Ho Van Ban

Trinh Thi

Thanh Van

Vu Van Chien

Thi Nguyen

Pham Thi Hue

Nguyen Hang

Marc Le Tuan

Chau Litaudon

Pham Van Minh

Van Cuong

Quoc Nguyen

Prof. Ph.D Vuong

Nhiem Nguyen

Tuan L Hang

Marc Nguyen

Minh Chau Litaudon

Van Cuong Van

Nguyen Pham

Tran Quoc

Tuyen Minh

Nguyen

Nguyen Vuong
email: nguyenvuong@imbc.vast.vn

Xuan

Nguyen Thi Hue

Pham Thi Hang

Nguyen Le Tuan

Marc Litaudon

Chau Van Minh

Pham Van Cuong

Xuan Nguyen

Prof. Ph.D Nhiem
email: nxnhiem@yahoo.com

Hoang Quoc

Flavone C-glycosides from the leaves of Amesiodendron chinense

Keywords: Amesiodendron chinense, Sapindaceae, amesiflavone A, amesiflavone B, amesiflavone C, cytotoxic Taxonomy Flavone, Natural Product Biochemistry Amesiodendron chinense, Sapindaceae, amesiflavone A, amesiflavone B, amesiflavone C, cytotoxic Amesiodendron chinense, Sapindaceae, amesiflavone A, amesiflavone B, amesiflavone C, cytotoxic

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers,

Graphical abstract Flavone C-glycosides from the leaves of Amesiodendron chinense

Ho Van Ban, Trinh Thi Thanh Van, Vu Van Chien, Nguyen Thi Hue, Pham Thi Hang, Nguyen Le Tuan, Marc Litaudon, Chau Van Minh, Pham Van Cuong, Nguyen Quoc Vuong, Nguyen Xuan Nhiem

Introduction

The genus Amesiodendron Hu (Sapindaceae Juss.) includes only three species (A. integrifoliolatum, A. tienlinensis, and A. chinense), is distributed in China and South-east Asia. Amesiodendron chinense (Merr.) Hu was listed in 2006 IUCN Red List of threatened species. The chemical and biological investigation of this plant has not been studied yet. As part of our research on anticancer reagent candidates from over than 2,500 Vietnamese plants, the ethyl acetate extract of A. chinense was found to inhibit the growth of KB human cancer cell line with IC 50 value of of 20 μg/mL. Herein, we report the isolation and structure elucidation of three new flavone C-glycosides together with four known compounds from A. chinense and their cytotoxic effects.

Results and discussion

Compound 1 was obtained as a yellow amorphous powder. The HR-ESI-MS analysis revealed the molecular formula to be C 28 H 32 O 13 with a cluster ion peak at m/z 577.1910 [M + H] + (Calcd. for [C 28 H 33 O 13 ] + , 577.1916). The 1 H-NMR spectrum of 1 revealed signals of four aromatic protons of B ring at δ H 7.10 (2H, d, J = 9.0 Hz) and 8.00 (2H, d, J = 9.0), two singlet proton of A and C rings at δ H 6.74 and 7.06 (each 1H, s) assigned to a flavone aglycone, two anomeric protons at δ H 4.96 (d, J = 7.5 Hz) and 5.54 (dd, J = 3.0, 12.5 Hz) assigned to two monosaccharide moieties. The 13 C-NMR and HSQC spectra of 1 showed signals of one carbonyl carbons at δ C 184.2, seven non-protonated carbons at δ C 107. 0, 114.3, 124.3, 158.5, 160.1, 164.6, and 166.3, fifteen methines at δ C 67. 1, 69.5, 71.1, 71.6, 72.3, 75.0, 77.1, 78.7, 96.3, 103.7, 104.8, 115.7×2, and 129.5×2, one methylene at δ C 62.7, and two methyl carbons at δ C 17.5 and 56.1. Analysis of 1 H-and 13 C-NMR spectra indicated the structure of 1 was similar to those of apigenin 6-C-β-boivinopyranosyl-7-O-β-glucopyranoside [START_REF] Zhu | A new C-glycosylflavone from Pogonatherum crinitum[END_REF] except for the addition of a methoxy group at C-4′. The position of methoxy group at C-4′ was confirmed by HMBC correlations from H-2′/H-6′ (δ H 8.00), H-3′/H-5′ (δ H 7.10), and methoxy group (δ H 3.90) to C-4′ (δ C 164.6). The HMBC correlations between H-3 (δ H 6.74) and C-1′ The analysis NMR spectra of 2 indicated the flavone aglycone was identical to those of 1. The NMR features for the first sugar moiety of 2 allowed to identify to be 6-deoxyhexopyranosyl moiety. Furthermore, this was confirmed by the COSY correlations of Gul H-1′′ (δ H 5.41)/gul

H-2′′ (δ H 4.45)/gul H-3′′ (δ H 4.26)/gul H-4′′ (δ H 3.69)/gul H-5′′ (δ H 4.22)/gul H-6′′ (δ H 1.29).
The 6-deoxyhexopyranosyl at C-6 was proved by HMBC correlations between Gul H-1′′ (δ H 5.41) and C-5 (δ C 161.1)/C-6 (δ C 110.1)/C-7 (δ C 164.9). The axial orientation of Gul H-1″, Gul H-2″, and Gul H-5″ and equatorial orientation of Gul H-3″ and Gul H-4″ were confirmed by the large coupling constant between Gul H-1″ and Gul H-2″ (J = 9.8 Hz) and small coupling constants between Gul H-2″ and Gul H-3″ (J = 3.0 Hz) and between Gul H-3″ and Gul H-4″ (J = 3.5 Hz) as well as NOESY correlations between Gul H-1″ and Gul H-5″. Based on above evidence, the C-glycoside was proved as C-β-6-deoxygulopyranoside [START_REF] Carnat | Violarvensin, a new flavone di-C-glycoside from Viola arvensis[END_REF]. Acid hydrolysis of 2 gave L-rhamnose (identified as TMS derivative by a gas chromatography method). The position of α-L-rhamnopyranosyl at Gul C-2′′ was confirmed by HMBC correlations from Rha H-1′′′ (δ The known compounds, (+)-aptosimon (4) [START_REF] Yamauchi | Synthesis of (+)-aptosimon, a 4-oxofurofuran lignan, by erythro selective aldol condensation and stereoconvergent cyclization as the key reactions[END_REF], (+)isolariciresinol (5) [START_REF] Jutiviboonsuk | Bioactive constituents from roots of Bursera tonkinensis[END_REF], cleomiscosin A (6) [START_REF] Yang | Coumarinolignans isolated from the seeds of Brucea javanica[END_REF], and cleomiscosin C (7) [START_REF] Tanaka | Total synthesis of coumarinolignans, aquillochin (cleomiscosin C) and cleomiscosin D[END_REF] were identified by comparison of their NMR data with those reported in the literature.

All compounds were screened for cytotoxic activity against five human cancer cell lines, KB (human epidemoid carcinoma), SK-LU-1 (human lung cancer), MCF-7 (human breast cancer), HepG2 (human hepatocyte carcinoma), and SW-480 (Human colon adenocarcinoma) at the concentration of 200 µM. Compounds 3-5 did not show cytotoxic activity (% cell survival >50%). Thus, compounds 1, 2, 4, 6, and 7 were furthered evaluated at the smaller concentrations, 100, 10, 1.0 µM. As the results, compounds 1, 2, 4, 6, and 7 showed weak activity on tested human cancer cell lines with IC 50 ranging from 71.0 to 146.0 µM.

Experimental

General

All NMR spectra were recorded on a Bruker 500 MHz. HR-ESI-MS spectra were obtained using an AGILENT 6550 iFunnel Q-TOF LC/MS system. Column chromatography (CC) was performed on silica-gel (Kieselgel 60, 70-230 mesh and 230-400 mesh, Merck) or RP-18 resins (30 -50 μm, Fuji Silysia Chemical Ltd.). For thin layer chromatography (TLC), precoated silica-gel 60 F254 (0.25 mm, Merck) and RP-18 F254S (0.25 mm, Merck) plates were used.

Plant material

The leaves of Amesiodendron chinense (Sapindaceae) were collected at Sontra, Danang, Vietnam in June 2018 and identified by Dr. Do Van Hai, Institute of Ecology and Biological Resources, VAST. A voucher specimen (PTH15032018) was deposited at the Institute of Marine Biochemistry, VAST.

Extraction and isolation

Dried and powdered leaves (7.0 kg) of A. chinense were extracted with MeOH (25 L) at room temperature for three times (each time, three days) and removed the solvent in vacuo to yield MeOH extract (250 g). The MeOH extract was suspended with H 2 O (2 L) and successively partitioned with n-hexane, and ethyl acetate (EtOAc) to yield n-hexane (ACH, 70 g), EtOAc (ACE, 50 g) residues, and water layer (ACW).

