Berit Smestad Paulsen Department of Pharmacognosy

Bioactive polysaccharides from medicinal plants used in woundhealing and against inflammation

• Berit Smestad Paulsen

• Department of Pharmacognosy

• School of Pharmacy

• Oslo, Norway

MALI

Malian population:

• Population: ca 12 mill.

• Ethnic diversity: Sénoufo, Bambara, Sarakolé, Milinké, Peulh, Dogon, Bobo, Sonrhaï, Tamacheck……..

• Area

• 1 241 248 km2

• Expected living age: 47 years

• Age repartition:

• 47.2% : 0-14 years
• 5,5% : > 60 years

• Fertility rate: 6.7 children born/woman

• Child mortality: 12%

• Rate of alphabetization:

• Men – 31%
• Women – 15%

• Appr. 75% in rural areas

Curative urban based health service,

• Curative urban based health service,

• limited curative or preventive health service in the rural areas

• 59% of the population have healthservice within 15 km

• Local clinics:

• difficult to keep the personnel
• Funding not available for long time treatment

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 interdisiplinary scientific exploration of biologically active agents traditionally employed or observed by man.

Traditional preparation and adminiatration

• Collection of the plant part

• Use either fresh or dry

• Preparation

• Dry powder
• Infusion or macerate

• Normally wash the body or drink the extract

Preparation of the polysaccharides

• Ethanol extraction

• Water extraction 50oC

• Dialysis, freezedrying
• Anion exchange chromatography
• gelfiltration

• Water extraction 100oC

• Dialysis, freezedrying
• Anion exchange chromatography
• gelfiltration

Structure elucidation

• Methanolysis, TMS GC

• Carbohydrate composition

• Methylation studies GC-MS

• Linkage determination

• NMR anomeric configuration, sequence

• Mw - gelfiltration

• Enzymatic degradation

• Gives polygalacturonans, hairy regions, oligosaccharide sidechains etc.

Immunomodulation associated with activation of the complement system:

• Thymus dependant antibody response

• Regulation of specific cyclic antibody production

• Regulation of IgM-IgG switch

• Modulation of T and B cell proliferation

• Induction of supressor or helper T-cells

• Modulation of monokine or lympokine release

Structures of pectic type polysaccharides

• Arabinogalactan type I

• 1,4,gal

• Arabinogalactan type II

• 1,3,6 gal

• Rhamnogalacturonan I

• Hairy region, alternating rha / galA backbone

• Rhamnogalacturonan II

• Macro-oligosaccharide, 20 units, complex

Proposed structure of pectins, Perez et al 2003

Hairy region of rhamnogalacturonan I

• 2Rha14GalA12Rha14GalA12Rha14GalA12Rha14GalA1

• 4 4 4

•   

• 1 1 1

• Araf Gal Gal

• 5 6 6

•   

• 1 1 1

• Araf13Araf Araf13Gal Gal

• 5 6 6

•   

• 1 1 1

• Araf Gal Araf1g3Gal

• 5 6 6

•   

• 1 1 1

• Araf13Araf Araf13Gal Gal

• 5 6

•  

• 1 1

• Araf Araf

Are polysaccharides transported over membranes?

• Peyer´s patches

• Yamada et al. produced antibodies against bupleuran

• Detected polymers with binding sites for the antibodies in liver

Bupleuran epitopes

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.))

• Cochlospermum tinctorium Perr.

• Glinus oppositifolius (L.) A. DC.

• Biophytum petersianum Klotzch

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.

• GASTROCEDAL

Characteristics of A.2

Effect of A.2.1 after enzymatic degradation

Effect of A.2.2 after enzymic degradation

Mitogenic activity of fractions from V. kotchyana

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

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.

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

Effect of the different fractions on the complement system

Mitogenic activity on the original fraction and the degradation products

Conclusion

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

Cochlospermum tinctorium, root

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

Cochlospermum tinctorium activity on whole spleen cells

Cochlospermum tinctorium activity on B - cells

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.

• Plant polysaccharides reported to have immunomodulating activities.

Glinus oppositifolius (L.) A. DC. (Aizoaceae)

• 50°C water extract separated into one neutral (GON) and two acidic fractions (GOA1 and GAO2).

The complement fixating activities determined in vitro.

