N Belmiloud

I Dufour

A Colin

L Nicu

Vibrating Microcantilevers : Tools for Microrheology

Keywords: Microrheology, microcantilever, viscosity, density PACS : 8785-gf

The aim of this paper is to demonstrate that vibrating microcantilevers can be used to quantify fluid properties such as density and viscosity. Contrary to classical rheological measurements using microcantilevers, the development of the proposed microrheometer is based on the measurement of fluid properties over a range of vibration frequencies, without necessarily being restricted to resonant phenomena. To this end, an analytical model is implemented and, when combined with measurements, allows the determination of the viscosity as a function of frequency. The preliminary results are encouraging for the development of a useful microrheometer on a silicon chip for microfluidic applications.

INTRODUCTION

Simultaneous measurements of both phase and amplitude of a vibrating microcantilever in a fluid can lead to the rapid determination of both unknown fluid parameters (viscosity and density). First, a brief review of the theory of fluid-microcantilever interaction is made to explain the proposed method of fluid property extraction from the beam's dynamic response. This is then followed by a discussion of our first measurements and fluid parameter determination for Newtonian silicon oils. To finish, the same approach is presented for the determination of viscoelastic properties of complex fluids.

VISCOSIMETRY

Under the incompressible and Newtonian assumption, viscosity and density are intrinsic constants. When a microcantilever vibrates in a viscous fluid, the fluid offers resistance to the motion of the beam. The fluid loading can be interpreted as the sum of two forces: an inertial force that is proportional to the cantilever acceleration, and a viscous force that is proportional to the cantilever velocity. Thus, a viscous fluid exerts on the beam surface the drag force per unit length 1,2,3

: 2 1 2 2 ( ) ( ) ( ) ( ) fluid w t w t F g f g f t t        (1)
with w the deflection at the end of the beam, f the vibration frequency, and t is time.

An analogy between beam dynamics in viscous fluids and second order low-pass band allows the simplification of the mechanical transfer function. The assumption that the contribution of higher modes in the transfer function value is negligible has been made, i.e., the first mode is considered dominant. Referring to the differential equation governing the microcantilever deflection, the amplitude and phase measurements (│H│,) may be converted to the drag force components at each frequency:

2 0, 0 1 2 s i n vac L f H g m H f     (2) 2 0, 0 2 1 cos 1 vac L f H g m H f                           (3)
where m L is the cantilever mass per unit length, f 0 , vac the resonant frequency in vacuum, H 0 the static value of the mechanical transfer function H, and f the frequency. Thus, the determination of the two parts of the drag force allows one to extract analytically at each frequency the viscosity and density, i.e., the fluid's rheological properties:

Preprint: The XVth international congress on rheology, Monterey, CA, USA, 3-8 aout 2008, AIP Conf. Proc, Vol. 1027, pp. 1159-1161. (doi: 10.1063/1.2964501)

    2 2 1 2 2 1 2 1 2 2 1 2 1 2 1 1 2 2 2 1 1 2 2 1 2 (2 ) 2 8 ( ) fg a b a b a g a fg b a g fg g a b b fa a b a b             (4)           2 2 1 21 1 2 21 2 1 2 112 2 1 2 2 1 2 1 2 2 1 2 1 2 2 1 2 1 2 1 1 2 2 2 8 2 (2 ) 2 8 a fg b g a fg b a g fg g ab a b a b fg a b a b a g a fg b a g fg g a b                          (5)
where a 1 =1.0553, a 2 =3.7997, b 1 =3.8018 and b 2 =2.7364 are Maali's parameters 1 and b is the microcantilever width. The deflection amplitude and phase have been measured as functions of frequency on a range of 10 to 10000 Hz using an optical read-out scheme. An alternating current circulates on the beam, which, in presence of a magnet, generates the beam vibration by electromagnetic actuation. Measurements are done in a thermo-regulated room in which temperature is controlled at 20°C. The beam has been immersed in oils of known viscosities ranging from 10 cP to 30000 cP. In Fig. 1.a, no resonant peaks occur for viscosities above 100cP because of the excessive damping. Using Eqs. 4 and 5, viscosity and density can be calculated after extraction of the drag force components (Eqs. 2 and 3). Preliminary measurements (Fig. 1) are in good agreement with the rheological behaviour of the silicon oils for this frequency range 4 . As is implicit in the Newtonian and incompressible model, we observe viscosity and density as frequency-independent constants. The model works well for high viscosities despite the absence of a resonant peak. For that reason, this represents an alternative to classical microcantilever-based rheometer 5,[START_REF] Boskovic | Rheological measurements using microcantilevers[END_REF] . The viscosity measurements are more accurate than the density values. This shows that, for those cases in which the density is known, the proposed method may be used to accurately quantify the dynamic viscosity. The accuracy of both, the beam geometry and the static value of the transfer function determines the quality of the measurements.

VISCOELASTICITY

The significant frequency-dependence of the properties of viscoelastic fluids motivates us to consider the use of microcantilevers to measure their properties over a broad frequency range, i.e., to serve as an improved rheometer. The objective is to extract several fluid properties from the beam response and to identify viscous/elastic or vitreous transitions. In the previous section, we have seen that a microcantilever can be used to determine a fundamental Newtonian fluid property (viscosity). A similar approach is possible for viscoelastic fluids. To achieve such a goal, we introduce the imaginary shear modulus  j G  into the simplified expression of the complex hydrodynamic function      j proposed by Maali&al 1 for the case of a viscous fluid. Then, the obtained expression of      j as a function of the imaginary shear modulus is used for the study of a viscoelastic fluid (complex shear modulus) by replacing G by G'+jG''. This can be done because the cantilever is larger than the mesh size of the rheological network 7 . By extracting the real and imaginary parts of the resulting hydrodynamic function, the expressions for a viscoelastic fluid are as follows:

Preprint: The XVth international congress on rheology, Monterey, CA, USA, 3-8 aout 2008, AIP Conf. Proc, Vol. 1027, pp. 1159-1161. (doi: 10.1063/1.2964501)

    2 2 2 2 2 1 1 2 2 1 '' ' '' ' ' '' ' 2 4 2 f b G g b b a G G G a b G G G                    (6)     2 2 2 2 2 2 1 2 2 1 2 1 2 ' ' '' ' ' '' ' 4 2 4 2 f f b G g a b b a b G G G a b G G G                        (7)
These analytical expressions (6-7) have been used in Fig. 2 to simulate the special case of a Maxwellian viscoelastic fluid. An example of the simulation is plotted in Fig. 2 where it can be seen that resonant phenomena clearly occur as a result of the low viscosities associated with high frequencies (Fig. 2a), even if the fluid is a 'compact gel' in its static state. The characteristics of the resonance (frequency and phase slope) depend on the plateau elastic modulus G 0 . In Fig. 2b, the asymptotic values of amplitude depend only on G 0 while the microcantilever deflection depends also on the relaxation time , at frequencies near or less than 1/ . Another set of experimental data was generated and analyzed for three different fluids: dodecane, EHDT-silicon gel and air. EHDT-silicon gel results from a mixture of powder in dodecane. In Fig. 2c and Fig. 2d, we see that the EHDT and dodecane have different mechanical properties. This change in dynamic behavior reflects the new mechanical behavior of the fluid. CpCl solution at various mass concentration in salted water: 12% (G 0 =235Pa, τ =1.02s), 8% (G 0 =106Pa, τ =1.07s), 6% (G 0 =62Pa,τ=0.99s). Experimental amplitude (c) and phase (d) of deflection for a silicon cantilever oscillating in dodecane, EHDT, and air.

CONCLUSIONS

The results are very encouraging as they demonstrate the potential for beam-based microstructures to serve as useful tools for rheological measurements at fixed frequency over a wide frequency range. The theoretical model developed in the present paper describes the dynamics of the beam-fluid interaction quite well. However, future work will be required to refine the model for viscoelastic fluids, to perform additional experiments on other complex fluids, and to assess in a more systematic manner the potential for microcantilever-based rheometers to serve as competitive devices for determining rheological properties of complex fluids.

1 .

 1 Measurements of amplitude (a) and phase (b) for a silicon cantilever (18μm x 600μm x 4mm) immersed in various silicon oils: 10cP, 20cP, 100cP, 288cP, 485cP and 1000cP. Oil densities are 1000kg.m -3 . Determination of viscosity (c) and density (d) corresponding to the spectra presented in (a) and (b).

2 .

 2 Theoretical amplitude (a and b) of deflection for a silicon cantilever (18μm x 600μm x 4000μm) oscillating in a

ACKNOWLEDGMENTS

We thank the "Région Aquitaine" for its financial sponsorship.


