0031-9422(94)00635-0 Pergamon Phytochemistry, VoL 38, No. 5, pp. 1245 1250, 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 00314422/95 $9.50 + 0.00 PHENOLIC GLYCOSIDES FROM ADONIS ALEPPICA GUIDO F. PAULI* and PETER JUNIOR Institut ffir Pharmazeutische Biologie, Geb. 26.23., Universit/itstrasse 1, Heinrich-Heine-Universitfit, 40225 Diisseldorf, Germany (Received 3 May 1994) Key Word Index--Adonis aleppica; Ranunculaceae; flavonoid glycosides; phenolic glycosides; phenylpropanoids; NMR; structure elucidation. Abstract--Isoetin- 4'-O-fl-glucuronide, a new derivative of the rare 2', 4', 5'-trihydroxyflavone isoetin, has been isolated from Adonis aleppica, along with the known vitexin-2"-O-fl-rhamnoside. These structures were established by means of DCI-NH3-mass spectral and NMR methods including full establishment of the CH-spin systems for both compounds. Furthermore, structural evidence is given for the presence of sinapoylglucose representing the first phenylpropanoid glycoside isolated from a member of the genus Adonis. 13C-IH HETCOR experiments and were determined as isoetin 4'-O-fl-glucuronide (1), vitexin 2"-O-fl-rhamnoside (2) and l'-sinapoylglucose (3). INTRODUCTION The botanical classification of annual Adonis species ( = sectio Adonis) is still full of contradictions. Recently, the form of the fruits has been pointed out as the main distinctive mark, although there are certain difficulties in its determination depending on the degree of ripeness [ 1]. From the phytochemical point of view, plants of the genus Adonis are well known for the occurrence of cardiac glycosides [2-4]. Only a few cardenolide studies have dealt with the isolation of phenolic constituents [5-7]. We have now developed an effective isolation procedure on the basis of vacuum liquid (VLC), column (CC), droplet counter current (DCCC) and middle pressure liquid chromatography (MPLC) allowing the isolation of both flavonoids, as well as cardenolide glycosides from the same extract [3]. This method has been successfully applied to Adonis aleppica Boiss., an annual endemic herb of the Mesopotamian region [1]. In previous communications, A. aleppica was shown to produce a large number of cardenolides, especially long-chained oligoglycosides ( = alepposides) [2] and sulphates [8], as well as the aliphatic alcohol glucoside aleppotrioloside HOH~ H H O ~ O Ho ~ / OCHa o H o 3 oc~ OH OH 0 ItO I.K)~~O ['9]. 0 10 ~/OH RESULTSAND DISCUSSION From the polar fractions of the total plant extract of A. OH 0 aleppica the two major flavonoid glycosides 1 and 2, as well as the phenylpropanoid glycoside 3 were isolated. Their structures have been elucidated by means of UV, DCI-NH3-mass spectra (DCI-MS), 1D and 2D IH NMR, tHBB and gated-decoupling 13CNMR, as well as The molecular formula of 1 was deduced as C21HIaOI3 from lSCNMR and neg. DCI-MS (m/z 302 *Author to whom correspondence should be addressed. Present address: Dept of Chemistry, Louisiana State University, Baton Rouge, LA 70803-1804, U.S.A. [M(agl)] -, 478 [M] -) proposing the presence of a hexuronic acid moiety ( ~ - 176 amu) linked to a flavone skeleton. A special feature of 1 is the 2' (para)hydroxylation of the B-ring causing a marked