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Bioactive polysaccharides from medicinal plants used in woundhealing and against inflammation Berit Smestad Paulsen Department of Pharmacognosy School.

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Presentation on theme: "Bioactive polysaccharides from medicinal plants used in woundhealing and against inflammation Berit Smestad Paulsen Department of Pharmacognosy School."— Presentation transcript:

1 Bioactive polysaccharides from medicinal plants used in woundhealing and against inflammation Berit Smestad Paulsen Department of Pharmacognosy School of Pharmacy Oslo, Norway

2 MALI http://www.xs4all.nl/~ilja/mali/mali-map-htm

3 Malian population: Population: ca 12 mill. Population: ca 12 mill. Ethnic diversity: Sénoufo, Bambara, Sarakolé, Milinké, Peulh, Dogon, Bobo, Sonrhaï, Tamacheck…….. Ethnic diversity: Sénoufo, Bambara, Sarakolé, Milinké, Peulh, Dogon, Bobo, Sonrhaï, Tamacheck…….. Area Area 1 241 248 km 2 1 241 248 km 2 Expected living age: 47 years Expected living age: 47 years Age repartition: Age repartition: 47.2% : 0-14 years 47.2% : 0-14 years 5,5% : > 60 years 5,5% : > 60 years Fertility rate: 6.7 children born/woman Fertility rate: 6.7 children born/woman Child mortality: 12% Child mortality: 12% Rate of alphabetization: Rate of alphabetization: Men – 31% Men – 31% Women – 15% Women – 15% Appr. 75% in rural areas Appr. 75% in rural areas Photo: C.S. Nergård

4 Curative urban based health service, Curative urban based health service, limited curative or preventive health service in the rural areas limited curative or preventive health service in the rural areas 59% of the population have healthservice within 15 km 59% of the population have healthservice within 15 km Local clinics: Local clinics: difficult to keep the personnel Funding not available for long time treatment Medical doctors 1:16,000 inhabitant (Norway: 1:272) TRADITIONAL HEALERS 1:500 inhabitants 80% use traditional medicine Department of Traditional Medicine (DMT) Traditional medicine is composed of: -Secret traditional medicine -Methods of diagnosis -Methods of treatment

5 Ethnopharmacology The observation, identification, description and experimental investigation of the ingredients and the effects of such indigeneous drugs is a truly interdisiplinary field of research which is very important in the study of traditional medicine The observation, identification, description and experimental investigation of the ingredients and the effects of such indigeneous drugs is a truly interdisiplinary field of research which is very important in the study of traditional medicine The interdisiplinary scientific exploration of biologically active agents traditionally employed or observed by man. The interdisiplinary scientific exploration of biologically active agents traditionally employed or observed by man.

6 Traditional preparation and adminiatration Collection of the plant part Collection of the plant part Use either fresh or dry Use either fresh or dry Preparation Preparation Dry powder Dry powder Infusion or macerate Infusion or macerate Normally wash the body or drink the extract Normally wash the body or drink the extract

7 Preparation of the polysaccharides Ethanol extraction Ethanol extraction Water extraction 50 o C Water extraction 50 o C Dialysis, freezedrying Dialysis, freezedrying Anion exchange chromatography Anion exchange chromatography gelfiltration gelfiltration Water extraction 100 o C Water extraction 100 o C Dialysis, freezedrying Dialysis, freezedrying Anion exchange chromatography Anion exchange chromatography gelfiltration gelfiltration

8 Structure elucidation Methanolysis, TMS GC Methanolysis, TMS GC Carbohydrate composition Carbohydrate composition Methylation studies GC-MS Methylation studies GC-MS Linkage determination Linkage determination NMR anomeric configuration, sequence NMR anomeric configuration, sequence Mw - gelfiltration Mw - gelfiltration Enzymatic degradation Enzymatic degradation Gives polygalacturonans, hairy regions, oligosaccharide sidechains etc. Gives polygalacturonans, hairy regions, oligosaccharide sidechains etc.

