Phytochemical Characterization of Low Molecular Weight Constituents


Jan 27, 2017 - Extract RE was obtained from the roots of Althaea officinalis in a yield of 8.1%, related to the dried plant material, by extraction wi...
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Phytochemical Characterization of Low Molecular Weight Constituents from Marshmallow Roots (Althaea of f icinalis) and Inhibiting Effects of the Aqueous Extract on Human Hyaluronidase‑1 Jandirk Sendker,*,† Ines Böker,† Isabelle Lengers,‡ Simone Brandt,† Joachim Jose,‡ Timo Stark,§ Thomas Hofmann,§ Careen Fink,⊥ Heba Abdel-Aziz,⊥ and Andreas Hensel† †

Institute of Pharmaceutical Biology and Phytochemistry and ‡Institute of Pharmaceutical and Medicinal Chemistry, University of Münster, Correnstrasse 48, D-48149 Münster, Germany § Chair of Food Chemistry and Molecular Sensory Science, Technical University of Munich, Lise-Meitner-Strasse 34, D-85354 Freising, Germany ⊥ Steigerwald Arzneimittelwerk GmbH, Bayer Consumer Health, Havelstrasse 5, D-64295 Darmstadt, Germany S Supporting Information *

ABSTRACT: Extract RE was obtained from the roots of Althaea of f icinalis in a yield of 8.1%, related to the dried plant material, by extraction with MeOH−H2O (1:1), followed by precipitation with EtOH to remove high molecular weight constituents. Phytochemical investigation of RE revealed the presence of N-phenylpropenoyl-L-amino acid amides 1−5, 8% glycine betaine 6, about 9% total amino acids with proline as the main compound, and about 61% mono- and oligomeric carbohydrates with sucrose as the main compound. Further fractionation revealed the presence of a hypolaetin diglycoside (12) and four hypolaetin glycosides (7−9 and 11) with Osulfocarbohydrate moieties; additionally, 4′-O-methylisoscutellarein-8-O-β-D(3″-O-sulfo)glucuronopyranoside (10) and the diglycosylated coumarin haploperoside D (13) were identified. The hypolaetin-O-sulfoglycosides 7−10 are new natural products. RE inhibited the enzymatic activity of surface-displayed human hyaluronidase-1 on Escherichia coli F470 cells with an IC50 of 7.7 mg/mL. RE downregulated mRNA expression of hyal-1 in HaCaT keratinocytes at 125 and 250 μg/mL, respectively. These data contribute to a deeper phytochemical understanding of marshmallow root extracts and to the positive influence of extracts used for therapy of irritated and inflamed buccal tissue and cough.

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immune-activating effects of aqueous marshmallow root extract and isolated polysaccharides.10 The mucilaginous and bioadhesive effects of the polysaccharides from aqueous marshmallow root extract are usually seen as the major pharmacodynamic effect, leading to the formation of a protecting and shielding polysaccharide layer on inflamed or destructed epithelial tissue. The formation of such bioadhesive layers has been shown in detail by fluorescence histology for pectin-like polygalacturonides with structural and physicochemical patterns similar to marshmallow polysaccharides.11 Additionally, strong adsorption of marshmallow polysaccharides to porcine buccal mucosal tissue has been shown.12 The polysaccharides from marshmallow roots also exert stimulating effects on the cell viability and cellular proliferation of epithelial KB cells.13 These findings lead to the hypothesis that the polysaccharides are forming a bioadhesive layer on the epithelial tissue that should lead to a better protection and rehydration of the tissue and an improved shielding of the mucosal barrier against

he roots of Althaea off icinalis L. (Malvaceae), also known as marshmallow roots, are used for the symptomatic treatment of oral or pharyngeal irritation associated with dry cough and for the symptomatic relief of mild gastrointestinal discomfort.1 The herbal material contains 5−11% water-soluble polysaccharides, mainly galacturorhamnans, arabinans, glucans, and arabinogalactans.2 Other constituents are about 0.2% flavone glycosides, phenolic acids, the coumarin scopoletin, starch, pectin, and tannins.3,4 The traditional oral use of aqueous extracts against dry cough caused by the irritation of oral, pharyngeal, or gastric mucosa is related to the bioadhesive effect of the polysaccharides to the epithelial mucosa, which protects the cells from local irritations.5 Phytochemical investigations, clinical particulars, and pharmacological properties provide evidence for a justifiable use of marshmallow extract, and the ESCOP monograph gives therapeutic indications for gastric and duodenal ulcers and for dry cough.6 Additionally, an antitussive effect of marshmallow root extract was found in an animal study using cats.7,8 Extract applied topically to UV-irritated rabbit ears had an antiinflammatory effect.9 Other animal experiments indicated slight © 2017 American Chemical Society and American Society of Pharmacognosy

Received: July 19, 2016 Published: January 27, 2017 290

DOI: 10.1021/acs.jnatprod.6b00670 J. Nat. Prod. 2017, 80, 290−297

Journal of Natural Products

Article

Figure 1. Structural features of compounds obtained from Althaea of f icinalis.



RESULTS AND DISCUSSION Dried and pulverized roots from A. off icinalis were extracted twice with 50% aqueous MeOH. High molecular weight material was removed by precipitation with EtOH, resulting in a raw extract, hereafter referred to as RE (yield: 8.1%, related to the dried plant material). Since preliminary TLC studies of RE indicated the presence of carbohydrates, amino acids, and highly polar caffeic acid derivatives, a direct investigation of RE with specific screening methods was applied to obtain a proper insight into the composition of RE prior to systematic chromatographic fractionation. N-Phenylpropenoyl-L-Amino Acid Amides (1−5). Using a stable isotope dilution assay19 five N-phenylpropenoyl-L-amino acid amides were identified (Figure 1): N(E)-caffeoyl-L-dopa (1), N-(E)-caffeoyl-L-tyrosine (2), N-(E)coumaroyl-L-dopa (3), N-(E)-coumaroyl-L-tyrosine (4), and N(E)-cinnamoyl-L-aspartate (5). Absolute quantification indicated a content of 60 μg/g dried plant material for 1, whereas a content of 0.3 μg/g was determined for each of 2−4 and of 0.1 μg/g for 5. Mono- and Oligosaccharides. Using carbohydratespecific IEC-HPLC with pulsed amperometric detection (PAD),20,21 the following mono- and oligomeric carbohydrates were identified from RE and quantified against the respective reference substances: sucrose (59%, related to RE dry mass), glucose (1.4%), fructose (1.0%), raffinose (0.4%), xylose (0.1%), galactose (0.1%), and rhamnose (20 kDa) exerts a multitude of physicochemical effects on the tissue, such as water-binding, regulation of osmotic pressure, and also anti-inflammatory effects by influencing the migration of leucocytes and macrophages. Induction of growth factors for epithelial cells, cell proliferation, and differentiation are also described for this polymer.14 In this regard, high molecular weight HA seems to be relevant for wound healing and tissue regeneration. Low molecular weight HA (