irradiation of H-3 and resulting in the pure singlet absorptions for 1245 1246 G. F. PAULI and P. JUNIOR Table 1. One and 2D ~H and t3C NMR data of isoetine-4'-O-glucuronide (1) 1-200/50 MHz, DMSO-d6, (5 in ppm, J in Hz] Pos. 6 H (Mult., 2 3 -7.05 (s) 4 5 6 7 8 9 10 Corr 6c (APT) IJc. n "Jc.n (n > 2) ~ 161.6 (C) 104.9 (CH) -160.3 2Jc2.Hv = 5.3, 3Jc2,H 6, = 1.6 Av = 4 2dc,.n 3 < 1 2 J c s , H 6 = 3.5 -- 182.1 (C) 13.00 (s) 6.17 (d, 2.2) -6.43 (d, 1.8) --- 1' -- 2' 3' 4' -- 5' 6' 1" 2" 3" 4" 5" 6" J) -- 161.4 (C) 98.9 (CH) 164.4 (C) 94.0 (CH) 157.6 (C) 103.9 (C) ,--, ,--, 162.1 -165.4 --- 110.7 (C) 6.77 (s) -- 151.2 (C) 108.2 (CH) 148.9 (C) ~ -7.33 (s) 4.90 (br d, 7.0) 3.41 (dd, 6/5) 3.373 (t, 5) 3.365 (t, 6) 3.83 (d, 8.5) ~ 139.9 (C) 114.2 (CH) 101.3 (CH) 73.2 (CH) 75.5 (CH) 71.8 (CH) 75.9 (CH) 170.6 A (C ~ ,-, ,--, ,--, ~ ~ 2Jc6,H 8 2Jc7.H 6 3Jc8,H 6 2Jc9,tl8 1.6 19.6 3.8 2JcT,H 8 = 3.1 4.4 4.2 3Jclo,ns = 5.3 3 J c I o , H 6 = 4.4, 3Jc10.H3 = 4.4 3Jc12.H3' = 6.2 3 J c I 2 . H 3 ~,~ 2 J c 1 . H 6, ~ 0 3Jc2,.H 6, = 9.6, 2Jc2.H 3, = 3.8 -171.4 -- -160.2 163.8 145.0 172.8 144.7 145.9 Av= 5 3Jc4..H 6, = 8.9 3Jc4, rll, = 3.6, 2Jc4,,H 3, = 3.6 3Jc5, ny = 7.3, 2Jc5, n6, = 4.4 Av= 5 Av = 10 3Jc2,, n4,, = 3.4 Av = 14 Av = 9 Av = 8 ',1t'*"_.....____~ 3 J c 6' 2._..~, 3" 2Jc6",HS'-- Z -1 8.9~ ~4.4 ~ ~.H~A~.~o = = = = _--2-'1 /3.6---~ ' I I;~ HO~OH ~,,J.~~ H~. 3H4OJ~H~~.2 • 3(IBm,°) .3 I 1 \0 '~4.4' "'4.4 / Fig. 1. Heteronuclear long-range coupling constants and pathways ofisoetin-4'-O-glucuronide (1) derived from 2D LR-CH and 1D gated-decoupling experiments (values given in Hz). the B-ring protons H-3' and H-6' (Table 1). N M R measurements included the determination of ~J and longrange ~3C-tH-correlations (Table 1, Fig. 1) and confirmed all assignments. In case of the H - 3 / H - 3 ' [10] they should be revised as far as C-3/C-3' can be clearly distinguished from their long-range coupling behaviour. In this context, another remarkable detail is the large size of the geminal coupling constant 2j (C-2, H-3) typically occurring in unsaturated y-pyrones [11]. 4'-O-Linkage of the glucuronic acid moiety is apparent from the heteronuclear splitting of the C-4' signal of the gated-decoupling ~ 3 C N M R (Table 1), from the ~H glycosylation effect affecting H-Y ( + 0.20 ppm) and from U V shift measurements. Addition of N a O M e gives rise to a very large bathochromic shift (113 nm) due to a free T - O H group; there is also degradation of the spectrum because of the hydroquinone partial structure. In the N a O A c and AICI 3 spectra bathochromic effects of free Y,4' (ortho)dihydroxy groups are lacking. Finally, the relatively low maximum absorbance of band I in methanol correlated with a non-planar conformation of the A / C and B-rings. This finding is consistent with the result of torsion-forcing calculations of I as shown in the molecule plot in Fig. 2. Flavonoid 2 according to D C I - M S and t 3C N M R data is a flavone C-diglycoside bearing two hexoses. In analogy to 1, interpretation of comprehensive N M R experiments (1H, I H - t H C O S Y , 1HBB and G D 13C, 1 3 C _ l H H E T C O R ) led to the structure of vitexin 2"-0-[3- Phenolic glycosides from Adonis aleppica 1247 Fig. 