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Composition and antiviral activities of a sulfated polysaccharide from schizymenia dubyi (rhodophyta, gigartinales)
Bioorgmic Q hfedicti ChemistryLetters, Vo1.3.No.6, pp. I 141-l 146.1993 Rited in Great Britain
0960-894x/93 $6.00 + .oo 0 1993 Pergamon Press Ltd
COMposTIoN
AND ANTIWRa NTNITIWOFASULFATBD POLYSACCHARIDB FROM DclB3T (RHODOI’EYTA, GIGARTINAUS)
Nathalie BOUKGOUGNON ‘, Marc LAHAYJJ b, Jean-Claude CHERMANN ’ and Jean-Michel KOKNPKOBST rz ; ISOMer, Groupe SMAB, Rtcultb de Phamack, I, rue G. Veil, 44035 Names Cedex Ol, Prance. Laboratoire de Biochimie et Technohqie des Ghtcides, INKA, BP 527,44026 Names Cedex 03, France. ’ INSEKM U 322, Campus Universitaire de Luminy, BP 33, I3273 Mar.sdle Ccdcx 09, Fkance.
(Received in USA 29 December 1992)
Matract. &&qmctia dubpi (Gig-s, Gymnophlaeaceae) contains an unusual sulfated heteropolysaccharide with uranic acids that is active against several viruses in&ding human immunodeficiency virus type-l (NIV-l), Herpes simplex bominis type 1 and type 2 (HSV-1 and HSV-2) and vesicular stomatitis virus (VSV).
Some polysaccharhles have shown io via0 activity against animal viruses incl~ adenovirus type 5 (Ad5), African swine fever virus (ASP), vesicular stoma&is virus (VSV), polio virus type 2, Sembki forest virus (SPV)~ * and Hcq.rcs sin@exllamiois virus (HSV)3. In recent years, sulfated po&darides infecti~r$ have also demonstrated in tifro inhibitory effects on human immumxMiciency virus (HIV) . Ked seaweeds contain large amounts of cell-wall polysac&arides, most of which are sulfated galactans These galacums are generally built on repeated alternating l$3-linked c&galactopyranose and l&-linked FDgalactopyranose units and differ in the level and pattern of suJfation, in the substitution of methoxyl and/or pyruvate groups and in other sugar residues (galactose, xylose). They also differ in 3,6anhydrogalactose content and the confiiation of the L3-linked a-galactopyranose residue, Among these agars are widely used as gelling or thickening additives by the food industry w, carrageenans , A sulfated polysacchatide is&ted from the red alga VpacifJca and in biotechnologies $“d has . The chemical displayed an inhibitory effect on HJV reverse transcriptase and io virro replication characteristics of this sulfated gahMan suggest that it belongs to the carrageenan family. The purpose of this work was to study the chemical composition of water-soluble polysaccharide from Sc&wm& dubyi (Chauvin ex Duby) J. Agardh (Rhcdophyta, Gigartinales, Gymnophlaeaceae) and its &I vitro activity against human immunodefii virus @IV), Hwpcs s@&x&amiais virus, vesicular stoma&is virus and polio virus. Extract.iattedu@uaand~analy!&methabL Gametophytic w dubyisamples were collected in May I991 on the east coast of Sicily. Water extractiom Sulfated polysac&ar& from air-dried seaweed powder (5 x 20 g, < 5 mm particle sire) was extracted in hot distilled water (5 x LS L) at 8o’C for 4 h with magnetic stirring, Separation of the soluble extract from insoluble debris was doue by filtration on diammaceou s earth, The filtrate at 5-c was poured into 2 vohtmes of absolute ethanol, with stirring. The precipitate was recovered and washed with 95’ ethanol, dehydrated with diethyl ether, dried ovemight at 5o’C, weighed and ground to a powder. The polysaccharide was redissolved in distilled water, dialysed against d.istilM water and B (l82O%yieldw/wontheinitialalgalweightbasis). Alkali extraction: Algal powder (LOOg) was treated with 12 L containing NaOH (0.3 M) and KCl (I6 I@ in distilled water without sodium borohydride at 8o’C for 4 h Polysaccharide was recovered from the alkali sohttion as above.
1141
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N. BOURGOUGNON et al.