Samia Bendamene

Naima Boutaghane

Charlotte Sayagh

Alabdul Abdulmagid

Zahia Magid

Chawki Kabouche

Laurence Bensouici

Abdulmagid Alabdul Magid

Zahia Kabouche

Chawki Bensouici

Laurence Voutquenne-Nazabadioko

Bis-iridoids and other constituents from Scabiosa semipapposa

Keywords: Scabiosa semipapposa Salzm. ex DC., Caprifoliaceae, Bis-iridoid glucosides, Semipapposiridoids A-F, Antioxidant activity

teaching and research institutions in France or abroad, or from public or private research centers.

Introduction

The Caprifoliaceae, also known as the honeysuckle family, is a dicotyledonous flowering plant consisting of about 860 species in approximately 42 genera, with a nearly cosmopolitan distribution, with the centers of diversity in eastern North America and eastern Asia, while absent in tropical and southern Africa (Xuand Chang, 2017). Plants of this family are usually shrubs and vines and rarely herbaceous. Many genera of this family like Cephalaria [START_REF] Tabatadze | Cytotoxic triterpenoidsaponins from the roots of Cephalaria gigantea[END_REF][START_REF] Pasi | Biologically active triterpenoids from Cephalaria ambrosioide[END_REF][START_REF] Mustafayeva | DNA-Damaging, mutagenic, and clastogenic activities of gentiopicroside isolated from Cephalaria kotschyi roots[END_REF][START_REF] Sarikahya | Antimicrobial triterpenoid glycosides from Cephalaria scoparia[END_REF][START_REF] Sarikahya | Saponins from twenty-two Cephalaria Species[END_REF], Valeriana [START_REF] Ming | The structures of three novel sesquiterpenoids fromValeriana jatamansi Jones[END_REF][START_REF] Tang | Iridoids from the rhizomes and roots of Valeriana jatamansi[END_REF][START_REF] Dong | Iridoids and sesquiterpenoids of Valeriana stenoptera and their effects on NGF-induced neurite outgrowth in PC12 cells[END_REF][START_REF] Quan | Iridoids and bisiridoids from Valeriana jatamansi and their cytotoxicity against human glioma stem cells[END_REF][START_REF] Liu | Iridoids from Valeriana jatamansi with anti-inflammatory and antiproliferative properties[END_REF], Lonicera [START_REF] Lin | Two new triterpenoid saponins from the flowers and buds of Lonicera japonica[END_REF][START_REF] Liu | Iridoid glycosides from the flower buds of Lonicera japonica and their nitric oxide production and α-glucosidase inhibitory activities[END_REF][START_REF] Li | Lonicera japonica flos and Lonicera flos: a systematic review of ethnopharmacology, phytochemistry and pharmacology[END_REF][START_REF] Qiu | Phytochemical and networkbased chemotaxonomic study of Lonicera[END_REF], and Scabiosa (Lehbili et al., 2018a;Lehbili et al., 2018b;[START_REF] Bendamene | Semipapposides A-M, triterpenoid bidesmosides saponins from the roots of Scabiosa semipapposa[END_REF][START_REF] Kılınç | Phytochemical investigation of Scabiosa sicula guided by a preliminary HPLC-ESIMS n profiling[END_REF] have attracted the attention of various authors for their phytochemical and biological properties. The Scabiosa genus, belonging to this family, is widespread in the Mediterranean area, including 12 species in Algeria [START_REF] Quezel | Scabiosa stellata L. phenolic content clarifies its antioxidant activity[END_REF]. Many Scabiosa species are used in folk medicine for the treatment of various illnesses such as asthma, influenza, bronchitis, bronchial pneumonia, liver diseases, neurodegenerative diseases, and to treat certain dermatoses, in particular scabies [START_REF] Girre | Connaitre et reconnaitre les plantes médicinales[END_REF][START_REF] Bammi | Les plantes médicinales dans la forêt de L'Achach (Plateau Central, Maroc)[END_REF][START_REF] Rigat | Studies on pharmaceutical ethnobotany in the high river Ter valley (Pyrenees, Catalonia, Iberian Peninsula)[END_REF][START_REF] Kose | Ethnobotanical survey of medicinal plants used in the Maseru district of Lesotho[END_REF][START_REF] Moteetee | Medicinal plants used in Lesotho for treatment of reproductive and post reproductive problems[END_REF][START_REF] Pinto | Scabiosa genus: a rich source of bioactive metabolites[END_REF]. Numerous Scabiosa species yielded a great variety of secondary metabolites such as triterpene saponins [START_REF] Alimbaeva | Triterpene glycosides of some representatives of the Dipsacaceae amily[END_REF][START_REF] Baykal | Triterpene saponins from Scabiosa rotata[END_REF][START_REF] Zheng | New biologically activeTriterpenoid saponins from Scabiosa tschiliensis[END_REF]Lehbili et al., 2018a;[START_REF] Kılınç | Phytochemical investigation of Scabiosa sicula guided by a preliminary HPLC-ESIMS n profiling[END_REF][START_REF] Bendamene | Semipapposides A-M, triterpenoid bidesmosides saponins from the roots of Scabiosa semipapposa[END_REF], flavonoids, coumarins [START_REF] Garaev | Flavonoids and oleanolic acid from Scabiosa caucasia[END_REF][START_REF] Al-Qudah | New flavonol glycoside from Scabiosa prolifera L. aerial parts with in vitro antioxidant and cytotoxic activities[END_REF]Lehbili et al., 2018b), mono-and bis-iridoid glucosides [START_REF] Papalexandrou | Iridoids from Scabiosa variifolia (Dipsacaceae) growing in Greece[END_REF][START_REF] Polat | Iridoids from Scabiosa atropurpurea L. subs p. maritime Arc. (L.)[END_REF]Lehbili et al., 2018b) and phenolic compounds (Akar,2022). In our continuous researches on the chemical constituents of Scabiosa species growing in Algeria (Lehbili et al., 2018a;Lehbili et al., 2018b;[START_REF] Bendamene | Semipapposides A-M, triterpenoid bidesmosides saponins from the roots of Scabiosa semipapposa[END_REF], we have investigated Scabiosa semipapposa Salzm. Ex DC. roots and aerial parts. After previous isolation of triterpenoid saponins from the roots of this plant [START_REF] Bendamene | Semipapposides A-M, triterpenoid bidesmosides saponins from the roots of Scabiosa semipapposa[END_REF], new phytochemical investigations were performed for its iridoids content. In this paper, we thus describe the isolation and structure elucidation of six undescribed bis-iridoids (1-6), namely semipapposiridoids A-F (Fig. 1), along with seventeen (7-23) known compounds.

Chemotaxonomy of this species was discussing. In addition, the antioxidant capacity of the 80% methanol extracts and fractions were evaluated using DPPH, ABTS and CUPRAC methods.

Results and discussion

The 80% methanol extract of S. semipapposa roots was fractionated by vacuum-liquid chromatography (VLC) on RP-C18 to give five fractions (RI to RV) which were purified by successive chromatographic techniques, including flash chromatography, as well as semipreparative and preparative high performance liquid chromatography (HPLC) to yield eleven known compounds (9-19). In the same manner, the 80% methanol extract of the aerial parts afford six undescribed compounds (1-6) (Fig. 1) and six known ones (7,8,(20)(21)(22)(23). Their structures were established by 1D-and 2D-NMR analysis ( 1 H, 13 C, COSY, TOCSY, NOESY, HSQC, HMBC) in combination with mass spectrometry ESI-and HR-ESI-MS, and by comparison of their physical and spectral data with those of literature values. The known compounds were identified as sylvestroside II (7) [START_REF] Jensen | Novel bis-iridoid glucosides from Dipsacus sylvestris[END_REF], sylvestroside I (8) [START_REF] Jensen | Novel bis-iridoid glucosides from Dipsacus sylvestris[END_REF]Abdallah,1991), triplotoside A (9) [START_REF] Graikou | Cantleyoside-dimethyl-acetal and other iridoid glucoside from Pterocephalus perennis -antimicrobial activities[END_REF][START_REF] Gülcemal | Monoterpenoidglucoindole alkaloids and iridoids from Pterocephalus pinardii[END_REF], sweroside (10) [START_REF] Jensen | Novel bis-iridoid glucosides from Dipsacus sylvestris[END_REF], loganin (11) (Calis et al., 1984a), secoxyloganin (12) (Calis and sticher. 1984b), vanilloloside (13) [START_REF] El-Ghazooly | Chemical constituents of Helichrysum conglobatum growing in Egypt[END_REF], vanillyl-β-Dglucopyranoside (14) [START_REF] Kanho | Biotransformation of benzaldehyde-type and Acetophenone-type derivatives by Pharbitis nil hairy Roots[END_REF], caffeic acid methylester (15) (Rani et Devanan.,2013), 3,4,5- [START_REF] Kanchanapoom | Iridoid and phenolic glycosides from Morinda coreia[END_REF][START_REF] Qingwei | Phenolic and lignan glycosides from the butanol extract of Averrhoa carambola L. Root[END_REF], (+)-8hydroxypinoresinol (17) (Tsukamoto et al.,1984a;Tsukamoto et al.,1984b;[START_REF] Cowan | Lignans from Strophanthus gratus[END_REF], (+)-8-hydroxypinoresinol-4ʹ-O-β-D-glucopyranoside) (18) [START_REF] Deyama | The constituent of Eucommia ulmoides OLIV.III. Isolation and structure of a new lignan glycoside[END_REF] (Tsukamoto et al., 1984a;[START_REF] Tsukamoto | Lignans from bark of Olea plants[END_REF], apigenin (20) [START_REF] Wawer | 13C CP/MAS NMR studies of flavonoids[END_REF], luteolin (21) [START_REF] Okamura | Flavonoids in Rosmarinus officinalis leaves[END_REF], lutéoline-7-O-β-D-glucopyranoside (22) [START_REF] Chiruvella | Phytochemical and antimicrobial studies of methyl angolensate and luteolin-7-Oglucoside isolated from callus cultures of Soymida febrifuga[END_REF], and kaempferol 3-O-(3ʹʹ,6ʹʹ-di-O-E-pcoumaroyl)-β-D-glucopyranoside (23) [START_REF] Yang | Synthesis of kaempferol 3-O-(3ʹʹ,6ʹʹ-di-O-E-p-coumaroyl)-β-D-glucopyranoside, efficient glycosylation of flavonol 3-OH with glycosyl O-alkynylbenzoatesas donors[END_REF].

