Chemical and biological characterization of

Journal of Ethnopharmacology 139 (2012) 350–358

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Chemical and biological characterization of polysaccharides from wild and cultivated roots of Vernonia kotschyana K.T. Inngjerdingen a,∗ , S. Meskini a , I. Austarheim a , N. Ballo b , M. Inngjerdingen c , T.E. Michaelsen a,d , D. Diallo a,b , B.S. Paulsen a a

Department of Pharmaceutical Chemistry, School of Pharmacy, University of Oslo, P.O. Box 1068 Blindern, 0316 Oslo, Norway Department of Traditional Medicine, BP 1746, Bamako, Mali c Institute of Immunology, Rikshospitalet University Hospital, 0027 Oslo, Norway d The Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, 0403 Oslo, Norway b

a r t i c l e

i n f o

Article history: Received 17 August 2011 Received in revised form 30 September 2011 Accepted 25 October 2011 Available online 15 November 2011 Keywords: Vernonia kotschyana Pectic polysaccharides Inulin Immunomodulation Cultivation of medicinal plants

a b s t r a c t Ethnopharmacological relevance: In Malian traditional medicine the roots of Vernonia kotschyana are used for treating gastric ulcer and gastritis. In 2006, 9000 kg of roots from Vernonia kotschyana were used to produce Gastrosedal, an ameliorated traditional medicine in Mali. Harvesting from the wild, the main source of raw material, is causing a growing concern of diminishing populations of the plant, and Vernonia kotschyana is now being cultivated in several areas around Mali. In the current study the structures and bioactive properties of isolated polysaccharides from wild and cultivated Vernonia kotschyana were compared. Materials and methods: Pectin- and inulin-type polysaccharides were isolated from the roots of cultivated and wild Vernonia kotschyana. The isolated polysaccharides were investigated regarding their chemical compositions, and for their abilities to fixate human complement and activate macrophages from a mouse macrophage cell line. Results: No significant differences in the carbohydrate composition of the fractions isolated from the cultivated versus the wild roots were observed. A previously reported pectic arabinogalactan Vk2a was found in both the cultivated and the wild roots in this study, and exhibited potent complement fixation activity, and a moderate activation of macrophages. Conclusions: The present study has shown that the cultivated roots of Vernonia kotschyana contain the same types of bioactive polysaccharides as the wild roots. It is therefore preliminarily feasible for the cultivated roots of Vernonia kotschyana to be used as a herbal medicine to replace the wild roots. © 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The roots of Baccharoides adoensis var. kotschyana (Sch. Bip. ex Walp.) M. A. Isawumi, G. El-Ghazaly and B. Nordenstam (Asteraceae), mainly described in the literature as Vernonia kotschyana Sch. Bip. ex Walp. or Vernonia adoensis Sch. Bip., are used against gastric ulcer in Malian traditional medicine. The name Vernonia kotschyana will be used throughout this paper as the new name has never been cited in the scientific literature.

Abbreviations: AG-I, arabinogalactan-I; AG-II, arabinogalactan-II; Ara, arabinose; FPLC, fast protein liquid chromatography system; Gal, galactose; GalA, galacturonic acid; Glc, glucose; GlcA, glucuronic acid; Man, mannose; NO, nitric oxide; RG-I, rhamnogalacturonan-I; Rha, rhamnose. ∗ Corresponding author. Tel.: +47 22 85 7504; fax: +47 22 85 4402. E-mail address: [email protected] (K.T. Inngjerdingen). 0378-8741/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2011.10.044

Gastric ulcer is regarded as an important public health problem in Mali, with a prevalence of 4.2% for men and 2.4% for women (Maïga et al., 1995). Pharmaceutical products, like relevant antibiotics for the treatment of gastric ulcer caused by Helicobacter pylori, are normally not available. In many cases the high cost of the medicines make them unaffordable for the population. It is therefore of interest for the Governmental Health Department of Mali, with Department of Traditional Medicine (DMT) as the active part, to ensure availability of effective and secure medicines based on Malian resources against, among other diseases, gastric ulcer. A decoction of the powdered roots of Vernonia kotschyana is recognized by the government in Mali as an ameliorated traditional medicine, Gastrosedal, for the treatment of gastritis and gastro duodenal ulcers, and this medicine is on the National List of Essential Drugs. Promising results regarding the efficiency in treatment of gastric ulcer was shown in an open, small clinical trial on 16 outpatients (Touré, 1989). In 50% of the patients symptoms were relieved, and in 6 patients lesions disappeared after