Uranic acids in polysaccharide were reduced according to the method of Taylor and Conrad”. The acid form of the polysaccharide was obtained as follows: polysaccharide (50 mg) was dissolved in distilled water (5 mL), and the solution was eluted through a column of ion exchange resin (Amberlite IRl20 plus). The column was then washed with distilled water and the polysaccharide was recovered and freeze-dried. After sulfuric acid hydrolysis of the polysaccharide (2N, lOOC, 2 h), neutral sugars were derived into alditol acetate’ and identified and quantified by gas liquid chromatography (GC) on a DB225 (IW Scientific) fused silica capillary column eluted with hydrogen at 21o’C. 3,Qmhydrogalactose content was determined by calorimetry according to the method of Yaphe and Arsenault . Uranic acids of the acid hydrolysates resulting from the neutral sugar analysis by GC were measured by calorimetry according to the method of Ahmed and I.&witch”. Identification of the uranic acids in the trifluoracetic acid polysaccharide hydrolysate (2N, l2o’C, 2 h) was attempted by high pressure liquid chromatography (HPLC) using an Aminex 87H column (Biorad) eluted with 10 M sulfuric acid and with differential refractive index detection. Protein content was measured by the Kjeldahl method (Nx 6.25). Sulfate content in polysaccharide (lg) was performed gravimetrically after complete hydrolysis (HCl IN, 200 ml, lb, SO’C followed by HCl lN, H202 110 vol. (8: 1 v/v, S h, 8o’C) followed by quantitative precipitation of the released sulfate by BaCl . Infrared spectra were recorded from a KBr pellet of the acid polysaccharide on a IRS 25PT (Bruier Instrument). Proton broadband decoupling ‘C NMR of the acid-degraded water-soluble polysaccharide (HCl lN, 90 mitt, 8o’C) in D20 was recorded on a Bruker AM 300 WB spectrometer at 75.5 MHz (room temperature). Chemical shtfts in ppm were measured in relation to TMS as external reference. Dete.rmillatkmofantiviraIa&vityofauRk4tedpoaysaccbari&. Monkey kidney cells (VRRO cells Plow Lab. ref 03230) were cultured at 37’C in Eagle’s @imal essential medium (MEM) with Rarle’s saline supplemented with l0 % inactive fetal calf serum . HSV type l, HSV type 2, polio-2 virus and VSV were isolated at Pontchaillou Hospital, Rennes, Prance. Virus stocks were prepared by inoculating VERO monolayers at low multiplicity of infection and incubation at 37’C. Two days after infection, cultures were froxen and thawed, and stocks were maintained at -70’C. Virus titration was performed by the Reed and Muench dilution method”. Viral titers were estimated from cytopathogenicity and expressed at 50 9b of the tissue culture infective dose (TCID, test: The method used was essentially that described by Van Den The cytotoxic concentration (CyD ) value was defined as the maximum drug concentration causing cytotoxicity effects in 50% of the cultured cells after 96 h of incubation at 37’C. Values lower than those obtained in these tests were used in antiviral tests. Antiviral activity was detected by measuring the inhibition of cytopathic effect (CPE), with estimation of a 50% antivirally effective concentration (RD ) and therapeutic index (TI)22. Cell monolayers were infected with virus at low multiplicity [lD2 and lO@DI (50% infective doses) per microtiter plate] in the presence of polysaccharide at various concentrations. k%er several virus replication rounds (72 h for HSV, 32 h for polio and 28 h for VSV at 37-C), the cytopatbic effects in infected and uninfected cells from 6 wells for each virus concentration of polysaccharide were examined under a phase-contrast microscope. To assess the effect of polysacharide on uninfected MT4 cells, dilutions ranging from 1 mg/mL to @g/n& in the maintenance medium were added to MT4 celI line at 37’C with 5% CO, during lh on 96 well microtiter plate containing 3x105 MT4 cells for lOOI.&of compounds (in duplicate). The cells were then reajusted to 3x10’ MT4/mL on 24-well microtiter plate containing various concentrations of polysaccharide. After 4 days the cells were diluted to l/3 with the corresponding dilution of compound. Reverse Transcriptase (RT) assay: 1 mL sample of cell supematants were concentrated 100 fold by ultracentrifugation at 95000 rpm at 4-C for 5 minutes in a TLlOO rotor (Beckman). The pellet was resuspended in lOpL of NTR buffer contaiuing 0.1% Triton X-100. The enxymatic reaction was performed in a 5Op.L of a reaction mixture containing: Tris 50mM, pH 7.8 ; MgC$ 20 mM ; KC1 20 mM ; dithiothreitol2 mM ; Oligo(dT) l2-ltS 0.25 OD/mL ; poly(rA) 0.25 OD/mL and 3HdTI’P 2.5pCi. After lb at 37-C, the reaction products were precipitated with 20% trichloracetic acid, filtered on MilIipore 0.45t1rr1 and the 8 radioactivity was measured.
A sulfated polysaccharide from Schizymeniadubyi
1143
Determination of antiviral activity with HIV-l was based on measuring the protective effect of the sulfated polysaccharide against this virus-induced cytopathogenicity in MT4 cells. Four to six days after infection, multinucleated giant cell (syncitium) formation was observed, preceding cell death. MT4 cells were incubated in RPMI l64O culture medium with lO% fetal calf serum (PCS) and 1% PSN antibiotics, 1% glutamine and 2pg/mL polybrene at 37-C during 1 h on 96-well microtiter plate containing various concentrations of sulfated polysaccharide (3xlO” cells for lOO l.tL of compound). DO I.tL of HIV-1 (dilution lO,“) suspension was added in wells. One hour after incubation, infected cells were washed three times with RPM1 and then cocultured at 3x10’ cells/mL on 24-well microtiter plate containing various concentrations of the sulfated polysaccharide. Every three or four days, cells were diluted three times and the cell concentration was adjusted at 3~10~ cells/mL RPMI, 10% PCS, 1% glutamine and 2pg/mL polybrene in the presence of sulfated polysaccharide. Three days after cultures, infected MT4-HIV-1 cells with and without sulf a@ polysaccharide and uninfected MT4 cells were observed for syncitial formation every one or two days . The cheakxl composition of the water-soluble polysaccharhk is shown in Table L Table 1
Fig. 1
Chemical composition of the sulfated polysacchar& from .9&&n&a dubyf
Water-extracted polysaccharide
Neuid!: xylose mannose
29.9
m&k-a
of phycocolloid sch&mc&dubyi
Reduced polysaccharideb 25.2 L6
;3”
glUCOSC
3danhydrogalacto~ Uranic acids Sulfate Protems Ash
E 33.7 15.5
::; 2.8
lE
Neither galacturonic, mannuronic nor glucuronic acid was detected by HPLC analysis of an acid hydrolysate of the polysaccharide. Two successive reductions of the polysaccharide decreased the uranic acid content from 33.7 to 2.8%. Neither marked increase of a parkular sugar (except a small increase in glucose) nor the appearance of a new sugar was detected gas chromatography after reduction. The 9 C NMR signal at I77 ppm for carboxylic presence of uronides was confirmed by the characteristic carbons and by typical absorbance at 1732 cm-’ on the infrared spectrum _y the polysaccharide in acid form. The latter spectrum also confim& the presence of sulfate (1234 cm ), and the absorbance at 8% and 820 cm-1 indicated that sulfate groups might be located on carbon 4 and 6 of the galactose units (Pig. 1). Sulfate content (M.O%) and infrared spectrum Of the alkali-extracted polysaccharide Were similar to those of the water-extracted polymer. No methyl from methoxyl or pyruvate groups was detected by DC NMR specuoscopy. The in vitro antiviral activities of the water-extracted polysaccharkk from m dub* are shown in Table 2. The maximal non-toxic concentration of the polysac&ar& for the VBRO cell line was 93.7 q.g/mL, giving a cytotoxic concentration of CyD at 187.5 Pg/mL. The polysaccharide was most *?! virus. The compound was toxic on the MT4 cell effective against HSV-1 and least effective against poll0 lines within the concentration range of hng to SOcrg/mL while toxicity was much less at 25wmL, Y)crgtmL and ll@mL doses showed no appamnt cytotoxichy.
