60 Planta Med. 58(1992)
Chemical Structure and Biological Activity of Polysaccharides from Hibiscus sabdariffa BerndM. Muller1'2 and Gerhard Franz1'3 1 2
Faculty of Pharmacy, University of Regensburg, D(W)-8400 Regensburg, Federal Republic of Germany Present address: Complex Carbohydrate Research Center, The University of Georgia, 220 Riverbend Road,
Athens, GA 30602, U.S.A.
Address for correspondence Received: January 14, 1991
Three water-soluble polysaccharides
Hibiscus sabdariffa L. (Malvaceae) is known to contain large quantities of viscous polysac-
have been isolated from flower buds of Hibiscus sab-
charides (1). Several other species of Hibiscus are used in
darWa L. (HIB 1, 2,3). The neutral polysaccharides (HIB
1 and 2) are composed of arabinans and arabinogalactans of low relative molecular mass. The major fraction was investigated by methylation analysis, pectinasetreatment, mild acid hydrolysis, and NMR studies, and it was shown to be a pectin-like molecule (Mr =
io d). The
main chain is composed of a-1,4-linked GalA (24% methyl-esterified) and a-1,2-linked Rha. Side chains are built of Gal and Ara and are connected to the main chain
via C-4 of every third Rha. Its structure seems to be different from polysaccharide structures described in other species of the Hibiscus genus and the Malvaceae family. All fractions were assayed for possible immunemodulating effects. All fractions showed some activity,
but the main acidic fraction was contaminated with lipopolysaccharide, and therefore its shown activity has to be discussed carefully. Key words ________________________________________
oriental medicine and were shown to contain polysaccharides with marked physiological activities (2). Hibiscus sabdariffa flower buds are mainly used for refreshing infusions. Their medical value is still uncertain although there are some reports about physiological activities of aqueous extracts of this drug: Sharaf(3, 4) reported the decrease of blood pressure, relaxation of rat uteri, inhibition of taenia motility and bacterial growth for aqueous extracts, Little is known about the exact chemical na-
ture of the polysaccharides in Hibiscus sabdariffa flower buds (1, 5, 6). Up to now the mentioned physiological activities of the aqueous extracts could not be attributed to known constituents (7). It seemed to be interesting to investigate the structure ofthe polysaccharide fraction and to test them for specific physiological activities.
Materials and Methods Materials
Hibiscus sabdarffa, Malvaceae, polysaccharides, pectin, immunological activity.
Flower buds of Hibiscus sabdarffa L. were obtained from SALUS (Bruckmuhl, FRG). All chemicals were of analytical grade quality. DEAE-Sephacel, Sephadex G 50, and
Abbreviations and Symbols
Superose p.g. were purchased from Pharrnacia. Aspergillus niger pectinase and Salmonella typhimurium lipopolysaccharide were obtained from Sigma.
Ara: Mr: MrCO:
Gal: GalA: LPS: Glc: G1cA:
Man: Rha: Xyl:
arabinose relative molecular mass molecular mass cut off galactose galacturonic acid lipopolysaccharide glucose glucuronic acid mannose rhamnose xylose
General methods Total carbohydrate, uronic acid, and protein were assayed by the anthrone (8), m-hydroxybiphenyl (9), and Lowry methods (10), respectively, using neutral standard sugars, GalA, and bovine serum albumin as the respective standards. Methyl and acetyl group content was assayed by the methods of Wood et al. (11) and McComb et al. (12), respectively, using methanol and D-glucose pentaacetate standards. The molecular weight distribution of polysaccharides were estimated by GPC with the aid of standard pullulans (Shodex).
Polysaccharides were hydrolysed with 2 M tnfluoroacetic acid (TFA) at 121°C for 90 mm. Sugars were converted into their corresponding alditol acetates by the method of Blakeney et al. (13). GLC was performed at 207°C using a Varian 3700 gas
chromatograph with FID on a glass capillary column (0.25mm x
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Introduction
Abstract
Planta Med. 58(1992) 61
Chemical Structure and Biological Activity of Polysaccharides from Hibiscus sabdar/'fa
30m) packed with 3% of OV 225, using He as carrier gas. The molar ratio of the neutral sugars was calculated from the peak areas and molecular weight of the corresponding alditol acetates. The molar ratio of uronic acid and neutral sugars was calculated from the uronic acid content. Reduction of acidic sugars was done in the presence of carbodiimide using NaBH4 (14).
pies were reduced with Li(Et)3BD/THF (16) and dialysed again. The
premethylated and deutero reduced poiysaccharides were methylated using Dimsyl-K/CH3I according to (15) except that the sampies were purified by dialysis. The methylated samples were by-
drolysed, the products were reduced with NaBH4, followed by acetylation. The resulting partially methylated alditol acetates
were analysed by GLC-MS on a Hewlett Packard model 5890 A with a DB 1701-30W capillary column (0.25 .tm film, 0.25mm x 30 m).
Dried plant material was powdered (0.5 mm) and preextracted by percolation with MeOH until the extract was colourless. For the isolation of the polysaccharides, the dried preextracted powder was stirred with water as a 5 % suspension at 20 °C for 12 h. The crude polysaccharide fraction was obtained by pouring the water extract into the five-fold volume of 99% EtOH at 4°C.
NMR-analysis 1H-NMR was performed on a Bruker WM at 250 Hz and 297K with D20 as internal standard (6 = 4.67 ppm). 13C-NMR was run at 297—307 K at 62.896 Hz with TMS as internal standard. Samples were dissolved in D20.
The precipitate was dissolved in water, dialysed against water
Biological tests
d) and freeze-dried. Purification and fractionation was carried out on DEAE-Sephacel (P043-, pH = 6.0). Fractions were subsequently eluted with water, a linear phosphate-buffer(MrCO = 3.500
gradient (0-1 M) as shown in Fig. 1 and finally 0.2 M NaOH. Further purification was achieved by GPC on Sephadex G-50 and Superose 12 pg., respectively.
Determination of iipopoiysaccharide contamination For quantitation of endotoxins in the poiysaccharide samples the LAL-kit of Kabi vitrum was used. LPS deriving from Salmonella typhimurium was used as reference.
Digestion with pectinase
Test for mitogenic activity (17)
HIB 3 (50 mg) was digested with pectinase (1.0 U) in 50mM acetate buffer (pH = 4.2, 10—20 ml) for three days at 30°C
in a shaker. After neutralisation, the mixture was heated to 100°C for 10 mm to inactivate the enzyme, centrifuged, and separated on Sephadex G-50.
Mild acid hydrolysis
were removed (Skatron cell harvester 7020/7019), washed with distilled water and dried at 60°C for 1 h. 2 ml of scintillation
The polysaccharide fractions were subjected to
cocktail were added (Packet Szinti Emulsifier 299) and the samples were counted for 5mm
hydrolysis with increasing strength, using TFA at concentrations of 0.05 M and 0.5 Mat 100°C for 1 h. The products were separated by GPC on Sephadex G-50.
at 121°C for 90 mm. 0.1 ml of ascites liquid (5 i06 cells) of sar-
Acidic polysaccharides were methylated once by the method of Waeghe et al. (15), followed by dialysis and freeze drying. In order to facilate permethylation, the premethylated sam-
HIB HIB RIB RIB HIB HIB HIB RIB RIB RIB RIB RIB RIB RIB
in
lalA 1f3 213
2131A
3u 3j3
313pecct 313pecjl
3jipecy
3/iA
3/lB 3/iC
RIB 3/2A RIB 3/2B RIB 3/2C RIB
3/la
RIB 3/lb RIB 3/ic RIB 3/2a RIB 3/2b
Yield°
UA
Rha
Ara
5.3
85.2
1.7
7.4
1.2 45.2
4.7 0.4
0.3
0.0
67.8 0.0
2.3
0.3 0.0 0.0
0.9
76.1
1.0
67.8
0.0
86.0 85.2 43.9 51.9 79.2 44.2 91.3
2.4
48.9 5.4
2.5 54.2 12.3 21.7 3.0 2.7 46.5 5.0 63.2 2.8 10.4
31.6 4.1
17.2 11.4 78.9 12.5 41.1
CD-i mice. After randomisation into groups of 10 mice polysaccharide solutions were injected i.p., starting 24 h after tumor in-
Xyl
Man
0.6 1.2
0.1
3.9
1.5
4.1 3.1
iRS
7.4 6.2 7.8
0.6
Gal
2.6 1.3 0.3
4.4 11.4
34.8
4.9 1.1 0.9
4.2
0.3
4.6
0.5 0.8
5.1
11.4 1.2
0.6 1.1 0.6 0.7
8.9
3.2
1.7
2.7 56.1
0.9 0.3 0.0
5.5 0.2
0.5
0.2
0.3 0.0 1.9
1.5 0.3 3.5 8.3
4.9
0.0 37.8
2.6
1.8 3.1 12.6 0.6
4.4
1.5
3.9
3.4
GIc
91.1 4.4 19.9
0.2
96.4 91.0
Calculated as weight % of applied material.
coma 180 were s.c. injected into the right side of allogenous female
7.2 11.5 18.2 11.7
2.6 2.7
33.5
0.0 80.9 83.0 73.7 83.4 82.5
Test of antitumor activity (18) Poiysaccharide solutions in PBS were autociaved
Methylation analysis
Fract.