trimethoxyphenyl-1-O-β-apiofuranosyl-(1''→6')-β-glucopyranoside (= kelampayosideA) (16)
, (+)-8- hydroxypinoresinol-4-O-β-D-glucopyranoside) (19)
Compound 1 was obtained as a yellow amorphous powder, with a molecular formula of C44H56O22 based on HR-ESI-MS (m/z 959.3170 [M+Na] + , calcd for 959.3161). The infrared (IR) spectrum indicated the presence of the hydroxyl functions (3356 cm -1 ), and α,β unsaturated ester carbonyl (1695 and 1629 cm -1 ). The 1 H and 13 C NMR spectra (Table 1) in combination with the HSQC and 1 H-1 H COSY correlations showed two distinct parts, indicated as units A and B (Fig. 1). The signals corresponding to unit A included one olefinic methine [δH7.52, s (H-3a); δC153.9 (C-3a)], one vinyl [δH5.80, ddd (J=17.4, 10.4, 8.6 Hz,H-8a); δC135.7 (C-8a), δH5. 28,dd (J=10.4,1.5 Hz,5.33,d(J=17.4 Hz, H-10a); δC119.6 (C-10a)], one methylene oxy [δH 4.11, 4.14, (H-7a); δC64.1 (C-7a)], one methylene [δH 1.86, td (J=13.8, 7.4 Hz, H-6a), 2.12, dd (J=13.8, 7.2 Hz, H-6a); δC30.1(C-6a)], two methines [δH2.90, q (J=6.3 Hz, H-5a); δC 31.3 (C-5a), δH2.70, dt (J=8.3, 6.1 Hz, H-9a); δC 45.4 (C-9a)], one acetal [δH5. 58, d (J=6.6 Hz, H-1a); δC97.7 (C-1a)], one carboxy group [δC168.2 (C-11a)]and one carbomethoxy group [δc 173.0 (C-12a)].

This 1 H and 13 C NMR spectroscopic data of 1 showed that unit A contained a secoiridoid moiety, almost identical to secologanic acid [START_REF] Kocsis | New Bis-Iridoids from Dipsacus laciniatus[END_REF], except that compound 1 had a carbomethoxy group at C-7a [δC64.1 (C-7a)] instead of the aldehyde group of secologanic acid, which was also detected from the HMBC correlations from H-5a and H-6a to C-7a (Fig.

2).

The remaining resonances in the 1 H and 13 C NMR spectra of 1 attributed to unit B, particularly those arising from an olefinic methine [δH7.47, s (H-3b); δC152.5 (C-3b)], one methylene [δH1. 78,ddd (J=14.1,7.8,5.1 Hz),2.33,ddd (J=14.1,7.7,1.5 Hz)(H-6b); δC40.3 (C-6b)], three methines [δH3.15, q(J=7.9 Hz,H-5b); δC32.6 (C-5b), δH2.18, m (H-8b); δC41.0 (C-8b); δH2. 13,ddd (J=17.4, 8.8, 4.8 Hz,H-9b); δC47.1 (C-9b)], one oxygenated methine [δH 5.24, t (J=4.5 Hz, H-7b); δC78.4 (C-7b)], one acetal [δH5.33, d (J=4.6 Hz, H-1b); δC97.6 (C-1b)], one secondary methyl [δH1.10, d (J=6.8 Hz, H-10b); δC13.8 (C-10b)], one methoxy [δH3.71, s (MeO-12b); δC51.7 (MeO-12b)] and one carboxy group [δC169.3 (C-11b)], indicated the second unit to be loganic-type iridoid [START_REF] Kocsis | New Bis-Iridoids from Dipsacus laciniatus[END_REF].The major differences between them is the downfield shifts of H-7b (δH5.24) and C-7b (δC78.4). The attachment site between unit A and B was found to be ester linkage between the C-7 of unit B and the carboxyl group (C-11a) of unit A as deduced by the HMBC correlation observed between the oxygenated methine proton (H-7b) and the carboxy carbon (C-11a).

Furthermore, two anomeric protons resonances corresponding to O-linked sugars were observed in the 1 H NMR spectrum of 1 as two doublets at δH4.74 (J = 7.9 Hz) and 4.76 (J =7.9 Hz). The sugar units were identified after analysis of COSY and HSQC spectra as two glucoses [glc-A (δH-1′ 4.74 and δC-1′100.1) and glc-B (δH-1ʺ4.76 and δC-1ʺ100.2)] (Table 1). The anomeric configurations were defined by the 3 JH1,H2 coupling constants and the comparison of 13 C-NMR chemical shifts with those in the literature [START_REF] Agrawal | Chemical compositions by using LC-MS/MS and GC-MS and antioxidant activities of methanolic extracts from leaf and flower parts of Scabiosa columbaria subsp. columbaria var. columbaria L.Saudi[END_REF] (Table 1).The glc-A unit was linked to C-1a as deduced from the long-range correlation observed between H-1′ and C-1a in the HMBC spectrum, and the glc-B unit was linked to C-1b as deduced from the longrange correlation observed between H-1′′ and C-1b in the HMBC spectrum(Fig. 2). These 1 H and 13 C NMR spectroscopic data of 1 are very similar to sylvestroside II [START_REF] Soeren | Novelbis-iridoidglucosides from Dipsacus sylvestris[END_REF], except for the signals for an additional trans-p-coumaroyl unit in 1 with two coupled trans double-bond protons [δH6.39, H-8ʹʹʹ and 7.69, H-7ʹʹʹ, each d, J = 15.9 Hz] and four aromatic protons at δH7. 49 and 6.83 (each 2H,d,J = 8.6 Hz) assignable to H-2ʹʹʹ/6ʹʹʹ and H-3ʹʹʹ/5ʹʹʹ respectively, as well as to an ester carbonyl (δC168.5) in the 13 C NMR spectrum. The chemical shifts of C-4′ʹ in the glucopyranosyl moiety (glc-B) were downfield shifted from δC71.6 to δC72.4 relative to those of sylvestroside II [START_REF] Soeren | Novelbis-iridoidglucosides from Dipsacus sylvestris[END_REF]. Additionally, the location of the E-p-coumaroyl unit was established by the key cross-peaks between the H-4ʹʹ (δH4.87) of glucosyl and carbonyl carbon C-9ʹʹʹ (δC168.5) of the coumaroyl in the HMBC spectrum. The relative configuration of 1 was further determined by the NOESY experiment. The NOE correlations observed between δH 2.90 (H-5a) with 2.70 (H-9a), 2.13 (H-9b) with 1.10 (H-10b), 3.15 (H-5b) with 2.13 (H-9b) and 5.24 (H-7b) with 2.18 (H-8b) reveal that they were β-oriented, as is usually observed in the iridoid skeleton [START_REF] Jensen | Novel bis-iridoid glucosides from Dipsacus sylvestris[END_REF][START_REF] Kocsis | New Bis-Iridoids from Dipsacus laciniatus[END_REF][START_REF] Tomassini | Bis-iridoid glucosides from Abelia chinensis[END_REF][START_REF] Dinda | A new bisiridoid glucoside from Mussaenda incana[END_REF][START_REF] Ji | A new iridoid glycoside from the roots of Dipsacus asper[END_REF]. Based on the above findings, compound 1 was identified as 4′′-O-E-p-coumaroyl-sylvestroside II, named semipapposiridoid A shown in Fig. 1 and differs from sylvestroside II, reported in Dipsacus sylvestris [START_REF] Soeren | Novelbis-iridoidglucosides from Dipsacus sylvestris[END_REF], by an additional E-p-coumaroyl group at C-4ʹʹ. Semipapposiridoid B (2) was isolated as a yellow powder. The HR-ESI-MS, m/z 959.3169 [M+ Na] + was identical to that obtained for compound 1, suggesting that both compounds were isomers with molecular formula C44H56O22. The IR spectrum indicated the presence of the hydroxyl functions (3352cm -1 ) and α,β unsaturated ester carbonyl (1688 and 1630 cm -1 ), which are responsible for the maximum UV absorption of the compound at 231 nm [START_REF] Tian | Two novel tetrairidoid glucosides from Dipsacus asper[END_REF].

