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Ellagitannins from Phyllanthus muellerianus (Kuntze) Exell.: geraniin and furosin stimulate cellular activity, differentiation and collagen synthesis of human skin keratinocytes and dermal fibroblasts.

ABSTRACT

Leaves from Phyllanthus muellerianus (Kuntze) Exell. are traditionally used for wound healing in Western Africa. Aqueous extracts of dried leaves recently have been shown to stimulate proliferation of human keratinocytes and dermal fibroblasts. Within bioassay-guided fractionation the ellagitannins geraniin (1), corilagin (2), furosin (3), the flavonoids quercetin-3-O-[beta]-D-glucoside (isoquercitrin), kaempferol-3-O-[beta]-D-glucoside (astragalin), quercetin-3-O-D-rutinoside (rutin), gallic acid, methyl gallate, caffeic acid, chlorogenic acid. 3.5-dicaffeoylquinic acid and caffeoylmalic acid (phaselic acid) have been identified in P. muellerianus for the first time. Geraniin was shown to be the dominant component of an aqueous extract.

Suitable analytical methods for quality control of geraniin in P. muellerianus extract (methanol/water, 70/30) have been developed and validated based on ICH guidelines (ICH-compliant protocol).

Geraniin and furosin increased the cellular energy status of human skin cells (dermal fibroblasts NHDF, HaCaT keratinocytes), triggering the cells towards higher proliferation rates, with fibroblasts being more sensitive than keratinocytes. Highest stimulation of NHDF by geraniin was found at 5 [micro]M. and of keratinocytes at 50-100 [micro]M. Furosin stimulated NHDF at about 50 [micro]M, keratinocytes at about 150-200 [micro]M. Necrotic cytotoxicity of geraniin, as measured by LDH release, was observed at 20 [micro]M for NHDF and 150 [micro]M for keratinocytes. Toxicity of furosin - less than that of geraniin - was observed at >400 [micro]M.

Furosin and geraniin stimulated the biosynthesis of collagen from NHDF at 50 [micro]M and 5-10 [micro]M respectively. Geraniin at 105 [micro]M significantly stimulated the differentiation in NHEK while furosin had a minor influence on the expression of involucrin and cytokeratins K1 and K10. The study proves clearly that hydrophilic extracts from P. muellerianus and especially the lead compound geraniin exhibit stimulating activity on dermal fibroblasts and keratinocytes, leading to increased cell proliferation, barrier formation and formation of extracellular matrix proteins. From these findings the traditional clinical use of such extracts for wound healing seems to be justified.

[C]2010 Elsevier GmbH. All rights reserved.

ARTICLE INFO

Keywords:

Phyllanthus muellerianus (Kuntze) Exell.

Euphorbiaceae

Keratinocytes

Skin fibroblasts

Geraniin

Furosin

Collagen

Differentiation

Proliferation

Introduction

Since ancient times, various medicinal plants or herbal remedies have been used as wound healing agents and for treatment of skin disorders. Medicinal plants for wound healing are widely used for antibacterial, anti-inflammatory effects and for induction of cell proliferation and skin cell differentiation towards an intact skin barrier. From this point of view, such traditional medicinal plants can effectively serve for identification of highly active new lead structures.

Within systematic ethnopharmacological studies on the rational use of wound healing plants polysaccharides and tannins have been shown to act specifically on proliferation and differentiation of human keratinocytes and dermal fibroblasts (Zippel et al. 2009; Houghton et al. 2005; Deters et al. 2001, 2005 a, b). Within a recent ethnopharmacological survey on African wound-healing plants in today's clinical use in Ghana (Agyare et al. 2009) an aqueous extract of dried leaves of Phyllanthus muellerianus (Kuntze) Exell. (Euphorbiaceae) has been reported many times to be used for old, deep and chronic wounds as well as for boils and skin eruptions. Target-oriented preclinical in vitro investigations indicated that the extract stimulates cell viability and proliferation of skin keratinocytes and primary dermal fibroblasts (Agyare et al. 2009). The aim of the following study was a phytochemical characterization of the extract towards the active principle(s) from P. muellerianus and a more detailed characterization of the skin cell activity under in vitro conditions in order to obtain information if the clinical use of extracts from P. muellerianus, which is still very common in West Africa, can be rationalized by in vitro experiments on human skin cells.