ACE layer was applied on a silica gel column eluting with CH 2 Cl 2 /EtOAc (10/1, v/v) to give ACE1 (6 g), ACE2 (8 g), ACE3 (13 g), ACE4 (11 g), and ACE5 (7 g). The ACE1 fraction was chromatographed on a silica gel column eluting with CH 2 Cl 2 /EtOAc (4/1, v/v) to yield compound 4 (30 mg). The ACE2 fraction was applied on a silica gel column eluting with nhexane/EtOAc (1/4, v/v) to give two smaller fractions, ACE2.1 (2.5 g) and ACE2.2 (1.5 g). The ACE2.1 fraction was chromatographed on a silica gel column with solvent eluent of nhexane/EtOAc (1/4, v/v) then purified on a sephadex LH-20 column to give 5 (7.0 mg). The ACE2.2 fraction was subjected on an RP-18 column with solvent eluents of MeOH/H 2 O (1/1, v/v) then purified by a sephadex LH-20 column eluting with MeOH to yield compound 1 (12.0 mg). ACE3 fraction was applied on a silica gel column eluting with CH 2 Cl 2 /EtOAc (4/1, v/v) then purified on an RP-18 column eluting with MeOH/H 2 O (1/1, v/v) to yield 2 (8.0 mg). The ACE4 fraction was chromatographed on a silica gel column eluting with CH 2 Cl 2 /EtOAc (1/1, v/v) then on an RP-18 column eluting with MeOH/H 2 O (1/1, v/v) to yield compound 3 (50 mg). The ACE5 fraction was applied on a silica gel column eluting with EtOAc/acetone (4/1, v/v) to give two smaller fractions, ACE5.1 (1.7 g) and ACE5.2 (2 g). The ACE5.1 fraction was chromatographed on an RP-18 column eluting with MeOH/H 2 O (1/1, v/v) and then purified on a sephadex LH-20 column eluting with MeOH to give compound 6 (9.0 mg). The ACE5.2 fraction was applied on a silica column eluting with CH 2 Cl 2 /acetone (6/1 v/v) and then purified on a sephadex LH-20 column to give compound 7 (10 mg).

Amesiflavone A (1)

Yellow amorphous powder; [α] D 25 =-76.0 (c 0.1, MeOH); UV λ max (MeOH, nm) -1 : 3413, 3306, 2940, 1731, 1650, 1623, 1605, 1261, 1136, 1102, 1056, 1027; 1 H-NMR (DMSO-d 6 , 500 MHz) and 13 C-NMR (DMSO-d 6 , 125 MHz): see Table 1.

Acid hydrolysis

Each compound (1 and 3, 2.0 mg) was separately dissolved in 1.0 N HCl (dioxaneH 2 O, 1:1, v/v, 1.0 mL) and heated to 80 °C in a water bath for 3 h. The solution was thoroughly driven out under N 2 overnight then partitioned in water : CHCl 3 (1:1), the aqueous layer was concentrated to dryness using N 2 . The residue was dissolved in 0.1 mL of dry pyridine, followed by addition of L-cysteine methyl ester hydrochloride in pyridine (0.06 M, 0.1 mL).

The reaction mixture was heated at 60 °C for 2 h and then added trimethylsilylimidazole solution (0.1 mL) at 60 °C for 1.5 h. The dried product was partitioned with n-hexane and water (0.1 mL each), and the organic layer was analyzed by GC: DB-5 column (0.32 mm ID × 30 m length), detector FID, column temp 210 °C, injector temp 270 °C, detector temp 300 °C, and carrier gas He (2 mL/min). Under these conditions, the standard sugars gave peaks at t R (min) 14.11 and 14.26 for D-and L-glucose, and 4.50 for L-rhamnose, respectively. Peaks at t R (min) 14.11 of D-glucose for 1 and 4.50 of L-rhamnose for 3 were observed.

Cytotoxic assays: See reference (Thu et al. 2015)

Disclosure statement

No potential conflict of interest was reported by the authors.

Table 1. 1 H-and 13 C-NMR spectroscopic data for compounds 1 -3. The concentration that inhibits 50% of cell growth was calculated (IC 50 ). Compounds were tested at a maximum concentration of 200 μM. Data are means of three experiments. b) Ellipticine was used as a postive control.

C 1 2 3 δ C a) δ H a) (mult., J in Hz) δ C a) δ H a) (mult., J in Hz) δ C b) δ H b) (mult., J in

  (δ C 124.3)/C-2 (δ C 166.3)/C-4 (δ C 184.2)/C-10 (δ C 107.0) and between H-8 (δ H 7.06) and C-6 (δ C 114.3)/C-7 (δ C 164.8)/C-9 (δ C 158.5)/C-10 (δ C 107.0) confirmed the two protons at C-3 and C-8. Acid hydrolysis of 1 revealed D-glucose (identified as trimethylsilyl (TMS) derivative by a gas chromatography method). In addition, the 13 C-NMR spectral data of 1 showed the presence of one O-β-D-glucose and one C-β-boivinose unit (Zhu et al. 2010). The HMBC correlations from Boi H-1′′ (δ H 5.54) to C-5 (δ C 160.1)/C-6 (δ C 114.3)/C-7 (δ C 164.8) of the aglycone and from Glc H-1′′′ (δ H 4.96) to C-7 (δ C 164.8) (Figure 2) indicated the position of C-β-D-boivinopyranosyl and O-β-D-glucopyranosyl moieties at C-6 and C-7, respectively. Consequently, the structure of 1 was determined to be acacetin-6-C-β-Dboivinopyranosyl-7-O-β-D-glucopyranoside and named as amesiflavone A. Compound 2 was also obtained as a yellow amorphous powder and its molecular formula was determined to be C 28 H 32 O 13 by HR-ESI-MS at m/z 577.1915 [M + H] + (Calcd. for [C 28 H 33 O 13 ] + , 577.1916). The 1 H-NMR spectrum of 2 revealed C-6-substituted of apigenin protons: δ H 7.09 (2H, d, J= 7.5 Hz) and 7.93 (2H, d, J= 7.5 Hz), 6.54 (1H, s), and 6.63 (1H, s).

  H 4.82) to Gul C-2′′ (δ C 70.8). Consequently, the new structure of 2 was elucidated as acacetin-6-C-(2ʺ-α-L-rhamnopyranosyl)-β-6deoxygulopyranoside and named as amesiflavone B. Amesiflavone C (3) possessed a molecular formula of C 28 H 30 O 13 as deduced from HR-ESI-MS at m/z 575.1761 [M + H] + (Calcd. for [C 28 H 31 O 13 ] + , 575.1759). The 1 H-NMR spectrum of compound 3 showed the presence of 6-C-substituted acacetin aglycone with the typical proton signals at δ H 8.02 (2H, d, J= 9.0 Hz), 7.12 (2H, d, J= 9.0 Hz,), 6.56 (1H, s), 6.80 (1H, s), and 3.87 (3H, s). Two anomeric protons at δ H 4.88 (d, J = 10.0 Hz) and 4.69 (br s) together with two secondary methyl group at 0.74 (3H, look like s) and 1.33 (d, J = 6.0 Hz), suggested the presence of two monosaccharide moieties. The 1 H-and 13 C-NMR spectral data of 3 were almost the same to those of cassiaoccidentalins A (Hatano et al. 1999) except for the addition of methoxy group at C-4′. The position of a methoxy group at C-4′ was also confirmed by HMBC correlation from methoxy group (δ H 3.87) to C-4′ (δ C 162.2). The signals in 1 H-and 13 C-NMR spectra of unexpectedly separated (some cases showed the broaden signals) at normal temperature (25 o C) but duplication collapsed at high temperature (60 o C) (See Supporting information), which indicated the existence of rotational isomers, caused by the restricted rotation around the C (sp 3 )-C (sp 2 ) bond between C-glucosyl and flavone (Zhou et al. 2019). The HMBC correlations between Rib H-1″ (δ H 4.88) and Rib C-2″ (δ C 75.4)/rib C-3″ (δ C 205.7)/rib C-5″ (δ C 77.8) and between Rib H-6″ (δ H 1.33) and Rib C-4″ (δ C 77.2)/Rib C-5″ (δ C 77.8) and also COSY correlations of Rib H-1″/H-2″; H-4″/H-5″/H-6″ confirmed the presence of 6-deoxy-ribo-hexos-3-ulopyranosyl moiety. The HMBC correlations between Rha H-1‴ (δ H 4.69) and Rib C-2″ (δ C 75.4); Rib H-1″ (δ H 4.88) and C-5 (δ C 161.5)/C-6 (δ C 107.4)/C-7 (δ C 161.8) determined the sugar linkages as 6-C-(2ʺ-α-L-rhamnopyranosyl)-β-6deoxy-ribo-hexos-3-ulopyranosyl. Thus, the structure of 3 was elucidated and named as amesiflavone C.