Comparison of 1 H and 13 C NMR values and the analysis of the 1 H-1 H-COSY, NOESY, HSQC and HMBC revealed that compound 2 is structurally closely related to 1 and contains the same skeleton (sylvestroside II) (Table 1). The only significant difference is the location of the E-pcoumaroyl group. The E-p-coumaroyl group was linked to the C-6ʺof glc-B unit in 2 instead of the C-4ʺof glc-B in 1. The HMBC spectrum gave further evidence of this by showing longrange correlations between the C-6ʺ methylene protons at δH4.45 and 4.54 of glucosyl and the carbonyl carbon C-9ʹʹʹ at δC168.9. The relative configuration of 2 was established as identical to 1 by the NOESY data of 2. Hence, the structure of 2 was elucidated as 6ʹʹ-O-E-p-coumaroylsylvestroside II (Fig. 1).

Semipapposiridoid C (3) was isolated as a mixture with semipapposiridoid A (1) in the ratio 1:1(Z:E), based on the integral intensity of the corresponding signals in their 1 H NMR. Both components of the mixture had the same molecular formula C44H56O22 [HR-ESI-MS, m/z 959.3169 [M+ Na] + ]. The 1 H and 13 C NMR spectra (Table 1) of 3 were extremely close to those of compound 1. The only significant difference was the value of the vinyl proton coupling constants [δH5.83 and 6.94 (each d, J = 12.8 Hz)]. HMBC correlations of the ester carbonyl at δC167.3 to the Z-vinyl protons [δH5.83 and 6.94 (each d, J = 12.8 Hz)] and Glc-H-4′′ [4.86 (t, J =9.5 Hz)] indicated that the structure of semipapposiridoid C (3) was 4′′-O-Z-p-coumaroylsylvestroside II (Fig. 1).

The 1 H-NMR and 13 C-NMR spectra (Table1) of semipapposiridoid D (4) were very similar to those of semipapposiridoid B (2) and again showed that they were a mixture of sylvestroside II esterified at the C-6′′ position and revealed the presence of Z-and E-p-coumaroyl groups. They had the same molecular formula, C44H56O22, according to their HR-ESI-MS. The Z-pcoumaroyl was located at the C-6′′ position in 4, based on HMBC correlations from the ester carbonyl at δC 170.8 to the Z-vinyl protons [δH5.80 and 6.90 (each d, J =12.9 Hz)] and Glc-H-6′′ [4.37 (dd, J =12.0, 6.5 Hz) and 4.50 (dd, J =12.0, 2.1 Hz)]. The relative configuration of 4 was established as identical to 2 by the NOESY data of 4. Therefore, the chemical structure of semipapposiridoid D was determined to be 6′′-O-Z-p-coumaroyl-sylvestroside II (Fig. 1).

Semipapposiridoid E (5) was obtained as a yellowish amorphous powder. Its molecular formula C26H40O12 was obtained from its HR-ESI-MS (m/z 567.2413; [M + Na] + , calcd 587.3298). The IR spectrum of 5 showed absorption bands of the hydroxy group at 3377cm -1 and the carbonyl groups at 1692 and 1629cm -1 , respectively. The UV spectrum showed absorption maxima at  abelioside B [START_REF] Murai | Abeliosides A and B, secoiridoid glucosides from Abelia grandiflora[END_REF] except for the presence of signals at δH3.42 (3H, s, 3-OCH3), δC56.0 attributed to the methoxy group, instead of the signal for an ester carbonyl group in abelioside B. In the HMBC spectrum, a correlation between the protons at δH3.42 and carbons at δC102.9 (C-3) were observed, indicating that this methoxy group was located at C-3. The relative configuration of 5 was established by analysis of the NOESY spectrum showing correlations between H-5 and H-9β axial and between H-1β axial and CH3-10 which indicated that H-5, H-9 and CH3-10 were all β-oriented, and between H-7 and H-8 α-oriented. The NOESY correlations observed between H-3 and H-1 β axial indicated a β-axial orientation of H-3 and an α-axial orientation of methoxyl group at the C-3 position. Based on the above evidence, the structure of semipapposiridoid E (5) was established as depicted in Fig. 1.

Semipapposiridoid F (6) was isolated as a yellowish amorphous powder. It has the same molecular formula as compound 5 (C26H40O12) and differs from the latter only by the orientation of the methoxy group at C-3. The IR spectrum displayed absorption bands at 3376, 1693 and 1629 cm -1 attributed to the hydroxyl and carbonyl functions. The 13 C NMR spectra (see Table 2) of 6 showed great similarities to those of 5, except for the signals of C-1 δC59.3 (Δ-4.9 ppm), C-3 δC99.3 (Δ-3.6 ppm) and C-4 δC33.1 (Δ-2.9 ppm). The lack of correlation between H-3 and H-1 in the NOESY spectrum indicates the β-axial orientation of the methoxyl group at C-3 [START_REF] Breitmaier | Carbon-13 NMR spectroscopy[END_REF]. Therefore, the structure of 6 (semipapposiridoid F) was elucidated as shown in Fig. 1.

The total phenolic and flavonoid contents of the 80% MeOH extracts of the roots (R)and aerial parts (A) of S. semipapposa and their fractions RI to RV (for the roots), AI to AV (for the aerial parts), obtained after VLC fractionation on RP-C18, were quantitatively estimated using a calibration curve established in terms of gallic acid (GAE) and quercetin equivalents (QE), respectively (Table 3). Moderate phenolic and flavonoid contents were found in the 80% MeOH extracts with values of 109.0 μg GAE/mg and 12.8 μg QE/mg for the roots and 169.6 μg GAE/mg and 19.5 μg QE/mg for the aerial parts. The highest contents of total phenolics and flavonoids were found in the fraction RI with values of 361.7 μg GAE/mg and 74.6 μg QE/mg, while the fraction RV containing saponins [START_REF] Bendamene | Semipapposides A-M, triterpenoid bidesmosides saponins from the roots of Scabiosa semipapposa[END_REF] was the poorest (32.3 μg GAE/mg and 5.7 μg QE/mg). Previous research carried out on organic extracts of the species S. tschiliensis [START_REF] Wang | Rapid micropropagation system in vitro and antioxidant activity of Scabiosa tschiliensis Grunning[END_REF], S. arenaria [START_REF] Hlila | Acetylcholinesterase inhibitory and antioxidant properties of roots extracts from the Tunisian Scabiosa arenaria Forssk[END_REF] and S. stelatta [START_REF] Mouffouk | Acute toxicity and in vivo anti-inflammatory effects and in vitro antioxidant and anti-arthritic potential of Scabiosa stellate[END_REF], indicated lower levels of phenol and flavonoid contents compared to the results of the present study. Here, we identified in the roots (fractions RI-RIV), three phenyl glucosides (13-14, 16), caffeic acid methyl ester (15), three lignans (17-19) and four iridoids (9-12), and in the aerial parts (AIII to AV), eight bis-iridoids (1-8) and four flavonoids (20-23), thus justifying these results.