P. muellerianus (local Asante-Twi name in Ghana is 'Awobe') is a glamorous or woody climber, often with recurved thorns leaves. The plant is growing in deciduous and secondary forests and is widespread in tropical Western Africa. Beside the documented clinical use of leaves and stem bark for wound healing in Africa the rare literature on this species indicates in vitro antibacterial activity of aqueous and acetone extracts which may be connected to a potential data wound healing activity (Doughari and Sunday 2008). Additionally antiplasmodial effects of decoctions from P. muellerianus are documented (Zirihi et al. 2005).

The phytochemical characterization of the species is insufficient: triterpenes, phtalates and methyl gallate have been isolated from the bark and leaves (Saleem et al. 2009; Adesida et al. 1972) while the occurrence of not defined tannins, flavonoids, alkaloids, anthraquinones from the leaves and stem bark have been reported (Doughari and Sunday 2008). Therefore fractionation of the leave material was performed in order to define details on the active principle and to obtain more details on phytochemical aspects of the plant.

The aim of the study was a phytochemical characterization of hydrophilic extracts, the development of analytical methods for quality assurance of plant material and extracts from P. muellerianus and investigations if the frequent clinical use of the species for wound healing in Western Africa can be rationalized by in vitro experiments to get a clearer insight into the potential mode of action.

Materials and methods

General procedures

If not stated otherwise solvents and reagents were used in analytical quality grade and purchased from Merck (Darmstadt, Germany) or from Sigma-Aldrich (Deisenhofen, Germany). NMR spectra were recorded on a Varian AS 400 Mercuryplus spectrometer. Mass spectra were obtained on a Finnigan MAT ESI mass spectrometer, HR-MS on Micro TOF (Bruker Daltronics). Optical rotation was measured on Perkin-Elmer 341 digital polarimeter in MeOH.

Plant materials and chemicals

The dried leaves of Phyllanthus muellerianus (Kuntze) Exell were collected in July 2007 in Kumasi, Ghana and were identified by Dr. Alex Asase, Department of Botany, University of Ghana, Ghana, where a voucher specimen is deposited (AA 102).

Tannin content

Tannin content was determined according Ph. Eur.VI, monograph 2.8.14 "Tannin content of herbal materials". Calculation was performed against pyrogallol (Merck, purity 99.5% HPLC) as reference compound.

Extraction and isolation

Dried and powdered leaves (1 kg) were extracted with cold 20 L of methanol/water (7:3 V/V) by rotor-stator extraction (Ultra-Turrax[R]). The combined extracts were concentrated at 40 C to 1 L of aqueous extract, which was allowed to stand for 12 h at RT, followed by filtration of the precipitated chlorophyll. Lipophilic compounds and remaining chlorophyll were removed by liquid-liquid partition with dichloromethane. The aqueous solution was acidified to pH 2 and partitioned between ethyl acetate (EtOAc). Both, the aqueous and the organic phases were concentrated and lyophilized to yield 78 g [H.sub.2]O-soluble fraction (W) and 59 g EtOAc-soluble fraction (E). E was further fractionated on Sephadex[R] LH-20, 730 mm x 55 mm (General Health Care, Munich, Germany) using methanol/water 1:1 (5L), followed by methanol/water 7:3 (2.5 L), 100% methanol (2.5 L) and acetone/water 7:3 (2.5 L) to yield 20 fractions which were monitored by TLC and analytical HPLC. Analytical TLC on silica gel 60 [F.sub.254] (Merck, Darmstadt, Germany), mobile phase ethyl acetate/water/formic acid (90:5:5), detection at A. 366 nm before and after spraying with 1% diphenylboryloxyethylamine in methanol. Analytical HPLC for investigation of the fractions obtained was performed on a Phenomenex Luna[R] C18, 5[micro], 100 A 250mm x 3 mm, (Phenomenex. Aschaffenburg, Germany) by gradientelution with acetonitrile/trifluoroacetic acid 0.1 % in water; UV detection by Waters 996 photodiode array at [lambda] 280 and 320 nm.