Figure 1 .Figure 2 .Figure S5 .Figure S6 .Figure S7 .Figure S8 .Figure S9 .FigureFigureFigure S8 .Figure S9 .Figure

 12S5S6S7S8S9S8S9 Figure 1. Chemical structures of compounds 17.

FigureFigure S25 .Figure S26 .S14Figure S27 .

 S25S26S27 Figure S20. 1 H-NMR spectrum of compound 3 at 30 o C

Table 2 .

 2 Cytotoxic effects of compounds 1

-7 IC 50 (µM) a) Compound

		KB	SK-LU-1	MCF7	HepG2	SW480
	1	89.3±6.6 118.2±9.4 95.6±9.3 71.0±6.2 102.8±8.9
	2	94.6 ± 5.0 132.2± 9.1 138.1±8.6 104.5±7.4 109.4±2.0
	3	>200	>200	>200	>200	>200
	4	88.5 ±8.4 143.5±10.7 140.1±6.3 120.1±6.9 99.0±6.2
	5	>200	>200	>200	>200	>200
	6	118.0±10.0 141.3±14.8 146.0±8.8 96.8±12.7 113.0±11.9
	7	92.6±6.9 139.6±13.5 153.9±13.7 127.9±8.9 124.7±13.8
	Ellipticine b) 1.6±0.2	1.8±0.1	2.0±0.2	1.8±0.2	1.5±0.2

a)

Funding

This work was supported by Vietnam Academy of Science and Technology under grant code VAST 04.04/18-19. The authors would like to thank NATPROCHEMLAB for the evaluation of cytotoxic activity.

Ho Van Ban, Trinh Thi Thanh Van, Vu Van Chien, Nguyen Thi Hue, Pham Thi Hang, Nguyen Le Tuan, Marc Litaudon, Chau Van Minh, Pham Van


Tran Nguyen

Bich Vu Le

Ngan Truong

Thi Phuong Le

Marc Litaudon

Tran Dang

Thach

Minh Van

Huong Chau
email: huongdm@imbc.vast.vn

Thi Doan

Van Cuong Mai

Pham

Thach Dang

Tran

Chau

Huong Doan

Thi Mai

Van Cuong Pham

Prof Assoc Huong Doan

Prof Assoc Van

Cuong Pham
email: phamvc@imbc.vast.vn

Cytotoxic phenolic compounds isolated from the fruits of Macaranga denticulata

Keywords: Macaranga denticulata, Euphorbiaceae, prenylated flavonoids

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Macaranga is a large genus of the Euphorbiaceae family, consisting in more than 300 species found mainly in the southern part of Asia [1,2]. In Vietnam, this genus comprises 13 different species. Macaranga denticulata Muell. Arg. (Euphorbiaceae) is a small to medium-sized tree. The roots were used in traditional medicine for treating icteric hepatitis [START_REF] Fan | Chemical Constituents of the roots of Macaranga denticulata[END_REF]. The leaves or stem water decoctions can be used traditionally for washing wounds and drunk as tonic by women after childbirth [START_REF] Pham | The Flora of Vietnam[END_REF][START_REF] Vo | Vietnamese Medicinal Plants Dictionary[END_REF]. [START_REF] Phupattanapong | Thai Medicinal Plants, Chutima[END_REF]Vo 2012) Previous studies on this species have resulted in the isolation of prenylated flavonoids from the leaves [START_REF] Sutthivaiyakit | [END_REF], stilbenediterpene heterodimer and prenylated flavonoids from the frond [6], terpenoids from the root [7] and C-methylated and isoprenylated chalcones from the twigs and leaves [8][9][10]. As a part of our study in the search for new anticancer compounds from Vietnamese medicinal plants, the ethyl acetate extract of the fruits of M. denticulata was found to inhibit the growth of KB cancer cell line. In this paper, we report the isolation and structural elucidation of a new modified geranyl-type side chain substituted flavonoid, 6-(8″-hydroxy-3″,8″-trimethyl-oct-2″-enyl)-kaempferol (1) from the fruits of M. denticulata along with 11 prenylated flavonoids (2)[START_REF] Pham | The Flora of Vietnam[END_REF][START_REF] Vo | Vietnamese Medicinal Plants Dictionary[END_REF][START_REF] Sutthivaiyakit | [END_REF](6)(7)(8)(9)(10)(11)(12), a prenylated stilbene [START_REF] Yoder | Isolation and structure elucidation of cytotoxic natural products from the rainforests of Madagascar and Suriname[END_REF], two diterpenoids (14 and 15), a sesquiterpenoid (16), and two triterpenoids (17 and 18) from the fruits of Macaranga denticulata Muell. Arg. (Euphorbiaceae). The cytotoxic activity of isolated compounds was also discussed.

Results and discussion

Compound 1 was isolated as a yellow powder and its molecular formula of C25H28O7 was established by HRESIMS at m/z 439.1779 [M-H] -(calcd. 43.1757), requiring 12 unsaturation degrees. The FT-IR showed absorption bands at nmax 3419, 1650, 1624, 1486 cm -1 indicating the presence of hydroxy, α,β-unsaturated carbonyl and aromatic ring functionalities, respectively. The UV absorption maximum at 349 and 257 nm was typical for a flavonol skeleton [9]. The presence of a substituted flavonol skeleton (more precisely substituted kaempferol) was suggeted by 1 H-NMR and 13 C-NMR (table 1). In the 1 H NMR spectrum, the presence of an A2B2 system at δH 6.93 (2H, d, J = 9.0 Hz), 8.09 (2H, d, J = 9.0 Hz) and a singlet proton at δH 6.44 was observed at the aromatic region. Additionally, the appearance of 3 methyl groups at dH 1.16 (2 x CH3) and 1.80 (CH3), a benzylic methylene proton at dH 3.35, three methylene protons at dH 1.49, 1.80, 2.00, and a vinyl proton at dH 5.28, in the 1 H NMR spectrum suggested the presence of a 8″-hydroxy-3″,8″-trimethyl-oct-2″-enyl moiety [11]. The analysis of 13 with C-6 (δC 112.3) determined the linkage of the modified geranyl-type side chain at C-6 of ring A. The double bond in the side chain for 1 was assigned as E, based on the 1 H NMR and 13 C-NMR chemical shifts of the vinylic methyl group in 8″-hydroxy-3″,8″-trimethyl-oct-2″-enyl [11,12]. Thus, the structure of new compound 1 was identified as 6-(8″-hydroxy-3″,8″-trimethyl-oct-2″-enyl)-kaempferol and named 3′dehydroxy-solophenol C (figure 1).

The known compounds were identified as denticulatain D (2) [9], bonannione A (3) [START_REF] Yoder | Isolation and structure elucidation of cytotoxic natural products from the rainforests of Madagascar and Suriname[END_REF]14], diplacol (4) [14], macarangin (5) [START_REF] Sutthivaiyakit | [END_REF]15], 3-O-methylmacarangin (6) [START_REF] Sutthivaiyakit | [END_REF]15], 5,7,3',4'-tetrahydroxy-3-methoxy-6-geranylflavone (7) [9], 3,5,7,3',4'pentahydroxy-6-geranylflavonol (8) [9], macarhizinoidin A (9) [16], robipseudin A 10) [17], denticulatain E (11) [9], 8-dimethylallylisosakuranetin (12) [18], 4'deprenylmappain (13) [19], deheiculatin H ( 14) [20], poilaneic acid (15) [21], derivative phaseic acid ( 16) [22], taraxerone (17) [23,24], epitaraxerol ( 18 Since prenylated flavonoid and prenylated stilbenes from Macaranga genus are reported to have potent cytotoxic activities [26,27], the cytotoxic activities of compound 1-13 were evaluated against seven cancer cell lines: MCF-7 (human breast cancer cells), HepG-2 and Hep3B (human liver cancer cells), Lu-1 (human lung adenocarcinoma cells), Hela (human cervical cancer cells), KB (human epithelial carcinoma cells), A549 (human non-small cell lung cancer) by MTT-based colorimetric assay [28,29]. The results showed that compound 1 showed moderate cytotoxic activity against A549 and Hela cell lines with IC50 values of 16.0 and 36.9 µM, respectively. Compound 9 showed the highest cytotoxic activities against KB, HepG2, Lu-1, and MCF7 cell lines with IC50 values of 0.6, 0.8, 1.3, and 1.2 µM, respectively.