K.T. Inngjerdingen et al. / Journal of Ethnopharmacology 139 (2012) 350–358

ingestion of 6 g powdered roots per day, formulated as tablets, for 30 days. In an animal model an aqueous extract has demonstrated to exhibit gastroprotective activity (Sanogo et al., 1996). Various mechanisms have been suggested, like coating and protection of gastric mucosa, decreasing the rate of extrusion of epithelial cells, and maintaining epithelial integrity. The aqueous extract contains, among other substances, steroidal saponins, inulin and pectic type polysaccharides. Saponins are known for their antiulcer properties, probably by facilitating the microcirculation and thus protect the mucosa (Germano et al., 1996). Polysaccharides have been found to exhibit immunomodulating and anti-inflammatory properties which may be linked to protective mechanisms. The biology of wound healing is a dynamic and complex regenerative process, and involves soluble mediators, extracellular matrix components, resident cells and circulating immune cells. Immune cells are critical for the outcome of healing, and agents that modulate immune function may have a significant impact on the reparative process of both dermal wounds and gastrointestinal ulcers (Cho et al., 2002; Wei et al., 2002). Immunomodulating polysaccharides have been isolated from several medicinal plants and are suggested to contribute to the wound healing capacity or anti-ulcer activity of these plants (Sun et al., 1991; Matsumoto et al., 2002). In a previous study a potent immunomodulating pectic arabinogalactan, Vk2a, was isolated from a hot water extract of the wild roots of Vernonia kotschyana. Vk2a showed a high complement fixation activity (human complement) and a T cell independent induction of B-cell proliferation (C3H/HeJ mice), in addition to the promotion of chemotaxis by human macrophages, T cells and NK cells (Nergard et al., 2005a). The structure of the pectic arabinogalactan is highly complex, but it seems that Vk2a is composed of both ␣-(1 → 4)-polygalacturonan- (homogalacturonan regions) and rhamnogalacturonan-I regions with about 60% of the rhamnose (Rha) units branched in a highly random fashion by single galactose (Gal) units, arabinans, or arabinogalactan type I and II. The bioactive sites seem to be located both in the more peripheral parts of the molecule, but also in the inner core of the RG-I region (Nergard et al., 2005b). The isolated pectic arabinogalactan shown to modulate immune function may play a role in the reparative process in gastrointestinal ulcers. In addition to pectic type polysaccharides, inulin-type fructans have found to be abundant in the crude water extract of the wild roots of Vernonia kotschyana. The extraction yield of inulin was shown to be about 40% based on the dried, powdered roots (Nergard et al., 2004). Inulin can be defined as polydisperse fructans, where the fructans are typically linked by ␤-2,1-linked fructofuranosyl linkages with a glucose (Glc) moiety typically resident at the end of almost each fructose chain (Watzl et al., 2005). Inulin-type fructans appears to beneficially affect the immune system, especially the intestinal immune functions by targeting the gut-associated lymphoid tissue and especially the Peyer’s patches. Consequently they have been shown to reduce the risk of diseases related to dysfunction of the gastrointestinal defense’s functions (Roberfroid, 2007). In 2006, 9000 kg of raw material (roots) of Vernonia kotschyana was used to produce 37,770 bags of Gastrosedal in Mali. Harvesting from the wild, the main source of raw material, is causing a growing concern that it might diminish the populations and lead to local extinctions. In order to prevent these problems Vernonia kotschyana is now being cultivated in the medicinal plant garden of DMT in Mali. This paper reports on the structures and bioactive properties of isolated polysaccharides from wild and cultivated Vernonia kotschyana. Safe medicines based on local plants that can be

