Echinocystic Acid Bidesmoside Saponins from Microglossa afzelii O. Hoffm and Their Cytotoxic Activity against the CAL-27 Oral Squamous Carcinoma Cell Line

This paper describes eight new triterpenoid saponins, including afzeliioside A (1), four acetylated afzeliiosides as pairs of inseparable regioisomers, called afzeliiosides B/C (2/3) and D/E (4/5), afzeliiosides F-H (6–8), and a known impatiprin C (9), which were isolated from the n-BuOH fraction of the liana of Microglossa afzelii. Their structures were established mainly by extensive spectroscopic analysis, including 1D and 2D NMR, HRFAB-MS, tandem ESI-MS/MS, and chemical methods, as well as a comparison of their spectral data with those of related compounds. All the isolates were screened for their cytotoxic activity against the CAL-27 oral squamous carcinoma cell line. Only compounds 4/5 (EC50 = 36.0 μg/mL (32.7 μM)) exhibited moderate cytotoxic activity. This work presents the first chemical and biological investigation of Microglossa afzelii and reports, for the first time, on the isolation of saponins in the genus Microglossa.


Introduction
Triterpenoid saponins, which are common in a large number of plant species, represent a class of secondary metabolites showing a remarkable structural variety, as well as notable biological activities [1]. Indeed, plants synthesize saponins as part of their normal program of growth and development and, in particular, as preformed chemical barriers functioning in defense mechanisms against insects, fungi, nematodes, and weeds [2]. The interest in saponins stems from their medicinal properties and their importance in the human and animal diet, since they have immunostimulant, hypocholesterolaemic, and anticarcinogenic properties and act as antifungal and antiviral agents [2].
An extremely rich chemical diversity characterizes the Microglossa species, as well as the other genera in the Asteraceae family. Microglossa (Asteraceae) is a genus of about 10 species, mainly distributed between South Africa, tropical Africa, and eastern Asia [3]. The plants of this genus are commonly used in folk medicine to treat several diseases. M. pyrifolia and M. angolensis are used in Kenya, Ghana and Madagascar to manage malaria, reduce the effects of malaria-related fevers, and treat gastro-intestinal disorders and microbial infections, and they are also used as abortifacients and antipyretics [4][5][6]. M. afzelii and other classes of compounds. The enriched saponin fraction (4.3 g) was subjected to silica gel CC eluted with EtOAc−MeOH−H 2 O (95:5:2, v/v/v) to obtain three main subfractions, Fr1 (1.3 g), Fr2 (720 mg), and Fr3 (925.3 mg). Fr1 was submitted to CC eluted with the ternary system EtOAc−MeOH−H 2 O (18:2:1, v/v/v) to obtain two main fractions, Fr11 (233 mg) and Fr12 (112 mg). Fr11 was submitted to successive RP-18 recycling HPLC in isocratic conditions (MeOH−H 2 O, 3:2, v/v) to obtain 2/3 ( Tables 2 and 3

Acid Hydrolysis and GC-MS Analysis of Saponins 1-9
Each saponin (2 mg) was refluxed with HCl (1 N, 2 mL) for 2 h. The hydrolyzed product was extracted with CH 2 Cl 2 . The residue obtained from the usual workup of the aqueous layer was derivatized by adding 100 µL methoxylamine hydrochloride in pyridine (20 mg·mL −1 ), vortexed and incubated at 60 • C for 45 min. Then, 200 µL of N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) was added and incubated at 60 • C for 45 min. The derivatized sample was centrifuged at 6000 rpm for 5 min at 25 • C to remove any solid debris. A GC-MS analysis was performed using a 7890A gas chromatograph (Agilent technologies, Santa Clara, CA, USA), equipped with an Agilent Technology GC autosampler 120 (PAL LHX-AG12) and coupled with an Agilent 7000 Triple Quad system (Agilent technologies, USA). An HP-5MS 30 m-250 mm (i.d.) fused-silica capillary column (Agilent J &W Scientific, Folsom, CA, USA), chemically bonded with a 5% diphenyl and 95% dimethylpolysiloxane cross-linked stationary phase (0.25 mm film thickness), was used. Separation was achieved using a temperature program of 80 • C for 2 min, which was then ramped at 5 • C min −1 to 300 • C and held for 1 min, at a constant flow of 1.0 mL·min −1 . The MS interface and ion source were set to 280. The configuration was determined by comparing the retention time with the derivatives prepared in a similar manner from standard sugar (t R D-xylose 27.2 min; t R L-rhamnose 32.3 min; t R D-glucuronic acid 29.2 min; t R D-apiose 4.2 min) [16].