9 Immunomodulation associated with activation of the complement system: Thymus dependant antibody response Thymus dependant antibody response Regulation of specific cyclic antibody production Regulation of specific cyclic antibody production Regulation of IgM-IgG switch Regulation of IgM-IgG switch Modulation of T and B cell proliferation Modulation of T and B cell proliferation Induction of supressor or helper T-cells Induction of supressor or helper T-cells Modulation of monokine or lympokine release Modulation of monokine or lympokine release

10 Structures of pectic type polysaccharides Arabinogalactan type I Arabinogalactan type I 1,4,gal 1,4,gal Arabinogalactan type II Arabinogalactan type II 1,3,6 gal 1,3,6 gal Rhamnogalacturonan I Rhamnogalacturonan I Hairy region, alternating rha / galA backbone Hairy region, alternating rha / galA backbone Rhamnogalacturonan II Rhamnogalacturonan II Macro-oligosaccharide, 20 units, complex Macro-oligosaccharide, 20 units, complex

11 Proposed structure of pectins, Perez et al 2003

12 Hairy region of rhamnogalacturonan I 2Rha1 4GalA1 2Rha1 4GalA1 2Rha1 4GalA1 2Rha1 4GalA1 2Rha1 4GalA1 2Rha1 4GalA1 2Rha1 4GalA1 2Rha1 4GalA1 4 4 4 4 4 4 1 1 1 1 1 1 Araf Gal Gal Araf Gal Gal 5 6 6 5 6 6 1 1 1 1 1 1 Araf1 3Araf Araf1 3Gal Gal Araf1 3Araf Araf1 3Gal Gal 5 6 6 5 6 6 1 1 1 1 1 1 Araf Gal Araf1g3Gal Araf Gal Araf1g3Gal 5 6 6 5 6 6 1 1 1 1 1 1 Araf1 3Araf Araf1 3Gal Gal Araf1 3Araf Araf1 3Gal Gal 5 6 5 6 1 1 1 1 Araf Araf Araf Araf

13 Are polysaccharides transported over membranes? Peyer´s patches Peyer´s patches Yamada et al. produced antibodies against bupleuran Yamada et al. produced antibodies against bupleuran Detected polymers with binding sites for the antibodies in liver Detected polymers with binding sites for the antibodies in liver

14 Bupleuran epitopes

15 Medicinal plants used for woundhealing in Mali Baccharoides adoensis var. kotschyana (Sch.Bip.ex.Walp.) M.A.Isawumi, G.El- Ghazaly & B.Nordenstam (Vernonia kotschyana (Sch.Bip.ex.Walp.)) Baccharoides adoensis var. kotschyana (Sch.Bip.ex.Walp.) M.A.Isawumi, G.El- Ghazaly & B.Nordenstam (Vernonia kotschyana (Sch.Bip.ex.Walp.)) Cochlospermum tinctorium Perr. Cochlospermum tinctorium Perr. Glinus oppositifolius (L.) A. DC. Glinus oppositifolius (L.) A. DC. Biophytum petersianum Klotzch Biophytum petersianum Klotzch

16 Baccharoides adoensis var. kotschyana (Vernonia kotschyana) Powdered roots are recognised by the government in Mali as a natural drug for the treatment of gastroduodenal ulcers. Powdered roots are recognised by the government in Mali as a natural drug for the treatment of gastroduodenal ulcers.GASTROCEDAL

17 Characteristics of A.2 Acacia gum (std.) Vk100 A1 A2 A2.1 A2.2 Gelfiltration of A.2 from V.kotschyana. Effect on the Yariv reagent, separation after enzymic degradation of A.2.2 A.2.1 A,2,2

18 Effect of A.2.1 after enzymatic degradation

19 Effect of A.2.2 after enzymic degradation

20 Mitogenic activity of fractions from V. kotchyana Alamar blue Acid phosphataeAlamar blue

21 Stimulation of macrophages with Vk100A2 fractions, chemotaxis in response of diff. conc. in 2 diff. patients

22 Yield (%, w/w), total carbohydrate content (%, w/w) and monosaccharide composition (mol.%) of the fractions Vk100A2a and Vk100A2b obtained after size exclusion chromatography on Sephacryl S-400 of Vk100A2. Polysaccharide fraction Vk100A2 ab Yield (%, w/w) a0.050.34 Total carbohydrate (%, w/w)86 68 Monosaccharide composition (mol.%) Arabinose31.33.9 Rhamnose11.04.9 Galactose24.44.9 Xylose 0.7trace Mannose 0.3 trace Glucose 5.71.8 Fucose -- Glucuronic acidtracetrace Galacturonic acid26.484.6 Reaction with Yariv-reagent+- Protein content (%, w/w) b< 0.5< 0.3 a based on dried and pulverized root b based on the Lowry method