A single compound-containing fraction 15, eluting with 1.25 L of methanol/water 7:3 (yield 12.0 g) was directly analyzed by NMR leading to the identification of two geraniin isomers (1a and b). Phenazine-geraniin derivative (1c) was synthesized according to Foo (1993) from (1) in order to confirm geraniin structure: Formation of 1c resulted in less simplified NMR spectra and identification was in accordance with data reported (Foo 1995; Okuda et al. 1982; Yoshida and Okuda 1980). Exact mass: 1005.1175 [neg. mode, M-[H.sup.+]] and 1029.1066 [pos. mode, M+[H.sup.+]].

From fraction 11, which was eluted from Sephadex LH20 with 1.45 L elution volume of methanol/water (1:1) (yield 790 mg) corilagin (2, yield 18.6mg) and kaempferol-3-O-[beta]-D-glucoside (7.8 mg) were isolated by preparative HPLC (Hypersil[R]ODS, 5 [micro]m, 250 mm x 16 mm, acetonitrile-water linear gradient, flow 5 mL/min, [lambda] 280nm).

From fraction 13, which was eluted from Sephadex with 2.45 L elution volume of 50/50 methanol/water (1:1) (yield 660 mg) furosin (3, yield 146 mg) and quercetin-3-O-P-D-glucoside (19.8 mg) were isolated by preparative HPLC (conditions see above).

Structural elucidation by ESI-MS and NMR and comparison with published data proved the structures of (2) (Yoshida et al. 1992; Yamada et al. 2008), (3) (Miguel et al. 1996; Kumaran and Karunakuran 2006), kaempferol-3-O-[beta]-D-glucoside (Wind et al. 1998), quercetin-3-O-[beta]-D-glucoside (Fernandez et al. 2005).

Fraction 9, which was eluted from Sephadex (yield 0.19g) with 1.0 L elution volume of methanol/water (1:1), was further fractionated by MCI[R] gel CHP 20P (Mitsubishe Kasei Corporation) (25 mm x 400 mm) by a linear gradient 20-80% (2L, 6mL/min) yielding 5 subfractions. The third subfraction 9c (23.2 mg) was identified and characterized as caffeoylmalic acid (phaselic acid) and its ESI-MS and NMR data were identical with published data (Wang et al. 1998; Hahn and Nahrstedt 1993).

For identification of compounds from fraction 8 eluted with 1.0 L elution volume of methanol/water (1:1) analytical HPLC separation was performed (Phenomenex Luna[R] CI8, 5[micro], 100A. 250 mm x 3 mm; gradient elution with 2% acetonitrile/98% 0.1% trifluoroacetic acid (TFA); flow rate 0.8mL/min; 50[degrees]C). Spiking experiments with the authentic reference compounds rutin (Carl Roth GmbH, Karlsruhe, Germany), chlorogenic acid (Carl Roth GmbH, Karlsruhe, Germany), gallic acid (Fluka Chemie AG, Switzerland) and methyl gallate (Fluka Chemie AG, Switzerland) revealed the identification of these compounds by comparison of the respective retention times, the UV spectra. Similar experiments were performed with fraction 9 (eluted with 1.0 L elution volume of methanol/water 1:1) using 3.5-dicaffeoylquinic acid reference compound from Institute of Pharmaceutical Biology and Phyto-chemistry, Munster, Germany) and fraction 10 (eluted 1.2 L 50/50 methanol/water) using caffeic acid (Fluka Chemie AG, Switzerland) as reference compound for peak identification. Additionally TLC of the purified fractions and spiking experiments with the reference compounds proved the identity of these compounds unambiguously.

Methods of cell biology

HaCaT keratinocytes were provided by Prof. Fusenig (DKFZ. Heidelberg, Germany). Primary keratinocytes and fibroblasts were obtained from surgical resectates (University Hospital of Munster, Germany, Departments of Dermatology and Paediatrics) of various Caucasian subjects and were cultivated either as NHEK (primary normal human epidermal keratinocytes) or NHDF (primary normal human dermal fibroblasts). Approvals of the studies were made by the local ethical committee of University of Munster (acceptance no. 2006-117-f-S). Decontamination of skin and isolation of keratinocytes was carried out according to (Zippel et al. 2009; Houghton et al. 2005; Deters et al. 2005a,b, 2008). For isolation and propagation of fibroblasts, the dermis was washed with PBS and incubated for 2 and 3 weeks in cell culture flasks in fibroblast growth medium (MEM high glucose. FCS (10%) and L-glutamine (1%) (PAA; Pasching, Austria). Fibroblasts growing out from the tissue tended to form monolayers within the flask bottom, from which cells were isolated and used for further passages.