Culture Conditions
The human tongue squamous cell carcinoma cell line (CAL-27) (ATCC CRL-2095) is an adherent cell line that was provided by the cell culture biobank of PCMD, ICCBS, which was used in the study. The cells with early passage numbers (P10-P12) were detached using trypsin-EDTA (Thermo Fisher Scientific, Waltham, MA, USA) and grown in Dulbecco's modified Eagle medium (DMEM) (Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, USA) and 1% antibiotic-antimycotic (Thermo Fisher Scientific, USA) in a humidified incubator supplemented with 5% CO 2 at 37 • C. The stock solutions of the tested compounds were prepared at 250 µg/µL by solubilizing the compounds in DMSO. The stock solutions were diluted to 500 µg/mL in DMEM.

Cytotoxic Assay
The cytotoxic effect of the isolated saponins was evaluated using an MTT(3-(4,5-Dimethylthiazol-2-yl)-2,5-dipheynyltetrazolium bromide) in vitro cell viability assay. This assay enabled a general evaluation of the cytotoxic activity of the tested samples. Briefly, CAL-27 cells were grown in DMEM on 96-well plates up to 70% confluence. Each compound was preliminarily screened at the concentrations of 250 µg/mL and 125 µg/mL in triplicate. A compound that showed 50% inhibition or more at 125 ug/mL was further evaluated for the EC 50 value. When evaluating the EC 50 values of the active compounds, the compounds were tested within the concentration range from 250 µg/mL to 7.813 µg/mL, followed by incubation for 48 h at 37 • C. After incubation, the medium was removed, 200 µL of MTT reagent (500 µg/mL) was added, and the samples were incubated for 1.5 h. The MTT reagent was removed, and DMSO was added to dissolve the formazan crystals formed by a reduction in the MTT reagent. A micro-plate reader was used to measure the absorbance at 570 nm [17]. Each sample analysis was performed in triplicate. The EC 50 was calculated by plotting a non-linear regression curve using GraphPad Prism 9 software.

Results
The n-BuOH fraction of the methanol extract of M. afzelii was subjected to Sephadex LH-20 column chromatography to obtain an enriched saponin fraction. Sephadex LH-20, open normal column chromatography, and the RP-18 HPLC of the enriched saponin fraction afforded one previously unreported triterpenoid saponin, named afzeliioside A The IR spectrum showed characteristic absorption bands for the hydroxyl groups (3377 cm −1 ) and ether groups (1037 cm −1 ), as well as a strong absorption due to the carbonyl groups (1728 cm −1 ) [15,19]. The analysis of the 1 H and 13 C NMR data (Table 1) 30), and an olefinic proton at δH 5.31 (br t, J = 3.7 Hz, H-12) characteristic of olean-12-ene-type triterpenoids [20]. The 13 C NMR spectrum showed 61 signals, among which 30 were assigned to a triterpenoid moiety and 27 to the saccharide moiety (see Table 1). The four additional signals were ascribable to an n-butyl (66.1 (CH2), 31.7 (CH2), 20.1 (CH2), and 14.0 (CH3)) unit, which was confirmed by the combined interpretation of the 1 H− 1 H COSY, HSQC, and DEPT 135 spectra [18]. The NMR data of the aglycone moiety of 1 were in good agreement with those of echinocystic acid [18,21], except for the downfield shift of C-3 (δC 91.1) and for the upfield shift of C-28 (δC 177.2), suggesting that 1 was a bidesmosidic saponin [22,23].  