Fitoterapia 82 (2011) 1265–1271

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Fitoterapia

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Constitutive phenolics of Harpephyllum caffrum (Anacardiaceae) and theirbiological effects on human keratinocytes

Mahmoud Nawwar a,⁎, Sahar Hussein a, Nahla Ayoub b, Amani Hashim a, Reham El-Sharawy a,Urlike Lindequist c, Manualle Harms c, Kristian Wende c

a Department of Phytochemistry and Chemosystematics, National Research Center, Dokki, Cairo, Egyptb Department of Pharmacognosy, Faculty of Pharmacy, Ain-Shams University, Egyptc Institute for Pharmacy, Pharmaceutical Biology, Ernst-Moritz-Arndt-University,Greifswald, D-17487 Greifswald, Germany

a r t i c l e i n f o

⁎ Corresponding author. Tel.: +20 2 2752711; fax:E-mail address: heshamnesrienmahmoud@yahoo.c

0367-326X/$ – see front matter © 2011 Elsevier B.V. Adoi:10.1016/j.fitote.2011.08.014

a b s t r a c t

Article history:Received 26 June 2011Accepted in revised form 17 August 2011Available online 31 August 2011

Assessment of the UV protecting potential of an aqueous methanol leaf extract of Harpephyllumcaffrum proved that it possesses a distinct radical scavenging effect and inhibits the productionof the proinflammatory cytokine IL-6 by human keratinocytes (HaCaT cells) following UV radi-ation. Phytochemical investigation of this extract led to isolation and structural determinationof the hitherto unknown phenolics, kaempferol 3-O-(2″-sulphatogalactopyranoside), its quer-cetin analogue and 3-methoxyellagic acid 4-O-galactopyranoside in addition to 18 known phe-nolic compounds. The structures were determined by spectroscopic and conventional methodsof analysis. Flavonoid sulphatoglycosides which have been rarely found in nature were majorphenolic constituents of this plant, and this is the first report of the isolation of any of themfrom Anacardiaceae. The extract was found to diminish UV phototoxic reaction of keratino-cytes. However, the isolated kaempferol sulphatogalactopyranoside did not interact with UVBtriggered IL-6 production of HaCaT keratinocytes.

© 2011 Elsevier B.V. All rights reserved.

Keywords:Harpephyllum caffrumAnacardiaceaePhenolicsFlavonol sulphatogalactosides3-methoxy ellagic acid 4-O-β-galactoside

1. Introduction

In recent years, the incidence of various diseases and disor-ders related to solar ultraviolet radiation has increased alarming-ly and continues to grow. Chronic exposure of human skin to UVradiation induces a number of biological responses, includingdevelopment of erythema, edema, sunburn cell formation, hy-perplasia, immune suppression, DNA damage, photoaging andmelanogenesis. These alterations are directly or indirectly in-volved in the development of skin cancer [1–3]. In biological sys-tems, the absorbed UV light can interact with endogenousphotosensitive molecules or/and with exogenous photosensi-tizers originating from drugs or cosmetic ingredients, thus pro-ducing reactive oxygen species (ROS). Consequently, theseinteractions can directly or indirectly produce deleterious,

+20 2 3370931.om (M. Nawwar).

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cytotoxic and genotoxic effects resulting in sunburn, skin cancerand other diseases.

A successful approach to prevent and treat the harmful ef-fects of UV irradiation is afforded by the high capability ofplant phenolics on absorbing UV electromagnetic radiationand by their strong antioxidant properties as well [4,5].

Harpephyllum caffrumBernh., commonly known in English aswild plum or bush mango, belongs to the family Anacardiaceae(mango family) which includes 82 genera [6]. H. caffrum is alarge, evergreen tree that grows up to 15 m tall. The mainstem is clean and straight, but the forest form often has sup-porting buttress roots. The bark is smoothwhen young, becom-ing rough, dark gray-brown as it grows older. Branches arecurved bowed upwards, with leaves crowded towards theends, forming a thick crown at the top of the tree. The shinydark green and glossy leaves are pinnate with sickle-shapedleaflets, and are sometimes interspersed with the odd redleaves. The whitish green flowers are borne near the ends ofthe branches with male and female flowers on separate trees,

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throughout summer. The tasty plum-like fruits first appeargreen and then turn redwhen they ripen in autumn. They con-tain a single seed and are enjoyed by people, mammals andbirds [6].