2. Molecular conformation of isoetin-4'-O-glucuronide (1) resulting from torsion-forcing calculations with DISCOVER. glucoside (Table 2). Signals of the two fl-glucose moieties were completely assigned from 1H-~H COSY and ~aC-~H HETCOR maps, the latter proving the C-glycosidic junction by shift value (71.6 ppm) and 1Jcn value of C-I" (142.6 Hz). Absolute evidence for C - C linkage in position 8 is obtained from the vicinal coupling C-9,--~H1" (5.9 Hz) representing the only heteronuclear correlation for C-9. A significant downfield shift of the H-2" signal in addition to 13C glycosidation effects for C-2"/3" (otlfl) indicate a 1'"---2" sugar linkage. As far as all assignments have been verified by a IaC-1H HETCOR experiment, revision of the 13CNMR data in ref. [12] concerning C-2"/C-5" of vitexin-2"-O-rhamnoside seems appropriate. The symmetrical 4'-monohydroxy substitution of ring B gives rise to a symmetrical CH coupling behaviour: C-4' itself produces a triplet of triplets due to equal 2j and 3j couplings, and the 1j couplings of all CH carbons are identical (161 Hz). Following the vicinal coupling of 4 Hz between C-2 and H-2'/6', the dihedral angle between ring B and A/C is different to 1 indicating a more planar conformation (q~ ca 45°). Distinction of C-2 and C-7 signals was achieved by their different coupling pattern with only small 2js for C-7. Finally, it can be stated that 1248 G. F. PAULI and P. JUNIOR Table 2. One and 2D ~H and ~3C NMR data of vitexin-2"-O-glucoside (2) [200/50 MHz, DMSO-d6, 6 in ppm, J in Hz] Pos. 6n (Muly., J) Corr -- ,5c (APT) 1j C,H 163.7 (C) -- "JC.H (n > - - 2) 2j C2.H3 3j 3 4 5 6 7 8 9 10 1' 7.05 (s) ~ -13.15 (s) 6.17 (s) ~ 104.9 (CH) 181.9 ( C ) 160.6 (C) 98.4 (CH) 167.4 5.5 C2.H2' ~ 3j C2,H6' = 4.1 -160.3 Av = 6 2Jc4.1t3 < 1 2j C5,H6 ~ 2j C2.H5 = 3.6 3j C6,H5 = 5.3 -- -- 163.5 (C) -- 2 J c T , a 6 ~.~ 3 J c 7 , H I , , .~ 2 --- 103.7 (C) 156.3 (C) 103.6 (C) ---- zj C9.H 1" -- - 5.9 -- 121.7 -- (C) * * aj 2j 8.00 (dd, 9/1) 6.90 (dd, 9/1)) ~ ~ CI',H3' -_ CI',H2 ~ 3j CI',HS' 2j CI',H6' 7.7 = 2.1 128.9 (CH) 115.9 (CH) 161.3 161.2 2Jcl,n a ~ 0 3Jc2,,H6, = 6.7, 2 J c 2 , , H 3, 3j C3'.H5' = 4.4 161.2 ( C ) -- 2jc4,.1t3. 3 J c 3 . H4, < < 1 1, 2 J c a , . H 2, < 1 2j C4,H5' = 2.0 4' -- 5' 6.90 (dd, 9/1) ~ 115.9 (C) 161.2 6' 8.00(dd, 9/1) 4.84 (d, 9.9) 4.90 (t, 9.3) 3.50 (t, 8.3) 3.46 (t, 8.9) 3.48 (m) 3.78 (br d, 11) 3.56 (dd, 5/13) 3.94 (dd, 7 5) 2.79 (dd, 7/8) 2.9 2.96 2.84 3.13 (br d, 13) 2.97 ~-* ,--. ,--, ~ ,--, ~ ~ 128.9 (CH) 71.6 (CH) 81.3 (CH) 78.4 (CH) 70.2 (CH) 81.7 (CH) 61.0 (C) 161.3 142.6 144.5 141.3 144.0 141.0 140.5 3j C4',H2' ~ 3j C4'.H6' = 9.2 3j C5',H3' = 4.4 3Jc5, H4, < 1, 2Jcs.n 2, < 1 aJc6.H2, 6.7, 2Jc6,H 5, < 1 Av = 14 Av = 14 Av = 12 Av = 12 Av = 10 Av = 9 ~ ,--, ~ ~ ~ ,--, 105.2 (CH) 74.4 (CH) 76.3 (CH) 69.5 (CH) 76.0 (CH) 60.4 (C) 156.8 142.0 140.2 143.7 136.9 141.3 Av = Av = Av = Av = Av = Av = 1" 2" 3" 4" 5" 6~ 6~ 1 r- 2'" 3"' 4" 5 rrr 6~, 6~ = 14 7 17 11 8 7 * Not determined because of signal overlap. wherever partial structures of 1 and 2 are comparable, analogous 13C signal multiplicities were registered (e.g. signals of C-2, C-5, C-6 and C - I ' / H - 3 coupling). C o m p o u n d 3, according to U V spectral