Indian Journal of Chemistry Vol. 43B, July 2005, pp. 1494-1496

1,8-Dihydroxyanthraquinone derivatives from rhizomes of Rheum emodi Wall

Sudhir S Singh1*, Satish C Pandey1, Rajesh Singh2 & Santosh K Agarwal2 1Chembiotek Research International Block-BN, Plot-7, Sector-V, Salt Lake, Kolkata 700 091, India 2Phytochemical Technology Division, Central Institute of Medicinal and Aromatic Plants, PO-CIMAP, Lucknow 226 015, India Email : [email protected] Received 25 July 2003; accepted (revised) 17 January 2004

Two 1,8-dihydroxyanthraquinones isolated from rhizomes of Rheum emodi Wall. have been characterized as 6-methyl- 1 and 6-methyl-aloe- 2 by spectral data and chemical studies.

Keywords: 1,8-Dihydroxyanthraquinone, rhizomes, Rheum emodi Wall, 6-methyl-rhein, emodin

IPC: Int.Cl.7 A 61 K 31/122

Rheum emodi Wall. (Fam : Polygonaceae, English compounds rhein, emodin, aloe-emodin, physcion and Name Rhubarb) is a stout herb and distributed in the chrysophanol from the rhizomes of the same plant4-5. temperate and sub-tropical regions of Asian countries, viz. India (Kashmir, Assam, Sikkim), Nepal, Bhutan Result and Discussion and China. Indian Rhubarb, which is official in Indian Silica gel column chromatography of the MeOH Pharmacopoeia, consists of the dried rhizomes of R. extract afforded compounds 1 and 2. Compound 1 emodi1,2. In a recent study3 the bioassay-guided chemical examination of the rhizomes of R. emodi Table I ⎯ 13C NMR data of compounds 1, 1a, 2 and 2a (75 resulted in the isolation of two new oxanthrone esters, MHz), multiplicities were determined by DEPT revandchinone-1, revandchinone-2, a new anthrax- Carbon 1 1a 2 2a quinone ether revandchinone-3 and a new oxanthrone ether, revandchinone-4. The present communication C-1 161.6 s 157.6 s 160.8 s 156.2 s describes the separation and identification of two new C-2 121.7 d 127.4 d 119.9 d 125.8 d natural products as 6-methyl-rhein 1 and 6-methyl- C-3 136.4 s 135.6 s 143.7 s 142.4 s aloe-emodin 2 from the methanolic extract of C-4 124.0 d 128.2 d 123.2 d 126.4 d rhizomes of R. emodi. The compounds were isolated C-5 123.5 d 127.8 d 124.1 d 128.2 d chromatographically and identified by their spectral C-6 138.5 s 137.6 s 139.6 s 138.4 s data and chemical studies (Table I). In our previous C-7 120.8 d 126.4 d 121.6 d 127.8 d reports, we have described the isolation and C-8 160.8 s 156.8 s 159.4 s 155.8 s identification of two rheinal and C-9 187.8 s 184.6 s 188.2 s 184.4 s rhein-11-O-β-D-glucoside in addition to known C-10 182.2 s 182.4 s 182.8 s 182.9 s C-11 171.6 s 171.8 s 70.2 t 74.5 t OR O OR C-12 21.8 q 21.2 q 21.4 q 20.6 q 8 1 8a 9a C-4a 138.2 s 137.2 s 141.0 s 140.2 s 7 9 2 C-8a 121.2 s 128.4 s 124.6 s 131.2 s 6 10a 4a 3 12 10 C-9a 119.5 s 125.8 s 127.2 s 132.6 s H C R 3 5 4 1 C-10a 136.8 s 134.6 s 140.7 s 139.7 s O Ar-OAc - 168.0 s - 169.2 s

1 R = H, R1 = COOH - 17.2 q - 16.2 q 1a R = Ac, R1 = COOH Aliph-OAc - - - 171.6 s 2 R = H, R = CH OH 1 2 - - - 17.9 q 2a R = Ac, R = CH OAc 1 2 SINGH et al.: 1,8-DIHYDROXYANTHRAQUINONES FROM RHIZOMES OF RHEUM EMODI WALL 1495