Compound 8 showed potent cytotoxic activity against KB cell line with an IC50 value of 1.4 µM. Compound 3 showed selectively cytotoxic activity against the Hep3B cell line with an IC50 value of 3.63 µM. Compound 5 showed a selective cytotoxic activity against the A549 cell line with an IC50 value of 4.5 µM (Table 2).

Experimental

General experimental procedures

Melting points were recorded on a Buchi B-545 instrument (Ontario, Canada). Optical rotations were recorded on a Polax-2L polarimeter in CHCl3 (ATAGO Co. Ltd.,Tokyo, Japan). IR spectra were recorded on a Nicolet Impact 410 FT-IR spectrometer (SpectraLab Scientific Inc., Ontario, Canada), and NMR spectra on a Bruker AM 500 MHz spectrometer (Bruker BioSpin AG, Fällanden, Switzerland) operating at 125.76 MHz for 13 C NMR, and at 500. [START_REF] Yoder | Isolation and structure elucidation of cytotoxic natural products from the rainforests of Madagascar and Suriname[END_REF] MHz for 1 H NMR. 1 H chemical shifts were referenced to CD3OD at δ 3.31, while the 13 C chemical shifts were referenced to the central peak at δ 49.0. The HMBC measurements were optimized to 7.0 Hz long-range couplings.. HR-ESI-MS were recorded on a FT-ICR 910-MS TQFTMS-7 T mass spectrometer (Agilent Technologies, Inc., California, USA).

Plant material

Extraction and isolation

Dry powdered fruits of M. denticulata (3.0 kg) was successively extracted with EtOAc (4 x5 L) and then with methanol (3 x 5 L) at room temperature. The resulting EtOAc and MeOH extracts were evaporated under reduced pressure to give 330 g and 150 g of crude extracts, respectively. The ethyl acetate extract (330 g) of fruits of M. denticulata was subjected to a silica gel column chromatography (CC) and eluted with n-hexane/EtOAc gradient resulting in 20 fractions (F1-F20). Fraction 5 (2.5 g) was washed with n-hexane, the solid residue followed by recrystallization from acetone to yield compound 18 (150 mg) and the soluble crude was purified by CC on silica gel, eluting with n-hexane/acetone (97/3) provided compound 14 (20 mg).

Fraction 7 (3.0 g) was washed with n-hexane, the solid residue obtained was subjected to column chromatography on silica gel, eluted with a mixture nhexane/acetone (97/3) to yield compound 19 (36.0 mg). Fraction F11 (12 g) was separated by silica gel column chromatography, eluted with a mixture of n-hexane/acetone (0 to 100% acetone in n-hexane) to give 12 subfractions. Subfraction 11.7 (0.8 g) was purified by column chromatography over silica gel, eluting with CH2Cl2/acetone (0 to 80% acetone in CH2Cl2) to obtained seven subfractions. Subfraction 11.7.4 (178 mg) was subjected to column chromatography on silica gel, eluted with a mixture CH2Cl2/acetone (95/5) to yield compound 9 (20.0 mg). Subfraction 11.7.6 (400 mg) was purified by column chromatography (CC) over silica gel, eluted with a mixture CH2Cl2/acetone (5 to 20% acetone in CH2Cl2) and then subjected on a Sephadex LH-20 column using a mixture of CH2Cl2/MeOH (1/9) to yield compound 16 (5.0 mg). Subfraction 11.9 (0.5 g) was washed with n-hexane/CH2Cl2 (9/1), the solid residue was recrystallized in EtOH, affording compound 12 (30.0 mg). Subfraction F11.11 (2.76 g) was separated on a silica gel column, eluted with CH2Cl2/EtOAc (5 to 50% EtOAc in CH2Cl2), then re-purified by Sephadex LH-20 column (100% MeOH) to give compound 3 (10.0 mg). Subfraction 11.12 (1.0 g) was purified by column chromatography over silica gel, eluting with CH2Cl2/acetone (0 to 100% actone in CH2Cl2) to obtained five subfractions. Subfraction 11.12.4 (0.3 g) was further separated on a Sephadex column, eluted with MeOH and then purified on a silica gel, eluted with a mixture of CH2Cl2/acetone (5 to 10% acetone in CH2Cl2) to give compounds 5 (15 mg), 6 (5 mg), and 10 (18 mg). Fraction 17 (43 g) was chromatographed on silica gel column, eluting with a mixture of CH2Cl2/MeOH (0 to 80% MeOH in CH2Cl2) to give 13 subfractions. Subfraction F17.9 (2.88 g) was purified on a Sephadex LH-20 column, eluted with MeOH to obtained 7 subfractions F17.9.1-17.9.7. Subfraction F17.9.6 (0.33 g) was then purified on a silica gel, eluted with a mixture of CH2Cl2/MeOH (5 to 50% MeOH in CH2Cl2), followed by crystallization to yield compounds 8 (15 mg) and 13 (10.0 mg). Subfraction F17.10 (3.7 g) was separated by silica gel column chromatography, eluted with a mixture of CH2Cl2/MeOH gradient to give 7 subfractions. Subfraction F17.10.3 (437 mg) was purified by CC on Sephadex LH-20 (CH2Cl2/MeOH: 1/9), followed by CC on silica gel (0 to 70% acetone in CH2Cl2), to provide compounds 4 (13.0 mg), 7 (6.0 mg), and 15 (9.0 mg). Subfraction F17.10.4 was separated on a silica gel column, eluted with a mixture of CH2Cl2/acetone/MeOH (100/2/1) to yield compounds 1 (6.0 mg) and 2 (5.0 mg).

The MeOH extract (150 g) was dissolved in 500 mL of acetone/MeOH (1/1), the soluble part was evaporated under reduced pressure to give 100 g of crude. This crude was subjected to a CC on silica gel (CH2Cl2/EtOH) to give 16 fractions (MF1-MF16).

Fraction MF10 (1.45 g) was purified on a Sephadex LH-20 column (100% MeOH), then purified by CC on silica gel column, eluted with a mixture of CH2Cl2/MeOH (5 to 50% MeOH in CH2Cl2) to yield compound 11 (6.0 mg).

3'-dehydroxy-solophenol C (1)

Yellow powder mp. 160-161ºC; Rf: 0.4 (CH Cl3-acetone-MeOH, 2:0.2:0.

Cytotoxic activity assay

The cytotoxicity assay was carried out in triplicate in a 96-well microtiter plates against 7 cancer cell lines: KB, HepG2, Lu-1, Hela, A549, MCF-7, Hep3B. An MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide] assay was used to measure the viability of cells after extract treatment. Cells were seeded in 96-well plates at a density of 1.0×105 cells/mL. Cells were treated with various concentrations of essential oils (0.3 1, 3, 10, and 30 μg/mL) and then incubated at 37 o C for 24 h in fresh RMPI1640 medium. Cells were subsequently incubated at 37 °C with MTT (0.5 mg/mL) for 4 h.

After removal of supernatant, formazan crystals were dissolved in isopropanol and the optical density was measured at 570 nm. The IC50 value was defined as the concentration of sample necessary to inhibit cell growth to 50% of the control.

Ellipticine and camptothecine was used as a positive control.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

We gratefully acknowledge the International Associated Laboratory (LIA "NATPROCHEMLAB") between the Centre National de la Recherche Scientifique

  C-NMR data of 1 aided by HSQC revealed the presence of 25 carbons, including three methyls, four methylenes, six sp 2 methines, four oxygenated tertiary and six quaternary sp 2 carbons, one sp 3 oxygenated tertiary carbon and one carbonyl carbon. The chemical shifts of carbons at dC 71.5 (C-8″), 137.0 (C-3), 147.7 (C-2), 156.2 (C-9), 159.1 (C-5), 160.4 (C-4′), 163.4 (C-7) suggested their linkage to oxygen. The HMBC correlations of H-6″ (δH 1.49) with C-8″ (δC 71.5) and the correlations of methyl protons at δH 1.16 (CH3-9″ and 10″) with carbon at δC 71.5 (C-8″), 44.2 (C-7″) indicated the location of hydroxy group at C-8″ of geranyl chain. Furthermore, HMBC correlations of H-1″ (δH 3.45) with C-5 (δC 159.1), C-6 (δC 112.3), C-7 (δC 163.4) and which of H-2″ (δH 5.28)

  ) [25] by detailed analyses of their NMR and ESI-MS data and their comparison with reported values. Compounds 9, 12, 13, 14, 15 were isolated from M. denticulata for the first time. Compound 10, 16 were isolated from Macaranga genus for the first time. Macaranga denticulata is phylogenetically very close to Macaranga tanarius. The two species that cohabites in same biotopes are particularly difficult to discriminate in the field even in case of the presence of fruits or flowers. However, from a phytochemical point of view it should be noted that unlike M. tanarius,(ref Peresse et al . JNP 2017, 80, 2684-2691), no prenylated stilbene possessing a hexahydroxanthene moiety could be identified in M. denticulata.

