Journal of Natural Products Vol. 5 5 , No. 2, pp. 179-183, February 1992
I79
INHIBITORY EFFECTS OF FLAVONOIDS ON MOLONEY MURINE LEUKEMIA VIRUS REVERSE TRANSCRIPTASE ACTIVITY SHU-CHENCHU, YIH-SHOUHSIEH, and JUNG-YAWLIN*
Institute of Biochemistry, College of Medicine, National Taiwan Univetsity, Taipei 10018, Taiwan, Republic of China Assmcr.-Several flavonoids were tested for their effects on Moloney murine leukemia virus reverse transcriptase activity. Four groups of flavonoids, namely flavones, flavanones, flavonols, and flavanonols, were studied, and it was found that flavonols and flavanonols were very active in this regard while flavones and flavanones displayed very low activity. Among the flavonoids tested, fisetin, quercetin, myricetin, kaempferol, morin, (+)-taxifolin, (+)catechin, and (-)-epicatechin were shown to be highly effective in inhibiting the reverse transcriptase activity. Structure-activity relationship analysis of these flavonoids revealed that the simultaneous presence of free hydroxyl groups at positions 3 and 4’ enhanced the reverse transcriptase inhibitory activity. Replacement of the 3-hydroxyl group with a monosaccharide or ofthe 4’-hydroxyl group with a methyl group reduced inhibitory activity. The double bond at position 2 and 3 of the flavonoid’s pyrone ring is not essential for inhibiting reverse transcriptase activity. The flavonoids studied demonstrated ability to inhibit the reverse transcriptase activity using either (rA)~(dT)12-l,or (rC)n(dG)12-ls as template-primers.
Antiviral drugs have mostly been directed against viral nucleic acid polymerases, and most of the active compounds have been found to be nucleotide analogues. By the use of reverse transcriptase (RT) as a target for antiviral chemotherapy, 3’-azido-2 ‘,3’dideoxythymidine (AZT) ( 1)and phosphonoformate (2) were demonstrated to be selective inhibitors of HIV RT. Suramin (3), HPA23 (4),and 2’,3’ dideoxynucleosides( 5 ) were also demonstrated to be potent RT inhibitors. However, clinical trials of these compounds in AIDS patients revealed side effects such as thrombocytopenia for HPA23 ( 4 )and leucopenia for AZT (6). Natural products represent a potential source of RT inhibitors. In the present study, evidence is provided to show that certain phenolic flavonoids, which are widely distributed in Chinese herbs (7), have an inhibitory effect on RT activity of Moloney murine leukemia virus (MMLV). EXPERIMENTAL MATERIhLs.-Flavonoids were purchased from Sigma Chemical Co., St. Louis, or from Aldrich Chemical Co., Milwaukee. Activated calf thymus DNA, (Riboadenylic acid), (deoxythymidylic acid),,-,,, and (ribccytidylic acid), (deoxyguanylic acid),,-,, were obtained from Pharmacia, Uppsala, Sweden. RT from MMLV is the product ofBethexla Research Laboratories Life Technologies, Inc., Gaithersburg, MD. 13H)dTTP (50 Ci/mmol) and C3H)dGTP (50 Ci/mmol) were obtained from New England Nuclear C o p . , Boston. Escherichia coli DNA polymerase I (DNAP I) was the product of Boehringer Mannheim GmbH, Penzberg, Germany. REVERSETRANSCRIPTASE AssAY.-The reaction mixture (30 ~ 1 contained ) 50 mM Tris-HCI buffer, pH 8.3; 75 mM KCI; 10 mM dithiothreitol; 3 mM MgCI,; 3 Fg nuclease-free bovine serum albumin; 0.5 Fg of (rA)n(dT)12-l, or (rC),(dG),2-l,; 2 mM dTTP or dGTP; 0.5 FCi L3H)dTTP or 13H]-dGTP; and 5 units of MMLV RT preparation (8). After incubation at 37’ for 30 min, 5 FI of 0.2 M EDTA was added to terminate the reaction. The reaction mixture (15 ~ 1 in, duplicate) was spotted on Whatman DE8 1 cellulose paper and washed 6 times with 5% Na,HPO4, 2 times each with H,O and EtOH, and once with Et,O (9).The radioactivity of the samples was measured with a Beckman 5801 liquid scintillation counter. DNA POLYMERASE I AssAY.-The reaction mixture (30 PI) contained 10 mM Tris-HC1 buffer, pH 8.0; 5 mM MgC1,; 10 Fg nuclease-free bovine serum albumin; 1.5 mM dithiothreitol; 5 ug ofactivated calf thymus DNA; 1 FM each ofdATP, dGTP, and dCTP; 0.5 FCi 13H]dTTP, and 1 unit DNAP I. The incubation was carried out at 37’ for 30 min, and the acid-insoluble precipitates were collected and processed for scintillation counting (10).