gave positive colour reactions6-8 for anthraquinones for four aromatic protons and two phenolic protons and was analysed for C16H10O6. The presence of the were also observed. hydroxyl groups and three types of carbonyls were The 13C NMR spectrum of 2 showed signals for revealed by the strong IR absorption bands at 3448 two carbonyl carbons at δ 188.2 (C-9) and 182.8 (C- (OH) and at 1705 (COOH), 1668 (free C=O) and 10)13, one aromatic methyl at δ 21.4 and one aromatic 1628 (chelated C=O) cm-1, respectively9. hydroxyl methylene at δ 70.2 besides other carbon The mass spectrum of 1 displayed the parent ion signals. Compound 2 on acetylation with Ac2O- + peak at 298 [M] and also strong peaks at m/z 270 and C5H5N gave the known 6-methyl-aloe-emodin-tri- 242 due to successive elimination of carbon monoxide acetate 2a as characterized by comparing the spectral 14 indicating the anthraquinoidal nature of the compound. data with that for an authentic sample . On the basis The fragment ion peak at m/z 135 supported the of the foregoing evidences the structure of 2 has been presence of one hydroxyl and one carboxylic group in established as 6-methyl-aloe-emodin. the same ring and that at m/z 107 showed the presence of one hydroxyl and a methyl group in the other ring10. Experimental Section The 1H NMR spectrum of 1 showed signals for four Melting points were determined on a Toshniwal aromatic protons. A three proton singlet at δ 2.50 melting point apparatus and are uncorrected. IR indicated the presence of an aromatic methyl in the spectra were recorded in KBr on a Perkin Elmer 399B compound. Two phenolic proton signals at δ 12.16 and spectrometer; UV spectra on a Pye Unicam SP8 100 11.86 were also present11. instrument; 1H and 13C NMR spectra on a Bruker WM The 13C NMR spectrum of 1 showed the carbonyl 300 spectrometer (chemical shifts in δ, ppm) with carbon signals at δ 171.6 (COOH), 187.8 (C-9) and TMS as internal standard. EIMS were scanned at 70 182.2 (C-10), the higher δ value for C-9 signal being eV on a JMXDX-300 spectrometer. Silica gel 60-120 due to strong intramolecular hydrogen bonding with mesh (Merck) was used for column chromatography hydroxyl groups4 at C-1 and C-8. Compound 1 on and silica gel G (Merck) for TLC. The spots were acetylation with Ac2O-C5H5N gave a di-acetate 1a as visualized by exposure to I2 vapours and /or by indicated by IR as well as 1H NMR spectra. spraying with 5 % vanillin-sulphuric acid solution 0 The known compound emodin isolated from this followed by heating at 100 C for a few minutes. Plant plant was converted to 6-methyl-rhein12 which was material, rhizomes of Rheum emodi were collected found to be identical with our isolated compound 1 by from the local market and identified in Botany comparing the spectral data and co-TLC. The Department, CIMAP, Lucknow, where a voucher foregoing observations along with specific colour specimen has been maintained. reactions established the structure of the compound 1 as 6-methyl-rhein. Extraction and isolation of compounds Compound 2, a yellow coloured solid also gave the The air dried and powdered rhizomes (2.0 Kg) characteristic colour reactions of anthraquinones6-8 were extracted with MeOH (3 x 3.5 L) in a percolator 9 and was analyzed for C16H12O5. The IR spectraum of at room temperature. After filtration, the dark brown 2 showed the presence of hydroxyl groups (strong methanolic extract was evaporated to dryness under peak at 3426 cm-1), free carbonyl (at 1672 cm–1), reduced pressure below 60°C to get a dark brown chelated carbonyl group (at 1634 cm–1) and aromatic mass (132 g). A portion of the methanol extract (30 g) unsaturation (at 1572 cm-1). Its UV absorption was chromatographed over a silica gel (600 g) column maxima were obtained at 221, 252 and 420 nm and eluted successively with hexane, hexane- characteristic for hydroxyl . The parent chloroform [(3:1), (2:1), (1:1), (1:2), (1:3) v/v], CHCl3 + ion peak was observed at 284 [M] in the EIMS and CHCl3-MeOH [(9:1), (8:2), (7:3) v/v]. The eluates spectrum of compound 2. Two prominent ion peaks at were monitored by TLC and grouped into 11 m/z 256 and 228 were generated due to successive fractions. Fraction no. 5 gave physcion and chryso- loss of CO which indicated the anthraquinoidal nature phanol, fr. no. 7 gave rheinal, fr. no. 8 gave aloe- of compound. The 1H NMR spectrum of 2 depicted a emodin, fr. no. 9 gave rhein and emodin, and fr. no. three proton broad singlet at δ 2.42 for aromatic 10 gave rhein-11-O-β-D-glucoside. Fr. no. 6 (2.38 g) methyl and a two proton signal at δ 4.79 for aromatic eluted with hexane-CHCl3 (1:3) was rechromato- hydroxyl methylene proton11. The presence of signals graphed over a silica gel column using hexane and 1496 INDIAN J. CHEM., SEC B, JULY 2005

increasing proportions of hexane-CHCl3 yielding two room temp. for 18 hr. On usual work up, a triacetate new compounds 1 (94 mg) and 2 (68 mg). 2a (32 mg) was obtained as dark yellowish viscous 6-Methyl-rhein 1: Orange crystalline solid (94 mg), liquid; IR : 1738, 1732, 1668, 1582, 1274 cm-1; EIMS mp 246-48°C; IR : 3448, 1705, 1668, 1628, 1576, 1430 m/z (rel. int.) : 410 [M+] (14); 1H NMR (300 MHz, -1 cm ; UV (MeOH) (log ε) : 232 (4.42), 248 (4.08)), 281 CDCl3) : δ 2.05 (s, 3H, Aliph-OAc), 2.36 (s, 6H, Ar- broad (4.18), 448 sh (4.06), 472 (4.08) nm; EIMS m/z OAc), 2.42 (s, 3H, Ar-CH3), 5.20 (s, Ar-CH2), 6.98 (rel. int.) : 298 [M]+ (18), 284 (48), 281 (100), 270 (d, J=2.6 Hz, 1H, ArH-7), 7.35 (brs, 1H, ArH-2), 7.45 (25), 242(32), 135 (10), 107 (18); 1H NMR (300 MHz, (d, J=1.8 Hz, 1H, ArH-5), 8.16 (d, J=3.4 Hz, 1H,