Permanent culture of HaCaT keratinocytes was performed in D-MEM high glucose medium supplemented with FCS (10%), penicillin/streptomycin (1%), glutamine (1%) and non-essential amino acids (1%) (PAA, Pasching, Austria).

Submerse cultivation of NHEK and NHDF was performed at 37[degrees]C. 5% [CO.sub.2]. HaCaT keratinocytes were cultivated at 35[degrees]C, 8% [CO.sub.2].

Investigations with NHEK and NHDF were carried out with cells from the 2nd to 6th passage. Prior incubation with the test compounds cells were directly adapted to serum- and BPE-free medium (NHEK: MCDB 153 complete. Biochrom, Berlin. Germany; NHDF: MEM high glucose. SerEx[R] (10%) and L-glutamine (1%) (PAA, Pasching, Austria). In vitro testing of test compounds was performed concerning mitochondrial activity by MTT test (Mosmann 1983), BrdU incorporation assay (Porstmann et al. 1985), LDH release (Deters et al. 2008; Brunold et al. 2004) and keratinocytes differentiation (Deters et al. 2008; Brunold et al. 2004) against untreated negative control and 1% FCS as positive control (Hamamra et al. 2005; Louis et al. 2010) Influence of test compounds on collagen production in NHDF: total protein was extracted from both treated and untreated cells with 0.5 M acetic acid. 50 [micro]-L extract was transferred into 96-well NuncImmuno plates (Nalge Nunc International, Denmark). Plates were blocked with 50[micro]L 1% bovine serum albumin (BSA) in PBS, 1 h, 37[degrees]C and probed with 50 [micro]L mouse anti human collagen 1 antibody (Biotrend, Cologne, Germany) for 1.5 h, 37[degrees]C. The plates were washed 5 times with 0.5% Tween[R]20 in PBS and incubated with 50[micro]L rabbit anti-mouse-[kappa] HRP conjugated secondary antibody (Rockland, Gilbertsville, USA) for 1.5 h, 37[degrees]C, washed, developed with tetramethyl-benzidine (TMB) solution (Roche Diagnostics, Mannheim, Germany) and measured at 450 nm against 650 nm. Positive control ascorbic acid 1% (Murad et al. 1981).

Preparation of water extract for cell testing

10 g of the powdered dried leaves of P. muellerianus in 100mL water was heated at 90[degrees]C/15min and centrifuged (6000 x g, 10min). The supernatant was concentrated at a temperature not exceeding 40[degrees]C and lyophilized (yield 1.46g). The % yield of geraniin and rutin in the aqueous extract was 5.5 and 1.73% respectively (m/m, related to the dried leaves) as determined by HPLC.

HPLC analysis of geraniin in P. muellerianus extract

Test solution for HPLC: 1.0 g powdered dried leaves are extracted for 15 min in 10mL methanol/water (7/3) by ultra sonic (Bandelin Electronics, Germany). After centrifugation (6000 x g, 10 min), the supernatant is used for HPLC.

HPLC equipment: Varian 9012 solvent delivery system, 9050 UV-V1S detector and 9100 auto sampler; stationary phase Chromolith[R] performance RP-18e Merck (100 mm x 4.6 mm); [lambda] = 280 nm; binary gradient (A: [H.sub.2]O with 0.1%TFA; B: [CH.sub.3]CN), [t.sub.0min] 98% A [right arrow] linear gradient to 60% A within 10 min [right arrow] linear gradient to 0% A/100% B within 4 min [right arrow] isocratic for 1 min [right arrow] linear gradient to 98% A within 5 min; flow rate 3.0 mL/min; injection volume 10 [micro]L Quantification of geraniin ([R.sub.t] 6.4 min) is calculated using external calibration.

Validation data (Validation 2010: Text on Validation 2010)

Specificity: Peak identification was done by spiking and co-injection with reference compound (geraniin). Linearity and range: Concentrations from 100 to 1000 [micro]g/mL showed linearity (6 measuring points): y = 384.01x-1191.1, [r.sup.2] = 0.9979.