180
Wol. 55, No. 2
Journal of Natural Products RESULTS AND DISCUSSION
INHIBITORY EFFECTS OF FLAVONOIDS O N M M L V RT.-The effects of flavonoids on RT activity are shown in Table 1 for template-primers (rA)n(dT)12-18 or
(Table values are ID5,J. Template-primer (rA),(dT)
Drug
pghl Flavonols Fisetin . . . . . Kaempferol . . . Morin . . . . . Quercetin . . . Myricetin . . . Myricitrin . . . Rutin . . . . . Flavanonols (+)-Taxifoh . . (+)-Catechin . . (-)-Epicatechin Flavanones Flavanone . . . Hesperetin . . . Flavones 3-Hydroxyflavone 7-Hydroxyflavone
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
. . . . . . .
*-
(KC)n(dG)12-1s
(pM)
pg/ml
WM)
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
0.08 0.35 0.35 0.06 0.08 4.67 4.83
(0.28) (1.22) (1.03) (0.20) (0.25) (10.05) (7.27)
0.07 0.37 0.34 0.05 0.09 4.50 4.87
(0.24) (1.29) (1.01) (0.17) (0.28)
. . . . . . . . . . . . . .
0.74 0.80 0.75
(2.43) (2.75) (2.58)
0.75 0.83 0.77
(2.46) (2.86) (2.65)
. . . . . . . . . . . . . .
. . . . . . . . . . . . . .
(9.69) (7.33)
. . . . . . . . . . . . . . >lo0 (>445.9) . . . . . . . . . . . . . . (20.9) 6.33
>lo0 6.67
(>445.9) (22.1)
. . . . . . . . . . . . . . >lo0 . . . . . . . . . . . . . . > 100
>lo0 > 100
(>419.7) (>419.7)
(M19.7) (>419.7)
(rC)n(dG)12-18.The flavonoids used in these experiments belong to four different structural groups, flavones, flavanones, flavonols, and flavanonols. As indicated in Table 1, with (rA)n(dT)12-18as a template-primer, the flavonols such as fisetin, quercetin, and myricetin were the most effective inhibitors, having ID,,’s between 0.06 and 0.08 pg/ ml. Two other flavonols, kaempferol and morin, have weaker inhibitory effects, with ID,, of 0.35 pg/ml. Flavanonols, such as (+)-catechin, (-)-epicatechin, and (A)taxifolin, exhibited similar inhibitory potencies (ID,,’s = 0.74-0.80 pglrnl), which were less than those of the flavonols. Hesperetin (a flavanone) has moderate inhibitory effects on the RT activity, with an ID,, value of 6.33 pg/ml. Flavones (3-hydroxyflavone and 7-hydroxyflavone) and flavanone have very low inhibitory effects on the RT activity. The extent of inhibition exhibited by these flavonoids was very similar for the two template-primers used.
EFFECTSOF FLAVONOIDS ON DNA POLYMERASE 1.-The inhibitory effects of flavonoids such as myricetin, morin, quercetin, kaempferol, and taxifolin on DNAP I were studied. Concentrations that inhibited DNAP I activity were observed to be tenfold to thirtyfold higher than those required to inhibit MMLV RT using activated calf thymus DNA template (Figure 1). Many flavonoids have been shown to possess antioxidant (12), antimutagenic (1315), and anticarcinogenic (16,17) properties. It is of particular interest to note that some flavonoids have also been shown to possess antiviral activities (18). The present in-
Chu et al. : Effects of Flavonoids
February 19927
181
100
a
50
4
I 0
I
1
I
J
1.0
2.0
3.0
4.0
Concentration
FIGURE 1 .
,
ug/ml
Effects of various flavonoids on the activity of DNA polymerase I. Quercetin (a -- a), myricetin .( -- )., morin ( + -- e), kaempferol (A -- A),and (+)-taxifolin (* -- *).
vestigation demonstrates that flavonoidshave an inhibitory effect on MMLV RT. These flavonoids are ubiquitous in vascular plants, including food plants, and are therefore consumed almost daily (11). Among the four groups of flavonoids tested, flavonols and flavanonols showed the highest activity in inhibiting the MMLV RT. It was observed that the flavones and flavanones show a lower inhibitory activity against RT. This strongly suggests that flavonoids that simultaneously have free hydroxyl groups at position 3 and 4’ exhibit a higher inhibitory activity against RT. When the hydroxyl group at position 3 is glycosylated with rutinose or rhamnose, for example, to form rutin or myricitrin, respectively, the inhibitory activity of these flavonoids is decreased moderately. A similar phenomenon was also observed for the flavonoids when the 4’-hydroxyl group was blocked with a methyl group, such as occurs in hesperetin. The double bond at position 2 and 3 seems not to be essential for the inhibitory activity, as similar high inhibitory activity is found for flavonols and flavanonols (Table 2). It was of great interest to observe that flavonoids were structurally different from U T or dideoxycytidine. In contrast to those compounds, the flavonoidsstudied can inhibit RT activity by using either (rA),,(dT)lz-18or (rC)n(dG)lz-18as template-primers. TABLE2.
Structural Formulae of the Flavonoids.
Flavonols Fisetin . . Kaempferol Morin . . Quercetin Myricetin Myricitrin Rutin . .
. . . .
. . . .
. . . . . . . . . . ...
. . . . . . .
. . . . . . . .. . . . . . . . . . . . .
