Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts.

Methanolic avocado (Persea americana Mill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes.

Anti-inflammatory activity of Eupatorium perfoliatum L. extracts, eupafolin, and dimeric guaianolide via iNOS inhibitory activity and modulation of inflammation-related cytokines and chemokines.

ETHNOPHARMACOLOGICAL RELEVANCE: Eupatorium perfoliatum L. has been used traditionally for the treatment of fever, malaria and inflammation-associated diseases. Nowadays it is mostly used as immune activating remedy. The following study was performed to evaluate extracts with different polarity and defined lead-compounds from the herbal material on potential in vitro activities concerning immune cell activation, phagocytosis, and inflammation-related processes. MATERIALS AND METHODS: MeOH-, EtOH-, and DCM extracts, beside several subfractions and isolated polysaccharides, sesquiterpene lactones and flavonoids were prepared and characterized analytically from the aerial parts of E. perfoliatum. Immunological activity was tested within lymphocyte transformation test on PBMC, test on enhancement of phagocytosis and of NO-production by murine RAW 264.7 macrophages. Anti-inflammatory effects were assessed from LPS-stimulated RAW 264.7 cells by NO/iNOS quantification, gene array, real-time PCR and ELISA. RESULTS: No stimulatory activity was found within lymphocyte transformation test, for phagocytic activity and NO formation in macrophages. MeOH-, EtOH- and DCM extracts showed anti-inflammatory activity against LPS-stimulated macrophages by inhibition of NO release (IC(50)>100, 89, 19 µg/mL resp.) with eupafolin and a dimeric guaianolide having prominent NO inhibiting activity (IC(50) 6 resp. 16 µM). Anti-inflammatory activity was found on gene and protein level by significant down-regulation of cytokines CSF-3, IL-1α, IL-1ß, and chemokines CCL2, CCL22 and CXCL10. Also TNF was down-regulated moderately (-17%). CONCLUSIONS: Although the postulated immunostimulating properties of E. perfoliatum have not been confirmed, the anti-inflammatory effects can be seen as a verification of the traditional use against inflammatory diseases.

Typha latifolia L. fruit polysaccharides induce the differentiation and stimulate the proliferation of human keratinocytes in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: In Northern America Typha latifolia L. (Typhaceae) fruits are used for more than 4000 years for treatment of skin disorders, burns and as wound dressing to absorb the ichors. AIM OF THE STUDY: The following studies attempted to characterize water-soluble polysaccharides from aqueous Typha latifolia extracts and to investigate the influence of the polymers on cell physiology of human dermal fibroblasts (NHDF) and epidermal keratinocytes (NHEK). MATERIALS AND METHODS: Water-soluble raw polysaccharides (RPS) were isolated from Typha latifolia fruits and fractionated by anion exchange chromatography (AEC) and size exclusion chromatography (GPC). Fractions obtained were characterized concerning monosaccharide composition by HPAEC-PAD. The bioactivity of the polysaccharides was investigated on cell viability, proliferation, differentiation and gene expression NHDF of NHEK. RESULTS: RPS was fractionated into 5 heterodisperse fractions (TL1-TL5). The polysaccharides were composed mainly of glucose (more than 50% in RPS and TL4), galactose, xylose, mannose, glucuronic acid, galacturonic acid, arabinose, ribose, fucose, rhamnose, and fructose with differing amounts concerning to RPS and AEC-fractions. Proteins were detected in the RPS (10%) and to a less extend in TL1-TL3 (1-3%). TL1-TL3 significantly increased the proliferation of keratinocytes, whereas TL4 was shown to be a potent inductor of the early differentiation process of keratinocytes. Gene expression analysis supported these results since Smad3 and PKC-α, known to be part of signal pathways leading to cell differentiation, were significantly up regulated. Effects on fibroblasts were not observed, indicating cell specific activity of the polysaccharides. CONCLUSION: The results clearly indicate a rationale for the traditional use of Typha latifolia fruits extracts for wound healing to the strong stimulatory activity of the polysaccharides on keratinocytes proliferation and early differentiation, major activities necessary for potent wound-healing agents.

Oligo- and polysaccharides exhibit a structure-dependent bioactivity on human keratinocytes in vitro.

In traditional medicine, a variety of plants with high carbohydrate contents were used for dermatological therapies. Contemporary investigations confirmed exogenous carbohydrates as biologically active. The recent study describes the characterization of oligo- and polysaccharides from medicinal herbs and evaluation of composite-dependent physiological activity of carbohydrates on human keratinocytes in vitro. Polysaccharide isolation was followed by size- and charge fractionation. Identification of monosaccharide components was performed by GLC/MS. Primary human keratinocytes (NHK) and cells of the cell line HaCaT were used for investigation of carbohydrate action on cellular proliferation (BrdU-uptake), differentiation specific enzymes (involucrin), cell viability (MTT-reduction) and cytotoxicity. Incubation of keratinocytes with a purified beta-glucan from Reed mace seeds resulted in an improved proliferation followed by an increased differentiation after contact inhibition. Fucosylated oligo- and polysaccharides of human milk and Sea weed induced involucrin expression as maker for early differentiation without an increase in proliferation. Cell viability and proliferation of keratinocytes were enhanced by an arabinogalactan of Kaki fruits. Okra fruit rhamnogalacturonans increased cell proliferation. Heart sease pectin-like polysaccharides reduced the proliferation significantly but improved the cell viability. These results led assume that the carbohydrates of traditional used herbs play a part in their efficacy.

Kiwi fruit (Actinidia chinensis L.) polysaccharides exert stimulating effects on cell proliferation via enhanced growth factor receptors, energy production, and collagen synthesis of human keratinocytes, fibroblasts, and skin equivalents.

Within physiological engineering exogenous carbohydrates were recently confirmed as pharmacologically active compounds. To investigate potential dermatological activity purified polysaccharides from kiwi fruits (Actinidia chinensis L., Actinidiaceae) were characterized concerning monomer composition, linkage types and molecular weights and were tested under in vitro conditions for regulating activities on cell physiology of human keratinocytes, fibroblasts, and skin equivalents. Ten micrograms per milliliter of raw polysaccharide, neutral type-II-arabinogalactans, and acidic arabinorhamnogalacturonans of kiwi fruits stimulated cell proliferation of human keratinocytes (NHK, HaCaT) up to 30% significantly while mitochondrial activity was stimulated for nearly 25% in regard to control cells. Fibroblasts were not as sensitive as keratinocytes but >130 microg/ml kiwi fruit polysaccharides increased proliferation and ATP-synthesis significantly, too. Proliferation-stimulating activity was dependent on terminal 1-alpha-l-arabinose residues since enzymatic release of these sugar moieties caused significantly decreased proliferation of HaCaT and fibroblasts of about 10% in regard to untreated cells. In three dimensional skin equivalents, it was shown that the polysaccharides led to a doubled collagen synthesis of fibroblasts compared to the normally strongly reduced biosynthetic activity.