1144
N. BOURGOUGNON er al.
Table 2 activity of water-soluble polysaccharide from &t&j against HSV-1, HSV-2, VSV and Polio2 viruses
In vitro antiviral
Sch+mc& viruses
TIb
EDJO wnWa IO’D$c /SOcLL”
loLDI, /50clLc
‘DLD’s /50pLc
lo&)
/50@
WV-1
l.5
5.0
125.0
37.5
HSV-2
25.0
6.0
75.0
31.2
vsv
lO.0
15.0
lg.7
l2.5
Polio-2
44.0
30.0
4.0
2.0
a antiviraUy efkctive ctmmccm
indcxz CyDsdgDso
b Tlwqmic
Evaluation of the anti-HIV effect of the polysaccharide (Table 3) indicated that syncitial formation was completely suppressed at 20 pg/mL and delayed at lOccg/ml. No toxicity was found at these concentrations. Furthermore, the sulfated polysaccharide is inhibitory to the HIV-associated Reverse TrarWliptase. Table 3 Evaluation of antiviral effect for polysaccharide with HIV-l D4*
D5
D6
D7
500
-frox
lo0
_ _
-fTox _ -
Tox - _
Tox - _
I781156
50
_ -
_ _
- _
- _
l40/5l2
20
_ -
_ -
- _
- _
4Wl34
lo
_ _
_ _
i-k/T u/r
7OOll666
2
++++
++ ++
++fr
i+
i-+/T u/r
39874P8632
0.2
uu
++ ++
++/r
++
u/r
0.0
+ (+1
u
++fl
++
0.0
_ -
po&acs
R;rEl$$$$$I
(cI.B/mL) MT4-HIV-1
MT4 =hY;
-:aba?oceofsyacytia;
++ _ _
+:prmumofsyacytia;~:Byncytia
+++
- _ ;T:
ufr
IWIW
u/r u _ _
2047o/lO984 l36Jm4
cdldcatt~;Tox:Toaicity.
The chemical composition of Sc&ymun~a dubs water-soluble polysaccharide~has never been descri$d in the literature, although that of S. pcilice has been reported by Whyte et al. and Nakashima etal. . Whyte etal. reported galactose, 3,6-mhydrogalactose and sulfate in a molar ratio of LO.04, and 0.75 respectively, with the presence of 0.5% methyl and the absence of uranic acids. Nakashima et&. reported galactose, 3,6_anhydrogahuztose and sulfate in a molar ratio of 1, 0.01 and 0.51 respectively and attributed this sulfate gahWan to a kumgeenan. B water-soluble polysac&aride from S. dut& another sulfated galactan encountered in many red algae ’ , differed because of its unusually high content of
A sulfated polysaccharide from Schizymenia dubyi
1145
uranic acids. Neither the methoxyl nor % pyruvate group was associated with these polymers. The presence of uranic acids was con6medby CNMRandIRspectroscopyand&micalreduction,butthe sugar-bearing carboxylic group remained unidentified. Uranic acids have been rarely reported in polysaccharides from marine red “1~ whereas D-glucur@c acid is present in the mucilage of several unicellular red algae. Craigie et al. and Okaaaki et al. reported guluronic-rich alginate-like polysaccharides in Serraticardia maxima and other coralline red algae. The galactan of Dilsea e&&3 contains 9.5 to ll% uranic acids’, although it has recently been reported that the Atlantic and Pacific gametophytes and tetrasporophytej,from Dilsea, Halymenia, Cryptonemia and Schizymenia species are very anatonomicallysimilar . The similarity in sulfate content and between IR spectra of the water- and alkali-extracted polysac&ride suggests that $e sulfate groups are alkali-stable and thus not located on C-6 or C-3 of 4linked galactose residues . Research is in progress in our laboratory to determine the identity of the uranic acid and the position of the sulfate groups on the water-soluble sulfated galactan from S. dub* Antiviral activity of sulfated polysaccharides has been recognixed for 0 e years. As early as 1947, polysaccharides from diverse sources were found to inhibit viral growth ,%B* The polysaccharide-rich fractions from two marine red algae, Cryptosrphtia woodii and Farlowia mollis (Dumontiaceae), were found to exhibit io t&o activity against HSV-1 and HSV-2, vaccinia virus and vsv== . In recent years sulfated polysaccharides have been shown to inhibit in vitro replication of many enveloped viruses: including HIV=! A sulfated polysaccharide isolated from the sea alga Sch@me&paci6ca may also suppress in vitro HIV infection by interfering with virus adsorption as well as inhibiting reverse transcriptasell’ a. Multinucleated giant cell formation induced by interaction between the glycoprotein expressed on the surface of cells infected with HIV-l and the receptor of u&f&ted cells may play an impor “-;” role in the depletion of T4 lymphocytes in acquired immune deficiency syndrome (AIDS) are inhibitors of giant cell patients . Pentosan polysulfate, fucoidan, mannan sulfate and karrageenan formation and hence protect target cells against destruction by killer HIV-1-infecte&cells. Their mode of actionhavebeenamibutedto~ninhibitionof~~ntotha~llmembram. In the present study, the water-soluble polysaccharide of &@ymenk dub9 showed activity against HSV-I, HSV-2 and VSV, but the relation between the chemical strucuue and antiviral activity remains to be determined. The antiviral mode of action of this polysacc hari&ais now under investigation. As the ,thefinechemicalstructureofthis nature of anionic groups are a decisive factor in anti-HIV process polysaccharide is currently being studied in our laboramry.