Table 1). The 1 H NMR spectrum also revealed the presence of one rhamnopyranosyle moiety amongst the sugars, and it was identified by the correlation between the methyl doublet at δH 1.26 (d, J = 6.2 Hz, H-6⁗) and the glycosidic proton at δH 3.76 (dd, J = 6.2; 9.5 Hz, H-5⁗). The acid hydrolysis of 1 afforded D-glucuronic acid, D-xylose, L-rhamnose, and D-apiose, which were identified by the comparison of the retention times of their trimethylsillyled derivatives with those of the authentic ones by GC-MS [16]. The β-configuration of the anomeric centers of glucuronic acid, xylose, and apiose, as well as the α of rhamnose, were determined based on the values of their coupling constants [23,24]. The complete assignments of the glycosidic protons and carbons (Table 1)   The IR spectrum showed characteristic absorption bands for the hydroxyl groups (3377 cm −1 ) and ether groups (1037 cm −1 ), as well as a strong absorption due to the carbonyl groups (1728 cm −1 ) [15,19]. The analysis of the 1 H and 13 C NMR data (Table 1) 30), and an olefinic proton at δ H 5.31 (br t, J = 3.7 Hz, H-12) characteristic of olean-12-ene-type triterpenoids [20]. The 13 C NMR spectrum showed 61 signals, among which 30 were assigned to a triterpenoid moiety and 27 to the saccharide moiety (see Table 1). The four additional signals were ascribable to an n-butyl (66.1 (CH 2 ), 31.7 (CH 2 ), 20.1 (CH 2 ), and 14.0 (CH 3 )) unit, which was confirmed by the combined interpretation of the 1 H− 1 H COSY, HSQC, and DEPT 135 spectra [18]. The NMR data of the aglycone moiety of 1 were in good agreement with those of echinocystic acid [18,21], except for the downfield shift of C-3 (δ C 91.1) and for the upfield shift of C-28 (δ C 177.2), suggesting that 1 was a bidesmosidic saponin [22,23].  Table 1). The 1 H NMR spectrum also revealed the presence of one rhamnopyranosyle moiety amongst the sugars, and it was identified by the correlation between the methyl doublet at δ H 1.26 (d, J = 6.2 Hz, H-6 ) and the glycosidic proton at δ H 3.76 (dd, J = 6.2; 9.5 Hz, H-5 ). The acid hydrolysis of 1 afforded D-glucuronic acid, D-xylose, L-rhamnose, and D-apiose, which were identified by the comparison of the retention times of their trimethylsillyled derivatives with those of the authentic ones by GC-MS [16]. The β-configuration of the anomeric centers of glucuronic acid, xylose, and apiose, as well as the α of rhamnose, were determined based on the values of their coupling constants [23,24]. The complete assignments of the glycosidic protons and carbons (Table 1)  to the loss of n-butyl, n-butylglucuronopyranosyl, and the C-28 tetrasaccharide chain formed, of two xylopyranosyls, one apiofuranosyl, and rhamnopyranosyl moieties, respectively. On the basis of the above analysis, the structure of 1 was thus elucidated as , attributed to the loss of an acetyl, a n-butylglucuronopyranosyl, a C-28 triglycosidic chain formed of two xylopyranosyls, one rhamnopyranosyl, and acetyl moieties, and the loss of the C-3,28 glycosidic chains, respectively. On the basis of the above analysis, the structure of 2/3 was thus elucidated as a mixture of  [18]. The signals at δC 95.5/95.4 (C-1‴), 98.4/101.1 (C-1⁗), 106.7/105.5 (C-1′′′′′), and 106.5/106.9 (C-1′) indicated that 4/5 possessed four sugar units. The duplication of the 1 H and 13 C signals indicated that 4/5 also constituted a mixture. The GC-MS analysis of the chiral derivatives of sugars from the acid hydrolysate of 4/5 afforded D-glucuronic acid, D-xylose, and L-rhamnose [16]. The comparison of the NMR spectra of 4/5 with those of 2/3 showed that they were almost superimposable. However, the extensive analysis of the 13 C NMR of 4/5 showed the disappearance of the signals attributable to an n-butyl unit, and this was further confirmed by the mass difference of 56 units between the two compounds, supporting the lack of a butyl unit. Tandem mass spectrometry (MS n ), which utilizes the collision-induced  Tables 1 and 2), and 2 to an acetyl group with the methyl signals at δ C 20.9/21.3 (s, MeCO), as well as the signals of ester carbonyls at δ C 172.2/172.5 (MeCO). The four additional signals were ascribable to an n-butyl (66.1 × 2 (CH 2 ), 31.7 × 2 (CH 2 ), 20.2 × 2 (CH 2 ) and 14.0 × 2 (CH 3 )) unit based on the interpretation of the 1 H− 1 H COSY, HSQC, and DEPT 135 spectra [18]. The comparison of the NMR data of the aglycone moiety of 2/3 with that of 1 showed that they are superimposable and indicated that 2/3 are also echinocystic acid glycosides [18,25]. Furthermore, the chemical shifts of C-3 (δ C 91.1 × 2) and C-28 (δ C 177.0/177.1) suggested that 2/3 were also bidesmosidic saponins [22,23].  