Spleen lymphocytes were obtained from C3H/ HEJ-mice and were cultivated in 96 well microtiter plates together with RPMI-medium (Gibco) in 10% FCS, 1% Pen-Strep, 2 mmol glutamine, and the test substance. After 48 h incubation, 30 zl [3JJ] thymidine was added to the cells (4 105/well. After 18h the cells
1.1
0.9
1.9
9.9 0.0 0.0 0.7 0.0 0.6
0.1
0.0 0.9 0.8 0.0 0.0
5.6
33.8
1.1
13.1
3.1
3.8
2.6
33.8
5.2 0.7
2.7 5.1
0.6 3.6 32.9 8.6 6.3 7.2 1.8 4.7
1.1
0.9 0.0 9.4 3.7 3.7 1.5 1.5
6.2
Table 1 Sugar composition (mol %) of polysaccharide fractions from Hibiscus sabdariffa.
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Isolation and pur(fication of water-soluble polysaccharides
BerndM. Muller and Gerhard Franz
62 Planta Mcd. 58(1992)
oculation, for 10 days at a concentration of 5 mg/kg. The control group was treated with PBS only. Tumor growth was measured
160
A 620 nm
buffer-mol.
every 10 days. After 30 days the mice were sacrificed and evaluated hy comparing each group's fresh tumor weight:
inhibition rate: (C-T/C) 100
C: average tumor weight in control group, T: average tumor weight in treated group.
Regression: number of tumor free mice/number of tumor bearing mice. Results
Isolation Preextraction with MeOH removed 45% of
amounts of Rha, Gal, and Ma. Only 1.8% was detectable as protein.
Fractionation
Three fractions of BIB were obtained by 0 50 100 150 250 200 300 DEAE-Sephacel chromatography with water and a phosfractions (2 ml) phate-buffer gradient (Fig. 1). BIB I was eluted by water (3.5% of applied material) and was mainly composed of Fraction 1 — 50 Ma. BIB 2 (2.4%) was eluted with the onset of the buffer H20 elution gradient and also contained mainly Ma (76 %). The main Buffer gradient Fraction 51 — 200 polysaccharide fraction HIB 3 (33%) was eluted at a buffer 0.2 N NaOH Fraction 201 — 250 strength of 0.45 M. By further elution with buffer BIB 4was obtained, similar in composition to BIB 3. With 0.2 N NaOH a hygroscopic and red coloured complex was obtained (BIB 5). BIB 3 was predominantly composed of uronic acid besides small amounts of Rha, Ara and Gal. Alter reduction of
Fig. 1 Fractionation of HIB on DE.AE-Sephacel.
A 620 nm
the native polysaccharide, the uronic acid could be identified as GalA.
HIB 1, 2, and 3 were further purified by
2.5
GPC. BIB 1 gave two subfractions after elution on Sephadex G-50 (Fig. 2). The first one, HIB icr (Mr 1 10) was mainly composed of Ma and Gal (Table 1). The second one, BIB 1/3 (Mr: 6 102) was red coloured and not further investigated.
BIB 2 turned out to be a neutral polysaccharide too. Fractionation on G-50 (Fig. 3) showed its main portion had a M of 6.3 103. Its sugar composition was related to BIB Ia. HIB 3 showed a high range Mr-distribution and was further
purified on Superose 12. The main fraction (BIB 3a) showed an Mr of 1.5 io5, the minor one (BIB 3/3) 5 10g. Both fractions had similar sugar compositions (Table 1)
consisting mainly of GalA which was 24% methyl-esterified. It is most likely that BIB 3 consists of one type of polysaccharide showing a broad range of Mr.
Pectinase-treatinent BIB 3 was digested with pectinase and the
resulting products were separated on Sephadex G-50,
yielding three subfractions (Fig. 4): BIB 3peca, a small fraction in the void volume, BIB 3pecfl, representing an inter-
mediate fraction, and the major fraction BIB 3pecy. BIB 3peca mainly consisted of GalA, Rha, Ma, and Gal, but the
0
20
40
60 80 fractions (1 ml)
Fig.2 GPC of HIB ion Sephadex G-50.
100
120
140
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the dry weight of Hibiscus sabdar[fa flower buds. The water extract of the crushed material yielded 5.3% of a crude polysaccharide fraction (BIB). It was composed of large amounts of uronic acid (Table 1) as well as small
Planta Med. 58(1992) 63
Chemical Structure and BiologicalActivity of Polysaccharides from Hibiscus sabdariffa
A 620
A 620
0.6
0.6
0.4
0
20
40
60 80 fraction (1 ml)
100
120
140
140
fraction (1 ml)
Fig.3 GPC of HIB 2 on Sephadex G-50.
Fig.4 GPO at HIB 3 after pectinase treatment.
GalA amount was diminished to 1/2 ofits initial content. Subfractions /3 and y showed increasing amounts of GalA.
position C-4. The small amount or 3,6-branched Gal decreased whereas 6-linked Gal increased, proving that at least some of the Ara residues had been connected to posi-
Methylation analysis (Table 2) showed that HIB 3peca contained terminal and 5-linked Ara; terminal, 2-linked, and 2,4-branched Rha; terminal, 4-, and 6-linked, and 3,6-branched Gal, and 4-linked GalA. HIB 3pecy was composed of terminal, 5-linked, and 2,5-disubstituted Ma
tion 3 of 3,6-branched Gal exclusively.
amount of terminal and 4-linked GalA. These results find an explanation in the fact that HIB 3pec/J and HIB 3pecy derive from a-1,4-linked GalA-rich regions of HIB 3. Thus, HIB 3 seems to he built up of two different carbohydrate blocks, represented by (1 —* 4)poly a-o-GaIA regions and neutral
nal, 5-linked, and 2,5-branched Ara. Gal was mainly 4linked apart from a minor amount of 3,6-branching. After
and terminal and 4-linked Gal besides a dominating
sugar enriched blocks (HIB 3peca) forming regions of branched structures. Mild acid hydrolysis
We have investigated the effect of hydrolysis with increasing TFA-concentrations on the M distribution and sugar composition of HIB 1, 2 and 3.
HIB la HIB Ia predominantly contained Ara, terminal (27%) and 5-linked (53 %), besides 4-linked Gal. Only 10% of the Gal amount was 3,6-branched. After hydrolysis with 0.05 N TFA nearly all Ara residues had been cleaved.