We thank Dr. M. CORMACI and G. FURNARI from the Istituto e Orto Botanico, Universiti di Catania, Italy for collecting the algal material, Dr. F. ARDRR from the Museum National d’Histoire Naturelle, Paris, France, for identification of v do&$, Dr. N. SINBANDHlT from the Centre R6gional de Mesures Physiques de l’ouest, Universiti de Rennes, France, J. A. MONTANHA from the team “Etudes et Bioproduction de Molecules Antivirales d’Origine Naturelle” of Rennes (France) and F. SILVY from the Unit& de Recherche sur les Retrovirus et Maladies Associk, INSRRM U 322, Campus de Luminy, Marseille, France, for excellent technical aksumce.
1. 2. 3. 4.
Shannon, WM. AntMa Agentsand I&al Diseases OfMan ; Galasso, G.J.; Menigan, T.e; Buchamm, R.A., m; Raven Press: New York, 1984; pp. 55l2L I987,3J l388-l393. Gonxakx, MB, Alarcon, B.; Cauascol, L. Antimic&.AgentsChemo&r. Rhresmann, D.W.; Deii, BP.; Hatch, MT. h4krineAIgae in Pham~ceutical Scienq Hoppe, H.A,; Levring, Y.; Tanaka, Y., l5-k W. De Gruyter, Rerlin, New York, 1979; pp. 293-302. Neusht& M. &&o&h&a, 1994 2081205,99-lO4.
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5. 6. 7. 8. 9. lo. 11. 12. 13. 14. 15. 16. n. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 3L 32. 33. 34. 35. 36. 37. 38.
N. BOURG~UGNON et al.
MC Chue, MO.; Moore, J.P.; Blanc, D.F.; Scott@, P.; Cook, G.M.W.; Keynes, R.J.; Weber, J.N.; Davies, D.; Weiss, R.A. Aids Reseru& and Human Re.troviruses, 1992,8, 19-26. Uryu, T.; Iku&ima, N.; Katsuruya, K.; Shoji, T.; Takahashi, N; Yoshida, T.; Kamto, K.; Murakami, T.; Nakashima, H.; Yamamoto, N. B&hem Pharnmcol l!%Z, 43.2385-2392. Percival, E.; MC Dowell, RH. C!!mis@yadl3agmolo~ ofMarine A&a Polysacchari&s; Academic Press Inc.: London and new York, 1967, pp. 73-98 aud X27-156. Craig& J.S. BiologyofRedA4qae; Cole K.M., Ed.; Cambridge University Press, 1990, pp. 232-236. Usov, A.I. FoodHjdrocoUoi&, 1992, 6, 9-23. MC La&b, M. PlaotandSoii, I985, 89, X37-157. Nakashima, H.; Kido, Y,; Kobayashi, N.; Motoki, Y.; Neushul, M.; Yamamoto, N. Anrimicrob. Agents and Chemotber., 1987, 34 1524-1528. Nakashima, H.; Kido, Y.; Kobayashi, N.; Motoki, Y.; Neushul, M.; Yamamoto, N.; J. Cancer Res. clin. Oncol., 1987, ll3,413-416. Yamamoto, N.; Nakashima, H.; Yoshida, 0.; Kaneko, Y.; Matsuzaki, K.; Uryu, T. Arch. ofAZDS l&s, m!I, 4 45-46. Taylor, R.L.; Conrad, H.E. Biochemiktry, S72, II, 1383-1388. Blakeney, A.B.; Harris, P.J.; Hemy, R.J.; Stone, B.A. [email protected]., 1983, ll3, 291-299. Yaphe, W.; Arsenault, G.P.C. Anal. Biochem, 1965, f3, l43-l48. Abmed, A.E.; Labavitch, J.M. J. FoodBiochem, 1977,A 361-365. Payment, P.; Trudel, M. i%onel de Tmues Vimlogiques, Payment, P.; Trudel, M., m, Presses de 1’Universiti du Quebec, 1989, pp. 5-19. Reed, L.J.; Muench, H. Amer. J. H@cne, l938,27,493-497. Van Den Berghe, D.A.; Ieven M, Mertens P.; Vlietinck A.J.; Lammens E. J. Nat, Prod., 1978, 4l, 463471. Hu, J.M.; Hsiung, G.D. AntiviralRes., 1989, 14 217-232. Amoros, M.; Fauconuier, B.; Girre, R.L. AntiviralRes., l987, 8, l3-25. Rey, F.; Barr&Sinoassi, F.; Scbmidtmayerova, H.; Chermann, J.C. J, Wol. M&hods, 1987, 16, 239249. Stancioff, D.J.; Stanley, N.F. Proc. oft&e Mth lotern, %aweed$ynposium, 1969, R. Margelalef, Ed., pp. 595-609. Whyte, J.C.C.; Foreman, RB.; De Wreede, RE. Hjdrobiolog& I984. w/117, 537-541. Okazaki, M.; Furuya, K.; Tsukayama, K.; Nisizawa, K. Bo&Mu,, l982, Xxt: X23-131. De Cew, T.C.; Silva, P.C.; West, J.A. J. Phycol., 1992, 28, 558-566. Rees, D.A. I. C%cm,SX., E&l, l2,5l68-5l7L Ginsberg, H.S.; Goebel, W.F.; Horsfall, FL. Jr. Proc, sot. l&p. Biol, M&t, 1w7, 6& 99-lO0. Green, R.H.; Wooley, D.W. J, EXp.~, l947, 86, 55-64. Deig, B.F.; Ehresmann, D.W.; Hatch, M.T.; Riedhnger, D.J. Aofimicrob, Agents Cbemofher.., 1914, 6, 524-525. Richards, J.T.; Kern, E.R.;Glasgow, L.A.; Overall, J.C. Jr.; Deig, B.F. and Hatch, M.T. AntinaicrobioL Agcxrts Chemother,, l978,l4,24-30. Baba, M.; Snoeck, R.; Pauwels, R.; De Clercq, B. Antimicrob. Agents Chernother., 1988, 32, l742l745. Sugawara, I.; Itoh, W.; Kimura, S.; Mori, S.; Shimada, K. Experientia, S89, 45, 996998. Ha&tine, W.B. J. Acq, linmun. Lkf syndr., 1988, l, 2%240. Mitsuya, A.H.; Looney, D.J.; Kuno, S.; Ueno, R.; Wrong Stall F.; Broder, S. Science, B&8, 240, 646-649. Hatch, M.T.; Bbresmann, D.W.; Deig, E.P. Marine Algae in Pharmaceutical Science, E& by HA. Hoppe, T. Levring, Y. Tanaka, W. De Gruyter, Berlin, New York, 1979, pp. 343-363. Baba, M.; Schols, D.; Pauwels, R.; Nakashima, H.; De Clercq, B. I. of&&. AimnuneDefkiency $pdronzs, 1990,3,493-499.
