Carbohydrate Research 391 (2014) 16–21
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Structural characterization of an acetylated glucomannan with antiinflammatory activity and gastroprotective property from Cyrtopodium andersonii José P. Parente a,⇑, Camila R. Adão a, Bernadete P. da Silva a, Luzineide W. Tinoco b a Laboratório de Química de Plantas Medicinais, Núcleo de Pesquisas de Produtos Naturais, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, PO Box 68045, CEP 21941-971 Rio de Janeiro, Brazil b Laboratório Multiusuário de Análises por Ressonância Magnética Nuclear, Núcleo de Pesquisas de Produtos Naturais, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, CEP 21941-902 Rio de Janeiro, Brazil
a r t i c l e
i n f o
Article history: Received 1 February 2014 Received in revised form 14 March 2014 Accepted 18 March 2014 Available online 27 March 2014 Keywords: Cyrtopodium andersonii Orchidaceae Acetylated glucomannan Antiinflammatory activity Gastroprotective property
a b s t r a c t A polysaccharide with an estimated weight-average molar mass of 5.35 105 was obtained from an aqueous extract of pseudobulbs of Cyrtopodium andersonii R. Br. It was composed of D-glucose and D-mannose in 1:3 molar ratio. Chemical and spectroscopic analyses revealed a linear structure of the polymer with a backbone composed of (1 ? 4)-linked b-D-glucopyranosyl and mannopyranosyl units slightly branched at C-2, C-3, and C-6 by side chains, as terminal non reducing residues of D-mannopyranose and D-glucopyranose. It was found to contain 14.6% of acetyl groups substituted at C-2 of (1 ? 4)-linked b-D-mannopyranosyl units. The acetylated glucomannan demonstrated antiinflammatory and antiulcerogenic activities. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The glucomannans are widespread in nature and can be extracted from different botanical sources, whose main function is acting as storage macromolecules. These polysaccharides have been largely consumed as nutritional supplements and some biological effects suggest its potential as bioactive polymers.1 Important health benefits are described for these compounds, which are currently used to reduce cholesterol, normalize triglyceride content and improve blood sugar levels, promoting intestinal activity and immune function.2 Orchidaceae is the largest and most evolved family of the flowering plants and comprises 25,000–35,000 species under 750–850 genera. Several orchid species have been used in different countries for therapeutic properties and are rich sources of bioactive polysaccharides.3 The Cyrtopodium genus comprises about 45 species of epiphytic and terrestrial orchids, some of which are used in the traditional medicine, in particular Cyrtopodium punctatum, which in Brazil is used against inflammatory disorders and commercialized as a phytopharmaceutical formulation for the treatment of abscesses and burnings.4 In a preliminary research, a ⇑ Corresponding author. Tel.: +55 21 2562 6791; fax: +55 21 2270 2683. E-mail address: [email protected] (J.P. Parente). http://dx.doi.org/10.1016/j.carres.2014.03.021 0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.
polysaccharide was isolated from Cyrtopodium cardiochilum and its therapeutic potential as a biological response modifier investigated.5 Cyrtopodium andersonii R. Br. (Orchidaceae) is a native terrestrial plant which grows wild in the coasts and cliffs. Sometimes this species is cultivated for ornamental purposes. In Brazil, the salep from the pseudobulbs of this plant is used in the traditional medicine for treating inflammatory symptoms, showing wound healing property, and antihemorrhagic activity.4 The present paper is concerned with the isolation, chemical characterization, and evaluation of the antiinflammatory property and gastroprotective activity of a glucomannan from the pseudobulbs of this medicinal species. 2. Results and discussion 2.1. Isolation and purification of the polysaccharide The crude neutral polysaccharide was extracted with hot water from the fresh pseudobulbs of C. andersonii, previously cut into small pieces. A sample of this fraction was fractionated by means of Sephacryl S-300 HR and desalted by Sephadex G-25 gel permeation chromatography, leading to the isolation of a neutral, protein free polysaccharide fraction. It showed negative specific rotation,