H. caffrum is a well known folk medicinal plant and has longbeen used as medicinal remedies for numerous ailments. Phar-macological studies by Buwa and Van Staden [7] revealed anti-bacterial and antifungal activities of its bark extracts which alsopossess hypoglycemic and hypotensive properties [8] and exhib-it anti-inflammatory activity as they inhibit cyclooxygenase andtherefore prostaglandin synthesis [9]. The total phenolic contentand antioxidant activity of themethanol extract of the stem barkof the plant have been previously evaluated [10] to prove that itprovides a source of natural antioxidants and may be used as al-ternatives to the available synthetic antioxidants, such as butyl-ated hydroxytoluene (BHT), butylated hydroxyanisole (BHA)and tertbutylhydroxyquinone (TBHQ) that are widely used inthe food and pharmaceutical industries. This extract showed, inaddition, a dose dependent acetylcholinesterase inhibitory activ-ity [10]. The leaf phenolics have been reported previously, on thebasis of chromatographic, UV spectral analysis and results of hy-drolysis to include protocatechuic acid, gallic acid,methyl gallate,kaempferol 3-rhamnoside, kaempferol 3-galactoside, apigenin7-glucoside, quercetin 3-rhamnoside, quercetin 3-glucoside,quercetin 3-arahinoside and the free aglycones quercetin andkaempferol [11].

The present study has been undertaken to investigate in de-tails, the constitutive phenolics of H. caffrum aqueous methanolleaf extract in association with its antioxidant and its capabilityon the UV protection of HaCaT human keratinocytes. The effectsof the extract and its column fractions for the radical scavengingcapacity using theDPPHmethod [12] and theORAC assay [13] aswell as the effect of the extract on the UV induced production ofthe proinflammatory cytokin IL-6 and of IL-8 by HaCaT (Humanadult low Calcium high Temperature) keratinocyte cells havebeen evaluated. The immortalized and spontaneously trans-formed nontumorigenic human epidermal cell line used in thisstudy offers a suitable in vitro model for the study of regulatorymechanisms involved in the differentiation of human epidermalcells. It does not require a 3T3 feeder layer and has the capacityfor normal differentiation in vitro [14]. UV radiation inducesthe production of the proinflammatory cytokine IL-6 in the UVe-posed cells [15,16]. Also, we report in the present work, aboutthe isolation and structure determination of 21 phenolic com-pounds. They include three new metabolites, which have notbeen reported previously to occur in plants, namely, kaempferol3-O-β-(2″-sulphatogalactopyranside) 10, quercetin 3-O-β-(2″-sulphatogalactopyranoside) 11 and 3-methoxyellagic acid 4-O-β- galactopyranoside 13 from the aqueous methanol extractof this H. caffrum. Compounds 10 and 11 are of special interestas they represent the first reported natural occurrence of flavo-nol sulphatoglycosides in higher plants [17,18].

2. Experimental

2.1. General experimental procedures

1H NMR spectra were measured by a Jeol ECA 500 MHzNMR spectrometer, at 500 MHz. 1H chemical shifts (δ) weremeasured in ppm, relative to TMS and 13C NMR chemical shiftsto DMSO-d6 and converted to TMS scale by adding 39.5.

FTESIMS spectra were measured on a Finnigan LTQ-FTMS(Thermo Electron, Bremen, Germany) (Department of Chemis-try, Humboldt-Universitat zu Berlin). ORAC experiment wasperformed on fluorometer, FLUOstar OPTIMA, Franka Ganske,BMG LABTECH, Offenburg, Germany. UV recording weremade on a Shimadzu UV–Visible-1601 spectrophotometer.Flame atomic absorption analysis was performed on a VarianSpectra-AA220 instrument, lamp current: 5 ma, fuel: acetylene,oxidant: air, slitwidth: 0.5 nm. Paper chromatographic analysiswas carried out on Whatman No. 1 paper, using solvent sys-tems: (1) H2O; (2) 6% HOAc; (3) BAW (n-BuOH–HOAc–H2O,4:1:5, upper layer).

2.2. Plant materials

Collection of the leaves of H. caffrumwasmade at El Ormangarden, Cairo, on April 2007. Authentication was performed byDr. M. El-Gebali, former researcher of Botany at the NationalResearch Centre (NRC) of Cairo, Egypt. Voucher specimen wasdeposited at the herbarium of the NRC.