The remaining polysaccharide HIB I alA (Fig. 2) was mainly composed of Gal (Table 1). Methylation analysis showed that 91 % of the Gal residues remained linked by
HIB 2/3
This polysaccharide fraction seemed to be very related to HIB la. Its high Ara content showed termihydrolysis with 0.05 N TFA, no 3,6-branched Gal could be detected. The percentage amount of 6-linked Gal remained unchanged. Maybe the amount of 3,6-branched Gal vanished by the release of the Ara residues connected to it as well. Not all Ara had been hydrolysed. HIB 2/31A showed terminal and 5-linked Ma (ratio 1: 2) and 4-linked Gal. This
might be due to the existence of arabinans as well as arabinogalactans in HIB 2/3. HIB 3
a), The first attempt to use mild acid hydrolysis for the structural elucidation of HIB 3 was the combination of two increasing hydrolysis methods using 0.05 N and 0.5 N TFA, sequentially. TheJirst step resulted in a de-
gradation of HIB 3 into three subfractions that could be eluted from Sephadex G-50 (Fig. Sa). HIB 3/lA was obtained in the void volume (5 % of applied material). Its sugar composition was related to HIB 3peca with GalA and Gal as major components. Only 50% of the fraction was suscepti-
ble to the pectinase treatment. The main fraction HIB 3/lB
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0.2
64 Planta Med. 58(1992)
Bernd M. Muller and Gerhard Franz
la
lalA
2f
1—
18.6
13.4
1.8
35.8 13.4 67.8
0 0 0 0
19.2
1,5— 1,2,5—
32.5 16.0 67.7
26.8 8.7 48.9
3.2 1.5 6.5
0 0
0 o 0 o 0
0 o 0 o 0
0 0 0 0 0
0 0
0 0
0.4
17.1
82.6
0
7.5
0 0 28.9 5.9 0 34.8 4.9 0 0 11.4 11.4
3/la
3/lA
3/lB
1.9 0.0 0.0 1.9
3.2 0.0 0.0 3.2
2.7 0.0
8.3
0.0 2.7
0.0 11.5
0 1.7 0 0.9 2.6
0
0.1 1.0
3.0
0 1.8 4.9
0 6.7 0 2.2 8.9
0.4 2.3 2.5 0.8 1.3 8.3
1.3 8.6 3.5 2.0 1.8 17.2
0 0 21.1 5.5 6.5 34.1
0
0
0
0
0
0 0 0 0
0 0 0 0
0 0 0
0
0 0 0 0
2lA
Table 2 Sugar linkages in the main polysac-
3$peca
charidefractionsofHibiscussabdariffa
Ara
3.2
Rha 1—
0
1,2—
0 0
1,4— 1,2,4—
3.1
2.2
0 0.5
1.2 1.2
1.6
7.6
0.7
3.3
0 0
0
1—
(1,4—) 1,4— 1,6—
1,3,6—
0
18.9
90.5
13.4 3.3 2.7 19.8
7.4
6.2
5.6
0 0
1.8
0
GIc 1,4—
Man 1—
1.4
1.5
1,2— 1,4—
0.8 2.7 4.9
0 1.6
3.1
4.4 4.4
0
1.0
4.9 0.9 2.0
2.7
11.1
0.9
0 0 0
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Gal
Represents 1,4-GalA after reduction prior to methylation.
A 620 nm
A 620 nm 1
0.8
0.6
0.4
0.2
0
0
20
40
60 80 fraction (1 ml)
100
120
140
0
20
40
60 80 fraction (1 ml)
100
120
140
Fig. 5a GPC of HIB 3 after hydrolysis (0.05 N TFA).
Fig. 5b GPC of HIB 3/lB after hydrolysis (0.5 N TFA).
(63 % yield) showed an increased GalA content besides Rha,
3/lB is composed mainly of a- 1,4-linked poly-Ga1A. HIB 3/
Gal, and Ara moieties. Pectinase treatment yielded an excessive degradation with only few percent of the fraction re-
1C was mainly composed of Ma which had been cleaved from the native polysaccharide by hydrolysis.
sisting the enzyme treatment. This is due to the fact that HIB
Planta Med. 58(1992)
Chemical Structure and BiologicalActivity ofPolysaccharides from Hibiscus sabdariffa
65
The degradation of HIB 3 resulted in a
A 620 nm
selective removal of Ara residues. This did not affect the ratio of 2-linked to 2,4-branched Rha, indicating that Ma was not bound to Rha directly. The ratio within the Gal-linkages changed. A decrease of 3,6-branched Gal was accom-
panied by an increase of terminal and 6-bound Gal. The cleaved Ma residues and the small amount of hydrolysed Gal residues must have been connected to Gal via position 3 and/or 6. Since no 3-linked Gal could be detected and nearly
all Ma residues had been hydrolysed, it seems to be likely that Ara was preliminary bound to position 3 whereas Gal residues were connected to position 6 of branched Gal. The
ratio of 2,4-branched Rha to Gal (1 : 5) remained unchanged. In conclusion it seems that the neutral sugar enriched regions of HIB 3 are composed of side chains connected to the backbone of pectin via C-4 of branched Rha. Only 1/3 of the Rha residues are branched. The side chains (6-): 2—3 (3,6-). No terminal GalA could be detected. The ratio of Rha : GalA in the presumed backbone (between 1 : 32 and 0
20
40
60 80 fraction (imi)
100
120
140
Elution profile with 0.05 N TFA
0.5 N TFA
1 : 54) is difficult to determine due to the possible formation of aldobiuronic acids and polysaccharide-degradation during reduction procedures (data not shown).
NMR-Studies
Fig. 6 GPC of HIB 3 after mild acid hydrolysis.
'3C-NMR: Weak signals at 16.83 and The second step of hydrolysis (0.5 N TFA) was carried out with HIB 3/lB (Fig. 5b). By this treatment a polysaccharide was precipitated (HIB 3/2A). It was NaOHsoluble and composed of a 1,4-linked GalA only. The high Mr fraction of the soluble residue of HIB 3/lB (HIB 3/2B) was composed of GalA (> 90%), Rha and Gal, 3.5% each. The low Mr fraction HIB 3/2C contained Ara and Gal which had not been cleaved off during the first hydrolysis. Also
these results indicated the presence of an a 1,4-GalA polymer (see HIB 3/2A and HIB 3/2B) and a second part containing neutral sugars (HIB 3/lA). The methylation results of the neutral sugar enriched fraction HIB 3/lA (Table 2) showed the presence of terminal and 5-linked Ara, 2-linked and 2,4-disubstituted Rha, 6- and 4-linked and 3,6-branched Gal. Alter reduction of the polysaccharide the GalA turned out to be bound via position 4. The methylation results of the other fractions showed only few neutral sugars. HIB 3/lB consists of terminal and 5-linked Ara, terminal, 2-linked and 2,4-
branched Rha, and terminal, 6-linked and 3,6-disubstituted Gal.
b) The second attempt of mild acid hydrolysis was done with each of the native polysaccharides for every hydrolysis. The hydrolysis with 0.5 M TFA resulted in a more pronounced degradation with a vanishing amount of high-Mr fraction HIB 3/2a (Fig. 6).
20.46 ppm indicated the presence of methyl groups of Rha and of acetyl groups. This is in accordance with weak signals at 1.48 and 1.96 ppm in the proton spectrum. An expressed signal at 53.12 ppm could derive from 0-methyl groups of methyl-esterified GalA. Signals between 68 and 80 ppm (86.31, 69.43, 79.10, 70,8 1) could derive from carbons C-2 to C-5 of GalA, the dominating sugar residue in HIB 3. In the area of anomeric C-atom signals, a signal at 100.52 ppm proved the a-configuration of GalA. Further signals at 175.60 and 170.98 ppm were identified as deriving from non-esterified and esterified C-6 of GalA. The intensity of the signal at 170.98 ppm is in good accordance with the one at 53.12 ppm standing for -OCH3.
Summary of overall structural
characteristics
NMR-analysis of HIB 3, pectinase-treatment, mild acid hydrolysis and methylation analysis gave rise to the following facts: HIB 3 represents a pectin with an average M of iO d. Its main sugar components are GalA (84%), methyl-esterified (24%), and smaller amounts of Rha, Ara, and Gal. The molecule is built by at least two different carbohydrate regions, i.e poly-a-(1,4)-GaIA and neutral sugar enriched blocks with Ara- and Gal-containing side chains. Rha is located in the main chain with every third unit being branched via C-4 bearing side chains. Rha is linked to Gal in the ratio 1 : 10—11. The non-reducing end of the side chains is built of Ma disaccharides linked 1,5;
Methylation analysis of HIB 3/la (Table 2)
probably connected to position 3 of branched Gal. The
branched Rha (unchanged ratio) and terminal, 4- and 6-
3,6-branched Gal in the ratio of 1 : 5 : 2 :2—3 and terminal, 5-linked and 2,5-branched Ara in the ratio 2 : 3: 1.
showed few terminal Ara besides 2-linked and 2,4- whole side chains consist of terminal, 4- and 6-linked and linked, and 3,6-branched Gal.
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are built of Ma and Gal in the ratio 1 (terminal):5 (4-): 2
66 Planta Med. 58 (1992)
BerndM. Muller and Gerhard Franz
tumor area fmm'i
Table 3 Treatment of sarcoma 180/CD-i with HIB3. dose (mg/kg)
average tumorweight (g)
control
—
—
HIB3
5
2.36
inhibition
regression
0
0/15
4/9
45
Inhibition: (C-TIC) x 100 where C: average tumor area of control; T: average tumor area of treated group (both at day 20). Regression: number of tumor-free mice/number of tumor-bearing mice.