0960-894x/93 $6.00 + .oo 0 1993 Pergamon Press Ltd
COMposTIoN
AND ANTIWRa NTNITIWOFASULFATBD POLYSACCHARIDB FROM DclB3T (RHODOI’EYTA, GIGARTINAUS)
Nathalie BOUKGOUGNON ‘, Marc LAHAYJJ b, Jean-Claude CHERMANN ’ and Jean-Michel KOKNPKOBST rz ; ISOMer, Groupe SMAB, Rtcultb de Phamack, I, rue G. Veil, 44035 Names Cedex Ol, Prance. Laboratoire de Biochimie et Technohqie des Ghtcides, INKA, BP 527,44026 Names Cedex 03, France. ’ INSEKM U 322, Campus Universitaire de Luminy, BP 33, I3273 Mar.sdle Ccdcx 09, Fkance.
(Received in USA 29 December 1992)
Matract. &&qmctia dubpi (Gig-s, Gymnophlaeaceae) contains an unusual sulfated heteropolysaccharide with uranic acids that is active against several viruses in&ding human immunodeficiency virus type-l (NIV-l), Herpes simplex bominis type 1 and type 2 (HSV-1 and HSV-2) and vesicular stomatitis virus (VSV).
Some polysaccharhles have shown io via0 activity against animal viruses incl~ adenovirus type 5 (Ad5), African swine fever virus (ASP), vesicular stoma&is virus (VSV), polio virus type 2, Sembki forest virus (SPV)~ * and Hcq.rcs sin@exllamiois virus (HSV)3. In recent years, sulfated po&darides infecti~r$ have also demonstrated in tifro inhibitory effects on human immumxMiciency virus (HIV) . Ked seaweeds contain large amounts of cell-wall polysac&arides, most of which are sulfated galactans These galacums are generally built on repeated alternating l$3-linked c&galactopyranose and l&-linked FDgalactopyranose units and differ in the level and pattern of suJfation, in the substitution of methoxyl and/or pyruvate groups and in other sugar residues (galactose, xylose). They also differ in 3,6anhydrogalactose content and the confiiation of the L3-linked a-galactopyranose residue, Among these agars are widely used as gelling or thickening additives by the food industry w, carrageenans , A sulfated polysacchatide is&ted from the red alga VpacifJca and in biotechnologies $“d has . The chemical displayed an inhibitory effect on HJV reverse transcriptase and io virro replication characteristics of this sulfated gahMan suggest that it belongs to the carrageenan family. The purpose of this work was to study the chemical composition of water-soluble polysaccharide from Sc&wm& dubyi (Chauvin ex Duby) J. Agardh (Rhcdophyta, Gigartinales, Gymnophlaeaceae) and its &I vitro activity against human immunodefii virus @IV), Hwpcs s@&x&amiais virus, vesicular stoma&is virus and polio virus. Extract.iattedu@uaand~analy!&methabL Gametophytic w dubyisamples were collected in May I991 on the east coast of Sicily. Water extractiom Sulfated polysac&ar& from air-dried seaweed powder (5 x 20 g, < 5 mm particle sire) was extracted in hot distilled water (5 x LS L) at 8o’C for 4 h with magnetic stirring, Separation of the soluble extract from insoluble debris was doue by filtration on diammaceou s earth, The filtrate at 5-c was poured into 2 vohtmes of absolute ethanol, with stirring. The precipitate was recovered and washed with 95’ ethanol, dehydrated with diethyl ether, dried ovemight at 5o’C, weighed and ground to a powder. The polysaccharide was redissolved in distilled water, dialysed against d.istilM water and B (l82O%yieldw/wontheinitialalgalweightbasis). Alkali extraction: Algal powder (LOOg) was treated with 12 L containing NaOH (0.3 M) and KCl (I6 I@ in distilled water without sodium borohydride at 8o’C for 4 h Polysaccharide was recovered from the alkali sohttion as above.