Table 2), confirming that each constituent of the mixture 2/3 contained four sugar units. The acid hydrolysis of 2/3 afforded glucuronic acid, xylose, and rhamnose, which were identified by the comparison of their retention times with those of the authentic trimethylsillyled derivatives by GC-MS [16,19]. Furthermore, the D-configuration of the glucuronic acid, xylose, and L of rhamnose were also determined by the GC-MS analysis of their trimethylsillylated derivatives. The β-configuration of the anomeric centers of the glucuronic acid and xylose and α of rhamnose were determined based on the values of their coupling constants [23,24]. An extensive analysis of the 1 H− 1 H COSY spectrum allowed us to assign the resonances observed in a lower field at δ H 5.12 (dd, J = 1.8; 3.5 Hz) and 5.04 (dd, J = 3.1; 9.2 Hz) to H-2 and H-3 of the two rhamnopyranosyl units, respectively, and suggested that they were acetylated. This was further confirmed by the HMBC correlation depicted between H-2 (δ H 5.12) and H-3 (δ H 5.04) and the acetyl carbonyl carbons at δ C 172.2 and 172.5, respectively, indicating that 2/3 were a mixture of 2-OAc and 3-OAc-rhamnopyranosyl regioisomers. The location of the glucuronopyranosyl unit at C-3 (δ C 91.1 × 2) was deduced based on the HMBC correlation of its anomeric proton (δ H 4.38 × 2) with C-3. The correlation of H-1 (δ H 4.18, t, J = 6.4 Hz) of the n-butyl w C-6 (170.9 × 2) of the glucuronopyranosyl unit allowed us to locate the n-butyl  [18]. The signals at δ C 95.5/95.4 (C-1 ), 98.4/101.1 (C-1 ), 106.7/105.5 (C-1 ), and 106.5/106.9 (C-1 ) indicated that 4/5 possessed four sugar units. The duplication of the 1 H and 13 C signals indicated that 4/5 also constituted a mixture. The GC-MS analysis of the chiral derivatives of sugars from the acid hydrolysate of 4/5 afforded D-glucuronic acid, D-xylose, and L-rhamnose [16]. The comparison of the NMR spectra of 4/5 with those of 2/3 showed that they were almost superimposable. However, the extensive analysis of the 13 C NMR of 4/5 showed the disappearance of the signals attributable to an n-butyl unit, and this was further confirmed by the mass difference of 56 units between the two compounds, supporting the lack of a butyl unit. Tandem mass spectrometry (MS n ), which utilizes the collision-induced dissociation (CID) of target ions, was used to confirm the sequences of the sugar chains. The negative ESI−MS 2 analysis of the parent peak at m/z = 1099. and suggesting the presence of isomers. We observed 134 mass units, which marked a difference from 4/5 and indicated the loss of one pentose unit. The IR spectrum showed characteristic absorption bands at 3300, 1050, and 1700 cm −1 , indicating the presence of the hydroxyl groups, ether linkage, and carbonyl groups, respectively [15,19,25]. The GC-MS analysis of the chiral derivatives of sugars from the acid hydrolysate of the sample afforded D-glucuronopyranosyl acid, D-xylopyranose, and L-rhamnopyranopyranosse [16], and the configurations of the anomeric centers were determined based on the values of their coupling constants [23,24]. An extensive analysis of the 1 H− 1 H COSY and HSQC spectra allowed us to assign the resonances observed in a lower field at δC/H 73.9/5.   19 : 967.4903) and suggesting the presence of isomers. We observed 134 mass units, which marked a difference from 4/5 and indicated the loss of one pentose unit. The IR spectrum showed characteristic absorption bands at 3300, 1050, and 1700 cm −1 , indicating the presence of the hydroxyl groups, ether linkage, and carbonyl groups, respectively [15,19,25].