Accuracy ("Trueness") and recovery: Spiking experiments were performed as follows: extraction of P. muellerianus was performed as described above. 100, 200, 400, 600, 800 [micro]L of stock solutions of geraniin (1 mg/mL) were added to 100[micro]L of the extract test solution. The solution was filled up to 1000 [micro]L The x-intercept (at y = 0) of the resulting calibration curve for (y = 379.21x +148,077, [r.sup.2] = 0.9914) gave the amount of geraniin in the extract. Comparison with results obtained from standard calibration procedure (cf. linearity) gave recovery of >99%. Limit of detection: 25 [micro]g/g. Limit of quantitation: 0.5%.

Precision: The intra-assay precision (repeatability) was 4.8% (% RSD), determined under the same conditions performed with double injections of 6 independent sample preparations. The intermediate precision (12 samples, different days, same analyst and same equipment) was 9.6% (% RSD).

Statistics

Statistical analysis of the influence of test compounds on cell physiology was performed by Student's t-test. p-values < 0.05 were considered significant (*p<0.05, **p<0.01). All data presented are the means of 24-32 random replicates from 4 independent experiments (errors bars: [+ or -]SD).

Results and discussion

The total tannin content of an aqueous extract from the dried leaves of P. muellerianus was determined with 14%, indicating the species to be strongly tannin-enriched herbal material. For detailed investigations of the nature of polyphenols a methanol-water (7:3) extract was partitioned between EtOAc and water (Bicker et al. 2009). The EtOAc extract was fractionated by Sephadex[R]LH-20 and purified by preparative HPLC on RP18 and MCI CHP200 gel.

The major ellagitannin geraniin (1) was isolated in yields of 2.9% and appeared to be the major constituent of the plant extract (Fig. 1). As known from literature isomerisation of la-lb occurred in solution, and resulted in a set of two NMR signals. Spectra were simplified after derivatization of the mixture with o-phenylene diamine to yield the phenazin derivative 1c (Foo 1993). All NMR data fit with literature data and high resolution mass spectroscopy continued these results. The presence of geraniin has been reported in other species of the Phyllanthus genus as for P. urinaria (Okuda et al. 1980), P. niruri (Ueno et al. 1988), P. flexuosus (Yoshida et al. 1992) and P. amarus (Foo and Wong 1992).

[FIGURE 1 OMITTED]

Furosin (2) and corilagin (3) were isolated as similar biosyn-thetic compounds in yields of 0.03 and 0.004% (Fig. 1). The presence of corilagin does not seem to be unusual for the genus and has also described for P. amarus (Foo 1995), P. urinaria (Foo and Wong 1992) and P.flexuosus (Yoshida et al. 1992).

By isolation and structure elucidation using NMR and MS techniques the flavonoids quercetin-3-0-[beta]-D-glucoside (isoquercitrin), kaempferol-3-O-P-D-glucoside (astragalin) and quercetin-3-O-D-[beta]-rutinosid (rutin) and caffeoyl-malic acid (phaselic acid) not described until now for P. muellerianus, were identified. By HPLC and TLC gallic acid, methylgallate, caffeic acid, chlorogenic acid (5-O-caffeoylquinic acid) and 3,5-O-dicaffeoyIquinic acid were identified by comparison with respective reference standards and spiking experiments.

A HPLC method for standard quality control was developed for identification and quantitation of geraniin and rutin as lead compounds of the extract. A typical chromatogram of a representative batch is shown in Fig. 2. The contents the marker compound geraniin was determined with 4.3% (m/m, related to the dried leaves). The method was validated according to ICH-compliant protocol (Validation 2010; Text on Validation 2010) regarding specificity, linearity, accuracy, precision, limit of detection and limit of quantitation (data see experimental part).