properties (see Experimental), was found to be a phenolic compound. The positive mode D C I - M S showed the signal of a quasimolecular ion at m/z 404 (in accord with C17H22Olo ) and a sequential ion at m/z 242 indicating the loss of one hexose unit. The glycosidic nature of 3 was also gleaned from the ~H N M R (Table 3). The doublet at 65.57 (J = 7.0 Hz) due to an anomeric proton and the AB-type signals assignable to two hydroxymethylene protons suggested the presence of fl-glucose (glc). Within the olefinic/aromatic region two signals of trans olefinic protons (66.53 and 7.72, J = 15.9 Hz) and a singlet absorption of two a r o m a t i c protons (67.01) were assigned. Taking into account the singlet absorption integrating for six protons at 63.86, the aglycone moiety of 3 should consist of a cinnamic acid skeleton bearing a symmetrical 3,5-dimethoxy-4-hydroxy substitution. Further evidence for the identification of a sinapoyl and a glc moiety came from the ~ac N M R (Table 3) showing good agreement with published data. As usually found in olefinic phenylpropanoids, both ~H and 1 3 C N M R of 3 show a second set of weaker signals due to isomerization of the at/fl double bond (A~/B). Thus, the acylgiycosidic linkage in 3 cannot only be proved by the shift values of C - I ' (695.6 in 3, ca 104.3 ppm in acetalglycosides) and C-2' (674.0 in 3, otherwise ca 75.6 ppm). It is also evident from the occurrence of a second signal due to the anomeric proton of the cis olefinic glycoside indicating the close neighborhood of the anomeric proton and A~/a. Finally, the structure of l'O-[E/Z]-sinapoyl-fl-glucose for 3 is in full concordance with the long-range C H couplings (2/a j , Table 3) observed in a gated-decoupling 13C N M R . Analysis of the "ddd' signal of the carbonyl carbon leads to 3j (H-I') = 3.0 Hz providing the ester linkage, while 3j(H-fl) = 6.7 Hz and 2 j ( H - a ) = 3.2 Hz are indicating Atrmn, configuration (14.6 and < 1 Hz in the cis isomer, respectively) [11]. Due to magnetic equivalence of H-2 and H-6 (symmetrical substitution pattern) the signal of C-4 ap- Phenolic glycosides from Adonis aleppica 1249 Table 3. *H and 13C NMR data of sinapoylglucose (3, trans-isomer) in comparison with the sinapic acid moiety (Sin) of methylleucosceptosid A and glucose (GIc) in the monoterpene esterglucoside digipenstrosid [25] (200/50 MHz, DMSO-dr-CDaOD (1 : 1), 6 in ppm, J in Hz) C 1 2 3 4 5 6 ct fl C =O 2*OMe 1' 2' 3' 4' 5' 6~, (~ tSn, Mult., J 7.01, s 7.01, s 6.53, d, 15.9 6.53, d, 15.9 6c 125.9 107.4 149.3 139.8 149.3 107.4 115.6 148.1 166.9 3.86,s 5.57, d, 7.0 3.79, br d, 12 3.60, dd, 5 57.1 95.6 73.9 77.9 70.9 79.0 62.1 JC,H 159 -- 2Jc.n 3Jc,rl 1.8, H-2 7.1, H-2/6 -159 165 155 -- 1.8, H-6 3.2, H-~ 145 164 145 142 149 141 141 pears as a triplet with 3j = 7.1 Hz, while the coinciding absorptions of C-3/5 are doublets of quartets overlapping each other and forming a quintet. With the isolation of sinapoylglucose (3) a phenylpropane glycoside has been detected in a member of the genus Adonis for the fist time. The structure was ascertained by detailed NMR studies, lacking so far [13]. Mona- and diesters of sinapic acid and glucose have recently been reported from Raphanus