Fruits

  of M. denticulata were collected in Sa Pa, Lao Cai, Viet Nam on July 2015, and identified by Dr. Nguyen The Cuong, Institute of Ecology and Biological Resources, VAST. A voucher specimen (VN-2497) was deposited at the Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology in Hanoi.

(

  CNRS, ICSN, France) and the Vietnam Academy of Science and Technology (Institute of Marine Biochemistry, VAST, Vietnam), which supports the project and The Ministry of Science and Technology of Vietnam for financial support [Grant number ĐTĐLCN.14/16].

Figure 1 .

 1 Figure 1. Structures of compounds 1-18 from M. denticulata.

Table 1 .

 1 1 H and13 C NMR spectral data of compounds 1 ( 1 H: 500 MHz and 13 C: 125

	MHz).
	Position dC	dH (J in Hz)	Position	dC	dH (J in Hz)
	2	147.7		4′	160.4
	3	137.0		1"	22.1	3.35 d (7.0)
	4	177.3		2"	123.7	5.28 t (6.5)
	5	159.1		3"	135.8
	6	112.3		4"	16.1	1.80 s
	7	163.4		5"	41.3	2.0 t (7.5)
	8	93.7	6.44 s	6"	23.6	1.49 m
	9	156.2		7"	44.2	1.40 m
	10	104.4		8"	71.5
	1′	123.9		9"	29.2	1.16 s
	2′,6′	130.6	8.09 d (9.0)	10"	29.2	1.16 s
	3′,5′	116.3	6.93 d (9.0)
	Measured in CD3OD

Table 2 .

 2 Cytotoxic activity of compounds 1-13 against seven cancer cell lines

	Compound

Supplemantal data

HR-ESIMS, 1D-and 2D-NMR spectra of compounds 1 are available as Supporting Information.


Jean-Jacques Bahain

Samer Farkh

Christophe Falguères

Qingfeng Shao

Pierre Voinchet

Bassam Ghaleb

David Herisson

Jean-Luc Locht

Nicole Limondin-Lozouet

Patrick Auguste

Julie Dabkowski

Laurent Deschodt

Pierre Antoine

Figure 1 -Location of the studied sites in Northern France ESR/U-series dating Eemian human occupations of Northern France

Introduction

Palaeolithic sites associated with the Eemian Interglacial (MIS 5e) are very rare in Northern France and their preservation is restricted to very specific geological contexts, in association with carbonated tufa (Caours) or peat deposits (Waziers) (Figure 1).

ESR/U-series dating method was hence applied on teeth recovered from different levels of the two sites, in the aim to evaluate the quality of the results obtained in very clearly chronologically constrained stratigraphic sequences. The results obtained will be displayed and discussed in this poster.

Caours

Material and method

ESR/U-series dating method was applied using the protocol of preparation and analyses described in [START_REF] Bahain | ESR/U-series dating of teeth recovered from well-stratigraphically age-controlled sequences from Northern France[END_REF].

Equivalent doses were determined using single exponential function. The dose rate was estimated from measurements by gamma-ray spectrometry, both in laboratory with a low background, high purity germanium detector and in situ using Canberra Inspector1000 spectrometer. Rn loss from each tissue was determined by combining alpha-ray and gamma-ray data [START_REF] Bahain | ESR dating of tooth enamel: a comparison with K-Ar dating[END_REF].The cosmic dose rate was estimated using the formula of [START_REF] Prescott | Cosmic ray contributions to dose rates for luminescence and ESR dating: large depths and long-term time variations[END_REF].

The following parameters were also used in the age determination:

-a k-value of 0.13 ± 0.02 [START_REF] Grün | An alpha irradiator for ESR dating[END_REF]; -water content of sediments was measured for each dated level by drying it in oven at 40°C during one week; -water contents of 0 wt% in the enamel and 7 wt% in the dentine ; -conversion factors of Adamiec and Aitken (1998); -beta attenuations values in dental tissues were calculated using a Monte Carlo approach [START_REF] Brennan | Beta doses in tooth enamel by ''One Group'' theory and the Rosy ESR dating software[END_REF].

For the Caours samples, the US-ESR model cannot be applied due to high 230 Th/ 234 U ratios higher to unity and relatively low equivalent doses, between 50 and 80 Gy. As a consequence, only the AU model was used to calculate ESR/U-series ages, indicating an initial U-uptake systematically followed by a U-leaching for quite all tissues of the five samples. This work demonstrates also the importance of the in situ dosimetry in the age calculation. Despite all these quite negative points, the obtained ages for N1 teeth are relatively homogeneous, leading to a mean age for level 1 of 124 ± 10 ka (±2σ), similar to the age obtained previously by [START_REF] Bahain | ESR/U-series dating of teeth recovered from well-stratigraphically age-controlled sequences from Northern France[END_REF]. The tooth analysed from level N3 provides an AU age of 129 ± 23 ka. Given these very homogeneous ages for the two dated levels, the attribution of the deposit to the Eemian interglacial is well confirmed with a mean age of 125 ± 9 ka.

In the Waziers teeth, we observe a very specific geochemical behavior for all the samples, with very low uranium contents and often higher in the enamels than in the dentines. Consequently the internal dose has a restricted impact in the age determination (<10%) compared to the external dose. The equivalent doses are low but very homogeneous for the N2 teeth, , leading to a mean age for this layer of 128 ± 20 ka (±2σ), while the DE value obtained for the N6 leads to a US age of 132 ± 8 ka Given these very homogeneous ages for the two dated levels, a weighted mean age of 129 ± 11 ka can be calculated from these four samples, confirming the attribution of the deposit to the Eemian interglacial and even, in agreement with the

Results

The ESR/U-series results are shown in Table 1. Several interesting points can be highlighted in these data. [START_REF] Bahain | ESR/U-series dating of teeth recovered from well-stratigraphically age-controlled sequences from Northern France[END_REF].

Meadow rhinoceros (Stephanorhinus cf hemitoechus)

Brown bear (Ursus arctos)

Red deer (Cervus elaphus)

Roe deer (Capreolus capreolus)

Site presentation and sampling

Since 2003 for Caours and 2011 for Wasiers, these two sites are studied by multidisciplinary teams including geologists, archaeologists, paleontologists, malacologists and geochronologists. The main results of these studies are summarized in Figure 2.

Conclusions

The combined ESR/U-series analyses realized on teeth carried out from two Eemian sites of Northern France, Caours and Wasiers, demonstrate the reliability of the method to date Middle Paleolithic sites and, despite particular geochemical characteristics of part of the analyzed tissues, the validity of the used analytical protocols. This work demonstrates in particular the importance of the in situ dosimetry in the age calculation.

At Caours, the obtained ESR/U-series ages are in excellent agreement with the other available geochronological data derived from U-series and luminescence studies. They permit to calculate new mean age for the lower part of the sequence (124 ± 8 ka for unit 5 (N1, n=7) and 127 ± 15 ka for units 6-11 (including N3, n=6). Associated to available paleo-environmental and archaeological data, they make of this site one of the best known Middle Palaeolithic sequence of France.

For Waziers, the new established ESR/U-series chronology, with a mean age of 129 ± 11 ka, confirms the record of Saalian Late Glacial deposits at the bottom of the sequence and of the subsequent Eemian interglacial stage, evidenced by malacological and palynological studies.

These results show the interest to integrate ESR/U-series dating analyses during the chronostratigraphical study of Middle Paleolithic sites when possible and demonstrate the necessity to involve geochronologists to the fieldworks and excavations, since its earliest stages.