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cultivated and still give the same biological effect as wild ones is important from a biodiversity point of view. 2. Materials and methods 2.1. Plant material Roots from Vernonia kotschyana, wild type, were collected in Mansatola, Kolokani, Mali, and identified by the Department of Traditional Medicine (DMT), Mali. Roots from cultivated Vernonia kotschyana were harvested from 6 month old plants and two year old plants from the Medicinal Plant Garden of DMT. The roots were washed, cut into small pieces, dried and pulverized to a fine powder by a mechanical grinder. Voucher specimens are deposited at the herbarium of DMT (Voucher No. 0929). The roots from the wild type of Vernonia kotschyana will be denominated Vkw throughout this paper, while the 6 month old and two year old cultivated roots of Vernonia kotschyana will be denominated Vky (young roots) and Vko (old roots), respectively. 2.2. Culture conditions The plants were cultivated in a sunny environment with a rich, fertile and well-drained soil. Seeds of Vernonia kotschyana were seeded in mounds. Mounds measuring 20–40 cm in height, being 20–40 cm wide and 3–5 m long were used. The distance between the mounds was 0.5 m to facilitate aeration and circulation of water. Seeding was performed in a proportion of 50 kg of seeds per hectare, and the yield of dried roots were 1–2 t per hectare. Vernonia kotschyana is a perennial plant, and can be cultivated during the whole season. 2.3. Polysaccharide extraction and fractionation In order to remove low molecular weight compounds the powdered roots of Vkw, Vky and Vko were pre-extracted with dichloromethane (DCM) and methanol (MeOH) using a soxhlet apparatus. When no more colored material could be observed in the extracts the procedure was ceased. Residues from the three plant materials were further extracted with 96% ethanol (EtOH) (1.2 l) for 2 h and 80% EtOH for 3× 1 h (in a total amount of 3.85 l), and filtered through Whatman GF/A glass fibre filter. Subsequently, the dried residues were extracted two times with distilled water (2× 3 l) at 100 ◦ C for 2 h, and filtered through Whatman GF/A glass fibre filter. Prior to further analysis the aqueous extracts were concentrated at 40 ◦ C in vacuum and dialysed at cut-off 3500 Da to give 100 ◦ C crude water extracts. The extracts were kept at −18 ◦ C or lyophilized. Precipitates of inulin were formed during dialysis and defreezing of the crude water extracts. The inulin were separated from the crude water extracts by centrifugation (10,000 × g for 20 min), washed two times in 96% EtOH, backwashed four times in distilled water and subsequently lyophilized. The inulin-reduced supernatants were purified by gel filtration on a HiLoadTM 26/60 SuperdexTM 200 prep grade column (GE Healthcare), coupled to a fast protein liquid chromatography system (FPLC, Pharmacia Äkta, Amersham Pharmacia Biotech) fitted with a 10 ml superloop. A total of 12 ml × 10 ml of each of the inulin-reduced supernatants were filtered through Acro 50A 0.45 ␮m filters, applied and eluted with 10 mM NaCl at 30 ml/h. Fractions of 3.8 ml were collected with a Fraction Collector Frac-900 (Amersham Pharmacia Biotech). The eluent was monitored with UV 214 nm, and the phenol–sulphuric acid assay (Dubois et al., 1956) was used to determine the carbohydrate elution profile. Three separate fractions were obtained from each of the three water extracts. The fractions were dialysed at cut-off 3500 Da and lyophilized.

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Fig. 1. Elution profiles of the crude water extracts Vkw100, Vky100 and Vko100 on Superdex 200 (gel filtration).

2.4. Fractionation of polysaccharide fractions on a Mono P column For analytical purposes the fractions obtained after purification on SuperdexTM 200 were further applied on a Mono-PTM HR 5/20 FPLC column (Pharmacia). The anion exchange column was coupled to an FPLC system (Pharmacia Äkta) consisting of two PUMP P-920 pumps, and fitted with a 500 ␮l loop. A 0.5 mg sample in 0.5 ml distilled water was filtered through Acro 50A 0.45 ␮m filters and injected on to the column. The column was eluted at 0.5 ml/min, using 0–1 M NaCl. The elution programme is shown in Figs. 2–4. Fractions of 1 ml were collected with a Fraction Collector Frac-900 (Amersham Pharmacia Biotech). The eluent was monitored with UV 214 nm, and the phenol–sulphuric acid assay was used to determine the carbohydrate elution profile (Dubois et al., 1956).