• The complement fixating activities determined in vitro.

• GOA1 shows an ICH50 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.

Conclusion

• Two different pectic polysaccharides isolated from Glinus oppositifolius (L.) A. DC. (Aizoaceae) with potent complement fixating activity.

• Further structure- and activity studies in progress.

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

Bp 100, Fr. III, and degradation fraction of Fr. III with the polygalacturonase, Fr.III 1 is the hairy region

• Monosaccharide Fr.III 1 2 3

• Arabinose 8,5 7,9 11,1 10,4

• Rhamnose 7,3 22,6 12,3 7,7

• Fucose 0,8 1,1 2,3 1,9

• Xylose 7,2 5,0 7.1 9,4

• Mannose 0,4 2,0 0,8 1,0

• Galactose 9,0 20,0 8,5 6,4

• Galacturonic acid 64,1 38,5 55,2 60,8

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 Orig B.P.I B.P.II B.P.III

• T-Ara 6,2 7 8,5 8

• 1->5 Ara 2,7 1,1 0 0

• T-Xyl l6,4 4,9 6,1 9,7

• 1->2 Xyl 0 0 1,3 0

• 1->4 Xyl 1,2 0,6 3,1 2,8

• T-Rha 1,2 2,4 5,2 2

• 1->2 Rha 3,4 14,2 2,5 3

• 1->3 Rha 0,8 0,8 2,9 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

• T-Fuc 1 1,3 2,4 2,1

• T-Gal 3,1 6,8 5 1,9

• 1->3 Gal 2,2 4,9 3,4 3,7

• 1->6 Gal 1,1 3,1 0 0

• 1->3,6 Gal 3,3 6,2 0,6 1,4

• T- GlcA tr tr.

• T- GalA 2,5 2,3 4,1 6,4

• 1->4 GalA 52,3 30,2 31,2 30,1

• 1->3,4 GalA 8,5 6,8 17,9 22,2

• 1->2,4 GalA 1,1 1,1 3,3 3

Major features of the regions of BP

• BP I HMW

• Hairy region, alternating GalA and Rha, sidechains of 1/3 of Rha

• BP II MMW

• High GalA, 1/3 branched

• BP II LMW

• High GalA, 2/3 branched

Conclusions

• 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.

• 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.

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

Traditional use in Thailand and other countries in the region

• Sea holly, a mangrove plant

• In Malaysia,

• seeds against cough and boils

• In China

• hepatitis, lymphoma and astma

• In Thailand

• Root and stem for skin diseases and longevity
• Crushed leaves on inflamed joints
• Decoction of the whole plant against inflammation

Effect of the fractions in the complement system

Linkages present

• Neutral polymers

• Arabinose, f (minor)

• T; 1,3; 1,5M; 1,3,5

• Galactose, p (major)

• T; 1,4M; 1,6; 1,4,6
• Incl. 3-O-methyl-Galactose

Conclusion

• High mol.weight fraction of 1002 highest activity

• May be one or two types of molecules,

• If so:
• One basically pectic like
• One rich in 3-O-methylgalactose
• Or one with all components

• The effect of the polysaccharides may explain the traditional use in inflammation

Plantago major

PM II

• De-esterification and de-acetylation

• Pectinase

• Oxalic acid hydrolysis

• TFA-hydrolysis

• Pectolyase

• Periodate oxidation

Structure/activity relations

• De-esterification and de-acetylation

• no change of activity

• Hairy regions more active than smooth areas

• HMW hairy regions with 1,3,6 linked galactose highly active

• Removal of arabinose on galactose

• increase activity

• Removal of arabinose on galacturonic acid

• reduce activity

Variation in individual effect

PMII protects against systemic Streptococcus pneumoniae Infection in Mice

• PMII protects against the infection when administered systematically prechallenge

• Protective effect is due to stimulation of the innate immune system, not the adaptive.

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.

• Possible agents or leads for safe medicines as no toxic effects yet shown for polysaccharides of these types.

Co-workers

• Cecilie Sogn

• Kari Inngjerdingen

• Drissa Diallo

• Haruki Yamada

• Hiroaki Kiyohara

• Terje Michaelsen

• Sanya Hokputsa

• Stephen Harding

• Anne Berit Samuelsen

• Hilde Barsett