23 Degradation of VKA2 by a, polygalacturonase followed by b. arabinofuranosidase followed by c. galactanase

24 Effect of the different fractions on the complement system

25 Mitogenic activity on the original fraction and the degradation products

26 Conclusion Bioactive parts on the VK1002a polysaccharide are situated both in the inner and in the external part of the pectic arabinogalactan Bioactive parts on the VK1002a polysaccharide are situated both in the inner and in the external part of the pectic arabinogalactan

27 Cochlospermum tinctorium, root The root is traditionally used agains gastric ulcer, often together with the powder from the root of V. kotchyana The root is traditionally used agains gastric ulcer, often together with the powder from the root of V. kotchyana

28 Cochlospermum tinctorium activity on whole spleen cells

29 Cochlospermum tinctorium activity on B - cells

30

31 Used in traditional medicine for the treatment of joint pains, inflammations, fever, malaria, skin disorders and wounds. Used in traditional medicine for the treatment of joint pains, inflammations, fever, malaria, skin disorders and wounds. The immune system an important factor in the wound healing process. The immune system an important factor in the wound healing process. Plant polysaccharides reported to have immunomodulating activities. Plant polysaccharides reported to have immunomodulating activities. Glinus oppositifolius (L.) A. DC. (Aizoaceae)

32 50°C water extract separated into one neutral (GON) and two acidic fractions (GOA1 and GAO2). 50°C water extract separated into one neutral (GON) and two acidic fractions (GOA1 and GAO2). The monosaccharide compositions of the crude water extract GO and the different fractions obtained after ion exchange chromatography.

33 The complement fixating activities determined in vitro. The complement fixating activities determined in vitro. GOA1 shows an ICH 50 value at 40 g/ml (the lowest concentration of sample needed to give 50% inhibition of lysis of antibody-sensitized SRBS). Plantago major L. was used as a positive control. GOA1 shows an ICH 50 value at 40 g/ml (the lowest concentration of sample needed to give 50% inhibition of lysis of antibody-sensitized SRBS). Plantago major L. was used as a positive control. Complement fixating activity I Complement activation associated with several immune reactions; activation of macrophages and lymphocytes, immunopotenation and regulation of cyclic antibody production, anti- inflammatory effects.

34 The main linkages in GOA1: GalA 1 4 Rha 1 2, 1 2,4 Gal 1 4, 1 3,4 Gal 1 3, 1 6, 1 3,6, 1 3,4,6 Araf T, 1 3, 1 5, 1 3,5 Linkage analysis

35 Chemotaxis of macrophages, B-cells, and T-cells. B-cells T-cellsMacrophages The migration of different leukocyte populations in response to GOA1.

36 Conclusion Two different pectic polysaccharides isolated from Glinus oppositifolius (L.) A. DC. (Aizoaceae) with potent complement fixating activity. Two different pectic polysaccharides isolated from Glinus oppositifolius (L.) A. DC. (Aizoaceae) with potent complement fixating activity. Further structure- and activity studies in progress. Further structure- and activity studies in progress.

37 A complement fixing polysaccharide from Biophytum petersianum Klotzch, a medicinal plant from Mali, West Africa.

38 Bp 100, Fr. III, and degradation fraction of Fr. III with the polygalacturonase, Fr.III 1 is the hairy region Monosaccharide Fr.III 123 Monosaccharide Fr.III 123 Arabinose8,57,911,110,4 Arabinose8,57,911,110,4 Rhamnose7,322,612,37,7 Rhamnose7,322,612,37,7 Fucose0,81,12,31,9 Fucose0,81,12,31,9 Xylose7,25,07.19,4 Xylose7,25,07.19,4 Mannose0,42,00,81,0 Mannose0,42,00,81,0 Galactose9,020,08,56,4 Galactose9,020,08,56,4 Galacturonic acid 64,138,555,260,8 Galacturonic acid 64,138,555,260,8