1992), in order to determine the composition of carbohydrate in the different polymer fractions. Mannitol as internal standard was included throughout the total procedure. Quantitative determination and identification of fructose could not be performed by methanolysis and GC, as fructose is readily decomposed into formic acid and acetic acid under acid hydrolytic conditions (van Loo et al., 1995). In order to determine the content of fructose the phenol–acetone–boric acid reagent (PABR) assay was used as described by Boratynski (1984) modified by Chaplin (1994). This method shows a high sensitivity (0.1–9 ␮g fructose in 100 ␮l), and interferences from non-ketose carbohydrates are slight (97% fructose, while Vko-inulin was shown to contain 84% inulin. Methanolysis revealed the presence of Glc in the Vk-inulin fractions, in addition to small amounts of GalA. In the Vko-inulin fraction Ara and Gal was also found. The monosaccharide compositions of the fractions obtained after fractionation of Vkw100, Vky100 and Vko100 on Superdex 200 are given in Table 2. The main components in the polysaccharide polymers in the VkI-fractions are the neutral monosaccharides Gal (30.6–33.6 mol%) and Ara (31.4–37 mol%). In addition to Gal and Ara, Rha (7.2–9.7 mol%) and GalA (13.1–15.2 mol%), and minor amounts of mannose (Man), glucuronic acid (GlcA) and 4-O-methyl glucuronic acid (4-O-Me GlcA) are also present. The Vk-I fractions are probably similar in structure as the pectic arabinogalactan Vk2a previously isolated from Vernonia kotschyana. Vk2a has been defined as a pectic arabinogalactan, and was shown to contain 24.4 mol% Gal, 31.3 mol% Ara, 11 mol% Rha, 26.4 mol% GalA and 5.7 mol% Glc. Pectic arabinogalactans are reported to mainly consist of a rhamnogalacturonan core substituted with neutral sugar chains. Vk2a was reported to fulfill the three criteria that are generally set to define arabinogalactanproteins (AGP). These are the presence of arabinogalactan chains, together with the ability to precipitate the ␤-glycosyl Yariv reagent, and finally a typical amino acid composition, with serine, alanine and hydroxyproline. Since the protein part of Vk2a was less than 0.5% it was chosen to define the polysaccharide as a pectic arabinogalactan (Nergard et al., 2005a). The monosaccharide compositions of Vky-I, Vko-I and Vkw-I are comparable, no significant differences can be seen between the three fractions. As shown by the PABR-assay Vky-I, Vko-I and Vkw-I contain 11.9%, 10.4% and 13% fructose, respectively. The finding of 11% fructose (inulin) in the very high molecular weight Vk2a was speculated to be caused by oligomers of inulin interacting noncovalently with the pectic arabinogalactan and partly be trapped or intertwined in the long and branched side chains of the polymer. The highest

2.10. Activation of macrophages The mouse macrophage cell line Raw 264.7 was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, antibiotics, l-glutamin, and 5 × 10−5 M 2-mercaptoethanol, and split every second day. Macrophages at a density of 5 × 105 cells/mL were seeded into 96-well flat-bottomed plates (5 × 104 cells/well) and stimulated for 24 h in duplicates with increasing concentrations of samples, LPS (from Pseudomonas aeruginosa 10, Sigma–Aldrich), and the pectic polysaccharide PM-II, from Plantago major L. (Samuelsen et al., 1996) as positive controls, or medium alone. Cells were then centrifuged at 1400 rpm for 2 min and the supernatants were harvested. The amount of NO was determined using a colorimetric method with nitrite (NaNO2 ) as a standard. Nitrite is a stable breakdown product of nitric oxide. The culture supernatant (50 ␮L) was mixed with an equal volume of Griess reagent A (1% [w/v] sulphanilamide in 5% [v/v] phosphoric acid) and incubated at room temperature in the dark for 10 min. After addition of 50 ␮L 0.1% (w/v) N-(1-naphthyl) ethylenediamine dihydrochloride in water (Griess reagent B), the absorbance was measured at 540 nm. A serial dilution of NaNO2 was used as a standard reference curve. The results are expressed as the mean ± SD. The difference between the control and the treatment in these experiments was tested for statistical significance by Dunnett’s Multiple Comparison Test. A value of p < 0.05 was considered to indicate statistical significance. 3. Results and discussion 3.1. Polysaccharide extraction and fractionation After the removal of precipitates of inulin by centrifugation and filtration the crude water extracts Vkw100, Vky100 and Vko100 were fractionated by gel filtration on a Superdex 200 HR column according to molecular weight. The gel filtration led to the isolation of three polysaccharide fractions for each of the three crude water extracts. These fractions are denominated Vkw-I, Vky-I and Vko-I, Vkw-II, Vky-II and Vko-II, and Vkw-III, Vky-III and Vko-III (Fig. 1). As can be seen from Fig. 1 the elution profiles of Vkw100, Vky100 and Vko100 are similar in nature. Further separation of the obtained fractions on the anionexchange column Mono P showed that the Vk-I fractions could not be separated further on an anion exchange column, and was eluted as single peaks in the range 0.4–0.75 M NaCl (Fig. 2). Regarding both the Vk-II and Vk-III fractions fractionation on the Mono P column led to broad peaks or several small


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Table 1 Monosaccharide compositions (mol%) of the crude water extracts Vkw100, Vky100 and Vko100.