The relative intensity of the proton signals and their combination with the 13 C NMR spectra suggested that 6-8, in 1:2:1 proportions, are also triterpenoid saponins with echinocystic as an aglycone. This was shown by the characteristic signals at δ C 2 × 90. 8 1 /H-1 ). The GC-MS analysis of the chiral derivatives of sugars from the acid hydrolysate of the sample afforded D-glucuronopyranosyl acid, D-xylopyranose, and L-rhamnopyranopyranosse [16], and the configurations of the anomeric centers were determined based on the values of their coupling constants [23,24]. An extensive analysis of the 1 H− 1 H COSY and HSQC spectra allowed us to assign the resonances observed in a lower field at δ C/H 73.9/5. Compounds 2/3, 4/5, and 6-8 are acetylated saponins, which are well known to be a typically unstable class of secondary metabolites that are extensively distributed in many species [15,19,26]. Their stability depends on the different solvents and separation materials used for their purification [26]. Due to the low activation energy of the acetyl migration reaction, it may be difficult to obtain the pure acetyl saponin [19,26]. According to Zeng et al. (2015) [26], the acetyl transfer reaction in saponins is faster in a mixture of solvents which contains water than in other solvents, and when comparing the normal/reverse silica gels, the reaction of acetyl migration almost did not occur during the process of purification by macroporous resin. Therefore, it can be assumed that compound 3 (or 5) may have resulted from compound 2 (or 4), and vice versa, due to the migration of the acetyl group from the 2 to the 3 position (vice versa) of the rhamnopyranosyl moiety, as shown by Zeng et al. (2015) [26] (Figure 3), thus resulting in the two inseparable mixtures of 2/3 and 4/5. Regarding the mixtures of 6-8, their appearance as three monoacetylated regioisomers is not surprising and could be due to the presence of three possible positions of acetyl migration in the rhamnopyranosyl unit, according to Zeng et al. (2015), compared to 2/3 and 4/5, in which we observed two. We could then recommend using a macroporous resin as a stationary phase and avoiding the use of water as a solvent for the phytochemical investigation of acetyl-saponin-rich plants. Compounds 2/3, 4/5, and 6−8 are acetylated saponins, which are well known to be a typically unstable class of secondary metabolites that are extensively distributed in many species [15,19,26]. Their stability depends on the different solvents and separation materials used for their purification [26]. Due to the low activation energy of the acetyl migration reaction, it may be difficult to obtain the pure acetyl saponin [19,26]. According to Zeng et al. (2015) [26], the acetyl transfer reaction in saponins is faster in a mixture of solvents which contains water than in other solvents, and when comparing the normal/reverse silica gels, the reaction of acetyl migration almost did not occur during the process of purification by macroporous resin. Therefore, it can be assumed that compound 3 (or 5) may have resulted from compound 2 (or 4), and vice versa, due to the migration of the acetyl group from the 2 to the 3 position (vice versa) of the rhamnopyranosyl moiety, as shown by Zeng et al. (2015) [26] (Figure 3), thus resulting in the two inseparable mixtures of 2/3 and 4/5. Regarding the mixtures of 6−8, their appearance as three monoacetylated regioisomers is not surprising and could be due to the presence of three possible positions of acetyl migration in the rhamnopyranosyl unit, according to Zeng et al. (2015), compared to 2/3 and 4/5, in which we observed two. We could then recommend using a macroporous resin as a stationary phase and avoiding the use of water as a solvent for the phytochemical investigation of acetyl-saponin-rich plants. Several