39

40 Linkage analyses of the different monosaccharides present in the fraction Bp 100 Fr.III and the fractions after polygalacturonase treatment Type of link B.P OrigB.P.I B.P.IIB.P.III Type of link B.P OrigB.P.I B.P.IIB.P.III T-Ara 6,2 7 8,5 8 T-Ara 6,2 7 8,5 8 1->5 Ara 2,7 1,1 0 0 1->5 Ara 2,7 1,1 0 0 T-Xyl l6,4 4,9 6,1 9,7 T-Xyl l6,4 4,9 6,1 9,7 1->2 Xyl 0 0 1,3 0 1->2 Xyl 0 0 1,3 0 1->4 Xyl 1,2 0,6 3,1 2,8 1->4 Xyl 1,2 0,6 3,1 2,8 T-Rha 1,2 2,4 5,2 2 T-Rha 1,2 2,4 5,2 2 1->2 Rha 3,414,2 2,5 3 1->2 Rha 3,414,2 2,5 3 1->3 Rha 0,8 0,8 2,9 1,6 1->3 Rha 0,8 0,8 2,9 1,6 1->3,4 Rha 0,5 0 1,8 1,6 1->3,4 Rha 0,5 0 1,8 1,6 1->2,4 Rha 2,5 6,3 0,7 0,5 1->2,4 Rha 2,5 6,3 0,7 0,5 T-Fuc 1 1,3 2,4 2,1 T-Fuc 1 1,3 2,4 2,1 T-Gal 3,1 6,8 5 1,9 T-Gal 3,1 6,8 5 1,9 1->3 Gal 2,2 4,9 3,4 3,7 1->3 Gal 2,2 4,9 3,4 3,7 1->6 Gal 1,1 3,1 0 0 1->6 Gal 1,1 3,1 0 0 1->3,6 Gal 3,3 6,2 0,6 1,4 1->3,6 Gal 3,3 6,2 0,6 1,4 T- GlcA tr tr. T- GlcA tr tr. T- GalA 2,5 2,3 4,1 6,4 T- GalA 2,5 2,3 4,1 6,4 1->4 GalA52,330,2 31,230,1 1->4 GalA52,330,2 31,230,1 1->3,4 GalA 8,5 6,8 17,922,2 1->3,4 GalA 8,5 6,8 17,922,2 1->2,4 GalA 1,1 1,1 3,3 3 1->2,4 GalA 1,1 1,1 3,3 3

41 Major features of the regions of BP BP I HMW BP I HMW Hairy region, alternating GalA and Rha, sidechains of 1/3 of Rha Hairy region, alternating GalA and Rha, sidechains of 1/3 of Rha BP II MMW BP II MMW High GalA, 1/3 branched High GalA, 1/3 branched BP II LMW BP II LMW High GalA, 2/3 branched High GalA, 2/3 branched

42 Conclusions Activity compared with structural differences indicate that the hairy region is important for the activity of the native polymer. Activity compared with structural differences indicate that the hairy region is important for the activity of the native polymer. It appears also that the regions of the original polymer containing sections of galacturonic acids that are highly branched, also are important for the activity when comparing those fractions with that of PMII not having long sections of branched uronic acid chains. It appears also that the regions of the original polymer containing sections of galacturonic acids that are highly branched, also are important for the activity when comparing those fractions with that of PMII not having long sections of branched uronic acid chains. Comparing the activity of Fr.III.1 with that of the native polymer, it is also obvious that the regions of the polymers giving rise to the other two fractions have an inhibitory effect on the activity of the total polymer. Comparing the activity of Fr.III.1 with that of the native polymer, it is also obvious that the regions of the polymers giving rise to the other two fractions have an inhibitory effect on the activity of the total polymer.

