plants with benzaldehyde as secondary component, and cluster III was of P. ulmifolia in which benzaldehyde was not detected. Further, the essential oil was assessed for cytotoxic and antimicrobial activities too, and it showed better cytotoxic but poor antimicrobial activities. Graphical Abstract


Introduction
Prunus ulmifolia Franch.(syn.Aflatunia ulmifolia (Franch.)Vassilcz.)belonging to family Rosaceae is a shrub or small tree which grows up to 3-5 m height (Figure S1).P. ulmifolia is endemic to Central Asia and was placed under endangered category in the Red Data Book of Kazakhstan (2014).Recently, this plant has been listed as least concerned species by the International Union for Conservation of Nature (IUCN 2022).The leaf flush does not occur until after the flowering of plant, the petals are bright pink and obovate, and the fruit is a drupe.The plant description and other information about P. ulmifolia have been described previously (Kirillov et al. 2022).It was noted that the phytochemical studies are not recently carried out on the plant, as only older literatures are available which reports the presence of flavanone, aromadendrin, catechin, and afzelin (Zapesochnaya et al. 1969;Zapesochnaya and Ban'kovskii 1971;Starovoitova and Zapesochnaya 1980).Other studies on genus Prunus suggested that these species are rich sources of essential oils, flavonoids, and phenolics, and some important species are P. mume Sieb.et Zucc.(Utsunomiya et al. 2005), P. armeniaca L. and P. domestica L. (Bonesi et al. 2018), and P. scoparia (Spach) CK Schneid (Taati et al. 2021).This genus also have plants with various therapeutic properties too e.g.P. cerasus L. has a protective effect against cardiovascular disease, Alzheimer's disorder, and chronic ailments caused by oxidative stress (Saleh et al. 2017).P. cerasoides D. Don is being used to cure kidney stones, asthma, thirst, and leucoderma, as well as it also has antipyretic and anti-inflammatory properties (Joseph et al. 2018).P. armeniaca L. has a carminative effect and is useful in the treatment of constipation (Shan et al. 2019), whereas P. persica (L.) Batsch has anti-inflammatory, anti-allergic, and antioxidant activities (Haleema et al. 2020).However, the information regarding biological activity is scanty for P. ulmifolia.
Thus, aim of the present study was divided in two aspects: (1) to investigate the chemical composition of leaves of P. ulmifolia by high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC/MS), and (2) to evaluate the antioxidant, cytotoxic, and antimicrobial activities of different extracts for the first time.

HPLC analysis
The chromatographic separation of P. ulmifolia extracts obtained as a result of ultrasonic extraction revealed that yields of dry extracts were 11-12%.The ultrasound method reduces the extraction time to 1.5 h, also no destructive effect of ultrasound on flavonoids was observed.When HPLC results were compared with rutin standard, the presence of rutin was confirmed in both the ethanol concentrations (Figures S2  and S3).Rutin is a pharmaceutically important compound with anti-cancer, antioxidant, anti-inflammatory activities, as well as have beneficial effects on diseases related to the central nervous systems, and neuroinflammation (Al-Dhabi et al. 2015).This flavonoid glycoside is commonly found in citrus fruits like orange, grapefruit, lemons, cranberries, and many Prunus species e.g.P. avium L., P. mahaleb L., P. padus L., P. spinosa L. (Mikulic-Petkovsek et al. 2016), P. cerasus L. (Oksuz et al. 2019), and P. avium L. (Antognoni et al. 2020).However, there was slight variation in rutin content between the two ethanol concentrations i.e. 0.88% rutin in 70% ethanol, and 0.85% rutin in for 96% ethanol (Table S1).Recently, Lohvina et al. (2021) also documented variation in total phenol content from Trigonella foenum-graecum L. when different ethanol concentrations were used, and 70% ethanol showed better extraction as compared to 96% concentration.A similar phenomenon was also observed for total flavonoid yield from Apis mellifera L. (Sun et al. 2015).The reason behind less recovery in 96% ethanol is competitive interactions of ethanol, water, and flavonol molecules.It has been suggested that the ethanol-water interactions are more favoured than ethanol-flavonol and flavonol-water interactions, which would decrease the solubility and in turn recovery of flavonols during extraction (Huaman-Castilla et al. 2019).