Table 4 Effects of polysaccharides on lymphocyte mitogenity. LPS-content (ng/mg(
conc.
control
—
—
LPS
—
10_i
—
0
5
10
15
20
25
ConA
—
HIB la
0.6
5
30
daya after tumor Inoculation
HIB2)3
6.0
HIB3
10
0.001 0.00 1
4
1.34 1.54
0.003 0.001 0.002 0.001
4
1.23
102
2.39
According Student's T-Test.
For the test of antitumor activity the sensitive allogenic transplanted tumor sarcoma 180/CD-i mice was used (Fig. 7). Mice were treated with polysaccharide solutions as described earlier (18). HIB 3 was applied in a concentration of 5mg/kg (19) (Table 3). Concerning the tumor area 20 days after tumor inoculation, tumor growth was inhibited to 50%. 10 days later the average tumor area had doubled, whereas the control group had to be sacrificed because of their physical affection by the tumor growth. It can be stated that HIB 3 only exhibits a tumor-growth-retarding effect in this test system as has been shown for other acidic polysaccharides (20, 21, 22). RIB 3, RIB icr, and RIB 2/3, were also tested for their mitogenic activity (17). In this assay the activation of lymphocytes by immunostimulating agents like ConA or other lectins is quantified. However, it must be kept in mind that substrate contamination with endotoxins (lipopolysac-
charide = LPS) can induce blast transformation by itself.
LPS contamination was detected for all RIB fractions (Table
4). In the neutral fractions it could be removed using Polymyxin-Sepharose (24). In the case of RIB 3 this proce-
dure was not feasible due to the strong binding of the
3.68 456.0 1.75
Stim. index: cpmSUbSl + lymphoc /cpmlymphoc.
Within the scope of a screening program for biologically active polysaccharides from fungi and higher plants the described polysaccharide fractions of Hibiscus sabdariffa were investigated for a possible antitumor and/ or immunomodulating effect.
—
0.001 0.001 0.001
1.52
3.94
mice).
Biological activity of the polysaccharide
1.000
4
Fig. 7 Test for antitumor activity of HIB 3 (growth of sarcoma 180/CD-i
fractions
significance'
102 102
control 0 HIB 3 (5 mg/kg)
stim.index'
ig/mI
polysaccharide to the column. Acid- or alkali-treatment was
not utilised because of their affect on the polysaccharide structure. HIB 3 was tested in comparison to the activity of commercially available Salmonella typhimurium LPS (Table 4). Additionally, lymphocytes were derived from female C3H/HFJ LPS-low responder mice. Both neutral fractions with a minor LPS-content exhibited a pronounced
mitogenic activity. Comparing the stimulatory indices of tested LPS-concentrations with the LPS in RIB 3 (max. iO .tg/ml) the stimulating activity of HIB 3 is significantly higher. However, assays of LPS-free polysaccharide fractions would be desirable. Further experiments on the evaluation of a physiological activity of RIB 3 are in progress (25). First re-
sults indicate a possible influence of RIB 3 on in vivo physiological processes too, as demonstrated in the R3327-MatLu prostate cancer metastasis test model, where RIB 3 effected a significant increase of lung metastases (26).
Discussion Three distinct polysaccharide fractions isolated from the flower buds of Hibiscus sabdariffa could be
demonstrated. The minor fractions RIB Ia and RIB 2/3 seem to represent neutral arabinans (Mr 2 i0) besides arabinogalactans (M. 6.3• 10). Since they could not be separated from each other their respective structures could not be further elucidated. RIB 3 showed the properties of a pectin-like polysaccharide with side chains of Gal and Ara. Ara is only present in the periphery of the molecule (non-reducing end of the side chains) and is connected to Gal.
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Substance
Planta Med. 58(1992) 67
Chemical Structure and Biological Acticity ofPolysaccharides from Hibiscus sabdariffa
References 2
6
Blumenkrantz, N., Asboe-Hansen, G. (1973) Anal. Biochem. 54, Is
12 13
Results of the biological activity of HIB 3 are
14
in agreement with earlier findings (31). In the tests for im-
15
munomodulating effects (25), for instance the test for mitogenic activity, HIB 3 proved to be active. However, the results are confused by the presence of LPS. The test for an
antitumor activity demonstrated a negligable activity for HIB 3. These results, combined with the cited effects of aqueous extracts of Hibiscus sabdariffa (3, 4) and the effect in the metastatic R-3327 MatLu test system (26), indicate that the pectin fraction HIB 3 seems to have physiological activities. Further tests will be required to clarify the possible medical value of this compound.
16
17
18 19
The authors are grateful to F. Roflkopf and J. Kraus for the skilful performance of the mitogenicity test and the discussions, to SALUS/Bruckmilhl for the gift of Hibiscus sabdar(ffa flower buds, the Deutsche Krebshilfe and the Fonds der Chemischeu Industrie for financial support.
484—489. Lowry, 0. H., Rosehrough, N. J., Farr, A. L., Randall, R. J. (1951) J. Biol. Chem. 193, 265—274. Wood, P., Siddiqul, J. (1971) Ana!. Biochem. 39, 418—428. McComh, F., Mdfieady, H. (1957) Anal. Chem. 29, 819—821. Blakeney, A.. Harris, P., Henry, H., Stone, B. (1983) Carbohydr. Res. 113, 291—299. Tay!or, R. T., Conrad, H. F. (1972) Biochem. 11, 1383—1388. Waeghe. T., Darvill, A., Albersheim, P., McNe!!, M. (1983) Car-
hohydr. Res. 123, 281—304. Stevenson, T., Darvill, A., Albersheim, P. (1988) Carbohydr. Res. 179, 269—288. Newton, P. C., Uh!, J. (1989) Pharmacological Methods in the Control of Inflammation, 83—99, Alan H. Liss, Inc., New York. Kraus, J. (1987) Thesis, Regensburg/FRG. Bruneteau, M., Fabre, I., Ricci, P., Joseleau, J.-P., Kraus, J., Bla-
schek, W., Schneider, M., Franz, G. (1988) Carbohydr. Res. 175, 137— 143. 20 21 22
Acknowledgements
Riaz, A., Rahman, R. (1968) Sci. md. 6, 74—86. Tomoda, M., Shimizu, N. Gonda, R., Kanari, M., Yamada, H., Hikino, H. (1989) Carbohydr. Res. 190, 323—328. Sharaf, A. (1962) Planta Med. 10,48—55. Sharaf,A., Gineidi, A. (1963) Planta Med. 11, 109—112. Haq, Q. N., Gomes, J. (1974) Banglad. J. Sd. md. Res. 9, 11—15. Riaz, A., Rahman, H. (1967) Sci. md. 435—441. Franz, G., Franz, M. (1988) Zeitschr. f. Phytoth. 9, 63—66. Morris, D. L. (1948) Science 107, 254.
23
Muller, B. M., Kraus, J., Franz, G. (1989) Planta Med. 55, 536—539. MUller, B. M., RoflkopL F., Paper, D., Kraus, J., Franz, G. (1991) Die Pharmazie 46, 557—663. Muller, B. M. (1990) Thesis, Regensburg/FRG. Niva, M., Umeda, M., Ohashi, K. (1988) Bacterial endotoxin: Chemical, Biological and Clinical aspects, (Homma, J. Y., Kanegasaki, S.,
Luderitz, 0., Shiba, T., Westphal, 0., eds.), Verlag Chemie, Weinheim, 383—394. Issekutz, A. C. (1983) J. Immunol. Meth. 61, 275—281. Ro0kopf, F., Personal communication. 26 Kager, M. (1990) Thesis, Regensburg/FRG. 27 Tomoda, M., Shimada, K., Shimizu, N., Kanari, M., Kaneko, F. (1986) Carbohydr. Res. 151, 29—35. 20 Tomoda, M., lchikawa, M. (1987) Chem. Pharm. Bull. 35 (6), 2360— 2367. 29 Shimizu, N., Tomoda, M., Adachi, M. (19861 Chem. Pharm. Bull 34 (10), 4133—4138. Tomoda, M., Gonda, H., Shimizu, N., Nakanishi, S., Hikimo, H. (1987) Phytochemistry 26, 2297—2300. 31 Kiyohara, H., Cyong, J.-C., Yamada, H. (1988) Carhohydr. Res. 182, 24 25
° 32
259— 272. O'Neill, M., Albersheim, P., Darvill, A. (1990) in: Methods in Plant
Biochemistry, Vol. 2, (Dey, P. M., ed), Academic Press, London.