[FIGURE 2 OMITTED]

Due to the fact that previous studies have indicated that aqueous extracts from P. muellerianus are capable to stimulate skin cell viability and proliferation (Agyare et al. 2009) the ellagitannins as major lead compounds were investigated on a potential influence on skin cells had to be determined. The influence of geraniin and furosin (corilagin was not tested due to low amounts) on skin cells was investigated using normal human fibroblasts from human skin and HaCaT keratinocytes, a non-malignant epithelial skin line. Both cell types represent the epidermal and dermal functionality of human skin barrier. To characterize the potential effects of geraniin and furosin on these skin cells cellular dehydrogenase activity (MTT Test), mitogenic cell proliferation rate (BrdU-incorporation ELISA) and keratinocyte differentiation (cytokeratins CK1, 10 and involucrin formation) were investigated. Potential toxic effects of the test compounds against fibroblasts and HaCaT cells were determined by the release of lactate dehydrogenase (LDH) as a typical marker for necrosis.

On both cell types, NHDF and HaCaT keratinocytes, geraniin exhibited strong stimulating effects, but at different concentrations, with fibroblasts being more sensitive than HaCaT keratinocytes (Fig. 3).

[FIGURE 3 OMITTED]

Within an optimum curve geraniin stimulated mitochondrial activity of NHDF significantly between 1 and 10 [micro]M, while higher concentrations were ineffective. The higher energy catabolism, observed within MTT test, was congruent with a significantly increased mitogenic cell proliferation between 1 and 5 [micro]M (Fig. 3A and B).

HaCaT keratinocytes were less sensitive towards geraniin compared to NHDF (Fig. 3C and D): Significant stimulation of the cells in MTT test was achieved at 20-150 [micro]M, which correlates well with stimulation values found for the respective proliferation rates. Within BrdU assay inhibition of proliferation was observed at > 20 [micro]M for NHDF and > 150 [micro]M for HaCaT keratinocytes. This was due to induction of cellular necrosis as was shown by substantial LDH release from NHDF at >10[micro]M and > 150[micro]M for HaCaT cells (Table 1).
Table 1
Influence of geraniin and furosin on LDH release from HaCaT
keratinocytes and pNHF. Measured values are related to the untreated
negative control; n.d., not determined (out of range).

 Cell type 1 [micro]M 5 [micro]M 10 [micro]M 21 [micro]M

Geraniin pNHF -2% -2% 12% 199%
 HaCaT n.d. n.d. 2% -2%

Furosin pNHF n.d. n.d. -2% -1%
 HaCaT n.d. n.d. -2% -2%

 Cell type 52 105 158 210 420
 [micro]M [micro]M [micro]M [micro]M [micro]M

Geraniin pNHF n.d. n.d. n.d. n.d. n.d.
 HaCaT 5% 9% 57% 118% n.d.

Furosin pNHF 1% 1% 3% 11% 119%
 HaCaT 2% 2% 3% 3% 55%


Different susceptibility of dermal fibroblasts and keratinocytes has been reported also for other skin active natural compounds (Deters et al. 2010) indicating different receptor signaling sensitivity. For practical use this implies that the potential dosing of geraniin or geraniin containing extracts has to be related to the effects to be induced: higher concentrations are necessary for induction of epithelial barrier while doses in the low micromolar range stimulate cellular compounds of connective tissue.

Similar results were obtained with furosin, but the concentrations necessary for significant activities were higher than those for geraniin (Fig. 4A and B). Furosin stimulated MTT activity of NHDF significantly within an optimum curve at 50 [micro]M. This correlated with a strong stimulation of proliferation rates to about 300%. It seems interesting that this stimulation of proliferation is assessed to be extraordinary high in comparison with other compounds known from literature. Again HaCaT keratinocytes were less sensitive than NHDF (Fig. 4B and C). Stimulatory activity of furosin on cellular energy status was observed at concentrations >100 [micro]M which correlated well with the respective proliferation rates.

[FIGURE 4 OMITTED]

It was interesting that the necrotic cell activity, as measured by LDH release, was less than that observed with geraniin (Table 1): up to concentration of 210 [micro]M furosin no LDH release was detected which is assessed to be typical for necrotic cell toxicity.

Summarizing these results indicate furosin to exhibits an extraordinary capacity to stimulate cell proliferation and energy status of dermal cells. Necrotic toxicity of furosin is less than that of geraniin.