J. P. Parente et al. / Carbohydrate Research 391 (2014) 16–21
½a20 D 85° (c 0.1 H2O). The weight-average molar mass of andersonii polysaccharide was estimated to be 5.35 105 based on the calibration curve of the elution volume of standard dextrans from gel filtration on Sephacryl S-300 HR. 2.2. Structural analysis of the polysaccharide Glucose and mannose were identified as the component sugars of andersonii polysaccharide by means of silica gel TLC of the acid hydrolysates and by GC–EIMS of the trimethylsilylated methyl glycosides derivatives prepared from the monosaccharides. Quantitative determination showed that the molar ratio of glucose, mannose was 1:3, respectively, with no other sugars. The absolute configurations of the glucose and mannose were determined by GC of their trimethylsilylated ()-2-butylglucoside and ()-2-butylmannoside. D-Glucopyranose and D-mannopyranose were identified. Thus, polysaccharide isolated from C. andersonii could be called as a glucomannan. The polysaccharide (andersonii glucomannan) was methylated by the method of Parente et al.6 The fully methylated product was hydrolyzed with acid, converted into the alditol acetates, and analyzed by GC and GC–EIMS. Andersonii glucomannan furnished 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl mannitol, 1,5-di-Oacetyl-2,3,4,6-tetra-O-methyl glucitol, 1,4,5-tri-O-acetyl-2,3,6-triO-methyl mannitol, 1,4,5-tri-O-acetyl-2,3,6-tri-O-methylglucitol, 1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl mannitol, 1,2,4,5-tetra-Oacetyl-3,6-di-O-methyl mannitol, 1,4,5,6-tetra-O-acetyl-2,3-di-Omethyl mannitol, in the relative molar ratios of 1:4:541:178:13:10:16, respectively. The branches explicitly join through C-2, C-3, and C-6 positions of D-mannopyranosyl units in main chain, which is composed of b-(1 ? 4)-linked D-mannopyranosyl and D-glucopyranosyl units. It is noted that the D-glucopyranosyl units do not participate in the branching as constituents of the main chain. The number of branches for every 100 monosaccharide residues (n) at C-2, C-3, and C-6 positions of D-mannopyranosyl units is calculated from the following equations:
n¼
ð2;3;4;6ManÞþð2;3;4;6GlcÞ 100 ð2;3;6ManÞþð2;3;6GlcÞþð3;6 or 2;6 or 2;3ManÞ
n¼
The absorption bands at 1734 and 1250 cm1 were attributed to C@O and CAO stretching bands of acetate, respectively. The characteristic peaks at 890, 878, and 812 cm1 were assigned to the b-pyranose form of glucose and mannose. The 1H (Fig. 1) and 13C NMR (Fig. 2) chemical shifts are presented in Table 1. The HSQC spectrum (Fig. 3) of the andersonii glucomannan was recorded, and the 1H-13C signals were assigned according to the literature.8–11 The HSQC spectrum clearly indicated that the andersonii glucomannan was composed of the sugar residues ?4)-b-D-Manp-(1?,?4)-b-D-Glcp-(1? and ?4)-2-Oacetyl-b-D-Manp-(1?. In order to determine the position of the acetyl groups at the mannopyranosyl units, the 1H and 13C NMR spectra of the andersonii glucomannan were very useful. The signals at d 99.9 and 70.8, belonged to C-1 and C-3 of 2-O-acetyl-(1 ? 4)-linked Manp, respectively. For the 2-O-acetyl derivative, C-1 and C-3 were shifted upfield significantly as compared with C-1 and C-3 of nonacetylated Manp. Due to the deshielding effect of acetyl group, one signal of H-2 in 2-O-acetylated b-D-Manp units occurred at d 5.45. Correlation of the signal at d 5.45 with the signal of C-2 in 2-Oacetylated b-D-Manp units at d 72.5 was found by HSQC. This is in accordance with the NMR data reported by Capek et al.9