studies have demonstrated that the polyhydroxylated triterpenes isolated from members of the Asteraceae family exhibit potent anti-inflammatory effects, as well as antitumor and cytotoxic activities [23−25]. Moreover, the work conducted by Wang et al. (2006) [27] reported that an oleanane triterpenoid possessing both 3-β and 6-α-hydroxyl Several studies have demonstrated that the polyhydroxylated triterpenes isolated from members of the Asteraceae family exhibit potent anti-inflammatory effects, as well as antitumor and cytotoxic activities [23][24][25]. Moreover, the work conducted by Wang et al. (2006) [27] reported that an oleanane triterpenoid possessing both 3-β and 6-α-hydroxyl groups exhibited anti-tumor activity against a diverse panel of tumor cell lines [27]. Taking into account the unusual structural features of Microglossa saponins, in order to determine their possible functional roles as cancer preventives, the isolated saponins (1−9) were screened for their cytotoxic activity against the CAL-27 oral squamous carcinoma cell line. The viability of the CAL-27 cell line incubated with the isolates was assessed using an MTT (3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide) assay [17]. Only 4/5 showed a moderate activity, with an EC 50 = 36 µg/mL (32.7 µM) (Figure 4), while the other saponins were found to be inactive. (See Supplementary Figure S51) It is well known that the linkage of the sugar moieties, as well as their number and nature, are very important for the cytotoxicity of triterpenoid saponins [25]. The previous results on the cytotoxicity of triterpenoid saponins against various cancer cell lines showed that the C-3,28-bidesmosides were noncytotoxic, in most cases, compared to the C-3 ones [25,28,29]. Additionally, the work conducted by Lee et al. (2002) [25] revealed that the linkages of sugars at the C-28 of echinocystic acid are essential for the non-cytotoxicity trait. Furthermore, the main differences between 4/5, which showed a moderate activity, and the other saponins are the lack of the n-butyl unit at the C-6 of the glucuronopyranosyl and the presence of one xylopyranosyl, which consequently, revealed that the carboxyl group and xylopyranosyl unit are essential for the observed activity. groups exhibited anti-tumor activity against a diverse panel of tumor cell lines [27]. Taking into account the unusual structural features of Microglossa saponins, in order to determine their possible functional roles as cancer preventives, the isolated saponins (1−9) were screened for their cytotoxic activity against the CAL-27 oral squamous carcinoma cell line. The viability of the CAL-27 cell line incubated with the isolates was assessed using an MTT (3-(4,5-dimethyl thiazolyl-2)-2,5-diphenyltetrazolium bromide) assay [17]. Only 4/5 showed a moderate activity, with an EC50 = 36 µ g/mL (32.7 µ M) (Figure 4), while the other saponins were found to be inactive. (See Supplementary Figure S51) It is well known that the linkage of the sugar moieties, as well as their number and nature, are very important for the cytotoxicity of triterpenoid saponins [25]. The previous results on the cytotoxicity of triterpenoid saponins against various cancer cell lines showed that the C-3,28-bidesmosides were noncytotoxic, in most cases, compared to the C-3 ones [25,28,29]. Additionally, the work conducted by Lee et al. (2002) [25] revealed that the linkages of sugars at the C-28 of echinocystic acid are essential for the non-cytotoxicity trait. Furthermore, the main differences between 4/5, which showed a moderate activity, and the other saponins are the lack of the n-butyl unit at the C-6 of the glucuronopyranosyl and the presence of one xylopyranosyl, which consequently, revealed that the carboxyl group and xylopyranosyl unit are essential for the observed activity.

