Fitoterapia 81 (2010) 1224–1227

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Fitoterapia

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Cytotoxic prenylated flavonoids from Morus alba

Nguyen Tien Dat a,⁎, Phung Thi Xuan Binh a,b, Le Thi Phuong Quynh b, Chau Van Minh a,Hoang Thanh Huong b, Jung Joon Lee c

a Institute of Marine Biochemistry, Vietnam Academy of Science and Technology, 18-Hoang Quoc Viet, Hanoi, Vietnamb Institute of Natural Product Chemistry, Vietnam Academy of Science and Technology, 18-Hoang Quoc Viet, Hanoi, Vietnamc Molecular Cancer Research Center, Korean Institute of Biosciences and Biotechnology, Ochang, South Korea

a r t i c l e i n f o

⁎ Corresponding author. Tel.: +84 4 38363375.E-mail address: ngtiend@hotmail.com (N.T. Dat).

0367-326X/$ – see front matter © 2010 Elsevier B.V.doi:10.1016/j.fitote.2010.08.006

a b s t r a c t

Article history:Received 16 June 2010Accepted in revised form 11 August 2010Available online 19 August 2010

A phytochemical fractionation of the methanol extract of the Morus alba leaves led to theisolation of eleven flavonoids (1–11). The structure of the new 3′-geranyl-3-prenyl-2′,4′,5,7-tetrahydroxyflavone (1) was elucidated by means of spectroscopic methods. The cytotoxicityof the isolated compounds against human cervical carcinoma HeLa, human breast carcinomaMCF-7, and human hepatocarcinoma Hep3B cells was evaluated. Of note, morusin (9) was themost potent with an IC50 value of 0.64 μM against HeLa cells.

© 2010 Elsevier B.V. All rights reserved.

Keywords:Morus albaMoraceaePrenylated flavonoidMorusinCytotoxicity

1. Introduction

Morus alba L. (Moraceae), known as white mulberry, is aspecies native to China and now widely cultivated in othercountries. The different parts of this plant have been used in thetraditional Chinese medicine for many purposes. The whitemulberry leaves, an important food for silkworm, are used totreat hypertension, arthritis, and the fruit is a diuretic and tonicagent. The root bark of the plant is considered as an importantmedicine to treat cough, inflammation, diabetes, cancer, hepatitisand heart diseases [1]. Previous studies showed that M. albamainly contained polyphenolic constituents including preny-lated flavonoids, benzofurans and Diels–Alder type adducts withvarious biological activities such as cytotoxicity, antioxidant,inhibition of NF-κB, LOX-1, cancer cell invasion and migration,andhepatoprotection [2–6]. The glycosidase inhibitory activity ofseveral alkaloids inM. alba has also been reported [7].

In our ongoing research on the chemistry and biologicalactivity of the traditional herbs, we found that the MeOH

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extract of theM. alba leaves showed strong cytotoxicity againstcancer cells. A phytochemical investigation of theMeOHextractof the M. alba leaves led to the isolation of a new (1) and tenknown (2–11) flavonoids (Fig. 1). The structure of newcompound 3′-geranyl-3-prenyl-2′,4′,5,7-tetrahydroxyflavone(1) were elucidated by spectroscopic methods and the knowncompounds were identified as 3′,8-diprenyl-4′,5,7-trihydroxy-flavone (2) [8], kuwanon S (3) [9], 8-geranylapigenin (4) [10],cyclomulberrin (5) [11], sanggenon J and K (6, 7) [12],cyclomorusin (8), morusin (9) [13], atalantoflavone (10) [14],and kaempferol (11) [15]. The isolated compounds showedstrong cytotoxicity against HeLa, MCF-7, and Hep3B cell lines.

2. Experimental

2.1. Instruments

UVspectrawere recordedon anUV-1601 spectrometer. NMRexperiments were performed on a Varianunity Ionva-400 instru-ment using TMS as internal standard. ESI-MS and HR-FAB-MSspectrawere recorded on a FinniganNavigator LC/MS/DS systemand a JMS-HX/HX110A tandemmass spectrometer, respectively.

Fig. 1. Structure of compounds 1–11 isolated from M. alba leaves.