1141
1142
N. BOURGOUGNON et al.
Uranic acids in polysaccharide were reduced according to the method of Taylor and Conrad”. The acid form of the polysaccharide was obtained as follows: polysaccharide (50 mg) was dissolved in distilled water (5 mL), and the solution was eluted through a column of ion exchange resin (Amberlite IRl20 plus). The column was then washed with distilled water and the polysaccharide was recovered and freeze-dried. After sulfuric acid hydrolysis of the polysaccharide (2N, lOOC, 2 h), neutral sugars were derived into alditol acetate’ and identified and quantified by gas liquid chromatography (GC) on a DB225 (IW Scientific) fused silica capillary column eluted with hydrogen at 21o’C. 3,Qmhydrogalactose content was determined by calorimetry according to the method of Yaphe and Arsenault . Uranic acids of the acid hydrolysates resulting from the neutral sugar analysis by GC were measured by calorimetry according to the method of Ahmed and I.&witch”. Identification of the uranic acids in the trifluoracetic acid polysaccharide hydrolysate (2N, l2o’C, 2 h) was attempted by high pressure liquid chromatography (HPLC) using an Aminex 87H column (Biorad) eluted with 10 M sulfuric acid and with differential refractive index detection. Protein content was measured by the Kjeldahl method (Nx 6.25). Sulfate content in polysaccharide (lg) was performed gravimetrically after complete hydrolysis (HCl IN, 200 ml, lb, SO’C followed by HCl lN, H202 110 vol. (8: 1 v/v, S h, 8o’C) followed by quantitative precipitation of the released sulfate by BaCl . Infrared spectra were recorded from a KBr pellet of the acid polysaccharide on a IRS 25PT (Bruier Instrument). Proton broadband decoupling ‘C NMR of the acid-degraded water-soluble polysaccharide (HCl lN, 90 mitt, 8o’C) in D20 was recorded on a Bruker AM 300 WB spectrometer at 75.5 MHz (room temperature). Chemical shtfts in ppm were measured in relation to TMS as external reference. Dete.rmillatkmofantiviraIa&vityofauRk4tedpoaysaccbari&. Monkey kidney cells (VRRO cells Plow Lab. ref 03230) were cultured at 37’C in Eagle’s @imal essential medium (MEM) with Rarle’s saline supplemented with l0 % inactive fetal calf serum . HSV type l, HSV type 2, polio-2 virus and VSV were isolated at Pontchaillou Hospital, Rennes, Prance. Virus stocks were prepared by inoculating VERO monolayers at low multiplicity of infection and incubation at 37’C. Two days after infection, cultures were froxen and thawed, and stocks were maintained at -70’C. Virus titration was performed by the Reed and Muench dilution method”. Viral titers were estimated from cytopathogenicity and expressed at 50 9b of the tissue culture infective dose (TCID, test: The method used was essentially that described by Van Den The cytotoxic concentration (CyD ) value was defined as the maximum drug concentration causing cytotoxicity effects in 50% of the cultured cells after 96 h of incubation at 37’C. Values lower than those obtained in these tests were used in antiviral tests. Antiviral activity was detected by measuring the inhibition of cytopathic effect (CPE), with estimation of a 50% antivirally effective concentration (RD ) and therapeutic index (TI)22. Cell monolayers were infected with virus at low multiplicity [lD2 and lO@DI (50% infective doses) per microtiter plate] in the presence of polysaccharide at various concentrations. k%er several virus replication rounds (72 h for HSV, 32 h for polio and 28 h for VSV at 37-C), the cytopatbic effects in infected and uninfected cells from 6 wells for each virus concentration of polysaccharide were examined under a phase-contrast microscope. To assess the effect of polysacharide on uninfected MT4 cells, dilutions ranging from 1 mg/mL to @g/n& in the maintenance medium were added to MT4 celI line at 37’C with 5% CO, during lh on 96 well microtiter plate containing 3x105 MT4 cells for lOOI.&of compounds (in duplicate). The cells were then reajusted to 3x10’ MT4/mL on 24-well microtiter plate containing various concentrations of polysaccharide. After 4 days the cells were diluted to l/3 with the corresponding dilution of compound. Reverse Transcriptase (RT) assay: 1 mL sample of cell supematants were concentrated 100 fold by ultracentrifugation at 95000 rpm at 4-C for 5 minutes in a TLlOO rotor (Beckman). The pellet was resuspended in lOpL of NTR buffer contaiuing 0.1% Triton X-100. The enxymatic reaction was performed in a 5Op.L of a reaction mixture containing: Tris 50mM, pH 7.8 ; MgC$ 20 mM ; KC1 20 mM ; dithiothreitol2 mM ; Oligo(dT) l2-ltS 0.25 OD/mL ; poly(rA) 0.25 OD/mL and 3HdTI’P 2.5pCi. After lb at 37-C, the reaction products were precipitated with 20% trichloracetic acid, filtered on MilIipore 0.45t1rr1 and the 8 radioactivity was measured.