Discussion
The present study reports, the first time, the isolation of triterpene saponins from the Microglossa genus. All the isolated compounds have echinocystic acid as an aglycone. The isolation of the triterpene saponins, as a major class of compounds from the n-BuOH fraction of this plant, was not surprising, since some triterpenoids from the less polar fraction of Microglossa pyrifolia have been reported [7]. This indicates that the chemical investigation of the polar fraction of this plant could yield related saponins. Moreover, previous works performed on other genera of the Asteraceae family led to the isolation of several triterpene-type saponins with various biological activities. Such is the case of Silphium radula, which afforded nine new saponins with cytotoxic activity against the breast cancer cell line [10]. From Lactuca scariola, one new saponin with antibacterial activity was isolated [30]. Furthermore, from Viguiera hypargyrea, four saponins were obtained, among which two new derivatives were isolated, but they exhibited no significant antiplasmodial or antibacterial activities [31]. Additionally, from Atractylis flava, three new saponins were isolated [32], while five saponins with one new saponin were isolated from Calendula arvensis [32], and from Aster sedifolius three new saponins were isolated [33]. Since all the isolated saponins from these genera are triterpenic with oleanane-based skeletons, it is noteworthy that this class has a broad spectrum of biological activities, including hepatoprotective, molluscicidal, anti-inflammatory, anti-tumor, immunomodulatory, anti-Alzheimer, hemolytic, and anti-allergic activities [11]. These chemical findings confirm the

Discussion
The present study reports, the first time, the isolation of triterpene saponins from the Microglossa genus. All the isolated compounds have echinocystic acid as an aglycone. The isolation of the triterpene saponins, as a major class of compounds from the n-BuOH fraction of this plant, was not surprising, since some triterpenoids from the less polar fraction of Microglossa pyrifolia have been reported [7]. This indicates that the chemical investigation of the polar fraction of this plant could yield related saponins. Moreover, previous works performed on other genera of the Asteraceae family led to the isolation of several triterpene-type saponins with various biological activities. Such is the case of Silphium radula, which afforded nine new saponins with cytotoxic activity against the breast cancer cell line [10]. From Lactuca scariola, one new saponin with antibacterial activity was isolated [30]. Furthermore, from Viguiera hypargyrea, four saponins were obtained, among which two new derivatives were isolated, but they exhibited no significant antiplasmodial or antibacterial activities [31]. Additionally, from Atractylis flava, three new saponins were isolated [32], while five saponins with one new saponin were isolated from Calendula arvensis [32], and from Aster sedifolius three new saponins were isolated [33]. Since all the isolated saponins from these genera are triterpenic with oleanane-based skeletons, it is noteworthy that this class has a broad spectrum of biological activities, including hepatoprotective, molluscicidal, anti-inflammatory, anti-tumor, immunomodulatory, anti-Alzheimer, hemolytic, and anti-allergic activities [11]. These chemical findings confirm the botanical identification of M. afzellii and further indicate its close relationship with other species of this genus. Additionally, echinocystic acid saponin derivatives, which constitute the main class of secondary metabolites isolated from the n-BuOH fraction of this plant, can thus be considered as chemotaxonomic markers of this species.

Conclusions
The chemical study of the n-BuOH fraction of Microglossa afzelii led to the isolation of eight new echinocystic-type triterpenoid saponins, trivially named afzeliiosides A−H (1-8), together with the known impatiprin C (9). Only the mixture of 4/5 displayed a moderate cytotoxic activity against the selected cancer cell lines. To the best of our knowledge, this study constitutes the first isolation of triterpene saponins from the Microglossa genus. The results obtained in this work indicated that echinocystic-type triterpenoid saponins represent one of the main classes of secondary metabolites derived from this plant and can thus be considered as its chemophenetic marker. Although the isolated saponins exhibited no significant cytotoxic activity, it would be interesting to re-isolate these saponins in large amounts and perform the cytotoxicity analysis using other cancer cell lines.

Data Availability Statement:
The data presented in this study are available in the main article and the supplementary materials.