. 3-, 7-, 3‘-, 4’-OH . 3-, 5-, 7 - , 4’-OH . 3-, 5 - , 7-, 2 ’ - , 4’-OH
. 3-, 5 - , 7 - , 3’-, 4’-OH
. 3-, 5-, 7 - , 3’-, 4’-, 5’-OH
. 5 - , 7 - , 3’-, 4 ‘ - , 5’-OH; 3-O-~-rhamnose . 5 - , 7-, 3’-, 4’-OH;3-0-rutinose
0
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TABLE 2.
Wol. 55, No. 2
Continued
fiavanonols (&)-Taxifolin . . . . . . (+)-Catechin . . . . . . (-)-Epicatechin . . . .
3-, 5 - , 7-, 3'-,4'-OH 3-, 5-, 7-, 3'-, 4'-OH 3-, 5 - , 7-, 3'-, 4'-OH
Flavanones Flavanone . . . . . . . Hesperetin . . . . . . .
5-, 7-, 3'-OH; 4'-Me
Flavones 3-Hydroxyflavone . . . . 7-Hydroxyflavone . . . .
3-OH 7-OH
This suggests that flavonoids can inhibit RT by competing with either (rA)n(dT)12-18 or (rC)n(dG)12-18as template-primers while AZT can compete only with (rA)n(dT)12-18 (8,19). Many natural flavonoids, including some studied in the present investigation, form part of the daily diet, particularly for Chinese people, who traditionally eat more vegetables than meat. ACKNOWLEDGMENTS This work was supported in part by grant NSC-80-0420-B002-27 from the National Science Council, Republic of China. LITERATURE CITED 1. 2. 3. 4. 5. 6.
7. 8. 9. 10. 11. 12. 13.
B. Oberg,J. Antimimb. Chemother., 17,549 (1986). H. Mitsuya, K.J. Weinhold, P.A. Furrnan, M.H. St. Clair, S.N. Lehrrnan, R.C. Gallo, D.P. Bolognesi, D. W. Barry, and S. Broder, Proc. Natl. Acad. Sri. USA, 8 2 , 7096 (1985). H. Mitsuya, M.Popovic, R. Yarchoan, S. Matsushita, R.C. Gallo, andS. Broder, Science, 226, 172 (1984). W. Rozenbaum, D. Dorrnont, B. Spire, E. Vilrner, M. Gentilini, C. Griscelli, L. Montagnier, F. Bard-Sinoussi, and J.C. Cherrnann, Lancet, 1,450 (1985). H . Mitsuya and S. Broder, Proc. Natl. Acad. Sri. USA, 83,19 11 (1986). D.D. Richrnan, M.A. Fischl, M.H. Grieco, M.S. Gottlieb, P.A. Volberding, O.L.Laskin, J.M. Leedorn, J.E. Grooprnan, D. Mildvan, M.S. Hinch, G.G. Jackson, D.T. Durack, D. Phil, S. Nusinoff-Lehrrnan, and the AZT Collaborative Working Group, N . Engl. ]. Med., 317, 192 (1987). E. Wollenweber and V.H. Dietz, Phytochemistry, 20, 869 (1981). L. Vrang, H . Bazin, G. Remand, J. Chattopadhyaya, and B. Oberg, AntiviralRes., 7, 139 (1987). Y.L. Shen, W.C. Ye, S.X.Zhao, Y.Z. Shu, M. Wataya, N. Kakiuchi, M. Hattori, K. Matsuura, and T. Namba, Shoyakugaku Zusshi, 42, 35 (1988). A. Schlabach, B. Fridlender, A. Bolden, and A. Weissbach, Biochem. Biophys. Res. Cwnm., 4 4 , 8 7 9 (197 1). K. Herman,]. FwdTarhnol., 11,433 (1976). J. Kuhnnan, Wwld Reu. Nutri. Diet, 24, 117 (1976). M.T. H u n g , A.W. Wood, H.L. Newrnark, J.M. Sayer, H . Yagi, D.M. Jerina, andA.H. Conney, Carcinogenesis, 4 , 163 1 (1983).
February 19921 14.
15. 16. 17. 18. 19.
Chu et a / .: Effects of Flavonoids
183
S. Ogawa, T. Hirayama, M. Nohara, M. Tokada, K. Huai, and S. Fukui, Mutut. Ra.,142, 103 (1985). C.M. Steeles, M.Lalies, and C. Ioannides, CumerRer.,45, 3573 (1985). R.L. Chang, M.T.Huang, A . W . Wood, C.Q. Wong, H.L. Newmark, H . Yagi, J.M. Sayer, D.M. Jerina, and A.H. Conney, Curcinogmcris, 6, 1127 (1985). J.E. Nixon, J . D . Hendriks, N.E. Pawlowski, C.B. Percera, R.O. Sinnhuber, and G.S. Baily, CurrinogmiJ, 5, 615 (1984). R. Vrijsen, L. Everaerr, and A. Boeye,J. Gm. Virol., 69, 1749 (1988). K. Ono, H. Nakane, M.Fukushima, J.C. Chermann, and F. Bark-Sinoussi, Ear.]. B i o c h . , 190, 469 (1990).
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