Several in vitro test procedures were carried out to estimate the antioxidant activity of crude plant extracts, but there is no simple universal method to assess antioxidant capacity accurately and quantitatively. Therefore, in the present study, three methods (DPPH, ABTS and CUPRAC assays), based on different principles, were applied to determine the antioxidant capacity of samples (Table 3). The antioxidant activity of the 80% MeOH extracts and fractions varied in a dose-dependent manner in all tests. The antioxidant activity of S. semipapposa samples ranged from 4.9 to 315.8 μg/mL when measured by the DPPH assay, from 8.9 to 124.0 μg/mL when measured by the ABTS •+ assay, and from 10.2 to 378.5 μg/mL when measured by the CUPRAC assay. These results are in agreement with those reported by [START_REF] Mouffouk | Acute toxicity and in vivo anti-inflammatory effects and in vitro antioxidant and anti-arthritic potential of Scabiosa stellate[END_REF]. The total antioxidant capacity values from ABTS •+ were higher than those of the DPPH • and CUPRAC assays. This may result from the presence of antioxidants that interact more selectively with fast reacting ABTS •+ radicals. Many studies reported that the solubility of the tested extract to different test systems and the stereoselectivity of radicals affect the ability of extracts to react against different radicals [START_REF] Yu | Free radical scavenging properties of wheat extracts[END_REF]. The moderate antioxidant activity of extract and fractions of the aerial parts is correlated with the presence of amounts of iridoids, which in their molecule have no (or few) phenolic OH groups, which neutralize free radicals. This is consistent with the study of [START_REF] Pacifico | Antioxidant efficacy of iridoid and phenylethanoid glycosides from the medicinal plant Teucrium chamaedrisin cell-free systems[END_REF] and [START_REF] Wu | Iridoids from Gentianaloureirii[END_REF] who reported that sweroside (10) and iridoids did not exhibit good radical scavenging ability.

Among the fractions tested, the fraction RI showed significant antioxidant activity in all tests.

Moreover, the results also indicated that this fraction showed higher activity (FRS50 4.9 μg/mL) (Table 3) than those of the antioxidant standards BHA and BHT (FRS50 8.7, 15.9 μg/mL, respectively) in the DPPH assays. The good antioxidant activity of the fraction RI could be associated with its high content of phenolic compounds (361.7±1.2 μg GAE/mg). Indeed, phenolic compounds have been the most studied for their antioxidant activity and have been reported to protect against many diseases through their ability to neutralize free radicals. [START_REF] Jayaprakasha | Antioxidant activity of grape seedVitis vinifera extracts on peroxidation models in vitro[END_REF][START_REF] Gali | Antioxidant and anticholinesterase effects of the ethanol extract, ethanol extract fractions and total alkaloids from the cultivated Ruta chalepensis[END_REF]. In the fraction RI, the iridoids sweroside (10) and secologanine (12) were purified as well as the phenolic glucosides (13, 14, 16).

Vanilloloside (13) was found to be a moderately active scavenger of DPPH FRS50 43.3 μg/mL) and moderate CUPRAC activity (0.397 mM TRg -1 ) [START_REF] Sarikahya | Isolation and characterization of biologically active glycosides from endemic Cephalaria Species in Anatolia[END_REF], while vanillyl β-D-glucopyranoside ( 14), showed DDPH scavenger activity (FRS50 30.88 μg/mL) (Shi et al., 2012).

Conclusions

In summary, the systematic phytochemical study on the roots and the aerial parts of S. semipapposa afforded six undescribed components, semipapposiridoids A-F (1-6) (Fig. 1), and three known bis-iridoid glucosides (7-9), three iridoids (10-12), three phenolic glucosides (13-14, 16), one caffeic acid methyl ester (15), three lignans (17-19) and four flavonoids (20-23) (Fig. S49 see supplementary data). These results expand our knowledge of chemical diversity and provide evidence to further analysis of their potential chemotaxonomic significance.

Compounds 7-12, 20-23, have been previously reported from different species of the genus Scabiosa [START_REF] Horn | Transformation of naturally-occurring 1, 9-trans-9,5-cis sweroside to all trans sweroside during acetylation of sweroside aglycone[END_REF][START_REF] Papalexandrou | Iridoids from Scabiosa variifolia (Dipsacaceae) growing in Greece[END_REF][START_REF] Christopoulou | Chemosystematic value of chemicalconstituents from Scabiosahymettia(Dipsacaceae)[END_REF]Wang etal.,2015;Lehbili et al.,2018a) and might reveal the close chemotaxonomic relationships between S. semipapposa and other Scabiosa species. It is also interesting to note that lignans (17-19) are described for the first time in the genus Scabiosa. They could serve as potential chemotaxonomic markers to differentiate Scabiosa from other genera in the Caprifoliaceae family.

Among the bis-iridoids, compounds (1-4, 7-9) isolated from S. semipapposa were found to possess secoiridoid/iridoid-subtype skeletons consisting of secologanic acid condensed to the 7-OH of loganin or loganin-like iridoids. These results also showed the close relationship between Scabiosa species [START_REF] Papalexandrou | Iridoids from Scabiosa variifolia (Dipsacaceae) growing in Greece[END_REF]Lehbili et al.,2018a).

In addition, the antioxidant activities of the 80% MeOH extracts and the fractions (RI to RV and AI to AIV) were evaluated by DPPH, ABTS and CUPRAC assays. Our results demonstrate that the fraction RI has particularly high antioxidant and free radical scavenging activities, probably due to the presence of vanillyl alcohol glucosides.

Experimental

General experimental procedures

UV spectra were measured on a Shimadzu UV/Vis U-2450 spectrophotometer. Optical rotations of pure compounds were measured in CH3OH using a MCP 5100 AntonPaar Polarimeter. IR spectra were recorded on a Nicolet Avatar 320 FT-IR spectrometer with KBr disks. The 1D and 2D NMR spectra ( 1 H and 13 C NMR, 1 H-1 H COSY, NOESY, HSQC and HMBC) were performed using a Bruker Avance III 600 spectrometer ( 1 H at 600 MHz and 13 C at 150 MHz) equipped with a 5 mm TCI cryoprobe. 2D-NMR experiments were performed using standard Bruker microprograms (TopSpin 3.5 software). ESI-MS and HR-ESI-MS experiments were performed using a Micromass Q-TOF micro instrument (Manchester, UK). Flash chromatography (FC) was carried out on a Grace Reveleris system equipped with dual UV and ELSD detection using Grace® cartridges (Silica gel or RP-C18). The medium-pressure liquid chromatography (MPLC) was employed using a Buchi pump system AP250/500 (Buchi, France), with a RP-C18 silica gel MERCK column (15 × 230 and 26 × 460 mm). Semipreparative HPLC was realized on a Dionex apparatus equipped with an ASI-100 automated sample injector, a STH 585 column oven, a P580 pump, a diode array detector UVD 340S and the Chromeleon ® software version 6.8. A prepacked RP-C18 column (Phenomenex 250 x 10 mm, Luna 5 µ) was used for semi-preparative HPLC. The eluting mobile phase consisted of H2O with TFA (0.0025%) and CH3CN with a flow rate of 5 mL/min and the chromatogram was monitored at 205 and 215 nm. Analytical HPLC experiments were performed using a Thermofisher Ultimate 3000 (Thermo Fischer Scientific, Villebon sur Yvette, France), equipped with a 4 ways pump LPG 3400 SD, an automatic injector WPS 3000 SL, a UV/visible diode array detector 3000 and the Chromeleon ® software version 6.8. A prepacked C18 column Uptisphere Strategy C18 (Interchim, 4.6×250 mm, 5μ) was used for analytical HPLC and the mobile phase consisted of H2O with TFA (0.0025% v/v) and CH3CN. A gradient elution method was applied from 5% to 80% of CH3CN in 30 min with a flow rate of 1 mL/min and the chromatograms were monitored at 205, 254, 300 and 360 nm.TLC was performed on pre-coated silicagel 60 F254 Merck and compounds were visualized by spraying the dried plates with 50% H2SO4, followed by heating.

The antioxidant activity assays were carried out at the Center of Biotechnology Research (Contantine, Algeria) on a 96-well microplate reader, Perkin Elmer Multimode Plate Reader EnSpire (Perkin Elmer, France). Butylated hydroxylanisole (BHA), butylated hydroxytoluene (BHT), 1,1-diphenyl-2-picrylhydrazyl (DPPH • ), 2,2′-azinobis (3-ethyl-benzothiazoline-6suphonic acid) diammonium salt (ABTS •+ ) and Ciocalteureagent (FCR) were obtained from Sigma Chemical Co. (Sigma-Aldrich GmbH, Sternheim, Germany). All other chemicals and solvents were of analytical grade.

Plant material

The whole plant Scabiosa semipapposa Salzm. ex DC. was collected in May 2017 from Alguemas, Constantine, North-Eastern Algerian (latitude 36.3479 and longitude 6.650773).The plant material was identified by Mr. Kamel Kabouche. A voucher specimen (LOST Ss.05/17) has been deposited at the herbarium of LOST Laboratory, University Frères Mentouri-Constantine, Algeria.