Monosaccharide compositiona Ara Rha Fuc Xyl Man Gal Glc GlcA 4-O-Me-GlcA GalA a

Vky100

Vko100

Vkw100

9.7 2.8 Traces Traces Traces 8.5 47.7 Traces 1.2 30.1

15.0 5.9 Traces Traces Traces 11.3 23.6 Traces 2.4 41.9

10.3 3.3 Traces Traces 1.2 10.7 32.2 Traces 1.1 41.2

mol% of total carbohydrate content.

degree of polymerization (DP) of inulin in the crude polysaccharide fraction was determined to about 50 (Nergard et al., 2005a). This may also be the case with the Vk-I fractions. The protein content in Vky-I, Vko-I and Vkw-I was determined by the Bio-Rad protein assay, based on the method of Bradford, and was shown to be 4.3%, 3% and 2.9%, respectively. In the previous studies on the pectic arabinogalactan Vk2a from Vernonia kotschyana the protein content was determined to be less than 0.5%, by the method of Lowry et al. (Lowry et al., 1951; Nergard et al., 2005a). Ferulic acids may be esterified to arabinose and galactose in pectic polysaccharides (Brett and Waldron, 1996). The phenolic content, expressed as ferulic acid equivalents were determined to be less than 1% for the Vk-I fractions. The Vk-II fractions are a heterogeneous mixture of polysaccharides, as can be seen from the fractionation on the Mono P column (Fig. 3). From the elution profiles it seems as though all three Vk-II fractions consist of three separate polysaccharide polymers. The polymers contain a mixture of Ara, Rha, Gal, Glc, GalA, and fructose. As can be seen from Fig. 4, the Vk-III fractions also contain a heterogeneous mixture of polymers. According to carbohydrate analysis the Vk-III fractions consists mainly of Glc and GalA (Table 2), with minor amounts of Ara, Rha, Man and Gal. The monosaccharides GalA, Rha, Ara and Gal are typical constituents in pectic polysaccharides, while the high content of Glc, especially for Vky-III and Vkw-III, is probably due to inulin co-eluting with the pectic polymers. The content of fructose in Vkw-III, Vky-III and Vko-III was shown to be 80.4%, 70.5% and 39.8%, respectively. The lower content of fructose and glucose in Vko-III compared to VkyIII and Vkw-III is probably due to less contamination of inulin in this fraction. The protein content in Vkw-III, Vky-III and Vko-III, determined by the Bio-Rad protein assay, was shown to be 1.2%, 1.1% and 0.4%, respectively, while the phenolic content, expressed as ferulic acid equivalents were determined to be less than 1%.

3.3. Enzymatic degradation of the polymers Vk-III by endo- and exo-inulinase The Vk-III fractions were shown to contain considerable amounts of fructose, and Glc, most probably coming from inulin. A combination of endo- and exo-inulinase from Aspergillus niger was used in order to remove the remaining inulin from the Vk-III fractions. Endo- and exo-inulinase will be able to hydrolyse inulin and fructo-oligosaccharides into glucose and fructose. The enzyme hydrolysates were applied to an ion-exchange ANX Sepharose 4 Fast Flow column and led to the isolation of one polysaccharide fraction denominated Vk-III.1, from each of the Vk-III fractions. The monosaccharide compositions of the Vk-III.1 fractions are given in Table 2. The PABR-assay showed a reduction in fructose content for Vky-III, Vko-III and Vkw-III of 78.2%, 80.5% and 84.8%, respectively. The degradation of the Vk-III fractions with inulinases led to an almost complete loss of Glc. Unfortunately, in addition to the breakdown of inulin the enzymatic treatment also resulted in the removal of arabinose and galactose subunits from the Vk-III fractions, so the Vk-III.1 fractions consists mainly of GalA. This was also seen for Vka2 when treated with inulinase, where a 60% removal of arabinose units was shown (Nergard et al., 2005a). The Vk-III.1 fractions are most probably similar in structure as the previously isolated pectic type polysaccharide, denominated Vk100A2b (Vk2b), consisting of 84.6% GalA, 1.8% Glc, 4.9% Gal, 4.9% Rha and 3.9% Ara. The polymer was shown to comprise large portions of a polygalacturonan region as the backbone chain, with a small portion of a rhamnogalacturonan core. Vk2b contained 4% fructose (Nergard et al., 2005a). 3.4. Pharmacological evaluation It is well accepted that normal wound repair involves a number of cell types, including macrophages, endothelial cells, fibroblasts, and keratinocytes, and also that wound repair is an immunemediated physiological mechanism. Consequently, several studies