2.3. Determination of the degree of acetylation The IR spectrum of andersonii glucomannan has absorptions at 1734 cm1 (C@O) and 1250 cm1 (CAO), suggesting the presence of ester linkages. In the 1H NMR spectrum of andersonii glucomannan (Fig. 1), signals showed at d 2.12–2.20 correspond to CH3 of acetyl groups. In the 13C NMR spectrum (Fig. 2) of andersonii glucomannan, the signals at d 20.9 and 21.2 ppm were attributed to CH3 and the signal at d 174.2 ppm attributed to C@O of acetyl groups. These features could be used as evidences for the presence of acetyl groups. Degree of acetylation (DA) of the andersonii glucomannan could be determined by using the data of 1H NMR spectroscopy. The DA value was estimated from the formula:
DA ¼
or
ð3;6 or 2;6 or 2;3ManÞ 100 ð2;3;6ManÞþð2;3;6GlcÞþð3;6 or2;6 or2;3ManÞ
where (2,3,4,6-Man), (2,3,4,6-Glc), (2,3,6-Man), (2,3,6-Glc), (3,6Man), (2,6-Man), and (2,3-Man) indicate relative molar ratios of partially methylated alditol acetates described above. The results obtained from the above equations n = 0.68 or 1.37, n = 0.68 or 1.77, n = 0.68 or 2.18 indicated that the branches at C-2, C-3, and C-6 positions of the D-mannopyranosyl units in the main chain exist for every 73–147, 56–147, and 46–147 monosaccharide residues, respectively. These results were estimated according to Katsuraya et al.7 The above results reveal the general structural features of the glucomannan. Thus, 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl mannitol and 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol in 1:4 molar ratio shows that the glucomannan possesses terminal non-reducing residues of D-mannopyranose and D-glucopyranose. The IR spectrum of the polysaccharide showed a wide band at 3000–3750 cm1 attributed to the OAH stretching of the glucomannan. The bands at 2932 and 2893 cm1 were attributed to the asymmetrical and symmetrical stretchings of CAH, respectively. The band at 1638 cm1 was ascribed to adsorbed water and the bands at 1422 and 1379 cm1 to the asymmetrical and symmetrical bending vibrations of CAH, respectively. The CAOAC system showed stretching at about 1150 cm1 while the CAO alcohol bond showed stretching bands at 1092, 1060, and 1028 cm1.
17
ðICH 100%Þ=3 IRH1
where ICH3 was the integral of the hydrogen atom in ACOCH3 group and I RH1 was total integral of the hydrogen atom of C-1 in ?4)-bD-Manp-(1?,?4)-b-D-Glcp-(1? and ?4)-2-O-acetyl-b-D-Manp(1? residues. As shown in Figure 1, the proton signal of acetyl group was separated well, so the value of acetyl content obtained could be quite accurate. The degree of acetylation of the andersonii glucomannan was determined to be DA = [(1 100%)/3]/ (0.64 + 1.21 + 0.43) 14.6. This result was obtained according to An et al.2 The numbers of the ?4)-2-O-acetyl-b-D-Manp-(1? (n1) and ?4)-b-D-Manp-(1? (n2) residues were calculated from the following equations, respectively:
n1 ¼ DA ½ð2; 3; 6 ManÞ þ ð2; 3; 6 GlcÞ=100 n2 ¼ ð2; 3; 6 ManÞ n1 where DA indicates the degree of acetylation and (2,3,6-Man) and (2,3,6-Glc) indicate their relative molar ratios described in section 2.2. The results obtained from the above equations n1 = 105 and n2 = 436 indicated that the numbers of the ?4)-2-O-acetyl-b-DManp-(1? and ?4)-b-D-Manp-(1? residues were 105 and 436, respectively (Fig. 4).
18
J. P. Parente et al. / Carbohydrate Research 391 (2014) 16–21
Figure 1. The 1H NMR spectrum of the glucomannan (500 MHz, D2O, 80 °C).
C-1 Man CH3
C-1 Glc
C=O
ppm Figure 2. The
Table 1 H (italics) and
1
13
13
C NMR spectrum of the glucomannan (125 MHz, D2O, 80 °C).
C NMR chemical shifts (d, ppm) for the constituent monomers of the acetylated glucomannan of Cyrtopodium andersonii
Sugar residue
?4)-b-D-Manp-(1? ?4)-b-D-Glcp-(1?