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This finding is different from other pectin structures demonstrated in different Hibiscus species (27, 28, 29, 30): HIB 3 posesses a higher amount of uronic acids and a lower one of neutral sugars compared to the pectins described for other Hibiscus species. No G1cA could be detected linked via C-3 of GalA (27). None of the GalA was hranched. Other pectin sidechains are mostly built of G1cA, terminal and 4-linked Gal; none of the Hibiscus polysaccharides described so far contained branched Gal and terminal, 5-linked and 2,5-branched Ara as constituents of the sidechains. However, the polysaccharide structures found in Hibiscus sabdarzfj'a flower buds are not essentially new. Related polysaccharides have been described in Angelica acutiloba for instance (31), and additionally the side chains structure is related to the one described for RG 1(32).
Chemical Structure and Biological Activity of Polysaccharides from Hibiscus sabdariffa BerndM. Muller1'2 and Gerhard Franz1'3 1 2
Faculty of Pharmacy, University of Regensburg, D(W)-8400 Regensburg, Federal Republic of Germany Present address: Complex Carbohydrate Research Center, The University of Georgia, 220 Riverbend Road,
Athens, GA 30602, U.S.A.
Address for correspondence Received: January 14, 1991
Three water-soluble polysaccharides
Hibiscus sabdariffa L. (Malvaceae) is known to contain large quantities of viscous polysac-
have been isolated from flower buds of Hibiscus sab-
charides (1). Several other species of Hibiscus are used in
darWa L. (HIB 1, 2,3). The neutral polysaccharides (HIB
1 and 2) are composed of arabinans and arabinogalactans of low relative molecular mass. The major fraction was investigated by methylation analysis, pectinasetreatment, mild acid hydrolysis, and NMR studies, and it was shown to be a pectin-like molecule (Mr =
io d). The
main chain is composed of a-1,4-linked GalA (24% methyl-esterified) and a-1,2-linked Rha. Side chains are built of Gal and Ara and are connected to the main chain
via C-4 of every third Rha. Its structure seems to be different from polysaccharide structures described in other species of the Hibiscus genus and the Malvaceae family. All fractions were assayed for possible immunemodulating effects. All fractions showed some activity,
but the main acidic fraction was contaminated with lipopolysaccharide, and therefore its shown activity has to be discussed carefully. Key words ________________________________________
oriental medicine and were shown to contain polysaccharides with marked physiological activities (2). Hibiscus sabdariffa flower buds are mainly used for refreshing infusions. Their medical value is still uncertain although there are some reports about physiological activities of aqueous extracts of this drug: Sharaf(3, 4) reported the decrease of blood pressure, relaxation of rat uteri, inhibition of taenia motility and bacterial growth for aqueous extracts, Little is known about the exact chemical na-
ture of the polysaccharides in Hibiscus sabdariffa flower buds (1, 5, 6). Up to now the mentioned physiological activities of the aqueous extracts could not be attributed to known constituents (7). It seemed to be interesting to investigate the structure ofthe polysaccharide fraction and to test them for specific physiological activities.
Materials and Methods Materials
Hibiscus sabdarffa, Malvaceae, polysaccharides, pectin, immunological activity.
Flower buds of Hibiscus sabdarffa L. were obtained from SALUS (Bruckmuhl, FRG). All chemicals were of analytical grade quality. DEAE-Sephacel, Sephadex G 50, and
Abbreviations and Symbols
Superose p.g. were purchased from Pharrnacia. Aspergillus niger pectinase and Salmonella typhimurium lipopolysaccharide were obtained from Sigma.
Ara: Mr: MrCO:
Gal: GalA: LPS: Glc: G1cA:
Man: Rha: Xyl:
arabinose relative molecular mass molecular mass cut off galactose galacturonic acid lipopolysaccharide glucose glucuronic acid mannose rhamnose xylose
General methods Total carbohydrate, uronic acid, and protein were assayed by the anthrone (8), m-hydroxybiphenyl (9), and Lowry methods (10), respectively, using neutral standard sugars, GalA, and bovine serum albumin as the respective standards. Methyl and acetyl group content was assayed by the methods of Wood et al. (11) and McComb et al. (12), respectively, using methanol and D-glucose pentaacetate standards. The molecular weight distribution of polysaccharides were estimated by GPC with the aid of standard pullulans (Shodex).
Polysaccharides were hydrolysed with 2 M tnfluoroacetic acid (TFA) at 121°C for 90 mm. Sugars were converted into their corresponding alditol acetates by the method of Blakeney et al. (13). GLC was performed at 207°C using a Varian 3700 gas
chromatograph with FID on a glass capillary column (0.25mm x
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Introduction
Abstract
Planta Med. 58(1992) 61
Chemical Structure and Biological Activity of Polysaccharides from Hibiscus sabdar/'fa
30m) packed with 3% of OV 225, using He as carrier gas. The molar ratio of the neutral sugars was calculated from the peak areas and molecular weight of the corresponding alditol acetates. The molar ratio of uronic acid and neutral sugars was calculated from the uronic acid content. Reduction of acidic sugars was done in the presence of carbodiimide using NaBH4 (14).
pies were reduced with Li(Et)3BD/THF (16) and dialysed again. The
premethylated and deutero reduced poiysaccharides were methylated using Dimsyl-K/CH3I according to (15) except that the sampies were purified by dialysis. The methylated samples were by-
drolysed, the products were reduced with NaBH4, followed by acetylation. The resulting partially methylated alditol acetates
were analysed by GLC-MS on a Hewlett Packard model 5890 A with a DB 1701-30W capillary column (0.25 .tm film, 0.25mm x 30 m).
Dried plant material was powdered (0.5 mm) and preextracted by percolation with MeOH until the extract was colourless. For the isolation of the polysaccharides, the dried preextracted powder was stirred with water as a 5 % suspension at 20 °C for 12 h. The crude polysaccharide fraction was obtained by pouring the water extract into the five-fold volume of 99% EtOH at 4°C.
NMR-analysis 1H-NMR was performed on a Bruker WM at 250 Hz and 297K with D20 as internal standard (6 = 4.67 ppm). 13C-NMR was run at 297—307 K at 62.896 Hz with TMS as internal standard. Samples were dissolved in D20.
The precipitate was dissolved in water, dialysed against water
Biological tests
d) and freeze-dried. Purification and fractionation was carried out on DEAE-Sephacel (P043-, pH = 6.0). Fractions were subsequently eluted with water, a linear phosphate-buffer(MrCO = 3.500
gradient (0-1 M) as shown in Fig. 1 and finally 0.2 M NaOH. Further purification was achieved by GPC on Sephadex G-50 and Superose 12 pg., respectively.
Determination of iipopoiysaccharide contamination For quantitation of endotoxins in the poiysaccharide samples the LAL-kit of Kabi vitrum was used. LPS deriving from Salmonella typhimurium was used as reference.
Digestion with pectinase
Test for mitogenic activity (17)
HIB 3 (50 mg) was digested with pectinase (1.0 U) in 50mM acetate buffer (pH = 4.2, 10—20 ml) for three days at 30°C
in a shaker. After neutralisation, the mixture was heated to 100°C for 10 mm to inactivate the enzyme, centrifuged, and separated on Sephadex G-50.
Mild acid hydrolysis
were removed (Skatron cell harvester 7020/7019), washed with distilled water and dried at 60°C for 1 h. 2 ml of scintillation
The polysaccharide fractions were subjected to
cocktail were added (Packet Szinti Emulsifier 299) and the samples were counted for 5mm
hydrolysis with increasing strength, using TFA at concentrations of 0.05 M and 0.5 Mat 100°C for 1 h. The products were separated by GPC on Sephadex G-50.
at 121°C for 90 mm. 0.1 ml of ascites liquid (5 i06 cells) of sar-
Acidic polysaccharides were methylated once by the method of Waeghe et al. (15), followed by dialysis and freeze drying. In order to facilate permethylation, the premethylated sam-
HIB HIB RIB RIB HIB HIB HIB RIB RIB RIB RIB RIB RIB RIB
in
lalA 1f3 213
2131A
3u 3j3
313pecct 313pecjl
3jipecy
3/iA
3/lB 3/iC
RIB 3/2A RIB 3/2B RIB 3/2C RIB
3/la
RIB 3/lb RIB 3/ic RIB 3/2a RIB 3/2b
Yield°
UA
Rha
Ara
5.3
85.2
1.7
7.4
1.2 45.2
4.7 0.4
0.3
0.0
67.8 0.0
2.3
0.3 0.0 0.0
0.9
76.1
1.0
67.8
0.0
86.0 85.2 43.9 51.9 79.2 44.2 91.3
2.4
48.9 5.4
2.5 54.2 12.3 21.7 3.0 2.7 46.5 5.0 63.2 2.8 10.4
31.6 4.1
17.2 11.4 78.9 12.5 41.1
CD-i mice. After randomisation into groups of 10 mice polysaccharide solutions were injected i.p., starting 24 h after tumor in-
Xyl
Man
0.6 1.2
0.1
3.9
1.5
4.1 3.1
iRS
7.4 6.2 7.8
0.6
Gal
2.6 1.3 0.3
4.4 11.4
34.8
4.9 1.1 0.9
4.2
0.3
4.6
0.5 0.8
5.1
11.4 1.2
0.6 1.1 0.6 0.7
8.9
3.2
1.7
2.7 56.1
0.9 0.3 0.0
5.5 0.2
0.5
0.2
0.3 0.0 1.9
1.5 0.3 3.5 8.3
4.9
0.0 37.8
2.6
1.8 3.1 12.6 0.6
4.4
1.5
3.9
3.4
GIc
91.1 4.4 19.9
0.2
96.4 91.0
Calculated as weight % of applied material.