Determination of in vitro antioxidant activity
The antioxidant activities of ethanol and hexane extracts of P. ulmifolia were determined using a ferric reducing antioxidant power (FRAP) assay.The results suggested that ethanol extract had high antioxidant activity in comparison to control, and 1 mg/ mL concentration showed maximum absorbance at 700 nm (3.824 ± 0.11), which was three times higher than control.In line with present results, ethanol extract of Bougainvillea buttiana Holttum & Standl.also showed potent antioxidant activity (Abarca-Vargas et al. 2019).Whereas, hexane extract showed less activity in comparison to ethanol extract and at par activity with control at all the concentrations (0.25-1 mg/mL) (Table S2).This is in accordance with results on Dendropanax morbiferus H.L ev.where ethanol extract showed better antioxidant activity than hexane extract (Cho et al. 2018).The variation in antioxidant activities of extracts may be due to the differences in type and amount of compounds present (Jana et al. 2012).Generally, ethanol extract is known to have more quantities of potent antioxidant agents like phenolics and flavonoids as compared to hexane extract (do Amaral et al.

2022
).In the present study, rutin was detected in ethanol extract which might be the reason for better antioxidant potential, as reports on Prunus serotina Ehrh (Luna-V azquez et al. 2016) and P. padus L. (Donno et al. 2018) documented rutin behind antioxidant potential of plants.

Essential oil composition
The essential oil of P. ulmifolia was a thick, pasty mass of white color with a pleasant smell, with total obtained yield of 0.5%.The composition of essential oil is presented in Table S3 and GC/MS chromatogram as Figure S4.The GC/MS analysis identified total 44 components which amounted to 99.94% of the oil.The main classes were oxygenated monoterpenes (51.06%), oxygenated sesquiterpenes (20.33%), non-terpene derivatives (18.71%), and sesquiterpene hydrocarbons (7.89%).Earlier reports on Argemone ochroleuca Sweet (Abd-ElGawad et al. 2020), Elettaria cardamomum (L.) Maton (Ashokkumar et al. 2021), and Artemisia austriaca Jacq.(Issayenko et al. 2022) also showed that most abundant components of essential oils are oxygenated monoterpenes.Among all the compounds detected in P. ulmifolia, major component was acyclic alcoholic monoterpenoid citronellol (36.58%).It is naturally abundant volatile constituent responsible for the pleasant aroma and flavor of Vitis vinifera L. fruits (El Hadi et al. 2013).Likewise, citronellol has been reported to be a main compound of the essential oils of Rosa alba L. (Verma et al. 2020), Cymbopogon citratus (DC.)Stapf, and Pelargonium graveolens L'H er.(Lecourt et al. 2022), whereas in Eucalyptus maculata Hook. it is present in small quantity (Almas et al. 2021).
The essential oil compositions from the leaves of different Prunus genus are represented in Table S4.Verma et al. (2017) suggested that benzaldehyde and/or its derivative could be characteristic constituents of the genus Prunus and their occurrence could be of chemotaxonomic significance.The hierarchical cluster analysis (HCA) constructed using present result and earlier studies on Prunus species formed three distinct groups based on the Euclidian distance (Figure S5).The first group (cluster I) consisted of P. arborea, P. domestica, P. padus, P. persica, P. laurocerasus, and P. phaeosticta, which was characterized by the presence of benzaldehyde as dominant component.Cluster II included P. armeniaca, P. scoparia, and P. serotina with benzaldehyde as secondary component, and cluster III consisted of P. ulmifolia in which benzaldehyde and/or its derivative is absence.Further, comparing the major essential oil components (>1%) obtained in the present experiment with previous reports revealed that only few compounds were common, like linalool is common with P. armeniaca L. (Bonesi et al. 2018;Nafis et al. 2020) and P. scoparia (Spach) CK Schneid (Taati et al. 2021), c-cadinene with P. armeniaca L. (Bonesi et al. 2018;Nafis et al. 2020), and hexyl benzoate with P. scoparia (Spach) CK Schneid (Taati et al. 2021).Whereas none of the species reported the main component obtained in present result i.e. citronellol.This variability in essential oil composition depends on many factors like plant species and environmental conditions (Zheljazkov et al. 2013).
Citronellol is mainly used in the perfume, cosmetics, softeners, and other scented products related industries due to its odor qualities (Ueno et al. 2020).It is considered to be safe for food use and the acceptable daily intake is 0.5 mg/kg body weight (WHO 2016).It has medicinal properties like analgesic, anti-inflammatory, antibacterial, antifungal, and anticonvulsant (Santos et al. 2019).Table S4 reports activities of different essential oils e.g.P. armeniaca and P. domestica oils have antioxidant and neuroprotective activities (Bonesi et al. 2018), P. armeniaca oil has antimicrobial (Nafis et al. 2020), and P. serotine oil showed antihypertensive (Ibarra-Alvarado et al. 2009) activities.