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2.2. Plant material

The leaves of M. alba were collected in March 2010 in MyDuc, Hanoi, Vietnam and identified by Dr. Tran Huy Thai,Institute of Ecology and Biological Resources, VietnamAcademy of Science and Technology. A voucher specimen(No. NCCB032010) has been deposited in the Department ofBioacive Products, Institute of Marine Biochemistry.

2.3. Extraction and isolation

The air-dried and powdered leaves of M. alba (3 kg) wereextracted with methanol (5 L×3 times) at room temperature.The combined extracts were concentrated to obtained crudemethanol residue (250 g)whichwas resuspended inwater (2 L)and successively partitioned in hexane (1 L×3 times) and ethylacetate (1 L×3 times). The organic layers were concentrated togive 74 and 55 g of hexane and ethyl acetate residues,respectively. A fractionation of the ethyl acetate extract on asilica gel column eluted by a gradient of 0–100% methanol inchloroformaffordedeight fractionsF1–F8. FractionF6waspassedover aSephadexLH20columnusing chloroform–methanol1:1asthe mobile phase to give 1 (32.4 mg), 8 (3.6 mg), and 6(70.0 mg). Fraction F3 was chromatographed on a silica gelcolumn eluted by hexane–ethyl acetate (4:1 v/v) to afford 7(6.2 mg), 5 (5.5 mg) and 9 (8.1 mg). Compounds 2 (3.0 mg), 3(18.5 mg),4 (3.6 mg)and10 (6.6 mg)were separated fromF4byusing a reverse phase column eluted with methanol–water(10:1 v/v). Compound 11 (11.0 mg) was purified from F7 on asilica gel column using a mobile phase of hexane–ethyl acetate(1:1 v/v).

3′-geranyl-3-prenyl-2′,4′,5,7-tetrahydroxyflavone (1): yellowamorphous powder; UV (MeOH) λmax (log ε) 216.5 (4.49),258.4 (4.37), 328.5 (4.20); 1H NMR (400 MHz, acetone-d6): δ13.10 (1H, br s, 5-OH), 7.01 (1H, d, J=8.4 Hz, H-6′), 6.57 (1H,d, J=8.4 Hz, H-5′), 6.30 (1H, d, J=2.0 Hz, H-8), 6.23 (1H, d,J=2.0 Hz, H-6), 5.32 (1H, t, J=7.2 Hz, H-2″), 5.08 (2H, m,H-7″, 12), 3.46 (2H, br d, J=7.2 Hz, H-1″), 3.08 (2H, br d,J=6.8 Hz, H-11), 2.05 (2H, m, H-6″), 1.97 (2H, m, H-5″), 1.78(3H, br s, H-4″), 1.61 (3H, br s, H-9″), 1.55 (3H, br s, H-10″,14), 1.37 (3H, br s, H-15); 13C NMR (400 MHz, acetone-d6): δ162.1 (C-2), 122.2 (C-3), 183.9 (C-4), 159.4 (C-5), 99.2 (C-6),164.8 (C-7), 94.3 (C-8), 163.4 (C-9), 105.4 (C-10), 24.6(C-11), 122.4 (C-12), 132.3 (C-13), 25.9 (C-14), 17.7 (C-15),113.2 (C-1′), 154.5 (C-2′), 117.0 (C-3′), 158.8 (C-4′), 108.8(C-5′), 128.8 (C-6′), 23.1 (C-1″), 123.6 (C-2″), 135.4 (C-3″), 16.4(C-4″), 40.6 (C-5″), 27.5 (C-6″), 125.3 (C-7″), 131.7 (C-8″), 25.9(C-9″), 17.7 (C-10″); ESI-MS m/z 489.5 [M−H]−; HR-FAB-MSm/z 491.2434 [M+H]+ (calcd. for C30H35O6, 491.2433).

2.4. Cell culture

Human cervical adenocarcinoma HeLa, human hepatocar-cinoma Hep3B, and human breast carcinoma MCF-7 cellswere obtained from American Type Culture Collection(Manassas, VA). HeLa and Hep3B cells were cultured inDulbecco's modified essential medium, and MCF-7 cells weremaintained in a RPMI-1640 medium in a humidified 5% CO2

atmosphere at 37 °C. All media were supplemented withpenicillin (100 U/mL), streptomycin (100 μg/mL) and 10%heat-inactivated fetal bovine serum (FBS).

Fig. 2. HMBC correlations of 1.