C-1
C-2
C-3
C-4
C-5
C-6
H-1
H-2
H-3
H-4
H-5
H-6
100.8 4.72 103.1 4.49
71.1 4.10 73.5 3.32 72.5 5.45
72.1 3.73 74.5 3.58 70.8 4.02
77.1 3.81 79.1 3.55 77.1 3.81
75.7 3.66 75.3 3.58 75.7 3.58
61.5 3.92, 3.78 61.5 3.82, 3.67 61.5 3.92, 3.78
?4)-2-O-acetyl)-b-D-Manp-(1 ? 99.9 4.88
2.4. Investigation of the antiinflammatory activity and gastroprotective property According to the literature, several glucomannans are used as components of nutritional supplements and demonstrate interesting pharmacological properties. These polysaccharides are known to equilibrate cholesterol levels, normalize triglyceride content and regulate the glycemic index.2 In order to confirm the utiliza-
C@O
CH3
174.2
20.9, 21.2 2.12–2.20
tion of this medicinal species in the treatment of inflammatory disorders, the antiedematogenic property of the glucomannan was investigated using an acute inflammation model. The results were measured by inhibition of carrageenan-induced mouse paw edema.12 The carrageenan-induced inflammation is a biphasic phenomenon. The early phase of edema is attributed to the release of histamine, serotonin, and similar substances. The later phase results mainly from the potentiating effects of prostaglandins on
19
J. P. Parente et al. / Carbohydrate Research 391 (2014) 16–21
Figure 3. The HSQC spectrum of the glucomannan isolated from Cyrtopodium andersonii.
O OH O
HO O HO
HO O HO
436
OH O
HO HO HO
HO HO D-Glc p - (1
HO
O OH
4
O
O HO
OH O
HO
O
O
105
O O HO
O
OH O
D - Man p - (1 or HO HO HO D-Glc p - (1
HO HO HO
2 O OH
2
OH O
HO O HO 16
13
10
HO HO
or
HO
O
HO
4
D - Man p - (1
O
OH O
D - Man p - (1 or HO HO HO D-Glc p - (1
HO HO HO
3 O OH
3
OH O
D - Man p - (1 or HO HO HO D-Glc p - (1
HO O HO
6 O OH
6
O OH
178
O
OH
OH
D-Glc p - ( 4 1 or OH HO O O OH HO D - Man p - ( 4
1
Figure 4. Schematic representation of the acetylated glucomannan of C. andersonii, where the acetyl groups are substituted at C-2 of (1 ? 4)-linked Manp. The values 10, 13, 16, 105, 178, and 436 were calculated on the basis of the weight-average molar mass of the glucomannan, molar carbohydrate composition and degree of acetylation.
mediator release.13 The polysaccharide showed significant antiinflammatory potential, controlling the initial phase of inflammation and provoking an inhibition of edema formation similar to the reference compounds indomethacin and dexamethasone (Fig. 5). However, the andersonii glucomannan exhibited lesser activity when compared with controls in the delayed phase of inflammation. The polysaccharide inhibited primarily the initial phase of edema, and it is known that soon after carrageenan injection there is a sudden elevation of tissue volume, correlating with the action
of inflammatory mediators on vascular permeability. Since several polysaccharides sharing the same backbone and branches possess the ability to inhibit the increase in vascular permeability,5,8,11 which is a typical model of first stage inflammatory reaction, probably this can be the mode of action of the isolated glucomannan. Generally, commercially available antiinflammatory drugs are associated with unwanted side effects especially ulceration, which is the most common and serious problem.14 Since there is popular information about the wound healing property and antihemor-
20
J. P. Parente et al. / Carbohydrate Research 391 (2014) 16–21
225 carrageenan
Paw oedema (mm x 10 -2)
200
dexamethasone
175
indomethacin glucomannan
150 125
*
*
*
100
*
*
75
**
50 25
** **
****
** **
1
2
** ** *
0 0
3
4
5
sists of a viscous, elastic, and adherent barrier formed by water and glycoproteins that covers the entire gastrointestinal mucosa.17 The protective properties of the mucous barrier depend not only on its structure but also on the amount or thickness of the layer covering the mucosal surface.18 Since literature reports indicated that polysaccharides of this type possess the ability to increase the mucosal defensive factors,19 inducing the turnover of glycoproteins in the mucosal cells, thus increasing the quantity of cellular mucus, probably this can be the mode of action of the glucomannan, preventing the penetration of the necrotizing agent or interacting with the macromolecules of the gastric mucosa.20 These results suggest that andersonii glucomannan may be the potential therapeutic agent involved in the gastroprotective property and the treatment of inflammatory conditions, justifying the use of C. andersonii in the traditional medicine.