coma 180 were s.c. injected into the right side of allogenous female
7.2 11.5 18.2 11.7
2.6 2.7
33.5
0.0 80.9 83.0 73.7 83.4 82.5
Test of antitumor activity (18) Poiysaccharide solutions in PBS were autociaved
Methylation analysis
Fract.
Spleen lymphocytes were obtained from C3H/ HEJ-mice and were cultivated in 96 well microtiter plates together with RPMI-medium (Gibco) in 10% FCS, 1% Pen-Strep, 2 mmol glutamine, and the test substance. After 48 h incubation, 30 zl [3JJ] thymidine was added to the cells (4 105/well. After 18h the cells
1.1
0.9
1.9
9.9 0.0 0.0 0.7 0.0 0.6
0.1
0.0 0.9 0.8 0.0 0.0
5.6
33.8
1.1
13.1
3.1
3.8
2.6
33.8
5.2 0.7
2.7 5.1
0.6 3.6 32.9 8.6 6.3 7.2 1.8 4.7
1.1
0.9 0.0 9.4 3.7 3.7 1.5 1.5
6.2
Table 1 Sugar composition (mol %) of polysaccharide fractions from Hibiscus sabdariffa.
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Isolation and pur(fication of water-soluble polysaccharides
BerndM. Muller and Gerhard Franz
62 Planta Mcd. 58(1992)
oculation, for 10 days at a concentration of 5 mg/kg. The control group was treated with PBS only. Tumor growth was measured
160
A 620 nm
buffer-mol.
every 10 days. After 30 days the mice were sacrificed and evaluated hy comparing each group's fresh tumor weight:
inhibition rate: (C-T/C) 100
C: average tumor weight in control group, T: average tumor weight in treated group.
Regression: number of tumor free mice/number of tumor bearing mice. Results
Isolation Preextraction with MeOH removed 45% of
amounts of Rha, Gal, and Ma. Only 1.8% was detectable as protein.
Fractionation
Three fractions of BIB were obtained by 0 50 100 150 250 200 300 DEAE-Sephacel chromatography with water and a phosfractions (2 ml) phate-buffer gradient (Fig. 1). BIB I was eluted by water (3.5% of applied material) and was mainly composed of Fraction 1 — 50 Ma. BIB 2 (2.4%) was eluted with the onset of the buffer H20 elution gradient and also contained mainly Ma (76 %). The main Buffer gradient Fraction 51 — 200 polysaccharide fraction HIB 3 (33%) was eluted at a buffer 0.2 N NaOH Fraction 201 — 250 strength of 0.45 M. By further elution with buffer BIB 4was obtained, similar in composition to BIB 3. With 0.2 N NaOH a hygroscopic and red coloured complex was obtained (BIB 5). BIB 3 was predominantly composed of uronic acid besides small amounts of Rha, Ara and Gal. Alter reduction of
Fig. 1 Fractionation of HIB on DE.AE-Sephacel.
A 620 nm
the native polysaccharide, the uronic acid could be identified as GalA.
HIB 1, 2, and 3 were further purified by
2.5
GPC. BIB 1 gave two subfractions after elution on Sephadex G-50 (Fig. 2). The first one, HIB icr (Mr 1 10) was mainly composed of Ma and Gal (Table 1). The second one, BIB 1/3 (Mr: 6 102) was red coloured and not further investigated.
BIB 2 turned out to be a neutral polysaccharide too. Fractionation on G-50 (Fig. 3) showed its main portion had a M of 6.3 103. Its sugar composition was related to BIB Ia. HIB 3 showed a high range Mr-distribution and was further
purified on Superose 12. The main fraction (BIB 3a) showed an Mr of 1.5 io5, the minor one (BIB 3/3) 5 10g. Both fractions had similar sugar compositions (Table 1)
consisting mainly of GalA which was 24% methyl-esterified. It is most likely that BIB 3 consists of one type of polysaccharide showing a broad range of Mr.
Pectinase-treatinent BIB 3 was digested with pectinase and the
resulting products were separated on Sephadex G-50,
yielding three subfractions (Fig. 4): BIB 3peca, a small fraction in the void volume, BIB 3pecfl, representing an inter-
mediate fraction, and the major fraction BIB 3pecy. BIB 3peca mainly consisted of GalA, Rha, Ma, and Gal, but the
0
20
40
60 80 fractions (1 ml)
Fig.2 GPC of HIB ion Sephadex G-50.
100
120
140
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the dry weight of Hibiscus sabdar[fa flower buds. The water extract of the crushed material yielded 5.3% of a crude polysaccharide fraction (BIB). It was composed of large amounts of uronic acid (Table 1) as well as small
Planta Med. 58(1992) 63
Chemical Structure and BiologicalActivity of Polysaccharides from Hibiscus sabdariffa
A 620
A 620
0.6
0.6
0.4
0
20
40
60 80 fraction (1 ml)
100
120
140
140
fraction (1 ml)
Fig.3 GPC of HIB 2 on Sephadex G-50.
Fig.4 GPO at HIB 3 after pectinase treatment.
GalA amount was diminished to 1/2 ofits initial content. Subfractions /3 and y showed increasing amounts of GalA.
position C-4. The small amount or 3,6-branched Gal decreased whereas 6-linked Gal increased, proving that at least some of the Ara residues had been connected to posi-
Methylation analysis (Table 2) showed that HIB 3peca contained terminal and 5-linked Ara; terminal, 2-linked, and 2,4-branched Rha; terminal, 4-, and 6-linked, and 3,6-branched Gal, and 4-linked GalA. HIB 3pecy was composed of terminal, 5-linked, and 2,5-disubstituted Ma
tion 3 of 3,6-branched Gal exclusively.
amount of terminal and 4-linked GalA. These results find an explanation in the fact that HIB 3pec/J and HIB 3pecy derive from a-1,4-linked GalA-rich regions of HIB 3. Thus, HIB 3 seems to he built up of two different carbohydrate blocks, represented by (1 —* 4)poly a-o-GaIA regions and neutral
nal, 5-linked, and 2,5-branched Ara. Gal was mainly 4linked apart from a minor amount of 3,6-branching. After
and terminal and 4-linked Gal besides a dominating
sugar enriched blocks (HIB 3peca) forming regions of branched structures. Mild acid hydrolysis
We have investigated the effect of hydrolysis with increasing TFA-concentrations on the M distribution and sugar composition of HIB 1, 2 and 3.
HIB la HIB Ia predominantly contained Ara, terminal (27%) and 5-linked (53 %), besides 4-linked Gal. Only 10% of the Gal amount was 3,6-branched. After hydrolysis with 0.05 N TFA nearly all Ara residues had been cleaved.