Table 2 Monosaccharide compositions (mol%) of the fractions obtained after fractionation of Vkw100, Vky100 and Vko100 on Superdex 200. Vky-I

Vko-I

Vkw-I

Vky-III

Vko-III

Vkw-III

Vky-III.1

Vko-III.1

Vkw-III.1

35.7 9.7 Traces Traces 1.4 33.6 1.7 Traces 4.3 13.7

31.4 7.2 Traces Traces 1.1 30.6 7.2 1.7 4.8 15.2

0.6 0.5 Traces Traces Traces 2.7 25.2 – – 70.9

1.9 2.1 – – 0.5 3.1 7.5 – 1.3 83.6

1.0 0.5 Traces Traces Traces 1.9 28.5 – 1.9 66.3

0.4 0.7 – – – 2.4 1.5 – – 95.0

0.9 2.1 – – – 2.6 0.9 Traces – 93.5

0.8 1.3 – – – 2.5 1.6 – – 93.9

a

Monosaccharide composition Ara 37 8.9 Rha Fuc Traces Xyl Traces 1.8 Man 30.9 Gal 0.9 Glc GlcA 1.1 4-O-Me-GlcA 5.6 13.1 GalA a

mol% of total carbohydrate content.

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Table 3 Complement fixation activity (IC50 ; concentration of the sample at 50% inhibition of hemolysis) of the crude water extracts, the inulin fractions and the fractions obtained after fractionation of the crude water extracts on Superdex 200, isolated from the roots of V. kotschyana. IC50 -values (␮g/ml) Vky100 Vko100 Vkw100 BP-IIa Vky-inulin Vko-inulin Vkw-inulin BP-IIa Vky-I Vko-I Vkw-I BP-IIa

253.9 53.8 57.0 14.6 >500 61.2 >500 14.6 31.7 9.2 13.7 20.2

Vky-III Vko-III Vkw-III BP-IIa

>500 378.3 226.6 12.4

Vky-III.1 Vko-III.1 Vkw-III.1 BP-IIa

>500 456.9 310.3 12.4

a

BP-II, a pectic polysaccharide from B. petersianum used as positive control.

have reported that agents that modulate immune function have a significant effect on the reparative process (Wei et al., 2002). Anti-ulcer polysaccharides have been isolated from several plants, including Panax ginseng C.A. Meyer (Sun et al., 1992), Decalepis hamiltonii (Srikanta et al., 2007), and Angelica sinensis (Oliv.) Diels (Ye et al., 2003). A suggested mechanism of anti-ulcer activity is that due to their anionic nature polysaccharides may bind effectively to positively charged amino acid residues of gastric mucin, and function as a protective coating (Srikanta et al., 2007). Pectic polysaccharide fractions isolated from Bupleurum falcatum L, a medicinal plant used in the treatment of gastric ulcer in traditional Chinese and Japanese herbal medicine, exhibit a potent inhibitory activity against HCl/ethanol-induced gastric lesions in mice. Furthermore, a pectin-type polysaccharide, bupleuran 2IIc, isolated from the roots of Bupleurum falcatum, is reported to significantly increase the contents of granulocyte colony-stimulating factor (G-CSF) secreted from murine normal colonic epithelial cells. G-CSF primarily modulates macrophage and dendritic cell function (Matsumoto et al., 2002, 2008). Pectic type polysaccharides isolated from the roots of Vernonia kotschyana have previously shown to fixate complement and stimulate macrophages (Nergard et al., 2005b). We therefore chose to compare the ability of pectins isolated from wild and cultivated roots of Vernonia kotschyana to fixate complement and to activate macrophages. 3.4.1. Effect on human complement in vitro The complement system is an important component of the immune defense against infection coordinating the interactions of inflammatory cellular responses against invading non-self antigens (Sim and Dodds, 1997). Complement fixating activity has previously shown to be a good indicator for effects in the immune system by polysaccharides from plants. The complement fixating activities of the isolated fractions from Vernonia kotschyana were repeated three times with similar results. The crude water extracts Vk100 showed dose dependent complement fixation activity, in particular Vko100 and Vkw100, with IC50 -values of 53.8 and 57 ␮g/ml (Table 3). The samples are compared with the positive control BP-II, a pectic fraction from

Fig. 5. Dose-dependent complement fixation activity of Vkw-I, Vky-I and Vko-I. The activity is expressed as % inihibition of lysis of sensitized sheep erythrocytes. BP-II, a pectic polysaccharide from B. petersianum is used as a positive control.