Extraction and isolation

The aerial parts and the roots of S. semipapposa were studied apart from each other. The dried and powdered roots of S. semipapposa (1 kg) were macerated in 80% MeOH (310 L, 24 h) at room temperature, followed by rotary evaporation at 45 °C under reduced pressure to give the crude extract (100 g). A part of this (55 g) was subjected to RP-C18vacuum liquid chromatography (VLC) eluted successively with MeOH-H2O (3:7, 4:6, 6:4, 8:2 and 10:0), to provide five major fractions (RI to RV, respectively). Fraction RI (1.3 g) was subjected to High Performance Flash Chromatography (HPFC), over silica gel, eluted by a gradient system of CH2Cl2-MeOH-H2O (10:0:0 to 7:3:0.5), in 39 min to afford 69 sub-fractions (RI-1 to RI-69). Subfractions RI-29 to RI-34were combined (27.5 mg) and purified by semi-preparative HPLC using a gradient (10-30% CH3CN, in 20 min) to furnish compounds 14 (1.4 mg, tR 5.9 min), 10 (3.5 mg, tR 11.1 min). Then the combined sub-fractions RI-36-37 (23 mg) were purified by semipreparative HPLC (10-30% CH3CN, in 20 min) to afford compounds 13 (1 mg, tR 4.5 min), 16

(1.9 mg, tR 13.7 min) and 12 (3.4 mg, tR 18.4 min). Fraction RII (1.3 g) was fractionated by silica gel CC, using a gradient of EtOAc-MeOH (1:0-6:4) to give 230 sub-fractions. Sub-fractions RII-50-55 contained the pure compound 11 (31.3 mg). Sub-fractions RII-59-63 (38.8 mg) was purified by semi prep HPLC using an isocratic elution of 15% CH3CN during 25 min to give compounds 10 (14.3 mg, tR 9.9 min) and 18 (6.4 mg, tR 19.4 min) whereas the purification of the RII-70-80 (58.2 mg) in the same condition gave 19 (2.9 mg, tR 22.1 min). Fraction RIII (1.2 g) was applied to HPFC on normal phase silica gel, eluted with the system CH2Cl2-MeOH-H2O

[10:0:0 to 60:40:7], in 48 min to afford 60 sub-fractions. The purification of the sub-fractions RIII-10-14 (24.8 mg) by semi prep HPLC in isocratic elution with 25% CH3CN during 20 min, gave compounds 17 (4.7 mg, tR 11.1 min), and 15 (6.9 mg, tR 8.1 min). Fraction RIV (198 mg) was purified by silica gel CC, eluted with the gradient of the mixture of CH2Cl2-MeOH-H2O

[95:5:0-70:30:5], to afford compound 9 (11.3 mg) as a white precipitate in pure form. Fraction RV was purified previously [START_REF] Bendamene | Semipapposides A-M, triterpenoid bidesmosides saponins from the roots of Scabiosa semipapposa[END_REF].

The same protocol was used for the extraction of the aerial parts of S. semipapposa (700 mg).

A part of the resulting 80% MeOH extract (50 g) was submitted to VLC over RP-C18 eluted successively with 4:6,6:4,8:2 and 10:0), to give 5 main fractions (AI to AV, respectively) and a pure compound 22 (3mg) obtained as a precipitate in fraction AIII. This latter (4 g) was fractionated by flash chromatography over silica gel, using a binary gradient of CH2Cl2-MeOH [10:0 to 6:4] in 55 min to afford 146 sub-fractions. Sub-fractions AIII-51-52 (51 mg), AIII-57-60 (50 mg) and AIII-72 (33.4 mg), were purified each one by MPLC on RP-C18 eluted by gradient system 10%-50% CH3CN, in 45 min to yield compounds 5 (4.4 mg) and 6 (2.6 mg), from AIII-51-52, 10 (8.5 mg), and a mixture of 3 and 1 (1.7 mg) from AIII-57-60, and 8 (15.2 mg) from AIII-72. Fraction AIV (1.9g) was purified by flash chromatography over silica gel, eluted by a gradient system of CH2Cl2-MeOH [10:0 to 6:4] in 45 min to afford 100 subfractions. Compounds 21(1mg), 1 (23.2 mg) and 7 (24.4 mg) were obtained as pure compounds in fractions AIV-25, AIV-45 and AIV-66-67, respectively, while the sub-fraction AIV-22 (31.5 mg) was purified by MPLC (20-55% CH3CN, in 18 min) to yield 3.4 mg of compound 20. Sub-fractions AIV-32 to AIV-35 (30 mg) were pooled and purified by MPLC (30-60% CH3CN, in 20 min) to afford 23 (6 mg). The remaining sub-fraction AIV-53-54 (28.5 mg) was chromatographed by MPLC (20-50% CH3CN, in 20 min) to furnish the pure compound 2 (8.5 mg), and the mixture of compounds 3 and 1 (1.9 mg), and 4 and 2 (1 mg).

Semipapposiridoid A (1)

Yellowish solid;   25 D  -97.7(c1, MeOH); IR (KBr) νmax (cm -1 ): 3356, 2924, 1695, 1629, 1603, 1514, 1440, 1371, 1260; UVλmax (MeOH) (log ): 231 (0.177), 313 (0.193) nm; 1 H (600 MHz, CD3OD) and 13 C NMR (150 MHz, CD3OD) data: see Table1. HR-ESI-MS m/z: 959.3170[M+Na] + (calcd for C44H56O22, 959.3161).

Semipapposiridoid B (2)

Yellowish solid;   25 D  -40(c 0.16, MeOH); IR (KBr) νmax (cm -1 ): 3352, 2925, 1688, 1629, 1605, 1515, 1440, 1380, 1282; UVλmax (MeOH) (log ): 228 (0.845), 308 (0.250) nm; 1 H (600 MHz, CD3OD) and 13 C NMR (150 MHz, CD3OD) data: see Table1. HR-ESI-MS m/z: 959.3169

[M+Na] + (calcd for C44H56O22, 959.3161).

Semipapposiridoid C (3)

Yellowish solid;   25 D  -66(c 0.17, MeOH); IR (KBr) νmax (cm -1 ): 3357, 2924, 1695, 1630, 1067; UVλmax (MeOH) (log ): 231 (1.266), 312 (0.630) nm; 1 H (600 MHz, CD3OD) and 13 C NMR (150 MHz, CD3OD) data: see Table 1. HR-ESI-MS m/z: 959.3156 [M+Na] + (calcd for C44H56O22,959.3161).

Semipapposiridoid D(4)

Yellowish solid;   25 D  13.3(c0.18, MeOH); IR(KBr) νmax(cm -1 ): 3358, 2920, 1695, 1630,1068; UVλmax (MeOH) (log ): 231 (0.724), 312 (0.304) nm; 1 H (600 MHz, CD3OD) and 13 C NMR (150 MHz, CD3OD) data: see Table1. HR-ESI-MS m/z: 959.3169 [M+Na] + (calcd for C44H56O22, 959.3161).

Semipapposiridoid E(5)

Yellowish solid:   25 D  -58 (c0.44, MeOH); IR (KBr) νmax (cm -1 ): 3377, 2926, 1692, 1629, 1449, 1386, 1277; UV λmax (MeOH) (log ): 235 (0.580) nm; 1 H (600 MHz, CD3OD) and 13 C NMR (150 MHz, CD3OD) data: see Table 2. HR-ESI-MS m/z: 567.2413 [M+Na] + (calcd for C26H40O12,567.2417).

Semipapposiridoid F (6)

Yellowish solid;   25 D  -14 (c0.26, MeOH);IR (KBr) νmax (cm -1 ): 3376, 2926, 1693, 1629, 1451, 1387, 1262; UV λmax (MeOH) (log ): 235 (1.761) nm; 1 H (600 MHz, CD3OD) and 13 C NMR (150 MHz, CD3OD) data: see Table 2. HR-ESI-MS m/z: 567.2408 [M+Na] + (calcd for C26H40O12, 567.2417).

Determination of total phenolic (TPC) and flavonoid contents (TFC)

Total phenolic content (TPC) of 80% MeOH extracts of aerial parts, roots, and fractions (RI-V and AI-IV) of S. semipapposa (Table 3) was determined spectrophotometrically using Folin-Ciocalteu method as previously reported [START_REF] Singleton | Colorimetry of total phenolics with phosphomolybdicphosphotungsticacid reagents[END_REF] with slight modifications (Muller et al., 2010). The absorbance was measured at 765 nm and the results were expressed as micrograms of gallic acid equivalent per milligram of extract (μg GAE/mg).

The total flavonoid content (TPC) was analyzed using an adapted aluminum chloride colorimetric reported by [START_REF] Topçu | A new flavone from antioxidant extracts of Pistacia terebinthus[END_REF]. The absorbance was measured at 415 nm and the results were expressed as mg of quercetin equivalents per milligram of extract (μg QE/mg).