The remaining polysaccharide HIB I alA (Fig. 2) was mainly composed of Gal (Table 1). Methylation analysis showed that 91 % of the Gal residues remained linked by
HIB 2/3
This polysaccharide fraction seemed to be very related to HIB la. Its high Ara content showed termihydrolysis with 0.05 N TFA, no 3,6-branched Gal could be detected. The percentage amount of 6-linked Gal remained unchanged. Maybe the amount of 3,6-branched Gal vanished by the release of the Ara residues connected to it as well. Not all Ara had been hydrolysed. HIB 2/31A showed terminal and 5-linked Ma (ratio 1: 2) and 4-linked Gal. This
might be due to the existence of arabinans as well as arabinogalactans in HIB 2/3. HIB 3
a), The first attempt to use mild acid hydrolysis for the structural elucidation of HIB 3 was the combination of two increasing hydrolysis methods using 0.05 N and 0.5 N TFA, sequentially. TheJirst step resulted in a de-
gradation of HIB 3 into three subfractions that could be eluted from Sephadex G-50 (Fig. Sa). HIB 3/lA was obtained in the void volume (5 % of applied material). Its sugar composition was related to HIB 3peca with GalA and Gal as major components. Only 50% of the fraction was suscepti-
ble to the pectinase treatment. The main fraction HIB 3/lB
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0.2
64 Planta Med. 58(1992)
Bernd M. Muller and Gerhard Franz
la
lalA
2f
1—
18.6
13.4
1.8
35.8 13.4 67.8
0 0 0 0
19.2
1,5— 1,2,5—
32.5 16.0 67.7
26.8 8.7 48.9
3.2 1.5 6.5
0 0
0 o 0 o 0
0 o 0 o 0
0 0 0 0 0
0 0
0 0
0.4
17.1
82.6
0
7.5
0 0 28.9 5.9 0 34.8 4.9 0 0 11.4 11.4
3/la
3/lA
3/lB
1.9 0.0 0.0 1.9
3.2 0.0 0.0 3.2
2.7 0.0
8.3
0.0 2.7
0.0 11.5
0 1.7 0 0.9 2.6
0
0.1 1.0
3.0
0 1.8 4.9
0 6.7 0 2.2 8.9
0.4 2.3 2.5 0.8 1.3 8.3
1.3 8.6 3.5 2.0 1.8 17.2
0 0 21.1 5.5 6.5 34.1
0
0
0
0
0
0 0 0 0
0 0 0 0
0 0 0
0
0 0 0 0
2lA
Table 2 Sugar linkages in the main polysac-
3$peca
charidefractionsofHibiscussabdariffa
Ara
3.2
Rha 1—
0
1,2—
0 0
1,4— 1,2,4—
3.1
2.2
0 0.5
1.2 1.2
1.6
7.6
0.7
3.3
0 0
0
1—
(1,4—) 1,4— 1,6—
1,3,6—
0
18.9
90.5
13.4 3.3 2.7 19.8
7.4
6.2
5.6
0 0
1.8
0
GIc 1,4—
Man 1—
1.4
1.5
1,2— 1,4—
0.8 2.7 4.9
0 1.6
3.1
4.4 4.4
0
1.0
4.9 0.9 2.0
2.7
11.1
0.9
0 0 0
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Gal
Represents 1,4-GalA after reduction prior to methylation.
A 620 nm
A 620 nm 1
0.8
0.6
0.4
0.2
0
0
20
40
60 80 fraction (1 ml)
100
120
140
0
20
40
60 80 fraction (1 ml)
100
120
140
Fig. 5a GPC of HIB 3 after hydrolysis (0.05 N TFA).
Fig. 5b GPC of HIB 3/lB after hydrolysis (0.5 N TFA).
(63 % yield) showed an increased GalA content besides Rha,
3/lB is composed mainly of a- 1,4-linked poly-Ga1A. HIB 3/
Gal, and Ara moieties. Pectinase treatment yielded an excessive degradation with only few percent of the fraction re-
1C was mainly composed of Ma which had been cleaved from the native polysaccharide by hydrolysis.
sisting the enzyme treatment. This is due to the fact that HIB
Planta Med. 58(1992)
Chemical Structure and BiologicalActivity ofPolysaccharides from Hibiscus sabdariffa
65
The degradation of HIB 3 resulted in a
A 620 nm
selective removal of Ara residues. This did not affect the ratio of 2-linked to 2,4-branched Rha, indicating that Ma was not bound to Rha directly. The ratio within the Gal-linkages changed. A decrease of 3,6-branched Gal was accom-
panied by an increase of terminal and 6-bound Gal. The cleaved Ma residues and the small amount of hydrolysed Gal residues must have been connected to Gal via position 3 and/or 6. Since no 3-linked Gal could be detected and nearly
all Ma residues had been hydrolysed, it seems to be likely that Ara was preliminary bound to position 3 whereas Gal residues were connected to position 6 of branched Gal. The
ratio of 2,4-branched Rha to Gal (1 : 5) remained unchanged. In conclusion it seems that the neutral sugar enriched regions of HIB 3 are composed of side chains connected to the backbone of pectin via C-4 of branched Rha. Only 1/3 of the Rha residues are branched. The side chains (6-): 2—3 (3,6-). No terminal GalA could be detected. The ratio of Rha : GalA in the presumed backbone (between 1 : 32 and 0
20
40
60 80 fraction (imi)
100
120
140
Elution profile with 0.05 N TFA
0.5 N TFA
1 : 54) is difficult to determine due to the possible formation of aldobiuronic acids and polysaccharide-degradation during reduction procedures (data not shown).
NMR-Studies
Fig. 6 GPC of HIB 3 after mild acid hydrolysis.
'3C-NMR: Weak signals at 16.83 and The second step of hydrolysis (0.5 N TFA) was carried out with HIB 3/lB (Fig. 5b). By this treatment a polysaccharide was precipitated (HIB 3/2A). It was NaOHsoluble and composed of a 1,4-linked GalA only. The high Mr fraction of the soluble residue of HIB 3/lB (HIB 3/2B) was composed of GalA (> 90%), Rha and Gal, 3.5% each. The low Mr fraction HIB 3/2C contained Ara and Gal which had not been cleaved off during the first hydrolysis. Also
these results indicated the presence of an a 1,4-GalA polymer (see HIB 3/2A and HIB 3/2B) and a second part containing neutral sugars (HIB 3/lA). The methylation results of the neutral sugar enriched fraction HIB 3/lA (Table 2) showed the presence of terminal and 5-linked Ara, 2-linked and 2,4-disubstituted Rha, 6- and 4-linked and 3,6-branched Gal. Alter reduction of the polysaccharide the GalA turned out to be bound via position 4. The methylation results of the other fractions showed only few neutral sugars. HIB 3/lB consists of terminal and 5-linked Ara, terminal, 2-linked and 2,4-
branched Rha, and terminal, 6-linked and 3,6-disubstituted Gal.
b) The second attempt of mild acid hydrolysis was done with each of the native polysaccharides for every hydrolysis. The hydrolysis with 0.5 M TFA resulted in a more pronounced degradation with a vanishing amount of high-Mr fraction HIB 3/2a (Fig. 6).
20.46 ppm indicated the presence of methyl groups of Rha and of acetyl groups. This is in accordance with weak signals at 1.48 and 1.96 ppm in the proton spectrum. An expressed signal at 53.12 ppm could derive from 0-methyl groups of methyl-esterified GalA. Signals between 68 and 80 ppm (86.31, 69.43, 79.10, 70,8 1) could derive from carbons C-2 to C-5 of GalA, the dominating sugar residue in HIB 3. In the area of anomeric C-atom signals, a signal at 100.52 ppm proved the a-configuration of GalA. Further signals at 175.60 and 170.98 ppm were identified as deriving from non-esterified and esterified C-6 of GalA. The intensity of the signal at 170.98 ppm is in good accordance with the one at 53.12 ppm standing for -OCH3.
Summary of overall structural
characteristics
NMR-analysis of HIB 3, pectinase-treatment, mild acid hydrolysis and methylation analysis gave rise to the following facts: HIB 3 represents a pectin with an average M of iO d. Its main sugar components are GalA (84%), methyl-esterified (24%), and smaller amounts of Rha, Ara, and Gal. The molecule is built by at least two different carbohydrate regions, i.e poly-a-(1,4)-GaIA and neutral sugar enriched blocks with Ara- and Gal-containing side chains. Rha is located in the main chain with every third unit being branched via C-4 bearing side chains. Rha is linked to Gal in the ratio 1 : 10—11. The non-reducing end of the side chains is built of Ma disaccharides linked 1,5;
Methylation analysis of HIB 3/la (Table 2)
probably connected to position 3 of branched Gal. The
branched Rha (unchanged ratio) and terminal, 4- and 6-
3,6-branched Gal in the ratio of 1 : 5 : 2 :2—3 and terminal, 5-linked and 2,5-branched Ara in the ratio 2 : 3: 1.
showed few terminal Ara besides 2-linked and 2,4- whole side chains consist of terminal, 4- and 6-linked and linked, and 3,6-branched Gal.