A sulfated polysaccharide from Schizymeniadubyi
1143
Determination of antiviral activity with HIV-l was based on measuring the protective effect of the sulfated polysaccharide against this virus-induced cytopathogenicity in MT4 cells. Four to six days after infection, multinucleated giant cell (syncitium) formation was observed, preceding cell death. MT4 cells were incubated in RPMI l64O culture medium with lO% fetal calf serum (PCS) and 1% PSN antibiotics, 1% glutamine and 2pg/mL polybrene at 37-C during 1 h on 96-well microtiter plate containing various concentrations of sulfated polysaccharide (3xlO” cells for lOO l.tL of compound). DO I.tL of HIV-1 (dilution lO,“) suspension was added in wells. One hour after incubation, infected cells were washed three times with RPM1 and then cocultured at 3x10’ cells/mL on 24-well microtiter plate containing various concentrations of the sulfated polysaccharide. Every three or four days, cells were diluted three times and the cell concentration was adjusted at 3~10~ cells/mL RPMI, 10% PCS, 1% glutamine and 2pg/mL polybrene in the presence of sulfated polysaccharide. Three days after cultures, infected MT4-HIV-1 cells with and without sulf a@ polysaccharide and uninfected MT4 cells were observed for syncitial formation every one or two days . The cheakxl composition of the water-soluble polysaccharhk is shown in Table L Table 1
Fig. 1
Chemical composition of the sulfated polysacchar& from .9&&n&a dubyf
Water-extracted polysaccharide
Neuid!: xylose mannose
29.9
m&k-a
of phycocolloid sch&mc&dubyi
Reduced polysaccharideb 25.2 L6
;3”
glUCOSC
3danhydrogalacto~ Uranic acids Sulfate Protems Ash
E 33.7 15.5
::; 2.8
lE
Neither galacturonic, mannuronic nor glucuronic acid was detected by HPLC analysis of an acid hydrolysate of the polysaccharide. Two successive reductions of the polysaccharide decreased the uranic acid content from 33.7 to 2.8%. Neither marked increase of a parkular sugar (except a small increase in glucose) nor the appearance of a new sugar was detected gas chromatography after reduction. The 9 C NMR signal at I77 ppm for carboxylic presence of uronides was confirmed by the characteristic carbons and by typical absorbance at 1732 cm-’ on the infrared spectrum _y the polysaccharide in acid form. The latter spectrum also confim& the presence of sulfate (1234 cm ), and the absorbance at 8% and 820 cm-1 indicated that sulfate groups might be located on carbon 4 and 6 of the galactose units (Pig. 1). Sulfate content (M.O%) and infrared spectrum Of the alkali-extracted polysaccharide Were similar to those of the water-extracted polymer. No methyl from methoxyl or pyruvate groups was detected by DC NMR specuoscopy. The in vitro antiviral activities of the water-extracted polysaccharkk from m dub* are shown in Table 2. The maximal non-toxic concentration of the polysac&ar& for the VBRO cell line was 93.7 q.g/mL, giving a cytotoxic concentration of CyD at 187.5 Pg/mL. The polysaccharide was most *?! virus. The compound was toxic on the MT4 cell effective against HSV-1 and least effective against poll0 lines within the concentration range of hng to SOcrg/mL while toxicity was much less at 25wmL, Y)crgtmL and ll@mL doses showed no appamnt cytotoxichy.
1144
N. BOURGOUGNON er al.
Table 2 activity of water-soluble polysaccharide from &t&j against HSV-1, HSV-2, VSV and Polio2 viruses
In vitro antiviral
Sch+mc& viruses
TIb
EDJO wnWa IO’D$c /SOcLL”
loLDI, /50clLc
‘DLD’s /50pLc
lo&)
/50@
WV-1
l.5
5.0
125.0
37.5
HSV-2
25.0
6.0
75.0
31.2
vsv
lO.0
15.0
lg.7
l2.5
Polio-2
44.0
30.0
4.0
2.0
a antiviraUy efkctive ctmmccm
indcxz CyDsdgDso
b Tlwqmic
Evaluation of the anti-HIV effect of the polysaccharide (Table 3) indicated that syncitial formation was completely suppressed at 20 pg/mL and delayed at lOccg/ml. No toxicity was found at these concentrations. Furthermore, the sulfated polysaccharide is inhibitory to the HIV-associated Reverse TrarWliptase. Table 3 Evaluation of antiviral effect for polysaccharide with HIV-l D4*
D5
D6
D7
500
-frox
lo0
_ _
-fTox _ -
Tox - _
Tox - _
I781156
50
_ -
_ _
- _
- _
l40/5l2
20
_ -
_ -
- _
- _
4Wl34
lo
_ _
_ _
i-k/T u/r
7OOll666
2
++++
++ ++
++fr
i+
i-+/T u/r
39874P8632
0.2
uu
++ ++
++/r
++
u/r
0.0
+ (+1
u
++fl
++
0.0
_ -
po&acs
R;rEl$$$$$I
(cI.B/mL) MT4-HIV-1
MT4 =hY;
-:aba?oceofsyacytia;
++ _ _
+:prmumofsyacytia;~:Byncytia
+++
- _ ;T:
ufr
IWIW
u/r u _ _
2047o/lO984 l36Jm4
cdldcatt~;Tox:Toaicity.