Waziers

  Caours (Somme) (excavations directed by J.-L. Locht, P. Antoine & J. Dabkowski) Waziers (Pas-de-Calais) (excavations directed by D. Hérisson & L. Deschodt)

  Waziers site is located close to Douai, in the Scarpe river valley. Discovered during archeological survey operation in 2011 et 2013, it was excavated in 2014-2015. The stratigraphic sequence encloses coarse fluvial deposits recovered by an alternation of silts and peat levels including archaeological levels with Middle Palaeolithic lithic industries, then by sandy loess deposits. Malacofauna study indicates at the bottom of the sequence both open landscape and cold environement, then an interglacial temperate environment with first peat bog then swampy forest. The associated mammal fauna is typical from forested environment also (cervids, aurochs, beavers,…). Four teeth from Waziers were analyzed during the present work, three from N2 and one from N6. The Caours site is located close to Abbeville in the Scardon Valley. Coarse fluvial deposits were here recovered by silts and a mm thick tufa sequence who fossilized several archeological levels containing abundant Middle Paleolithic industry and faunal remains associated with a forested temperate environment. U-series, TL and OSL dates constrain the tufa formation and Caours archeological level to ca 123 ka, allowing its clear attribution to Eemian interglacial stage (MIS 5e). Malacofauna study permits to place the climatic optimum of this stage in the lower part of the tufa formation. Five teeth, four from archaeological level N1 and one from N3, were analyzed in the present work by ESR/U-series. Another tooth was previously dated by the same method of 124 ± 15 ka

ESR/U-series ages (US or AU) (ka)

				dentine	8.235 ± 0.220 1.281 ± 0.032	318	1.342 ± 0.052	0.17					-0.0139 ± 0.0098
		N1	CA2005-01	enamel	0.332 ± 0.020 1.376 ± 0.094	12	0.927 ± 0.086	1.00	867 ± 108	27 ± 3	4 ± 1	79.31 ± 3.07	-0.0123 ± 0.0037	185 ± 143	185 ± 117	259 ± 21	629 ± 186	126 ± 37
				dentine	6.560 ± 0.219 1.232 ± 0.044	90	1.419 ± 0.080	0.32					+0.0390 ± 0.1431
		N1	CA2005-02	enamel	0.301 ± 0.017 1.247 ± 0.075	16	0.813 ± 0.078	0.55	993 ± 124	50 ± 6	20 ± 2	67.53 ± 2.66	-0.0128 ± 0.0051	41 ± 37	316 ± 196	210 ± 20	567 ± 201	119 ± 42
				dentine	10.270 ± 0.242 1.167 ± 0.026	133	1.150 ± 0.042	0.16					-0.0105 ± 0.0005
	Caours	N1	CA2005-03	enamel dentine	0.393 ± 0.029 1.063 ± 0.094 13.771 ± 0.316 1.225 ± 0.021	23 67	0.953 ± 0.102 1.135 ± 0.048	1.00 0.09	893 ± 119	59 ± 7	4 ± 1	53.39 ± 5.30	-0.0088 ± 0.0005 -0.0104 ± 0.0002	121 ± 3	159 ± 10	169 ± 22	449 ± 24	119 ± 15
		N3	CA2005-04	enamel	0.193 ± 0.012 1.222 ± 0.090	13	0.884 ± 0.087	1.00	1014 ± 127	43 ± 5	17 ± 2	73.16 ± 2.90	-0.0077 ± 0.0001	116 ± 1	74 ± 3	395 ± 20	585 ± 20	125 ± 5
				dentine	5.281 ± 0.165 1.229 ± 0.014	113	1.008 ± 0.052	0.14					-0.0102 ± 0.0008
		N1	CA2007-01	enamel	0.338 ± 0.016 1.274 ± 0.066	8	0.923 ± 0.055	0.92	957 ± 120	2 ± 0	29 ± 4	71.00 ± 10.62	-0.0312 ± 0.0057	59 ± 2	279 ± 21	210 ± 20	548 ± 29	129 ± 23
				dentine	0.097 ± 0.001 1.609 ± 0.004	128	0.547 ± 0.003	1.00					-0.0127 ± 0.0017
		2	WBT-19	enamel	0.036 ± 0.001 1.467 ± 0.017	46	0.600 ± 0.009	1.00	1250 ± 156	38 ± 5	135 ± 17	58.08 ± 1.70	-0.7551 ± 0.0608	9 ± 22	48 ± 9	408 ± 20	465 ± 33	125 ± 8
				dentine	0.028 ± 0.001 1.348 ± 0.005	16	0.655 ± 0.004	1.00					-0.8125 ± 0.0502
	Waziers	2	WBT-22-1	enamel	0.071 ± 0.001 1.246 ± 0.005.	213	0.775 ± 0.004	1.00	1209 ± 151	70 ± 9	84 ± 11	59.71 ± 1.03	-0.0106 ± 0.0010	21 ± 3	4 ± 42	408 ± 20	433 ± 26	138 ± 8
		2	WBT-22-2	dentine enamel	0.015 ± 0.001 1.508 ± 0.007 0.009 ± 0.001 1.386 ± 0.011	145 30	0.555 ± 0.004 0.672 ± 0.008	1.00 1.00	1113 ± 139	55 ± 7	39 ± 5	58.95 ± 1.66	-0.6443 ± 0.0608 -0.9384 ± 0.0438	2 ± 20	69 ± 10	408 ± 20	479 ± 30	123 ± 7
		6	WBT-318	enamel dentine	1.276 ± 0.005 0.068 ± 0.001 1.276 ± 0.005 0.070 ± 0.001	344 13	0.687 ± 0.003 0.679 ± 0.004	1.00 1.00	883 ± 110	39 ± 5	75 ± 9	76.47 ± 2.72	-0.9386 ± 0.0461 -0.9016 ± 0.0479	20 ± 32	89 ± 12	470 ± 23	579 ± 41	132 ± 8

Table 1

 1

. ESR/ U-series results obtained on teeth recovered from two Eemian sites of Northern France

Acknowledgements

We would like to thank the Caours and Waziers excavation teams for their disponibility, patience and helpful discussions.

The present study was financially supported by the UMR7194 HNHP which allowed the funding of the field missions and analyses., with the help of the PRC PaléHauts

The ESR and mobile gamma-ray spectrometers of the French National Museum of Natural History were bought with the financial support of the 'Sesame Île-de-France' program and the 'Région Centre' respectively.


Alice Fontbonne

Hervé Sauer

François Goudail

How to integrate digital post-processing algorithms in professional optical design software for co-designing complex optical systems?

Keywords: Image processing, piecewise-constant deconvolution, optical co-design, hybrid imaging system

We investigate hybrid imaging systems consisting of a lens and a fast post-processing algorithm, where the lens is designed or co-designed with professional systems optical design software. Two approaches have been investigated: adapt the optimization criterion to the use of a unique deconvolution filter by requiring uniformity of the modulation transfer functions (MTF) over the field of view, or use piecewise-constant deconvolution to adapt the processing algorithm to spatial variations of the MTF in conventionally designed optical systems. By studying the evolution of the deconvolution performance across the field of view in systems optimized with these two approaches, we highlight that the optimal use of post-processing algorithm leads to lenses whose intrinsic performance varies little in the field of view, with however a significant amount of appropriate aperture aberrations but well suited to be post-processed.

INTRODUCTION

Today, almost all imaging systems include both an optical part (the lens) and an image processing part in order to improve the final image quality. It therefore seems natural to optimize them simultaneously to obtain the best possible result. However, even if this "co-design" approach is more and more recognized at a conceptual level, it is still rarely used in practice for designing complex lenses with many adjustable parameters and constraints. This is due to the fact that the contribution of image processing is currently difficult to take into account in optical systems design software.

Until now, the field of co-design has thus mainly focused on simplest imaging systems, consisting for example of single co-optimized optical elements such as phase masks. ? One of the first attempts to co-design optical systems involving complex lenses has been done by Stork & Robinson, ? who integrated the image processingbased criterion known as mean square error (MSE) in the optical software Zemax OpticStudio. It is also possible to consider other optimization criteria than the MSE, such as a surrogate criterion built from more classical optical parameters (modulation transfer function, point spread function, etc.) ?

In the present paper, we investigate and compare two optimization criteria for designing hybrid systems with professional optical systems design software: a conventional criterion, based on minimizing the spread of spot diagrams, and a "codesign-based" criterion, that enforces small changes of the MTF over the field of view. The images obtained from the systems optimized with these two criteria can be post-processed with a single deconvolution filter, or with a piecewise-constant deconvolution algorithm. We study the impact of the optimization criterion and of the post-processing algorithm on the global performance of these different systems. This article is organized as follow: firstly, we define in detail the two optimization criteria for hybrid systems (Section 2) and then we evaluate the performance of the designed systems when the image is post-processed with a piecewise-constant deconvolution algorithm of adjustable number of zones, including one, i.e. the single filter case (Section 3). Finally, we conclude in Section 4 about these different approaches to design a hybrid system with a professional optical systems design software and to use it in practice.

DESIGN HYBRID IMAGING SYSTEMS IN PROFESSIONAL OPTICAL SYSTEMS DESIGN SOFTWARE

Specifications and goal

We consider hybrid optical-digital systems composed of two main parts: an imaging system (i.e. a lens and a sensor) and an image processing algorithm consisting of a deconvolution filter. The goal of the design (or codesign) in this paper is to obtain a hybrid system with the best mean performance possible for all the positions over the field of view. Both hybrid systems (the conventional and the codesigned one) are optimized with the same specifications. First, we consider a F/4 lens of 50 mm focal length made of two cemented doublets separated by a diaphragm. Limitations on the size of the lens are the same for the two systems. Secondly, the processing algorithm is the same of both systems. We use a fast algorithm that take approximately the same computation time as a single convolution.