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are built of Ma and Gal in the ratio 1 (terminal):5 (4-): 2
66 Planta Med. 58 (1992)
BerndM. Muller and Gerhard Franz
tumor area fmm'i
Table 3 Treatment of sarcoma 180/CD-i with HIB3. dose (mg/kg)
average tumorweight (g)
control
—
—
HIB3
5
2.36
inhibition
regression
0
0/15
4/9
45
Inhibition: (C-TIC) x 100 where C: average tumor area of control; T: average tumor area of treated group (both at day 20). Regression: number of tumor-free mice/number of tumor-bearing mice.
Table 4 Effects of polysaccharides on lymphocyte mitogenity. LPS-content (ng/mg(
conc.
control
—
—
LPS
—
10_i
—
0
5
10
15
20
25
ConA
—
HIB la
0.6
5
30
daya after tumor Inoculation
HIB2)3
6.0
HIB3
10
0.001 0.00 1
4
1.34 1.54
0.003 0.001 0.002 0.001
4
1.23
102
2.39
According Student's T-Test.
For the test of antitumor activity the sensitive allogenic transplanted tumor sarcoma 180/CD-i mice was used (Fig. 7). Mice were treated with polysaccharide solutions as described earlier (18). HIB 3 was applied in a concentration of 5mg/kg (19) (Table 3). Concerning the tumor area 20 days after tumor inoculation, tumor growth was inhibited to 50%. 10 days later the average tumor area had doubled, whereas the control group had to be sacrificed because of their physical affection by the tumor growth. It can be stated that HIB 3 only exhibits a tumor-growth-retarding effect in this test system as has been shown for other acidic polysaccharides (20, 21, 22). RIB 3, RIB icr, and RIB 2/3, were also tested for their mitogenic activity (17). In this assay the activation of lymphocytes by immunostimulating agents like ConA or other lectins is quantified. However, it must be kept in mind that substrate contamination with endotoxins (lipopolysac-
charide = LPS) can induce blast transformation by itself.
LPS contamination was detected for all RIB fractions (Table
4). In the neutral fractions it could be removed using Polymyxin-Sepharose (24). In the case of RIB 3 this proce-
dure was not feasible due to the strong binding of the
3.68 456.0 1.75
Stim. index: cpmSUbSl + lymphoc /cpmlymphoc.
Within the scope of a screening program for biologically active polysaccharides from fungi and higher plants the described polysaccharide fractions of Hibiscus sabdariffa were investigated for a possible antitumor and/ or immunomodulating effect.
—
0.001 0.001 0.001
1.52
3.94
mice).
Biological activity of the polysaccharide
1.000
4
Fig. 7 Test for antitumor activity of HIB 3 (growth of sarcoma 180/CD-i
fractions
significance'
102 102
control 0 HIB 3 (5 mg/kg)
stim.index'
ig/mI
polysaccharide to the column. Acid- or alkali-treatment was
not utilised because of their affect on the polysaccharide structure. HIB 3 was tested in comparison to the activity of commercially available Salmonella typhimurium LPS (Table 4). Additionally, lymphocytes were derived from female C3H/HFJ LPS-low responder mice. Both neutral fractions with a minor LPS-content exhibited a pronounced
mitogenic activity. Comparing the stimulatory indices of tested LPS-concentrations with the LPS in RIB 3 (max. iO .tg/ml) the stimulating activity of HIB 3 is significantly higher. However, assays of LPS-free polysaccharide fractions would be desirable. Further experiments on the evaluation of a physiological activity of RIB 3 are in progress (25). First re-
sults indicate a possible influence of RIB 3 on in vivo physiological processes too, as demonstrated in the R3327-MatLu prostate cancer metastasis test model, where RIB 3 effected a significant increase of lung metastases (26).
Discussion Three distinct polysaccharide fractions isolated from the flower buds of Hibiscus sabdariffa could be
demonstrated. The minor fractions RIB Ia and RIB 2/3 seem to represent neutral arabinans (Mr 2 i0) besides arabinogalactans (M. 6.3• 10). Since they could not be separated from each other their respective structures could not be further elucidated. RIB 3 showed the properties of a pectin-like polysaccharide with side chains of Gal and Ara. Ara is only present in the periphery of the molecule (non-reducing end of the side chains) and is connected to Gal.
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Substance
Planta Med. 58(1992) 67
Chemical Structure and Biological Acticity ofPolysaccharides from Hibiscus sabdariffa
References 2
6
Blumenkrantz, N., Asboe-Hansen, G. (1973) Anal. Biochem. 54, Is
12 13
Results of the biological activity of HIB 3 are
14
in agreement with earlier findings (31). In the tests for im-
15
munomodulating effects (25), for instance the test for mitogenic activity, HIB 3 proved to be active. However, the results are confused by the presence of LPS. The test for an
antitumor activity demonstrated a negligable activity for HIB 3. These results, combined with the cited effects of aqueous extracts of Hibiscus sabdariffa (3, 4) and the effect in the metastatic R-3327 MatLu test system (26), indicate that the pectin fraction HIB 3 seems to have physiological activities. Further tests will be required to clarify the possible medical value of this compound.
16
17
18 19
The authors are grateful to F. Roflkopf and J. Kraus for the skilful performance of the mitogenicity test and the discussions, to SALUS/Bruckmilhl for the gift of Hibiscus sabdar(ffa flower buds, the Deutsche Krebshilfe and the Fonds der Chemischeu Industrie for financial support.
484—489. Lowry, 0. H., Rosehrough, N. J., Farr, A. L., Randall, R. J. (1951) J. Biol. Chem. 193, 265—274. Wood, P., Siddiqul, J. (1971) Ana!. Biochem. 39, 418—428. McComh, F., Mdfieady, H. (1957) Anal. Chem. 29, 819—821. Blakeney, A.. Harris, P., Henry, H., Stone, B. (1983) Carbohydr. Res. 113, 291—299. Tay!or, R. T., Conrad, H. F. (1972) Biochem. 11, 1383—1388. Waeghe. T., Darvill, A., Albersheim, P., McNe!!, M. (1983) Car-
hohydr. Res. 123, 281—304. Stevenson, T., Darvill, A., Albersheim, P. (1988) Carbohydr. Res. 179, 269—288. Newton, P. C., Uh!, J. (1989) Pharmacological Methods in the Control of Inflammation, 83—99, Alan H. Liss, Inc., New York. Kraus, J. (1987) Thesis, Regensburg/FRG. Bruneteau, M., Fabre, I., Ricci, P., Joseleau, J.-P., Kraus, J., Bla-
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Muller, B. M., Kraus, J., Franz, G. (1989) Planta Med. 55, 536—539. MUller, B. M., RoflkopL F., Paper, D., Kraus, J., Franz, G. (1991) Die Pharmazie 46, 557—663. Muller, B. M. (1990) Thesis, Regensburg/FRG. Niva, M., Umeda, M., Ohashi, K. (1988) Bacterial endotoxin: Chemical, Biological and Clinical aspects, (Homma, J. Y., Kanegasaki, S.,
Luderitz, 0., Shiba, T., Westphal, 0., eds.), Verlag Chemie, Weinheim, 383—394. Issekutz, A. C. (1983) J. Immunol. Meth. 61, 275—281. Ro0kopf, F., Personal communication. 26 Kager, M. (1990) Thesis, Regensburg/FRG. 27 Tomoda, M., Shimada, K., Shimizu, N., Kanari, M., Kaneko, F. (1986) Carbohydr. Res. 151, 29—35. 20 Tomoda, M., lchikawa, M. (1987) Chem. Pharm. Bull. 35 (6), 2360— 2367. 29 Shimizu, N., Tomoda, M., Adachi, M. (19861 Chem. Pharm. Bull 34 (10), 4133—4138. Tomoda, M., Gonda, H., Shimizu, N., Nakanishi, S., Hikimo, H. (1987) Phytochemistry 26, 2297—2300. 31 Kiyohara, H., Cyong, J.-C., Yamada, H. (1988) Carhohydr. Res. 182, 24 25
° 32
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This finding is different from other pectin structures demonstrated in different Hibiscus species (27, 28, 29, 30): HIB 3 posesses a higher amount of uronic acids and a lower one of neutral sugars compared to the pectins described for other Hibiscus species. No G1cA could be detected linked via C-3 of GalA (27). None of the GalA was hranched. Other pectin sidechains are mostly built of G1cA, terminal and 4-linked Gal; none of the Hibiscus polysaccharides described so far contained branched Gal and terminal, 5-linked and 2,5-branched Ara as constituents of the sidechains. However, the polysaccharide structures found in Hibiscus sabdarzfj'a flower buds are not essentially new. Related polysaccharides have been described in Angelica acutiloba for instance (31), and additionally the side chains structure is related to the one described for RG 1(32).