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2.5. Cytotoxicity

Cytotoxic assay was carried out by the 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. TheHeLa, Hep3B and MCF-7 cells seeded in 96-well plates at aconcentration of 1×104 cells/well were treated with variousconcentrations of test compounds and incubated in a humid-ified 5% CO2 atmosphere at 37 °C. After 72 h incubation, 10 μL of5 mg/mL MTT was added to each well and incubated foranother 4 h. After removal of the supernatant, formazan crystalswere dissolved in 100 μL DMSO and the OD values weremeasured at 570 nm with a microplate reader. An antitumoragent, deguelin, was used as the positive control [16].

3. Results and discussion

Compound 1 was obtained as a yellow amorphous powderand itsUVspectrumshowed absorptionmaximaat216.5, 258.4and 328.5 nm. The positive HR-FAB-MS spectrum of 1 revealedthe [M+ H]+ atm/z 491.2434 corresponding to the molecularformula C30H35O6. The 1H and 13C NMR data of 1 showed thatthe compound was closely related to the co-occurring 6 and 7except for the appearance of a geranyl group instead of thechromenesmoiety. TheHMBC correlations (Fig. 2) showing the

Table 1Cytotoxicity (IC50 in μM) a of compounds 1–11 isolated from M. alba leaves.

Compounds HeLa

3′-Geranyl-3-prenyl-2′,4′,5,7-tetrahydroxyflavone (1)

1.32±0.51

3′,8-Diprenyl-4′,5,7-trihydroxyflavone (2)

1.66±0.27

Kuwanon S (3) 1.64±0.218-Geranylapigenin (4) 2.24±0.48Cyclomulberrin (5) 3.69±0.86Sanggenon J (6) 2.28±0.40Sanggenon K (7) 2.29±1.64Cyclomorusin (8) 1.66±0.27Morusin (9) 0.64±0.14Atalantoflavone (10) 1.25±0.46Kaempferol (11) 23.05±4.15Deguelin 6.4±1.68

a Data are means±SD from three distinguished experiments.

coupling from proton H-1″ (δH 3.46) of the geranyl groupcoupled to C-2′ (δC 154.5), C-3′ (δC 117.0) and C-4′ (δC 158.8)confirmed the geranyl group coupled to C-3′. In contrast, theprenyl methylenic protons H-11 (δH 3.08) correlated with C-2(δC 162.1), C-3 (δC 122.2) and C-4 (δC 183.9) indicating that theprenyl moiety is attached to C-3. Thus 1 was newly elucidatedas 3′-geranyl-3-prenyl-2′,4′,5,7-tetrahydroxyflavone. Com-pounds 6 and 7 may be biosynthetized from the precursor 1through the cyclization of the geranyl chain with the adjacenthydroxy groups 4′-OH and 2′-OH, respectively [17].

The cytotoxicity of the isolated compounds was evaluatedby the MTT method. As showed in Table 1, the prenylatedflavonoids exhibited strong cytotoxic effects against threecancer cell lines while kaempferol showed a weak effect. Thisindicated that the prenyl or geranyl group as well as theircyclization with the hydroxy group increased the cytotoxicityof flavone. Morusin (9) was the most potent against the HeLacell linewith an IC50 value of 0.64 μM. This effectwas significantin comparison with an antitumor agent, deguelin. It hasreported that morusin at 29 μM induced apoptosis of humancolorectal cancer HT-29 cells by activating caspase-3, 8 and 9proteins and inhibiting nuclear factor κB signaling [18]. Thusthe potent cytotoxic effect of morusin against HeLa cells in thepresent study could be due to similar mechanisms. Further

MCF-7 Hep-3B

3.92±0.91 5.22±0.95

5.27±1.02 4.71±1.04

7.02±1.66 8.47±2.073.21±0.87 3.65±0.817.19±0.77 6.64±1.234.56±0.71 5.30±1.453.51±0.59 3.09±0.677.85±1.30 7.55±2.147.88±1.89 9.21±2.066.54±1.23 4.33±0.75

21.47±3.92 36.18±6.865.3±1.32 29.3±3.14

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study is needed to clarify the anticancer ability of morusin onHeLa cells.

Acknowledgments

This work is supported by a grant from the Vietnam'sNational Foundation for Science and Technology Development(NAFOSTED-104.01.41.09) to one of the authors (N.T.D.).

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