43 Bioactive polysaccharides from the stems of Acanthus ebracteatus Sanya Hokputsa, Stephen Harding, Kari Inngjerdingen, Kornelia Jumel, Terje Michaelsen, Thomas Heinze, Andreas Kochella and Berit Smestad Paulsen

44 Traditional use in Thailand and other countries in the region Sea holly, a mangrove plant Sea holly, a mangrove plant In Malaysia, In Malaysia, seeds against cough and boils seeds against cough and boils In China In China hepatitis, lymphoma and astma hepatitis, lymphoma and astma In Thailand In Thailand Root and stem for skin diseases and longevity Root and stem for skin diseases and longevity Crushed leaves on inflamed joints Crushed leaves on inflamed joints Decoction of the whole plant against inflammation Decoction of the whole plant against inflammation

45 Separation on DEAE-Sepharose, 50 o Extract

46 Separation on DEAE-Sepharose, 100 o Extract

47 Table 1. Appearance, yield, protein content and sugar composition (mol%) of polysaccharide fractions obtained after separation of the crude extracts on the DEAE- Sepharose Fast- flow column 60 C water extract 100 C water extract A601A602A1001A1002 Appearance cream, fluffy white, fluffy Yields a 20.050.210.148.9 Protein content (w/w) 4.86.45.96.2 Sugar composition b Ara Ara9.111.013.36.1 Rha Rha0.912.30.87.5 Fuc Fuctrace0.8trace0.3 Xyl Xyl0.61.41.10.8 Man Man3.61.42.30.4 Glc Glc4.33.19.92.2 Gal Gal55.018.739.611.0 3-O-Me-Gal 3-O-Me-Gal26.51.333.01.9 GalA GalA-50.0-69.8 a calculated as weight % of applied material b mol% of total carbohydrate content

48 Effect of the fractions in the complement system

49 Table 2. Glycosidic linkage composition (mol%) of the polysaccharide fractions obtained after separation of the crude extracts on the DEAE-Sepharose Fast-flow column Sugar Type of linkage Fraction A601A602A1001A1002 Ara TfTfTfTf1.82.52.72.3 1,31.0-0.7- 1,54.97.26.03.8 1,3,51.41.32.1- 1,2,5--1.8- RhaT-3.1-- 1,2-7.30.87.5 1,2,4-1.9-trace FucT--trace0.3 XylT--1.10.8 ManT3.61.42.3- GlcTtracetrace0.9trace 1,44.33.17.9- 1,6-0.4- 1,4,6--0.6- Gal a TpTpTpTp7.83.58.51.7 TfTfTfTf4.1-1.5- 1,455.89.857.95.3 1,64.61.42.3- 1,4,69.53.94.45.3 1,2,41.40.5 GalAT-1.8-trace 1,4-45.4-69.8 1,3,4-2.0-- 1,2,4-0.8-trace a as combined amount of galactose and 3-O-Me-galactose

50 Linkages present Neutral polymers Neutral polymers Arabinose, f (minor) Arabinose, f (minor) T; 1,3; 1,5M; 1,3,5 T; 1,3; 1,5M; 1,3,5 Galactose, p (major) Galactose, p (major) T; 1,4M; 1,6; 1,4,6 T; 1,4M; 1,6; 1,4,6 Incl. 3-O-methyl-Galactose Incl. 3-O-methyl-Galactose Acidic polymers Acidic polymers Arabinose, f Arabinose, f T; 1,5 Rhamnose, p Rhamnose, p T; 1,2M; 1,2,4 Galactose, p Galactose, p T; 1,4m; 1,4,6 Incl. 3-O-methyl-Galactose Galacturonic acid, p Galacturonic acid, p T; 1,4M

51 Acetyl CH 3 -O-C 108,5 – C1 L-Araf 104,5 – 1-4 gal 105,3 – 1-4 3-O-megal 58,5 – OCH 3 21,2 and 174,8 – acetylgroups NMR of the Neutral Fraction

52 Residues Chemical shift (ppm) C-1C-2+3C-4C-5C-6 -(1 4)-GalpA -(1 4)-GalpA100,269.2-69.779.2-79.771.5175.8 6-Me-GalpA 101.069.2-69.779.2-79.771.5171.7 -(1 4)-Galp -(1 4)-Galp104.472.5n.dn.d61.8 -(1 5)-Araf -(1 5)-Araf108.6n.dn.dn.d- -(1 2)-Rhap -(1 2)-Rhapn.dn.dn.dn.d17.6 Summary of 13C chemical shifts for A1002