sativus (Brassicaceae) [14-16], while related compounds (e.g. l'-Osinapoyl-6'-O-gallloyl-glucose)occur in Cynanchum hancockianum (Asclepiadaceae) [17]. Isoetin-4'-O-fl-glucuronide(1) is a new natural product representing the first 4'-O-glycoside belonging to the series of 2', 4', 5'-trihydroxyflavones. This unusual flavonaid glycoside supports the exceptional position of A. aleppica within the genus Adonis as already inferred from the occurrence of novel types of cardenolides, e.g. alepposides and sulphates [2, 3, 8]. Isoetin has been found in the Lycopsida (Isoetes delilei) [10] and glycosides are known from some Asteraceae [18, 19], but this is the first report on its occurrence in a member of the Ranunculaceae. In contrast to this, C-glycosylflavones e.g. vitexin (v), orientin (o), homoorientin (h) and adonivernith (a) have previously been detected in Adonis plants, but only in perennial species belonging to the sectio Consilioo: A. amurensis ( o / h / a [20]), A. monoolica (o [7]), A. sibiricus (o/a [21]), A. tianschanicus (o/a [22]), A. turkestanica ( v / o / h [23]), A. venalis [5, 6] and A. woloensis (v [24]). EXPERIMENTAL Plant material. Authentic plant material of Adonis aleppica Boiss. (total plants, 3.5 kg dry wt) was collected in April 1990 near Urfa (Turkey) and identified by the 3.0, H-I' 6.7, H-fl DEPT Sin C CH C C C CH CH CH C 126.5 107.0 149.5 139.9 149.5 107.0 115.5 148.2 168.2 Me CH CH CH CH CH CH 56.8 Glc 96.0 74.0 78.1 71.1 78.8 62.3 authors. Voucher specimens are deposited at the Heinrich-Heine Universit/it, Diisseldorf, Germany. Instrumentation. NMR spectra were recorded at 300 K on a Bruker AC 200 spectrometer using 5 mm tubes and soins in D M S O - d 6 (99.5% D, 1 and 2) or DMSOd6-CD3OD (1 : 1, 3). The solvent shifts were used as int. standards (DMSO-dr: fin 2.49 ppm, 6c 39.5 ppm). Standard IH (gated-decoupling), laC, APT (Attached Proton Test), DEPT, COSY and HETCOR experiments were measured using the Brucker standard software. DCl-mass spectra were run on a Finnigan INCOS 50 system with NH3 as reactant gas (emitter heating rate 10 mA s -1, calibration with FC43). Middle pressure liquid chromatography (MPLC) prepns were carried out on selfbuilt glass columns (20 cm x 16 mm i.d.) with a Knauer HPLC pump (Model 64), a DuPont detector and additional TLC detection. Gradient elution was performed using corresponding vessels as described in ref. [3]. Droplet counter current chromatography (DCCC) was run on a Biichi 670 apparatus. Molecular calcns were carried out using the Biosym Software Package. Extraction and purification. Whole plants (3.5 kg, airdried) were successively extracted with 231 CHC13, 49 l MeOH and 241 M e O H - H 2 0 (70%) with the UltraTurrax apparatus. The combined extracts (1145 g) were evapd in vacua (40°) to give a brown gummy residue, divided into 7 portions, redissoived in H 2 0 and exhaustively extracted with CHC13-iso-PrOH (3:2) and nBuOH. The CHC13-iso-PrOH (3:2) layers were combined, the solvent was removed in vacua and the residue (162g) filtered over XAD-2 (I kg) in 3 portions by stepwise elution with H20, MeOH and Me2CO. Vacuum liquid chromatography (VLC) of the M e O H eluates (48 g) on cellulose (Avicel~ Merck, stepwise gradient elution with petrol-EtOAc-MeOH) followed by gel filtration on 1250 G.F. PAULIand P. JUNIOR I000 g Sephadex LH-20 (MeOH) afford 3.4 g of an enriched cardenolide mixt. fir. 1) also containing 3. VLC on silica gel 60 (stepwise gradient elution with petrolE t O A c - M e O H - H 2 0 , ---fir. la = 1380 mg) followed by DCCC (CHCI3-MeOH-iso-PrOH-H20 9: 12:1:8, descending mode) led to a crude fr. (140 mg) of 3. The latter was isolated after gel filtration on 120 g Sephadex LH-20 (MeOH) by means of MPLC on silica gel RP-18 using M e O H - H 2 0 gradient elution to yield 98 mg pure 3. The combined n-BuOH layers (105 g) were chromatographed on XAD-2 (1 kg) in 2 portions by stepwise elution with H20, M e O H - H 2 0 (1 : 1) and MeOH. VLC of the M e O H - H 2 0 eluates (33 g) on silica gel 60 and cellulose (Avicel R Merck, stepwise gradient elution with E t O A c - M e O H - H 2 0 gradients, both) led to a fr. (33 g) containing polar cardenolide glycosides, cardenolide sulphates and flavonoid glycosides [3]. Their sepn was achieved by gel chromatography on Sephadex LH-20 followed by DCCC in descending mode where the flavonoids remained in the stationary phase. MPLC on RP-18 silica gel with M e O H - H 2 0 gradient elution yielded pure 1 (50 mg) and 2 (90 mg). lsoetin-4'-O-glucuronide 1. C 2t H 1s O 13, yellow solid (50 mg); UV 2m.x rim: 264, 286(sh), 368 (MeOH); 262, 269, 337, 481 ( + NaOMe, degr.); 270, 291,401 ( + A1Cl3); 270, 291 ( + A1CI3-HCI); 264, 284 (sh), 369 ( + NaOAc); 254, 286, 371 ( + NaOAc-H3BOa); DCI-NHa-MS neg. mode m/z (rel. int.): 158 [glucuronic acid - 2 H 2 0 1 - , 176 [glucuronic a c i d - H 2 0 ] - , 302 [M(agl)]-, 478 [ M ] - ; IH and 13CNMR: Table 1. Vitexin-2"-O-glucoside 2. C27HaoOls , yellow solid (90 mg); UV 2m,x nm: 270, 303(sh), 335 (MeOH); 280, 330, 395 ( + NaOMe); 277, 305, 352, 386 ( + AICI3); 279, 303, 345, 383 ( + AICI3-HCI); 278, 299, 380 ( + NaOAc); 270, 302 (sh), 342 ( + NaOAc-HaBO3); D C I - N H a - M S m/z (rel. int.): 180 [Glc - H 2 0 + NH4] +, 271 [M(agl) + HI +, 325 [(Glc - Glc) - H 2 0 + H] 4, 343 [(Glc - Glc) + H] ÷, 415 [M - Glc + H ] - , 595 [M + H]+; 1H and 13CNMR: Table 2. Sinapoylglucose (3). C17H22Olo , pale-yellow needles (98 mg); mp (uncorr.) 95 o; [~]20 _ 71 ° (MeOH; c 1.00); UV 2m.x rim: 332, 241 228(sh), 206 (MeOH, MeOH + AIC13); 402, 270, 218 ( + NaOMe); 402, 348(sh), 258 (sh), 224 ( + NaOAc); IR !/ma xKBrc m - 1: 3320 (OH), 3060 (CH .... ), 1710 (C = O), 1580 (arom.); DCI-NH3-MS m/z (rel. int.): 404 [MNH4] + (20), 386 [ M N H , - H 2 0 ] ÷ (4), 259 [M - (Glc - H 2 0 ) + NH 4 + NH3] + (8), 242 [M (Glc - H 2 0 ) + N H , ] + (15), 198 [Glc + NH4] + (61), 180 [GIc - H 2 0 + NH4] + (100), 162 [Glc - 2H20 + NH4] + (12); IH and 13CNMR: Table 3. Acknowledgements--The authors are grateful to Dr U. Matthiesen, Heinrich-Heine Universit/it, Diisseldorf, for recording the DCI-mass spectra and to Dr H.-J. Hemmeding and Mr H. Mathew, Heinrich-Heine Universit/it, Diisseldorf, for NMR measurements. REFERENCES 1. Riedl, H. (1963) Ann. Naturhist. Mus. Wien 66, 51. 2. Pauli, G. F., Junior, P., Berger, S. and Matthiesen, U. (1993) J. Nat. Prod. 56, 67. 3. Pauli, G. F. 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