By considering a set of K differents positions in the field Ψ = {ψ 1 , ψ 2 , ...ψ K } (in ascending order), it as been established ? that the best linear filter to deconvolve an image is the averaged Wiener filter

w(ν) = 1 K k hψ k (ν) 1 K k | hψ k (ν)| 2 + Snn(ν) Soo(ν) , (1)
where hψ k (ν) is the local optical transfer function (OTF) at the position ψ k in the field of view ( hψ k being the Fourier Transform of the PSF h ψ k ), stands for the complex conjugate, S nn (ν) is the power spectral density (PSD) of the noise and S oo (ν) is the PSD of the scene. Note that for the sake of simplicity in the formulae, we use the symbol ν for the 2D spatial frequency vector (ν x , ν y ). We consider a power-law object PSD S oo (ν) ∝ ||ν|| -2.5 and a white noise PSD such that the signal-to-noise ratio (SNR) is equal to 34 dB. It corresponds to a standard digital camera under good illumination conditions.

This averaged Wiener filter is applied to the image provided by the imaging system. We consider two systems: a conventional one (Section 2.2) and a system co-optimized in a professional optical systems design software by taking into account the impact of the deconvolution on the final performance (Section 2.3).

Conventionally optimized hybrid imaging system

There are several ways to optimize a lens with professional optical systems design software. The most usual way is to minimize the quadratic sum of the root mean square (RMS) diameters of the spot diagrams for all the considered field positions. This method, which does not take into account any post-processing algorithm, allows to limit the aberrations of an optical system so that the spot diagrams are, on quadratic average, as small as possible. Even if the modulation transfer functions (MTF) are often used as a tool by the optical designer to check the performance of the system, we do not directly consider them in the optimization criterion. Let us consider for example an optimization, with this criterion, of a lens made of two cemented doublets separated by a diaphragm. Figure 1(a) shows the spot diagrams (extracted from Synopsys CodeV) of the optimized system at four positions in the field, from the on axis position (bottom of the figure) to the maximum object field position, of 20 • . We can notice that the conventional optimization led to spot diagrams whose sizes vary considerably over the field of view. Small sizes occur at positions in the field which are close to the axis. However, on the axis, the width of the spot is more important, due to curvature of field and best composite focusing. The spot diagram for the maximum position in the field is the largest: its RMS width is almost twice The evolution of the shape of the spot diagrams of this system (Figure 1(a)) and its MTF (Figure 1(b)) is usual in conventional optical design. This optical system is not co-designed, but it is still possible to add digital post-processing such as the averaged Wiener filter (Eq. 1), so that its performance can be compared with that of a co-designed hybrid system.

Co-optimized hybrid system

The idea of co-design is to design, in the same time, the optical system and the associated digital processing (for this article, the averaged Wiener filter calculated from the OTFs of the optical system). As for any optimization, a criterion to be minimized must be defined. The averaged Wiener filter (Eq. 1), built with a set of OTFs calculated for a representative set of positions in the field Ψ (the set Ψ comprising K positions in the field) is the linear filter allowing to minimize the MSE criterion, defined in the Fourier space by: ?

M SE(Ψ) = 1 K k ν [S oo (ν)| hψ k (ν) w(ν) -1| 2 + | w(ν)| 2 S nn (ν)]dν (2)
This optimization criterion is not natively implemented in professional optical systems design software such as Zemax or CodeV. A way around this problem has already been proposed in Zemax, ? but until now, no publication, to the best of our knowledge, claims to have implemented it in CodeV, the software we use here. Instead, a surrogate criterion to MSE has been proposed. ? It is a compound criterion taking into account both the size of the spot diagrams (like the conventional criterion) and the disparity between the MTFs over the field of view. Indeed, as a single deconvolution filter is used, the optimization on the MSE criterion naturally leads to the approximation that the MTFs have to be close to each other for all the positions in the field. ? It is this behavior that is reproduced with the implementation of such a surrogate criterion.

In this article, we use a similar algorithm which consists in equalizing the MTFs by constraining their value differences to zero at two or three spatial frequency values. It is possible to change the values of the frequencies and constraints during the design path, to facilitate equalization. Note that, with this method, only the MTFs are optimized and not the spot diagrams. Therefore, we had to constrain the level of the MTFs in addition to equalizing them so that they are not too low (because the size of the spot diagrams is not minimized).

The MTFs of the system optimized with this criterion are given in Figure 2(b). As expected, they are quite similar to each other. We can notice that they show no nulling up to the spatial frequency 50 lp.mm -1 . On the other hand, they are all rather weak (< 0.05) at this frequency, which was not the case for the conventional system. They drop very sharply up to 5 lp.mm -1 but, starting from this frequency (where all MTFs are about 0.3), the decay slope becomes relatively small. Optimizing on MTFs equalization is very interesting when using a single deconvolution filter as the hybrid system behavior is independent of the position in the field of view. It is also important that there is no nulling, because a nulling of the MTFs causes a loss of information that cannot be recovered by deconvolution. However, as the co-optimized MTFs are quite low (< 0.25 starting from 10 lp.mm -1 ), the reconstruction can be difficult if there is too much noise. We have considered from the beginning the case of a good SNR, which is favorable to this method of co-design by MTFs equalization. Let us now examine, Figure 2(a), the spot diagrams of this system co-designed by MTFs equalization. They are similar to each other, and are very large compared to those of the conventional system (the scale is the same for Figure 1(a) and Figure 2(a)). Globally, the RMS size of spot diagrams in the co-designed case is five to ten times larger than in the conventional case. This is an important difference between the conventional optimization method and the presented co-optimization method. Such large spot diagrams are typical of an imaging system corrupted by strong aperture aberrations, which is bad when used without digital post-processing, but its images can be efficiently restored with a unique deconvolution filter.

To conclude, we have obtained two imaging systems that can be used with digital post-processing. The first one, optimized in a conventional way, gives priority to the small size of the spot diagrams. However, as all its MTFs are different from each other, it is not very well suited for use with a single deconvolution filter. The second one is co-optimized by equalizing the MTFs in view of its use with an averaged Wiener filter. In the following section 3, we will evaluate the performance after post-processing of the two hybrid systems.

PIECEWISE-CONSTANT DECONVOLUTION FOR HYBRID SYSTEMS

In the previous section, the co-designed system has been optimized by enforcing similarity of the MTFs over the field of view. Deconvolution with a unique averaged Wiener filter is thus an appropriate post-processing algorithm for this system. In contrast, it is not appropriate for the optical system optimized in a conventional way since its MTFs vary significantly in the field of view. Hence, we will also consider in this section another post-processing algorithm that takes into account MTF variation over the field. Reconstruction algorithms for continuously varying blur exist, ? but in this paper, we will only consider non-recursive piecewise-constant deconvolution algorithms whose advantage is to keep the computational time equivalent to that of a single convolution.

Evolution of the MSE with piecewise-constant deconvolution

We propose here to use a method of piecewise-constant deconvolution. It means the entire field of view is divided into square areas and, for each area, a deconvolution by an averaged Wiener filter is performed. The used filter depends on the area since each filter is built from a set of Ψ A positions in this area A. Therefore, for each area A, it is possible to calculate the local value of the MSE, M SE(Ψ A ), by using the Eq. 2. The total MSE for the entire field of view of the lens is the mean of the MSE of the different areas (of equal size):

M SE(Ψ tot , N A ) = 1 N A A M SE(Ψ A ) , (3)
where Ψ tot denotes the set of positions in the field for the entire field of view and N A is the number of areas considered, such as

∪ A Ψ A = Ψ tot .
This section aims to evaluate how the quality of the deconvolution of hybrid systems evolves with the number of areas considered for the piecewise-constant deconvolution (N A = 1 gives the unique averaged Wiener filter). All simulations will be done considering a sensor of Nyquist frequency 100 lp.mm -1 . Note that, instead of the M SE(Ψ tot ), we will prefer to use the mean image quality as a performance metric for hybrid systems (expressed in dB):

IQ mean (N A ) = 10 log 10 σ 2 O M SE(Ψ tot , N A ) . ( 4
)
where σ 2 O denotes the variance of the sharp image. First, it is possible to see on Figure 3 that the mean performance before deconvolution is far better (+2.8 dB) for the conventional system than for the co-designed one. This is due to the fact that, on average and except for special cases (on the axis or extreme field), the MTFs of the conventional system (Fig. 1) are higher than those of the co-designed system (Fig. 2). After deconvolution with a unique averaged Wiener filter (N A = 1), the two systems have very equivalent performance, close to 10 dB. This represents a significant performance gain for the co-designed system (+3.7 dB), showing that the MTFs equalization criterion is very well suited for use with a single deconvolution filter. On the other hand, the gain is less important for the conventional system, only 0.9 dB: the strong variations of the MTFs across the field prevent the single deconvolution from working well.