Antioxidant activities

Evaluation of the antioxidant activities of 80% MeOH extracts of aerial parts, roots, and fractions (RI-RV and AI-AIV) was carried out by three different methods including scavenging of the free radicals of 2,2-diphenyl-1-pycrylhydrasyl (DPPH) and 2,2'-azinobis-(3ethylbenzothiazoline-6-sulfonate) (ABTS) and cupric reducing antioxidant capacity (CUPRAC) assays.

DPPH free radical scavenging activity

The free radical scavenging activity of the 80% MeOH extracts and the fractions of S. semipapposa was determined by the DPPH assay described by [START_REF] Blois | Antioxidant determinations by the use of a stable free radical[END_REF] with few modifications as previously described [START_REF] Boutaghane | Chemical constituents of Genista numidica Spach aerial parts and their antimicrobial, antioxidant and antityrosinase activities[END_REF]. Briefly, 40 μL of either standard or sample solutions was mixed with 160 μL of DPPH solution in microplate. The mixture was then incubated at room temperature in the dark for 30 min at 37 °C, and the absorbance was measured at λ 517 nm and expressed as percentage of DPPH radical scavenging calculated as follows: DPPH scavenging% = [(Abcontrol -Absample)/Abcontrol] × 100.

The sample concentration providing 50% free radical scavenging activity (FRS50) was calculated from the graph of DPPH scavenging effect percentage against sample concentration.

BHA and BHT were used as positive controls. All the tests were conducted in triplicate. The experimental data were expressed as mean ± standard deviation.

ABTS radical scavenging activity

The ABTS scavenging activity of the 80% MeOH extracts and the obtained fractions was performed spectrophotometrically according to a literature method [START_REF] Re | Antioxidant activity applying an improved ABTS radical cation decolorization assay[END_REF] with a little modification. The ABTS •+ was produced by the reaction between 7 mM ABTS in water and 2.45 mM potassium persulfate, stored in the dark at room temperature for 12 h. Before usage, the ABTS •+ solution was diluted to get an absorbance of 0.70 ± 0.02 at 734 nm with methanol.

Then 40 μL of either standard or sample solutions was added to 160 μL of ABTS •+ solution in microplate). After 10 min, the percentage inhibition at λ 734 nm was calculated for each concentration relative to a blank absorbance (methanol). The scavenging capability of ABTS •+ was calculated using the following equation: ABTS

•+ scavenging% = [(Abcontrol - Absample)/Abcontrol] × 100.
The sample concentration providing 50% cation radical scavenging activity (CRS50) was calculated from the graph of ABTS •+ scavenging effect percentage against sample concentration. BHA and BHT were used as positive controls. All the tests were conducted in triplicate. The experimental data were expressed as mean ± standard deviation.

Power cupric ion reducing (CUPRAC)

80% MeOH extracts and fractions were tested for their power cupric ion reducing (CUPRAC) capacity assay using the method of Apak et al. (2004) with some modifications. Sixty microliters of ammonium acetate buffer solution (1 M, pH = 7.0) were added to 40 μL of a solution of copper (II) chloride (10 mM) and 50 μL of the neocuprine solution (7.5 mM).

Different concentrations of extracts, fractions and standards were added to the initial mixture to make a final volume of 200 μL. After 1 h, the absorbance at 450 nm was recorded against a reagent blank by using a 96-well microplate reader. The power cupric ion reducing was calculated as follows: [1 -A0/ (A1 -A2)] x 100, where A0 is absorbance of the control (without sample), A1 is absorbance in the presence of the sample and A2 is absorbance of the blank. All the tests were conducted in triplicate and the reduction capacity of the extracts was compared with BHA and BHT standards. Fraction AIV 183.9 ± 0.9 62.2 ±2.7 46.9 ± 0.6 41.7 ± 0.9 40.9 ± 0.2 BHT a 15.9 ± 0.1 2.7±0.2 5.4±0.3 BHA a 8.7 ±0.1 1.8 ± 0.1 3.1 ±0.1

Fig. 1 .

 1 Fig. 1. Structures of undescribed bis-iridoids 1-6 isolated from S. semipapposa

Fig. 2 .

 2 Fig. 2. Selected HMBC and NOESY correlations of compound 1

Table 1

 1 13 C NMR and 1 H NMR spectroscopic data of compounds 1-4 in CD3OD. a a in ppm, J in parentheses in Hz.

	Position	1		2		3		4
		δCδH		δCδH		δC	δH	δC	δH
	Unit A
	1a	97.7	5.58, d (6.6)	97.7	5.56, d (6.5)	97.7	5.59, d (6.5)	97.7	5.56, d (6.6)
	3a	153.9	7.52, s	153.7	7.49, d (1.3)	153.7	7.52, (s l)	153.7	7.49, d (2.8)
	4a	111.6	-	111.6	-	111.6	-	111.6	-
	5a	31.3	2.90, q (6.3)	31.2	2.87, q (6.4)	31.3	2.90, q (6.3)	31.2	2.87, q (6.3)
	6a	30.1	1.86, td (13.8, 7.4)	30.1	1.81,dq (13.8, 7.2)	30.1	1.85, dq (13.7, 7.1)	30.2	1.82, dq (13.8, 6.7)
			2.12, dd (13.8,7.2)		2.02, m		2.04,m		2.01, m
	7a	64.1	4.11, m	64.1	4.08, m	64.1	4.11, m	64.1	4.08, m
			4.14, m		4.12, m		4.14, m		4.12, m
	8a	135.7	5.80, ddd (17.4, 10.4, 8.6)	135.7	5.78, ddd (14.4-10.4-8.9)	135.7	5.80, q (8.5)	135.7	5.78, dd (9.8, 8.8)
	9a	45.4	2.70, dt (8.3, 6.1)	45.3	2.68, dd (8.2-6.1)	45.3	2.69, dt (8.2, 5.8)	45.3	2.67, dd (17.2, 10.4, 6.3)
	10a	119.6	5.28, dd (10.4-1.5)	119.6	5.26, d (10.4)	119.6	5.28, d (10.7)	119.6	5.25, d (10.4)
			5.33, d (17.4)		5.31, d (14.4)		5.33, dd (14.7, 1.4)		5.32, d (17.2)
	11aC=O	168.2	-	168.1	-	168.3	-	168.2	-
	12aC=O	173.0	-	173.0	-	173.0	-	173.0	-
	13aCH3	20.9	2.04, s	21.0	2.02, s	20.9	2.04, s	21.0	2.18, s
	1a-O-glc					1A-		1A-
						O-glc		O-glc
	1ʹ	100.1	4.74, d (7.9)	100.6	4.68, d (8.0)	100.1	4.74, d (7.9)	100.1	4.71, d (8.0)
	2ʹ	74.7	3.23, dd (9.0, 7.9)	74.6	3.22, dd (9.0, 8.0)	74.8	3.22, t (8.7)	74.7	3.22, dd (8.8, 8.0)
	3ʹ	78.0	3.40, t (9.0)	78.0	3.39, t (9.0)	78.0	3.39, t (9.1)	78.0	3.39, t (8.8)
	4ʹ	71.6	3.30, t (9.2)	71.5	3.29, t (9.5)	71.6	3.29, t (9.2)	71.6	3.29, t (9.6)
	5ʹ	78.4	3.33, m	78.4	3.34, m	78.5	3.33, m	78.4	3.35, m
	6ʹ	62.8	3.69, dd (11.9, 6.0)	62.8	3.68, dd (11.9, 6.2)	62.8	3.68, dd (11.9, 4.6)	62.1	3.68, dd (12.0, 6.1)
			3.93, dd (11.9, 2.0)		3.92, dd (11.9, 1.9)		3.93, dd (11.9, 1.8)		3.92, d (12.0, 1.3)
	Unit B
	1b	97.6	5.33, d (4.6)	97.3	5.08, d (5.7)	97.6	5.34, d (4.8)	98.0	5.09, d (5.5)
	3b	152.5	7.47,d (1.3)	152.8	7.44, d (1.1)	152.5	7.45, d (1.2)	152.6	7.44, d (1.3)
	4b	113.3	-	112.9	-	113.3	-	112.9	-
	5b	32.6	3.15 q (7.9)	33.1	3.09, q (8.4)	32.6	3.15, q (7.9)	32.7	3.10, q (8.0)
	6b	40.3	1.78, ddd (14.1, 7.8, 5.1)	40.6	1.61, ddd (14.4, 8.9 ,4.8)	40.3	1.78, ddd (14.5, 7.7, 5.0)	40.5	1.61, ddd, (11.3, 9.0, 5.0)
			2.33, ddd (14.1, 7.7, 1.5)		2.26, dd (14.4, 7.7)		2.33, td (14.5, 7.7)		2.27, m
	7b	78.4	5.24, t (4.5)	78.2	5.22, t (4.7)	78.4	5.24, t (4.5)	78.4	5.23, t (4.6)
	8b	41.0	2.18, m	41.2	2.12, m	41.1	2.18, m	41.1	2.09, m
	9b	47.1	2.13, ddd (17.4, 8.8, 4.8)	46.9	2.02, m	47.1	2.12, ddd (13.8, 8.9, 5.0)	47.1	2.02, m
	10b	13.8	1.10, d (6.8)	14.1	1.02, d (7.1)	13.8	1.10, d (6.7)	13.9	1.02, d (7.1)
	11b-C=O	169.3	-	169.2	-	169.3	-	169.3	-
	12b-OCH3	51.7	3.71, s			51.8	3.72, s
	1b-O-glc					1B-O-glc
	1ʹʹ	100.2	4.76, d (7.9)	100.1	4.69, d (7.9)	100.2	4.77, d (7.9)	100.6	4.66, d (7.8)
	2ʹʹ	74.8	3.34, dd (9.3, 7.9)	74.6	3.25, dd (9.0, 7.9)	74.8	3.33, m	74.7	3.23, dd (9.5, 7.8)
	3ʹʹ	75.7	3.68, t (9.2)	77.8	3.42, t (9.0)	75.7	3.68, t (9.1)	77.8	3.40, t (8.9)
	4ʹʹ	72.4	4.87, t (9.5)	71.7	3.39, t 9.0)	72.0	4.86, t (9.5)	71.7	3.30, t (9.0)
	5ʹʹ	76.5	3.60, m	75.7	3.59, m	76.5	3.59, m	75.6	3.55, m
	6ʹʹ	62.5	3.60, m	64.0	4.45, dd (11.9, 6.4)	62.5	3.57, m	64.1	4.37, dd (12.0, 6.5)
			3.66, m		4.54, dd (11.9, 2.1)		3.66, m		4.50, dd (12.0, 2.1)
	4ʹʹ