Time after injection (h) Figure 5. Antiinflammatory activity of the glucomannan (100 mg/kg, p.o.) and reference compounds indomethacin (25 mg/kg, p.o.) and dexamethasone (25 mg/ kg, p.o.) against mouse paw edema induced by carrageenan. Results are mean ±SEM (n = 5); ⁄p
Contents lists available at ScienceDirect
Carbohydrate Research journal homepage: www.elsevier.com/locate/carres
Structural characterization of an acetylated glucomannan with antiinflammatory activity and gastroprotective property from Cyrtopodium andersonii José P. Parente a,⇑, Camila R. Adão a, Bernadete P. da Silva a, Luzineide W. Tinoco b a Laboratório de Química de Plantas Medicinais, Núcleo de Pesquisas de Produtos Naturais, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, PO Box 68045, CEP 21941-971 Rio de Janeiro, Brazil b Laboratório Multiusuário de Análises por Ressonância Magnética Nuclear, Núcleo de Pesquisas de Produtos Naturais, Centro de Ciências da Saúde, Universidade Federal do Rio de Janeiro, CEP 21941-902 Rio de Janeiro, Brazil
a r t i c l e
i n f o
Article history: Received 1 February 2014 Received in revised form 14 March 2014 Accepted 18 March 2014 Available online 27 March 2014 Keywords: Cyrtopodium andersonii Orchidaceae Acetylated glucomannan Antiinflammatory activity Gastroprotective property
a b s t r a c t A polysaccharide with an estimated weight-average molar mass of 5.35 105 was obtained from an aqueous extract of pseudobulbs of Cyrtopodium andersonii R. Br. It was composed of D-glucose and D-mannose in 1:3 molar ratio. Chemical and spectroscopic analyses revealed a linear structure of the polymer with a backbone composed of (1 ? 4)-linked b-D-glucopyranosyl and mannopyranosyl units slightly branched at C-2, C-3, and C-6 by side chains, as terminal non reducing residues of D-mannopyranose and D-glucopyranose. It was found to contain 14.6% of acetyl groups substituted at C-2 of (1 ? 4)-linked b-D-mannopyranosyl units. The acetylated glucomannan demonstrated antiinflammatory and antiulcerogenic activities. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction The glucomannans are widespread in nature and can be extracted from different botanical sources, whose main function is acting as storage macromolecules. These polysaccharides have been largely consumed as nutritional supplements and some biological effects suggest its potential as bioactive polymers.1 Important health benefits are described for these compounds, which are currently used to reduce cholesterol, normalize triglyceride content and improve blood sugar levels, promoting intestinal activity and immune function.2 Orchidaceae is the largest and most evolved family of the flowering plants and comprises 25,000–35,000 species under 750–850 genera. Several orchid species have been used in different countries for therapeutic properties and are rich sources of bioactive polysaccharides.3 The Cyrtopodium genus comprises about 45 species of epiphytic and terrestrial orchids, some of which are used in the traditional medicine, in particular Cyrtopodium punctatum, which in Brazil is used against inflammatory disorders and commercialized as a phytopharmaceutical formulation for the treatment of abscesses and burnings.4 In a preliminary research, a ⇑ Corresponding author. Tel.: +55 21 2562 6791; fax: +55 21 2270 2683. E-mail address: [email protected] (J.P. Parente). http://dx.doi.org/10.1016/j.carres.2014.03.021 0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.