For the conventional hybrid system, when the number of areas N A increases, IQ mean is slowly increases. It reaches 11.2 dB for N A = 25 areas, i.e. when the field is divided into 5 × 5 equal areas. On the contrary, for the co-designed system, IQ mean sharply increases (of 2 dB when N A = 4), and then tends towards an asymptote. It reaches 13.3 dB for N A = 25 areas. Thus, the conventional system and the hybrid system have a quite different behavior regarding the number of areas. This discrepancy can be explained by the differences of MTF variations with the position in the field, as will be shown in the following.

Disparity between MTFs and intrinsic difficulty of deconvolution

The MSE of an area A depends on the values of the OTFs hψ (ν) over this area. To facilitate the interpretation of the evolution of the MSE with the field of view, it is possible to split the expression of the MSE (Eq. 1), corresponding to a set of positions Ψ in the field of view, into two parts ? M SE D and M SE I , so that

M SE(Ψ) = M SE D (Ψ) + M SE I (Ψ).
The first part,

M SE D (Ψ) = ν S oo (ν) 2 [ 1 K k | hψ k (ν)| 2 -| 1 K k hψ k (ν)| 2 ] S oo (ν) 1 K k | hψ k (ν)| 2 + S nn (ν) dν , (5)
is characteristic of the disparity between the MTFs, and would be null if all the OTFs were equal across the field of view. Considering an area A, the more the local OTF varies with the position in the field (in Ψ A ), the more M SE D (Ψ A ) will be high.

The other term is

M SE I (Ψ) = ν S oo (ν)S nn (ν) S oo (ν) 1 K k | hψ k (ν)| 2 + S nn (ν) dν , (6)
which is equal to M SE(Ψ) if all the OTFs at all the positions in the field of view are equal. Therefore, this term is characteristic of the intrinsic difficulty of deconvolution, i.e. of the compromise made by the averaged Wiener filter between noise amplification and signal reconstruction with the average MTF

1 K k | hψ k (ν)| 2 .

Evolution of area-specific MSE with the number of areas for the conventionally optimized system

Figure 4 represents the piecewise-constant square root of M SE D (Ψ A ), M SE I (Ψ A ) and M SE(Ψ A ) for the conventional system and different divisions in the field of view. These divisions range from 1 (using a single deconvolution filter for the entire field) in the first row to 25 (using then 25 different averaged Wiener filters for 5 × 5 areas of equal size) in the last row.

We can first notice on Fig. 4 that, as the number of areas N A increases, the intrinsic difficulty of deconvolution (column a) increases for some areas. It remains constant with the increase in N A only for the areas located in the region corresponding to spot diagrams of small diameters, that is, around 10 • (see Fig. 1(a)). Secondly, the contribution of the disparity between the MTFs (Fig. 4(b)) decreases with the number of areas but in a way depending on the location of the area in the field. M SE D remains approximately constant with the number of areas N A in the areas of strong radial or azimuthal variations of MTFs and decreases for the other areas. It follows from the sum of these two contributions that the square root of the MSE (RMSE, Fig. 4(c)) globally decreases (with the number of areas) for the areas where the disparity between the MTFs decreases. However, it increases for the other areas, because the disparity between the MTFs has remained stable while the intrinsic difficulty of deconvolution increases since the spot diagrams in the field are wider.

Evolution of area-specific MSE with the number of areas for the co-optimized system

Figure 5 shows that the behavior of the co-optimized hybrid system is very different from the previous one. The first thing to note is that the intrinsic difficulty of deconvolution remains almost constant, regardless of the number of areas. This is due to the fact that the co-designed system has a very homogeneous performance in the field of view (see Fig. 2). However, the value of M SE I (Ψ A ) is larger on average than for the conventional system, since the average MTF of the co-designed system is quite low.

We can also observe that, as the number of areas increases, the RMSE related to the disparity between the MTFs (Figure 5(b)) approaches zero, as the closer the positions considered in the field are, the closer the MTFs are to each other. The extreme field area has the highest RMSE because the extreme field spot diagrams does no more exhibit a circular symmetry (Fig. 2(a)), which leads to a larger disparity between the different local MTFs in the area.

Therefore, the total RMSE (Fig. 5(c)) essentially follows the improvement due to the increasingly strong match between MTFs with the increase in the number of areas. This explains the rapid increase of IQ mean (N A ) (Fig. 3) for the co-designed system by MTFs equalization, and also the fact that there is an asymptote: M SE I (Ψ A ) is stable and M SE D (Ψ A ) cannot be smaller than 0.

As a general conclusion on the studied systems, the error due to the intrinsic difficulty of deconvolution tends to increase with the number of areas used to divide the field of view. This observation is particularly relevant for the conventional system, for which the spot diagram varies (and often grows) in the field of view, causing an increase in the associated intrinsic difficulty of deconvolution. In contrast, by construction, codesigned systems are little affected by this increase. On the other hand, the disparity between MTFs tends to decrease with the number of areas (i.e., as the areas become smaller), except in areas of rapid azimuthal or radial variation of the spot diagram. Since areas of strong radial or azimuthal variation are often correlated with the presence of a larger spot diagram (due to strong field aberrations), these areas see their intrinsic difficulty of deconvolution increase with the increase of the number of areas N A , whereas the disparity between the MTFs is not decreasing. Therefore, for these areas, the total RMSE increases. If we consider the whole image, this increase is counterbalanced by the decrease of the RMSE in the simpler areas so that the total RMSE continues to decrease with the number of areas, as seen on Fig. 3.

CONCLUSION

Today, it would be possible to implement the standard co-design criterion of MSE in professional optical systems design software such as Zemax and CodeV. However, this approach, difficult to implement, is still rarely used. To optimize a hybrid system, two simple approaches to implement are the conventional approach (optimize on spot-diagram size) and the co-design approach by MTFs equalization.

Fast processing algorithms, such as piecewise-constant deconvolution, can be added to optical systems. We studied the potential gain of this post-processing algorithm on conventionally and co-optimized systems. This study has shown that piecewise-constant deconvolution systematically allows to refine and improve the reconstruction with the increase of the number of considered areas (as they get smaller). This improvement levels off for a large number of areas, in particular for co-designed systems, which shows that in this case, a limited number of areas is sufficient to reach optimal performance. For a fixed number of areas, piecewise-constant deconvolution works better for a slow variation of the MTF in these areas. Therefore, it works well on a co-designed system because the disparity between MTFs is small by construction, but may not be able to compensate for the fast variation of the MTF observed in a conventional system.

To conclude, it is worth taking into account, from the optimization phase of the lens, that a deconvolution will be used. In fact, even if the processing algorithm is adapted as much as possible to shift variant blur, slow variations of the MTF in the field is more appropriate for use with a deconvolution filter. This study remains to be generalized on more complex optical systems or systems co-optimized with the MSE criterion.

Figure 1

 1 Figure 1. (a) Spot diagrams of the conventional optical system (b) MTFs of the conventional optical system.

Figure 2 .

 2 Figure 2. (a) Spot diagrams of the co-designed optical system (b) MTFs of the co-designed optical system.

Figure 3 .

 3 Figure3. IQmean as a function of the number of areas NA considered for the piecewise-constant deconvolution, for the conventional system and the co-designed system. Note that NA = 1 gives the post-processing with the unique averaged Wiener filter.

Figure 4 .

 4 Figure 4. Piecewise-constant RMSE after deconvolution for the conventional hybrid system. (First column) Intrinsic difficulty of deconvolution √ M SEI ; (Second column) Disparity between MTFs √ M SED; (Third column) Total √ M SE. The number of areas used for the piecewise-constant deconvolution depends on the row: (1) one area; (2) four areas; (3) nine areas; (4) sixteen areas; (5) twenty-five areas. The colorbar is the same for all the graphs.

Figure 5 .

 5 Figure 5. Piecewise-constant RMSE after deconvolution for the co-designed hybrid system. (First column) Intrinsic difficulty of deconvolution √ M SEI ; (Second column) Disparity between MTFs √ M SED; (Third column) Total √ M SE. The number of areas used for the piecewise-constant deconvolution depends on the row: (1) one area; (2) four areas; (3) nine areas; (4) sixteen areas; (5) twenty-five areas. The colorbar is the same for all the graphs.

ACKNOWLEDGMENTS

The work reported in this study is supported in part by the Agence de l'Innovation de Défense (AID) that provides half of a PhD fellowship to Alice Fontbonne. Authors have no conflicts of interest related to the work leading to the results presented in this paper.