Biophytum petersianum, previously shown to be highly active in the complement fixation assay. BP-II typically shows a 50% inhibition of hemolysis at 10–20 ␮g/ml (Inngjerdingen et al., 2006; Grønhaug, 2010). According to methanolysis the crude extract Vky100 contains less of the monosaccharides typical for pectins, and more of inulin compared to Vko100 and Vkw100 (Table 1). This might be an explanation for the lower complement fixation activity seen for Vky100. The inulin fractions Vky-inulin and Vkw-inulin did not achieve 50% inhibition of hemolysis in the range of concentrations tested (15.6–500 ␮g/ml), while Vko-inulin showed a IC50 -value of 61.2 ␮g/ml. In previous studies inulin isolated from Vernonia kotschyana did not show any complement fixation activity (Nergard et al., 2004). This was suggested to be due to a degree of polymerization of less than 50 of the inulin. The reason for the activity seen for Vko-inulin might be the presence the monosaccharides Ara, Gal and GalA, and thereby traces of pectin in the fraction. The Vk-I fractions showed potent fixation activity of human complement (Table 3 and Fig. 5). The Vk-I fractions are, as discussed above, probably similar in structure to the pectic arabinogalactan Vk2a previously isolated from Vernonia kotschyana. Vk2a was also shown to exhibit a high complement fixating activity (Nergard et al., 2005a). Compared to Vk-I, the Vk-III fractions did not have the same effect on fixation of human complement (Table 3). The fact that Vko-III and Vkw-III has a more potent activity compared to Vky-III might be due to a higher amount of pectic type monosaccharides in these fractions compared to Vky-III (Table 2). After removal of inulin from the Vk-III fractions the activity decreased (Table 3). The previously isolated pectin Vk2b, similar in monosaccharide composition as the Vk-III.1 fractions, did not show complement fixation activity in the concentration range tested (up to 500 ␮g/ml) (Nergard et al., 2005a). As stated in the previous study on Vernonia kotschyana the most evident difference between the inactive pectin, Vk2b, being similar in nature to the Vk-III.1 fractions, and the active Vk2a, being similar to the Vk-I fractions, are the presence of arabinogalactanI and arabinogalactan-II type structures in Vk2a. Vk2b is mainly composed of a homogalacturonan region with very small amounts of rhamnogalacturonan regions with short neutral side chains (Nergard et al., 2005a). For all the fractions tested in this study it seems that the extracts and polymers isolated from the cultivated young roots (Vky) have a slight lower activity than the extracts and fractions isolated from the cultivated old roots (Vko), and the wild roots (Vkw). There is however no difference between Vko and Vkw.