polysaccharide was isolated from Cyrtopodium cardiochilum and its therapeutic potential as a biological response modifier investigated.5 Cyrtopodium andersonii R. Br. (Orchidaceae) is a native terrestrial plant which grows wild in the coasts and cliffs. Sometimes this species is cultivated for ornamental purposes. In Brazil, the salep from the pseudobulbs of this plant is used in the traditional medicine for treating inflammatory symptoms, showing wound healing property, and antihemorrhagic activity.4 The present paper is concerned with the isolation, chemical characterization, and evaluation of the antiinflammatory property and gastroprotective activity of a glucomannan from the pseudobulbs of this medicinal species. 2. Results and discussion 2.1. Isolation and purification of the polysaccharide The crude neutral polysaccharide was extracted with hot water from the fresh pseudobulbs of C. andersonii, previously cut into small pieces. A sample of this fraction was fractionated by means of Sephacryl S-300 HR and desalted by Sephadex G-25 gel permeation chromatography, leading to the isolation of a neutral, protein free polysaccharide fraction. It showed negative specific rotation,
J. P. Parente et al. / Carbohydrate Research 391 (2014) 16–21
½a20 D 85° (c 0.1 H2O). The weight-average molar mass of andersonii polysaccharide was estimated to be 5.35 105 based on the calibration curve of the elution volume of standard dextrans from gel filtration on Sephacryl S-300 HR. 2.2. Structural analysis of the polysaccharide Glucose and mannose were identified as the component sugars of andersonii polysaccharide by means of silica gel TLC of the acid hydrolysates and by GC–EIMS of the trimethylsilylated methyl glycosides derivatives prepared from the monosaccharides. Quantitative determination showed that the molar ratio of glucose, mannose was 1:3, respectively, with no other sugars. The absolute configurations of the glucose and mannose were determined by GC of their trimethylsilylated ()-2-butylglucoside and ()-2-butylmannoside. D-Glucopyranose and D-mannopyranose were identified. Thus, polysaccharide isolated from C. andersonii could be called as a glucomannan. The polysaccharide (andersonii glucomannan) was methylated by the method of Parente et al.6 The fully methylated product was hydrolyzed with acid, converted into the alditol acetates, and analyzed by GC and GC–EIMS. Andersonii glucomannan furnished 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl mannitol, 1,5-di-Oacetyl-2,3,4,6-tetra-O-methyl glucitol, 1,4,5-tri-O-acetyl-2,3,6-triO-methyl mannitol, 1,4,5-tri-O-acetyl-2,3,6-tri-O-methylglucitol, 1,3,4,5-tetra-O-acetyl-2,6-di-O-methyl mannitol, 1,2,4,5-tetra-Oacetyl-3,6-di-O-methyl mannitol, 1,4,5,6-tetra-O-acetyl-2,3-di-Omethyl mannitol, in the relative molar ratios of 1:4:541:178:13:10:16, respectively. The branches explicitly join through C-2, C-3, and C-6 positions of D-mannopyranosyl units in main chain, which is composed of b-(1 ? 4)-linked D-mannopyranosyl and D-glucopyranosyl units. It is noted that the D-glucopyranosyl units do not participate in the branching as constituents of the main chain. The number of branches for every 100 monosaccharide residues (n) at C-2, C-3, and C-6 positions of D-mannopyranosyl units is calculated from the following equations:
n¼
ð2;3;4;6ManÞþð2;3;4;6GlcÞ 100 ð2;3;6ManÞþð2;3;6GlcÞþð3;6 or 2;6 or 2;3ManÞ
n¼
The absorption bands at 1734 and 1250 cm1 were attributed to C@O and CAO stretching bands of acetate, respectively. The characteristic peaks at 890, 878, and 812 cm1 were assigned to the b-pyranose form of glucose and mannose. The 1H (Fig. 1) and 13C NMR (Fig. 2) chemical shifts are presented in Table 1. The HSQC spectrum (Fig. 3) of the andersonii glucomannan was recorded, and the 1H-13C signals were assigned according to the literature.8–11 The HSQC spectrum clearly indicated that the andersonii glucomannan was composed of the sugar residues ?4)-b-D-Manp-(1?,?4)-b-D-Glcp-(1? and ?4)-2-Oacetyl-b-D-Manp-(1?. In order to determine the position of the acetyl groups at the mannopyranosyl units, the 1H and 13C NMR spectra of the andersonii glucomannan were very useful. The signals at d 99.9 and 70.8, belonged to C-1 and C-3 of 2-O-acetyl-(1 ? 4)-linked Manp, respectively. For the 2-O-acetyl derivative, C-1 and C-3 were shifted upfield significantly as compared with C-1 and C-3 of nonacetylated Manp. Due to the deshielding effect of acetyl group, one signal of H-2 in 2-O-acetylated b-D-Manp units occurred at d 5.45. Correlation of the signal at d 5.45 with the signal of C-2 in 2-Oacetylated b-D-Manp units at d 72.5 was found by HSQC. This is in accordance with the NMR data reported by Capek et al.9