Cytotoxic activity of essential oil
To use phytocompounds for medicinal purposes, it should have some properties, one of which is toxicity to eukaryotic cells.The brine shrimp (Artemia salina L.) bioassay is a suitable, accurate, and inexpensive alternative to pre-screening the chemical toxicity against mammals (Nunes et al. 2006).The result of testing the cytotoxic activity of P. ulmifolia essential oil is shown in Table S5.The essential oil at concentrations 1, 10, and 100 mg/mL exhibited cytotoxicity as the mortality of A. salina larvae was 10%, 10%, and 20%, respectively, and the LC 50 was found to be 38.0 mg/mL (Table S5).Similarly, Pectis brevipedunculata (Gardner) Sch.Bip.essential oil showed LC 50 of 36.0 mg/mL when tested against A. salina larvae (Oliveira et al. 2011).Meyer et al. (1982) suggested that LC 50 value lower than 1,000 lg/mL for extract is considered bioactive when cytoxicity was analysed using A. salina.Thus, from the present LC 50 value it was inferred that P. ulmifolia essential oil has high toxicity.Similarly, essential oils of Psila spartioides Cabrera, Hyptis mutabilis (Rich.)Briq.(Oliva et al. 2007), Aframomum melegueta [K.Schum].(Owokotomo 2018), and Peperomia circinnata Link var.circinnata (Mesquita et al. 2021) also showed cytotoxic properties.This activity may be attributed to specific chemical constituents or synergism between various components.The results also justified the fact main compound of essential oil is citronellol which have insect and pesticide repellent activities (Santos et al. 2019).

Antimicrobial activity of essential oil
The essential oil composition of P. ulmifolia is dominated by oxygenated monoterpenes especially citronellol, and both are known to have promising antibacterial activities (Kotan et al. 2007;Ghavam et al. 2021).Thus, in the present study antibacterial activity of essential oil was checked against different organisms.The results suggested that the essential oil showed weak antimicrobial activity against Gram-positive test microorganisms S. aureus and B. subtilis, and no activity against Gram-negative bacteria E. coli and P. aeruginosa as well as against fungus C. albicans (Table S6).Abers et al. (2021) also reported that the volatiles of clove, wild orange, ylang ylang, and fennel had least antimicrobial activity.Further, they have also reported that oils of lavender, peppermint, lemon, cedarwood, frankincense, white fir, oregano, cinnamon, and lemongrass are specific to some microbial species.Previously, Andrade-Ochoa et al. (2021) showed that essential oils of different plants when tested against different organisms, Gram-negative bacteria were found to be most resistant to the tested essential oils.The essential oils majorly contain terpenoids, and their hydrophobic nature is considered to be the reason for their attraction to microbial cells (Jaroque et al. 2019).The outer membrane of Gram-negative bacteria limits the diffusion of hydrophobic compounds.Whereas Gram-positive bacteria are surrounded by a thick peptidoglycan wall which facilitates infiltration of hydrophobic essential oils due to the lipophilic ends of lipoteichoic acid present in cell membrane (Cox et al. 2000).However, some studies suggested that antimicrobial activity of essential oils does not have only one mechanism of action (their hydrophobic nature), but it depends on various actions on the target organisms (Tariq et al. 2019).This can be correlated with the present result in which essential oil showed no activity against C. albicans.Fungi contain chitin and other carbohydrates in their cell wall which provides a rigid structure, but it is not entirely hydrophobic and is considered to be partially repellent to essential oils.Nevertheless, there are reports on essential oil's effect on cell membrane (Kong et al. 2019) which confirms that the antifungal activity can be related more to the regulation of enzymes bounded to cell membrane, and not directly due to their interaction with membrane (Nazzaro et al. 2017).

Experimental
All experimental procedures are described in the Supplementary material.

Conclusion
Present study reports quantification of rutin and essential oil composition along with biochemical test for the first time in P. ulmifolia.HPLC analysis revealed presence of rutin in ethanol extract, and GC/MS analysis showed 44 compounds in essential oil of which the main was citronellol.Further, the ethanolic extract showed antioxidant potential, and essential oil showed strong cytotoxic and weak antimicrobial activities.This study confirmed that P. ulmifolia is a rich source of volatile compound citronellol and hence can be used as an alternative to traditional plants like Cymbopogon and Eucalyptus species for its extraction.The finding also suggests that essential oil of P. ulmifolia might have a possible usage in food applications in order to reduce synthetic chemicals in food industries.Future work can be directed for conservation of the species as it is endangered in Kazakhstan and thus needs to be cultivated at large scale, also other medicinal activities of different extracts should be investigated.

Disclosure statement
No potential conflict of interest was reported by the authors.