The chemical composition of Sc&ymun~a dubs water-soluble polysaccharide~has never been descri$d in the literature, although that of S. pcilice has been reported by Whyte et al. and Nakashima etal. . Whyte etal. reported galactose, 3,6-mhydrogalactose and sulfate in a molar ratio of LO.04, and 0.75 respectively, with the presence of 0.5% methyl and the absence of uranic acids. Nakashima et&. reported galactose, 3,6_anhydrogahuztose and sulfate in a molar ratio of 1, 0.01 and 0.51 respectively and attributed this sulfate gahWan to a kumgeenan. B water-soluble polysac&aride from S. dut& another sulfated galactan encountered in many red algae ’ , differed because of its unusually high content of
A sulfated polysaccharide from Schizymenia dubyi
1145
uranic acids. Neither the methoxyl nor % pyruvate group was associated with these polymers. The presence of uranic acids was con6medby CNMRandIRspectroscopyand&micalreduction,butthe sugar-bearing carboxylic group remained unidentified. Uranic acids have been rarely reported in polysaccharides from marine red “1~ whereas D-glucur@c acid is present in the mucilage of several unicellular red algae. Craigie et al. and Okaaaki et al. reported guluronic-rich alginate-like polysaccharides in Serraticardia maxima and other coralline red algae. The galactan of Dilsea e&&3 contains 9.5 to ll% uranic acids’, although it has recently been reported that the Atlantic and Pacific gametophytes and tetrasporophytej,from Dilsea, Halymenia, Cryptonemia and Schizymenia species are very anatonomicallysimilar . The similarity in sulfate content and between IR spectra of the water- and alkali-extracted polysac&ride suggests that $e sulfate groups are alkali-stable and thus not located on C-6 or C-3 of 4linked galactose residues . Research is in progress in our laboratory to determine the identity of the uranic acid and the position of the sulfate groups on the water-soluble sulfated galactan from S. dub* Antiviral activity of sulfated polysaccharides has been recognixed for 0 e years. As early as 1947, polysaccharides from diverse sources were found to inhibit viral growth ,%B* The polysaccharide-rich fractions from two marine red algae, Cryptosrphtia woodii and Farlowia mollis (Dumontiaceae), were found to exhibit io t&o activity against HSV-1 and HSV-2, vaccinia virus and vsv== . In recent years sulfated polysaccharides have been shown to inhibit in vitro replication of many enveloped viruses: including HIV=! A sulfated polysaccharide isolated from the sea alga Sch@me&paci6ca may also suppress in vitro HIV infection by interfering with virus adsorption as well as inhibiting reverse transcriptasell’ a. Multinucleated giant cell formation induced by interaction between the glycoprotein expressed on the surface of cells infected with HIV-l and the receptor of u&f&ted cells may play an impor “-;” role in the depletion of T4 lymphocytes in acquired immune deficiency syndrome (AIDS) are inhibitors of giant cell patients . Pentosan polysulfate, fucoidan, mannan sulfate and karrageenan formation and hence protect target cells against destruction by killer HIV-1-infecte&cells. Their mode of actionhavebeenamibutedto~ninhibitionof~~ntotha~llmembram. In the present study, the water-soluble polysaccharide of &@ymenk dub9 showed activity against HSV-I, HSV-2 and VSV, but the relation between the chemical strucuue and antiviral activity remains to be determined. The antiviral mode of action of this polysacc hari&ais now under investigation. As the ,thefinechemicalstructureofthis nature of anionic groups are a decisive factor in anti-HIV process polysaccharide is currently being studied in our laboramry.
We thank Dr. M. CORMACI and G. FURNARI from the Istituto e Orto Botanico, Universiti di Catania, Italy for collecting the algal material, Dr. F. ARDRR from the Museum National d’Histoire Naturelle, Paris, France, for identification of v do&$, Dr. N. SINBANDHlT from the Centre R6gional de Mesures Physiques de l’ouest, Universiti de Rennes, France, J. A. MONTANHA from the team “Etudes et Bioproduction de Molecules Antivirales d’Origine Naturelle” of Rennes (France) and F. SILVY from the Unit& de Recherche sur les Retrovirus et Maladies Associk, INSRRM U 322, Campus de Luminy, Marseille, France, for excellent technical aksumce.
1. 2. 3. 4.
Shannon, WM. AntMa Agentsand I&al Diseases OfMan ; Galasso, G.J.; Menigan, T.e; Buchamm, R.A., m; Raven Press: New York, 1984; pp. 55l2L I987,3J l388-l393. Gonxakx, MB, Alarcon, B.; Cauascol, L. Antimic&.AgentsChemo&r. Rhresmann, D.W.; Deii, BP.; Hatch, MT. h4krineAIgae in Pham~ceutical Scienq Hoppe, H.A,; Levring, Y.; Tanaka, Y., l5-k W. De Gruyter, Rerlin, New York, 1979; pp. 293-302. Neusht& M. &&o&h&a, 1994 2081205,99-lO4.
1146
5. 6. 7. 8. 9. lo. 11. 12. 13. 14. 15. 16. n. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 3L 32. 33. 34. 35. 36. 37. 38.
N. BOURG~UGNON et al.
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