During development of human skin towards an intact barrier system keratinocytes will undergo cellular proliferation followed by a switch to cellular differentiation. The potential differentiation behaviour of keratinocytes in the presence of geraniin and furosin was investigated on the basis of terminal differentiation-specific protein expression, with cytokeratins CK1, 10 and involucrin (Deters et al. 2008). NHEK isolated from human skin resectates and treated with geraniin and furosin showed increased keratin and involucrin expression compared to the untreated negative control as evaluated by semi-quantitative dot blotting of protein extracts with the respective mouse-anti-human-antibodies (Table 2). Semi-quantitative evaluation was done by a 0-5 scoring system in a double-blinded evaluation from 3 independent experiments. The aqueous extract, geraniin and furosin had a positive influence on NHEK differentiation compared to the untreated cells. 105 [micro]M geraniin was found to significantly increase the expression of both involucrin and cytokeratins CK1/10 comparable to the positive control. These differentiation-inducing effects are not due to a follow-up effect of the cells due to an increased proliferation, triggering the cells into terminal differentiation. This effect can be excluded because evaluation of the experiment was done at about 40-50% of confluence of cells, which is below the cell density necessary for contact inhibition of proliferation and secondary induction of endogenous differentiation.
Table 2
Induction of NHEK differentiation by aqueous extract from
P. muellerianus. geraniin and furosin after 9 days of incubation.
Semi-quantitative determination from Dot Blots of involucrin and
cytokeratins CK1, 10 from 2 different experiments. Positive control:
calcium ionophore 13 [micro]g/mLA23187: negative control: untreated
cells. Numbers indicate the intensity of antibody coloured spoty on
blot membranes, evaluated under blindes conditions and asessed by a
score with 5 being the highest and 0 representing lowest spot
intensity.

 Marker protein Negative Positive Geraniin
 control control

 52 [micro]M 105 [micro]M

Involucrin 1.0 2.5 1.9 2.5
Cytokeratins CK 1/10 2.1 3.1 2.6 4.7

 Marker protein Furosin P. muellerianus Aq. extract

 210 [micro]M 50 [micro]g/mL

Involucrin 1.5 1.4
Cytokeratins CK 1/10 2.9 2.5


The differentiation-inducing effect on keratinocytes is in the same concentration range (50-100 [micro]M) as for the stimulation of cell vitality and proliferation, indicating that the epithelial cells can be stimulated by these doses of geraniin towards increased barrier formation.

Beside the stimulation of the epidermal keratinocytes barrier functionality also the formation of extracellular matrix markers from the fibroblasts as typical dermis cells was investigated. The influence of the aqueous extract, geraniin and furosin on the induction of collagen synthesis, an important texture marker protein for extracellular matrix, was investigated by ELISA from NHDF (Fig. 5).

[FIGURE 5 OMITTED]

The aqueous extract was found to increase the synthesis of collagen highly significantly almost the same as the positive control, ascorbic acid (about 3-fold compared to untreated control). Also furosin and geraniin had significant influence on collagen formation. This stimulation of collagen production may be attributed to the strong redox properties of the extract and the ellagitannins (Murad et al. 1981; Chojkieret al. 1989; Agyare et al. 2009). It seems interesting that the collagen inducing effect on dermal fibroblasts is regulated by concentrations of geraniin in the low micromolar range, very similar to the doses needed for stimulation of mitochondrial activity and proliferation of fibroblasts. Again it gets obvious that these low doses trigger dermal fibroblasts towards higher activity and increased ECM formation, while higher concentrations (50-100 [micro]M) act on the keratinocyte cell population.

Summarizing the aqueous extract of P. muellerianus, and especially the ellagitannins geraniin and furosin exhibit a strong induction of cellular proliferation of skin fibroblasts and ker-atinocytes. These effects are accompanied by stimulation of terminal differentiation of keratinocytes as well as by significant induction of collagen synthesis from fibroblasts. Additionally geraniin, the major compound from P. muellerianus, has been found to possess apoptose-inducing effects (Lee et al. 2008), antiviral (Yang et al. 2007; Li et al. 2008; Notka et al. 2003, 2004) and antimicrobial properties (Xiaoli et al. 2009; Li et al. 2008; Taguri et al. 2004, 2006) beside strong antioxidative properties under in vitro conditions (Agyare et al. 2009; Wu et al. 2010).