2.3. Determination of the degree of acetylation The IR spectrum of andersonii glucomannan has absorptions at 1734 cm1 (C@O) and 1250 cm1 (CAO), suggesting the presence of ester linkages. In the 1H NMR spectrum of andersonii glucomannan (Fig. 1), signals showed at d 2.12–2.20 correspond to CH3 of acetyl groups. In the 13C NMR spectrum (Fig. 2) of andersonii glucomannan, the signals at d 20.9 and 21.2 ppm were attributed to CH3 and the signal at d 174.2 ppm attributed to C@O of acetyl groups. These features could be used as evidences for the presence of acetyl groups. Degree of acetylation (DA) of the andersonii glucomannan could be determined by using the data of 1H NMR spectroscopy. The DA value was estimated from the formula:
DA ¼
or
ð3;6 or 2;6 or 2;3ManÞ 100 ð2;3;6ManÞþð2;3;6GlcÞþð3;6 or2;6 or2;3ManÞ
where (2,3,4,6-Man), (2,3,4,6-Glc), (2,3,6-Man), (2,3,6-Glc), (3,6Man), (2,6-Man), and (2,3-Man) indicate relative molar ratios of partially methylated alditol acetates described above. The results obtained from the above equations n = 0.68 or 1.37, n = 0.68 or 1.77, n = 0.68 or 2.18 indicated that the branches at C-2, C-3, and C-6 positions of the D-mannopyranosyl units in the main chain exist for every 73–147, 56–147, and 46–147 monosaccharide residues, respectively. These results were estimated according to Katsuraya et al.7 The above results reveal the general structural features of the glucomannan. Thus, 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl mannitol and 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl glucitol in 1:4 molar ratio shows that the glucomannan possesses terminal non-reducing residues of D-mannopyranose and D-glucopyranose. The IR spectrum of the polysaccharide showed a wide band at 3000–3750 cm1 attributed to the OAH stretching of the glucomannan. The bands at 2932 and 2893 cm1 were attributed to the asymmetrical and symmetrical stretchings of CAH, respectively. The band at 1638 cm1 was ascribed to adsorbed water and the bands at 1422 and 1379 cm1 to the asymmetrical and symmetrical bending vibrations of CAH, respectively. The CAOAC system showed stretching at about 1150 cm1 while the CAO alcohol bond showed stretching bands at 1092, 1060, and 1028 cm1.
17
ðICH 100%Þ=3 IRH1
where ICH3 was the integral of the hydrogen atom in ACOCH3 group and I RH1 was total integral of the hydrogen atom of C-1 in ?4)-bD-Manp-(1?,?4)-b-D-Glcp-(1? and ?4)-2-O-acetyl-b-D-Manp(1? residues. As shown in Figure 1, the proton signal of acetyl group was separated well, so the value of acetyl content obtained could be quite accurate. The degree of acetylation of the andersonii glucomannan was determined to be DA = [(1 100%)/3]/ (0.64 + 1.21 + 0.43) 14.6. This result was obtained according to An et al.2 The numbers of the ?4)-2-O-acetyl-b-D-Manp-(1? (n1) and ?4)-b-D-Manp-(1? (n2) residues were calculated from the following equations, respectively:
n1 ¼ DA ½ð2; 3; 6 ManÞ þ ð2; 3; 6 GlcÞ=100 n2 ¼ ð2; 3; 6 ManÞ n1 where DA indicates the degree of acetylation and (2,3,6-Man) and (2,3,6-Glc) indicate their relative molar ratios described in section 2.2. The results obtained from the above equations n1 = 105 and n2 = 436 indicated that the numbers of the ?4)-2-O-acetyl-b-DManp-(1? and ?4)-b-D-Manp-(1? residues were 105 and 436, respectively (Fig. 4).
18
J. P. Parente et al. / Carbohydrate Research 391 (2014) 16–21
Figure 1. The 1H NMR spectrum of the glucomannan (500 MHz, D2O, 80 °C).
C-1 Man CH3
C-1 Glc
C=O
ppm Figure 2. The
Table 1 H (italics) and
1
13
13
C NMR spectrum of the glucomannan (125 MHz, D2O, 80 °C).
C NMR chemical shifts (d, ppm) for the constituent monomers of the acetylated glucomannan of Cyrtopodium andersonii
Sugar residue
?4)-b-D-Manp-(1? ?4)-b-D-Glcp-(1?