53 Gelfiltration of the acidic fraction

54 Table 4. Yield, weight average molecular weight (M w ) and sugar composition (mol%) o f A1002 sub- fractions obtained after size exclusion chromatography of A1002 Fraction A1002aA1002bA1002cA1002dA1002eA1002f PM II a Yield b 8.68.624.016.031.43.4- Molecular weight (KDa) 1500 30 276 20 58 6 42 3 32 4 29 4 46-48 Sugar composition c Ara Ara7.37.15.64.33.94.38.8 Rha Rha5.95.75.65.55.55.74.2 Fuc Fuc0.60.30.30.3trace0.3- Xyl Xyl0.70.60.60.60.50.6- Man Man1.4------ Glc Glc5.12.11.51.61.52.37.3 Gal Gal16.213.110.08.26.98.18.0 3-O-Me-Gal 3-O-Me-Gal13.91.80.5tracetracetrace- Total neutral sugars 51.130.724.120.518.321.328.3 GalA (A) GalA (A)49.169.475.979.581.878.771.1 a data obtained from Samuelsen et al. 1996 b calculated as weight% of applied material c mol% of total carbohydrate content

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56 Effects on the complement system

57 Conclusion High mol.weight fraction of 1002 highest activity High mol.weight fraction of 1002 highest activity May be one or two types of molecules, May be one or two types of molecules, If so: If so: One basically pectic like One basically pectic like One rich in 3-O-methylgalactose One rich in 3-O-methylgalactose Or one with all components Or one with all components The effect of the polysaccharides may explain the traditional use in inflammation The effect of the polysaccharides may explain the traditional use in inflammation

58 Plantago major Traditional use in Scandinavia: Wound healing

59 PM II De-esterification and de-acetylation De-esterification and de-acetylation Pectinase Pectinase Oxalic acid hydrolysis Oxalic acid hydrolysis TFA-hydrolysis TFA-hydrolysis Pectolyase Pectolyase Periodate oxidation Periodate oxidation

60 Structure/activity relations De-esterification and de-acetylation De-esterification and de-acetylation no change of activity no change of activity Hairy regions more active than smooth areas Hairy regions more active than smooth areas HMW hairy regions with 1,3,6 linked galactose highly active HMW hairy regions with 1,3,6 linked galactose highly active Removal of arabinose on galactose Removal of arabinose on galactose increase activity increase activity Removal of arabinose on galacturonic acid Removal of arabinose on galacturonic acid reduce activity reduce activity

61 PM II activate the complement system Classical pathway activator Alterantive pathway activator not through C1Q (as IgG) not through the lectin pathway probably through C3 Variation in activity between different sera, 220 fold No difference in the level of antibodies against PMII in the sera

62 Variation in individual effect

63 PMII protects against systemic Streptococcus pneumoniae Infection in Mice PMII protects against the infection when administered systematically prechallenge PMII protects against the infection when administered systematically prechallenge Protective effect is due to stimulation of the innate immune system, not the adaptive. Protective effect is due to stimulation of the innate immune system, not the adaptive.

64 Conclusions Polysaccharides from different medicinal plants from various countries that traditionally are used as woundhealing agents or against inflammation have bioactivities that influence the immmunsystem in different ways in vitro, most probably also in vivo. Polysaccharides from different medicinal plants from various countries that traditionally are used as woundhealing agents or against inflammation have bioactivities that influence the immmunsystem in different ways in vitro, most probably also in vivo. Possible agents or leads for safe medicines as no toxic effects yet shown for polysaccharides of these types. Possible agents or leads for safe medicines as no toxic effects yet shown for polysaccharides of these types.

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66 Co-workers Cecilie Sogn Cecilie Sogn Kari Inngjerdingen Kari Inngjerdingen Drissa Diallo Drissa Diallo Haruki Yamada Haruki Yamada Hiroaki Kiyohara Hiroaki Kiyohara Terje Michaelsen Terje Michaelsen Sanya Hokputsa Sanya Hokputsa Stephen Harding Stephen Harding Anne Berit Samuelsen Anne Berit Samuelsen Hilde Barsett Hilde Barsett Financial support: NUFU,PRO 22/2002 Medicinal plants from Mali COST D28 Therapeutic polysaccharides


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