(CD3)2CO) : δ 2.50 (s, 3H, Ar-CH3), 7.25 (brs, 1H, ArH-4). ArH-7), 7.68 (brs, 1H, ArH-5), 7.87 (d, J=1.8 Hz, 1H, ArH-2), 8.34 (d, J=1.6 Hz, 1H, ArH-4), 11.86 and Acknowledgement 12.16 (s, 2 x OH); Anal. Found : C, 64.82; H, 3.24. The authors are grateful to the Director, CIMAP Calc. for C16H10O6 : C, 64.43; H, 3.38 %. for granting institution fellowship and to the Head, 6-Methyl-rhein-diacetate 1a. Compound 1 RSIC, CDRI, Lucknow for spectral data. (50 mg) was mixed with C5H5N (0.5 mL) and acetic anhydride (0.75 mL) and the reaction mixture was References stirred at room temp. for 18 hr. On usual work up, a rd diacetate 1a (26 mg) was obtained as yellowish solid, 1 Nadkarni K M, The Indian Materia Medica, 3 edn, (Popular Prakashan, Bombay), 1954, 1056. mp 164-66°C; IR : 1740, 1704, 1676, 1584, 1278, -1 + 1 2 Agarwal S K, Singh S S, Lakshmi V, Verma S & Kumar S, 1072 cm ; EIMS m/z (rel. int.) : 382 [M] (16); H J scient ind Res, 60(1), 2001, 1.

NMR (300 MHz, CDCl3) : δ 2.28 (s, 6H, Ar-OAc), 3 Suresh Babu K, Srinivas P V, Praveen B, Hara Kishore K,

2.48 (s, 3H, Ar-CH3), 7.16 (brs, 1H, ArH-7), 7.41 Suryanarayana Murty U &. Madhusudana Rao J, Phytochem, (brs, 1H, ArH-5), 8.23 (d, J=1.4 Hz, 1H, ArH-2), 8.48 62(2), 2003, 203. 4 Agarwal S K, Singh S S, Verma S & Kumar S, Indian J (d, J=1.6 Hz, 1H, ArH-4). Chem, 38B, 1999, 749. 6-Methyl-aloe-emodin 2. Yellow crystalline solid 5 Agarwal S K, Singh S S, Verma S & Kumar S, (68 mg), mp 234-35°C; IR : 3426, 1672, 1634, 1572, J Ethnopharma, 72, 2000, 43. 1436 cm-1; UV (MeOH) (log ε): 221 (2.51), 252 6 Karrer P, Organic Chemistry, Vol. 4, (Nordamann, New (4.81), 266 (3.4), 290 (1.6), 420 (3.6); EIMS m/z (rel. York), 1950, 591. int.) : 284 [M]+ (14), 270 (54), 267 (100), 256 (24), 7 Houben J D, Anthracen und die Anthraquinone in dem zuge 1 horigen, (Viel-Koeringen Svstemen, Leipzig), 1928. 228 (38), 121 (32), 107 (28); H NMR (300 MHz, 8 Robinson T, The Organic constituents of higher plants, (CD3)2CO) : δ 2.42 (s, 3H, Ar-CH3), 4.79 (s, 2H, Ar- (Bargles Publishing Co., USA), 1963, 107. CH2), 5.98 (s, CH2OH), 6.72 (d, J=2.3 Hz, 1H, ArH- 9 Muhatadi F J & Moss M J R, Tetrahedron Lett, 4, 1969, 3751. 7), 6.86 (d, J=1.6 Hz, 1H, ArH-5), 7.15 (d, J=1.8 Hz, 10 Gupta A, Siddiqui I R, Singh J & Sharma J P, Indian J Chem, 1H, ArH-2), 7.39 (brs, 1H, ArH-4), 11.96 and 12.06 37B, 1998, 615. (s, 2 x OH); Anal. Found: C, 67.98; H, 4.06. Calc. for 11 Lee S W, Kuo S C & Chen Z T, J Nat Prod, 57(9), 1994, 1313. C16H12O5 : C, 67.60; H, 4.25 %. 12 Fonteneau N, Martin P, Mondon M, Ficheux H & Gesson J P, 6-Methyl-aloe-emodin-triacetate 2a. Compound Tetrahedorn, 57, 2001, 9131. 2 (40 mg) was mixed with C5H5N (0.5 ml) and acetic 13 Cohen P A & Towers G H N, J Nat Prod, 58(4), 1995, 520. anhydride (1.5 ml) and the reaction mixture stirred at 14 Piattelli M & Nicola M G D, Phytochem, 7, 1968, 1183.