C-1
C-2
C-3
C-4
C-5
C-6
H-1
H-2
H-3
H-4
H-5
H-6
100.8 4.72 103.1 4.49
71.1 4.10 73.5 3.32 72.5 5.45
72.1 3.73 74.5 3.58 70.8 4.02
77.1 3.81 79.1 3.55 77.1 3.81
75.7 3.66 75.3 3.58 75.7 3.58
61.5 3.92, 3.78 61.5 3.82, 3.67 61.5 3.92, 3.78
?4)-2-O-acetyl)-b-D-Manp-(1 ? 99.9 4.88
2.4. Investigation of the antiinflammatory activity and gastroprotective property According to the literature, several glucomannans are used as components of nutritional supplements and demonstrate interesting pharmacological properties. These polysaccharides are known to equilibrate cholesterol levels, normalize triglyceride content and regulate the glycemic index.2 In order to confirm the utiliza-
C@O
CH3
174.2
20.9, 21.2 2.12–2.20
tion of this medicinal species in the treatment of inflammatory disorders, the antiedematogenic property of the glucomannan was investigated using an acute inflammation model. The results were measured by inhibition of carrageenan-induced mouse paw edema.12 The carrageenan-induced inflammation is a biphasic phenomenon. The early phase of edema is attributed to the release of histamine, serotonin, and similar substances. The later phase results mainly from the potentiating effects of prostaglandins on
19
J. P. Parente et al. / Carbohydrate Research 391 (2014) 16–21
Figure 3. The HSQC spectrum of the glucomannan isolated from Cyrtopodium andersonii.
O OH O
HO O HO
HO O HO
436
OH O
HO HO HO
HO HO D-Glc p - (1
HO
O OH
4
O
O HO
OH O
HO
O
O
105
O O HO
O
OH O
D - Man p - (1 or HO HO HO D-Glc p - (1
HO HO HO
2 O OH
2
OH O
HO O HO 16
13
10
HO HO
or
HO
O
HO
4
D - Man p - (1
O
OH O
D - Man p - (1 or HO HO HO D-Glc p - (1
HO HO HO
3 O OH
3
OH O
D - Man p - (1 or HO HO HO D-Glc p - (1
HO O HO
6 O OH
6
O OH
178
O
OH
OH
D-Glc p - ( 4 1 or OH HO O O OH HO D - Man p - ( 4
1
Figure 4. Schematic representation of the acetylated glucomannan of C. andersonii, where the acetyl groups are substituted at C-2 of (1 ? 4)-linked Manp. The values 10, 13, 16, 105, 178, and 436 were calculated on the basis of the weight-average molar mass of the glucomannan, molar carbohydrate composition and degree of acetylation.
mediator release.13 The polysaccharide showed significant antiinflammatory potential, controlling the initial phase of inflammation and provoking an inhibition of edema formation similar to the reference compounds indomethacin and dexamethasone (Fig. 5). However, the andersonii glucomannan exhibited lesser activity when compared with controls in the delayed phase of inflammation. The polysaccharide inhibited primarily the initial phase of edema, and it is known that soon after carrageenan injection there is a sudden elevation of tissue volume, correlating with the action
of inflammatory mediators on vascular permeability. Since several polysaccharides sharing the same backbone and branches possess the ability to inhibit the increase in vascular permeability,5,8,11 which is a typical model of first stage inflammatory reaction, probably this can be the mode of action of the isolated glucomannan. Generally, commercially available antiinflammatory drugs are associated with unwanted side effects especially ulceration, which is the most common and serious problem.14 Since there is popular information about the wound healing property and antihemor-
20
J. P. Parente et al. / Carbohydrate Research 391 (2014) 16–21
225 carrageenan
Paw oedema (mm x 10 -2)
200
dexamethasone
175
indomethacin glucomannan
150 125
*
*
*
100
*
*
75
**
50 25
** **
****
** **
1
2
** ** *
0 0
3
4
5
sists of a viscous, elastic, and adherent barrier formed by water and glycoproteins that covers the entire gastrointestinal mucosa.17 The protective properties of the mucous barrier depend not only on its structure but also on the amount or thickness of the layer covering the mucosal surface.18 Since literature reports indicated that polysaccharides of this type possess the ability to increase the mucosal defensive factors,19 inducing the turnover of glycoproteins in the mucosal cells, thus increasing the quantity of cellular mucus, probably this can be the mode of action of the glucomannan, preventing the penetration of the necrotizing agent or interacting with the macromolecules of the gastric mucosa.20 These results suggest that andersonii glucomannan may be the potential therapeutic agent involved in the gastroprotective property and the treatment of inflammatory conditions, justifying the use of C. andersonii in the traditional medicine.
Time after injection (h) Figure 5. Antiinflammatory activity of the glucomannan (100 mg/kg, p.o.) and reference compounds indomethacin (25 mg/kg, p.o.) and dexamethasone (25 mg/ kg, p.o.) against mouse paw edema induced by carrageenan. Results are mean ±SEM (n = 5); ⁄p