Additionally geraniin increases functionality of macrophages towards an increased phagocytosis (Ushio et al. 1991) and is described as anti-inflammatory compound by inhibition of TNF-[alpha] release (for review see Fujiki et al. 2003) and NO formation (Kumaran and Karunakaran 2006). Additionally inflammatory response provoked by bacterial infection can effectively diminished by geraniin (Park et al. 2007).

Using these data the effect of geraniin during wound healing can be assessed as follows:

i. stimulation of keratinocytes and dermal fibroblasts viability, energy production and proliferation for tissue regeneration,

ii. induction of keratinocyte terminal differentiation for initiation of barrier formation,

iii. induction of collagen for remodeling of tissue and regeneration of extracellular matrix,

iv. reduction of cellular damages due to oxidative stressors by radical-scavenging effects,

v. prevention of bacterial or viral infection, and vi. inhibition of inflammation processes during healing process.

Generally, it has been accepted that for an agent to be classified as a good wound healing agent, it should possess at least two of the following properties, namely antimicrobial and/or antiinflammatory properties, induction of proliferation of fibroblasts or keratinocytes or differentiation of keratinocytes (Houghton et al. 2005). From that point of view P. muellerianus extract and the ellagitannins fit well into this therapeutic regime. Detailed analysis for evaluation of structure-activity relation of the tannin should be carried out in future in order to figure out the relevance of the galloyl-dehydrohexahydroxydiphenoyl group linked to the sugar moiety in order to clarify also the molecular targets for these compounds on the cellular side.

In summary, the current in vitro findings may justify the rational use of the plant and respective plant extracts as wound healing agent, due to the relatively high amounts of geraniin found in the plant material (>4%). As geraniin is a relatively widespread compound, especially found within the genus Phyllanthus, it can be hypothesized that existing reports on in vivo wound healing properties determined in rat excision, incision models (Datta et al. 2009; Sumitra et al. 2009; Kumar et al. 2008) of complex Phyllanthus emblica preparations is due to this ellagitannin. From the mechanistical point it seems interesting that the tannins act on specific points of skin cell regulation and differentiation. In former literature it can be found that polyphenols are a kind of compounds with unspecific effects on protein structures, due to the astringent and denaturing properties of the molecules. From the data presented in this study on the effects of geraniin on cell physiology and of the antiinflammatory, anti TNF-[alpha] release and proapoptotic effects known from literature on this compound we assume that the wound healing activity should be due to multifactorial, but specific activities of this compound.

In summary this study proves clearly that hydrophilic extracts from P. muellerianus and especially the lead compound geraniin exhibit stimulating activity on dermal fibroblasts and keratinocytes, leading to increased cell proliferation, barrier formation and formation of extracellular matrix proteins. From these findings the traditional clinical use of such extracts for wound healing seems to be justified.

Acknowledgements

The authors are grateful to German Academic Exchange Service (DAAD) for the fellowship offered to C. Agyare for this project and Engelhard Arzneimittel GmbH, Frankfurt, Germany for financial support. We are also grateful to Dr. H. Lahl and Mrs. M. Heim, Munster, for recording NMR spectra and Dr. H. Luftmann, Muenster, for performing MS experiments.

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Christian Agyare, Matthias Lechtenberg, Alexandra Deters, Frank Petereit, Andreas Hensel *

Institute of Pharmaceutical Biology and Phytochemistry, University of Munster, Munster, Germany

Abbreviations: BrdU, bromodeoxyuridine; ESI, electrospray ionization: FCS. fetal calf serum: HaCaT. Human adult low calcium high temperature keratinocyte cell line: LDH, lactate dehydrogenase: MTT. 3-(4.5-dimethylthiazol-2-yl)-2.5-diphenyltetrazolium bromide: NHDF. primary normal human fibroblasts: NHEK. primary normal human epidermal keratinocytes.

* Corresponding author at: University of Munster. Institute of Pharmaceutical Biology and Phytochemistry. HittorfstraBe 56. D-48149 Munster. Germany. Tel.: +49 251 83 33380: fax: +49 251 8338341.

E-mail address: ahensel@uni-muenster.de (A. Hensel).

doi: 10.1016/j.phymed.2010.08.020
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Author:Agyare, Christian; Lechtenberg, Matthias; Deters, Alexandra; Petereit, Frank; Hensel, Andreas
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
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Date:May 15, 2011
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