-O-E-P-coumaroyl 6ʹʹ-O-E-P-coumaroyl 4ʹʹ-O-Z-P-coumaroyl 6ʹʹ-O-Z-P-coumaroyl

	1ʹʹʹ	127.1	-	127.0	-	127.5	-	127.5	-
	2ʹʹʹ	131.3	7.49, d (8.6)	131.3	7.48, d (8.5)	134.0	7.72, d (8.6)	133.9	7.68, d (8.7)
	3ʹʹʹ	116.9	6.83, d (8.6)	116.9	6.82, d (8.5)	115.8	6.77, d (8.6)	115.9	6.78, d (8.7)
	4ʹʹʹ	161.4	-	161.4	-	160.3	-	160.5	-
	5ʹʹʹ	116.9	6.83, d (8.6)	116.9	6.82, d (8.5)	115.8	6.77, d (8.6)	115.9	6.78, d (8.7)
	6ʹʹʹ	131.3	7.49, d (8.6)	131.3	7.48, d (8.5)	134.0	7.72, d (8.6)	133.9	7.68, d (8.7)
	7ʹʹʹ	147.3	7.69, d (15.9)	146.9	7.67, d (15.9)	146.2	6.94, d (12.8)	145.5	6.90, d (12.9)
	8ʹʹʹ	114.7	6.39, d (15.9)	114.9	6.37, d (15.9)	115.9	5.83, d (12.8)	116.2	5.80, d (12.9)
	9ʹʹʹ	168.5	-	168.9	-	167.3	-	170.8	-

Table 2

 2 13 C NMR and 1 H NMR spectroscopic data of compounds 5-6 in CD3OD. a a in ppm, J in parentheses in Hz.

	Position	5		6
		δCδH		δCδH
	Unit A
	1a	97.7	5.57, d (6.5)	97.7	5.56, d (6.5)
	3a	153.4	7.47 s	153.4	7.48, s
	4a	112.0	-	112.0	-
	5a	31.0	2.84, q (6.6)	31.0	2.84, q (6.4)
	6a	33.1	1.73, ddd (13.7-7.7-6.2)	33.1	1.72, m
			1.88 m		1.86, m
	7a	61.2	3.55, m	61.2	3.55, m
			3.60, m		3.60, m
	8a	135.9	5.80, ddd (17.4-10.4-8.7)	135.9	5.80, ddd (17.3, 10.7 ,8.7)
	9a	45.4	2.65, dt (8.2, 6.1)	45.4	2.65, dt (8.1-6.0)
	10a	119.3	5.26, dd (10.4-1.2)	119.3	5.25, d (10.7-1.3)
			5.31, dd (17.4-1.2)		5.30, d (17.3)
	11-C=O	168.7	-	168.7	-
	1A-O-glc
	1ʹ	100.1	4.71, d (7.9)	100.1	4.71, d (7.9)
	2ʹ	74.6	3.21, dd (9.1-7.9)	74.6	3.20, dd (9.0-7.9)
	3ʹ	78.0	3.38, t (8.8)	78.0	3.38, t (8.8)
	4ʹ	71.5	3.28, dd (9.7-8.7)	71.6	3.28, dd (9.7-8.7)
	5ʹ	78.4	3.32 m	78.4	3.31 m
	6'	62.8	3.68, dd (11.9-6.1)	62.8	3.68, dd (11.9-6.0)
			3.92, dd (11.9-2.0)		3.92, dd (11.9-2.0)
	Unit B
	1b	64.2	3.79, dd (12.1, 4.8)	59.3	3.52, dd (11.9-3.7)
			3.83, dd (12.1, 5.0)		3.99, dd (11.9-1.4)
	3b	102.9	4.42, dd (8.9, 3.3)	99.3	4.67, t (3.5)
	4b	35.4	1.28, ddd (13.3-9.7-8.9)	33.1	1.55, ddd (14.1 ,10.0, 3.9)
			1.84, m		1.68, ddd (14.2-6.2-3.2)
	5b	34.4	2.34, m	31.1	2.51, m
	6b	39.8	1.84, m	39.8	1.74, ddd (13.6-7.6-6.0)
			1.97, ddd (14.6-8.1-2.5)		1.86, m
	7b	79.1	5.29, m	78.7	5.32, m
	8b	39.3	2.13, dq (11.5, 6.3)	39.0	2.28, dq (11.1, 6.6)
	9b	44.8	1.83, m	44.6	1.98, m
	10b	12.8	0.88, d (6.8)	12.9	1.00, d (6.9)
	11b-OCH3	56.0	3.42, s	55.0	3.37, s

Table 3 .

 3 Phenolic, flavonoid contents and antioxidant activity by the DPPH • , ABTS •+ , and CUPRAC assays of extracts and fractions from Scabiosa semipapposa. FRS50, CRS50 and A0.50 values represent the means ± S.D of three parallel measurements

		TPC	TFC	DPPH	ABTS	CUPRAC
	Extracts	(μg GAE/mg)	(μg QE/mg)	FRS50	CRS50	A0.50
				(μg/mL)	(μg/mL)	(µg/mL)
	Roots 80% MeOH extract (R)	109.0±0.5	12.8±1.7	80.0±0.6	49.5 ±1.2	122.2±1.0
	Fraction RI	361.7±1.2	74.6±1.4	4.9 ±0.1	8.9±0.3	10.2±0.1
	Fraction RII	266.8±0.6	37.4±0.1	34.3±0.6	12.7±0.4	29.1±0.4
	Fraction RIII	150.8±1.4	10.5 ±0.5	64.6 ±0.1	23.3 ±0.9	54.8±0.8
	Fraction RIV	158.9±1.6	43.0±0.6	62.1±0.8	36.2±0.6	44.1±1.9
	Fraction RV	32.3 ±0.3	5.7±0.4	315.8±0.7	124.0±0.9	378.5 ±1.4
	Aerial parts 80% MeOH extract (A)	169.6±0.2	19.5±0.0	80.0±0.5	31.1 ±0.7	92.2 ± 1.7
	Fraction AI	190.8±0.9	20.9±1.8	45.1 ± 0.8	27.3±0.7	32.9 ± 1.1
	Fraction AII	243.9±0.8	44.2 ±1.6	46.0 ± 1.8	18.8 ±0.6	35.5 ± 0.5
	Fraction AIII	146.7 ±0.8	57.2 ±1.4	42.1 ± 0.1	23.5 ±1.3	37.1 ±0.8

a Used as a positive control

Acknowledgements

The authors are grateful to the DGRSDT of the Ministry of Higher Education and Scientific Research of Algeria for PRFU Project (B00L01UN250120180001), Biotechnology Research Center (CRBt) (Constantine-Algeria), Conseil Regional Champagne Ardenne, Conseil General de la Marne, Ministry of Higher Education and Research (MESR) in France, and to the PlANET CPER project for financial support.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary material related to this article can be found, in the online version.