3.4.2. Activation of macrophages It is known that macrophages are pivotal cells in wound repair, and macrophages have for a long time been considered as one of the main target cells for polysaccharide interaction. Several compounds have been shown to modulate their cytokine production and to enhance phagocytic activity of macrophages, and it has been suggested that polysaccharides activate macrophages through the pattern recognition receptor Toll-like receptor 4 (TLR4) (Yang et al., 2007). Glucans interact with pattern recognition receptors on macrophages, which are stimulated to modulate their functional activities. It has further been suggested that the effect of glucan on wound repair involves macrophage release of wound growth factors with subsequent modulation of fibroblast activity including collagen biosynthesis (Wei et al., 2002). Also pectic polysaccharides have been shown to affect macrophages. An acidic polysaccharide isolated from Angelica sinensis has shown to promote nitric oxide (NO) production by up-regulating the expression of TLR4 on macrophages (Yang et al., 2007). The production of NO after stimulation of macrophages with the isolated extracts and fractions from Vernonia kotschyana was measured through nitrite, which is a stable breakdown product of NO. The well-characterized mouse macrophage cell line Raw264.7 was used for these experiments. LPS and PM-II, a pectin from Plantago major (Samuelsen et al., 1996), were utilized as positive controls. LPS is a potent stimulator of cells of the monocytic lineages (Sweet and Hume, 1996). We were unable to detect nitric oxide release under a wide range of concentrations of the crude water extracts Vk100, the inulin fractions Vk-inulin, the Vk-II, Vk-III and Vk-III.1- fractions (data not shown). This result may indicate that the fractions are not acting as general activators of human macrophages. However, inulin-type polysaccharides have previously been reported to activate murine macrophages in vitro. Endotoxin-free inulin isolated from the roots of Chicorium intybus induced nitric oxide and tumor necrosis factor (TNF)-␣ secretion when the cells were pre-treated with IFN-␥ (Koo et al., 2003). Also, an inulin-type polysaccharide isolated from the roots of Platycodon grandiflorum has been reported to induce macrophage activation through TLR4/NF-␬B signalling pathway (Yoon et al., 2003). Release of NO was however observed from macrophages stimulated with the Vky-I fraction, in a dose dependent manner (Fig. 6). The fact that Vky-I was active might suggest that the presence of arabinogalactan side chains is part of the structural requirements for the induction of the macrophage response. In one of the previous studies on Vernonia kotschyana the purified pectic fractions Vk2a and Vk2b did not induce nitric oxide release from macrophages under a wide range of concentrations. It was concluded that the fractions are not acting as general activators of human macrophages. The fractions did, however, induce chemotaxis of macrophages, T cells and NK cells (Nergard et al., 2005a). The structure–activity relationship of the pectins isolated from Vernonia kotschyana has been thoroughly described in earlier papers (Nergard et al., 2005b, 2006). The conclusions made are that the bioactive sites in Vk2a seem to be located both in the more peripheral parts of the molecule, but also in the inner core of the molecule. Especially the AG-I and AG-II type structures in Vk2a are suggested to be involved in the bioactivities seen. Vk-inulin did not show any activity in the complement fixation assay or in activating macrophages. A range of immunological effects have however been reported from supplementation of fructan in animal models. These include anti-inflammatory effects in inflammatory bowel disease-models, enhanced mucosal antibody responses, and modulation of mucosal cytokine patterns and cell populations. There are also reports that fructans have effects on Peyer patch cells (Vos et al., 2007).

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Fig. 6. Measurement of nitric oxide release from Raw 264.7 macrophages after overnight stimulation with the Vk-I fractions from V. kotschyana. LPS and the pectin PM-II from P. major are used as positive controls. Data are presented as the mean of three independent experiments ±SD. *P < 0.05.

4. Conclusions Polysaccharides of both the pectin- and inulin-type were isolated from cultivated and wild roots of Vernonia kotschyana in this study. There are no significant differences between the polymers in the cultivated versus the wild roots. The pectic fractions isolated from the cultivated roots are also similar in nature as pectic polysaccharides isolated from wild roots in previous studies (Nergard et al., 2005a,b). The bioactive pectic arabinogalactan, Vk2a, previously reported isolated from wild roots of Vernonia kotschyana (Nergard et al., 2005b) was also present in the cultivated roots of Vernonia kotschyana. The complement fixation activity of the crude water extract and the polysaccharide fractions in the two year old roots of cultivated Vernonia kotschyana was similar to those isolated from the wild roots. The pectic fraction Vky-I from the young roots was the least active of the Vk-I fractions in fixating complement, while it was the most potent fraction in activation of macrophages. Studies have shown that pectic polysaccharides can form bioadhesive layers on top of irritated epithelia, leading to a protective coating and shielding of the epithelia. The bioadhesive acidic polysaccharides were shown to be mainly pectin-like galacturonides, the main adhesive structural part being the homogalacturonide region of the pectins (Schmidgall and Hensel, 2002). A polysaccharide fraction isolated from immature okra fruits containing rhamnogalacturonans with a considerable amount of glucuronic acid was highly active against Helicobacter pylori in an in situ adhesion model on sections of human gastric mucosa. Helicobacter pylori is known to play a major role in the development of chronic gastritis and gastric ulcerations (Lengsfeld et al., 2004). It would therefore be of interest in future studies to test if the Vk-I and Vk-III.1 fractions isolated from Vernonia kotschyana exhibit bioadhesive properties. The Vk-III.1 fractions consists of >90% GalA, while the Vk-I fractions contains rhamnogalacturonans with fair amounts of GlcA and 4-O-Me GlcA (Table 2). This study has shown that the cultivated roots of Vernonia kotschyana contain the same types of bioactive polysaccharides as the wild roots. It is therefore preliminarily feasible for the

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