2.3. Preparation of extract

The fresh leaves of H. caffrum, dried in the shade in an airdraft at room temp. (800 g) were refluxed in MeOH/H2O:(3:1) mixture (three extractions, each for 8 h with 1.25 L).The collected solution was filtered on and dried in vacuum toyield dark brown amorphous powder of the aqueousmethanolextract (53 g).

2.4. Isolation and identification of phenolics

The aqueous MeOH extract (43 g) was applied to a MCI gel(CHP20P, Supelco), (200 g) column (100×5 cm) and elutedwith H2O followed by H2O–MeOH mixtures of decreasing po-larities to yield seven fractions (I–VII), which were individuallysubjected to 2D-PC. Compounds 1 (101 mg) and 2 (115 mg)were isolated pure from fraction I (eluted with 10%) by columnfractionation (CF) of 1.12 g material over 15 g Sephadex LH-20using H2O for elution. Compounds 3 (88 mg), 4 (102 mg), 5(75 mg) and 6 (78 mg) were separated pure from a SephadexLH-20 column of fraction II (898 mg, eluted with 20%). Com-pounds 7 (44 mg), 8 (63 mg), 9 (70 mg) and 10 (11 mg) wereobtained from 308 mg of fraction III (eluted with 30%) throughprep. PC of using 6% AcOH as solvent. Repeated CF of 565 mg offraction IV (elutedwith 40%) on Sephadex LH-20, using n-BuOHsaturated with H2O yielded pure samples of 10 (122 mg) and11 (29 mg). Compounds 12 (34 mg) and 13 (62 mg) were iso-lated from 275 mg of fraction V (eluted with 60%) by repeatedprep. PC using 6%AcOH as solvent. 602 mgof fraction VI (elutedwith 80%), dissolved in ethanol while hot, filtered and left tocool to room temp. The formed light yellow precipitate was fil-tered off. The mother liqueur thus left was subjected to repeat-ed prep. PC, using BAW as solvent, whereby pure samples ofcompounds 14 (85 mg), 15 (34 mg) and 16 (42 mg) were iso-lated. Compounds 17 (45 mg) and 18 were obtained pure byfractional crystallization of the previously obtained precipitate.Compounds 19 (28 mg), 20 (19 mg) and 21 (39 mg)were indi-vidually isolated from 292 mg of the last major column fractionVII (eluted byMeOH) through repeated prep. PC, using BAW assolvent.

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2.4.1. Kaempferol 3-O-β-(2″-sulphatogalactopyranoside)(10)A light yellow amorphous powder, Rf-values: 0.92 (H2O),

0.87 (HOAc), 0.45 (BAW). Electrophoretic mobility: 2.6 cm, onWhatman no. 3 MM paper, buffer solution of pH 2, H2O–HCOOH–AcOH (89:8.5:2.5), 1 1/2 h, 50 v/cm. UV λmax nm inMeOH: 267, 292 shoulder, 350; +NaOMe: 273, 324, 395;+NaOAc: 273, 310, 385; +NaOAc–H3BO3: 266, 300 shoulder,355; +AlCl3: 272, 302, 348, 392; AlCl3–HCl: 272, 300, 345,392. ESIMS (negative mode): m/z=527 [M−Na]−, m/z=447[M−SO3Na]−, ESIMS (positive mode): m/z=551 [M+H]+,573 [M+Na]+; molecular formula: C21O14H20SNa. Normalacid hydrolysis gave galactose and kaempferol (CoPC); thehydrolysate gave white BaSO4 precipitate with BaCl2; flameatomic absorption of the hydrolysate: sodium line at589 nm. β-galactosidase enzymic hydrolysis: 10 was recov-ered unchanged after being incubated for 24 h with the en-zyme. Controlled acid hydrolysis, (10% AcOH, 100 °C,10 min): yielded kaempferol 3-O-galactoside (CoPC, UV, 1HNMR and β-galactosidase enzymic hydrolysis). 1H NMR of10: δ ppm (500 MHz, DMSO-d6): aglycone: 8.12 (2H, d,J=8.5 Hz, H-2′ and H-6′), 6.82 (2H, d, J=8.5 Hz, H-3′ andH-5′), 6.41 (1H, s(br), H-8), 6.16 (IH, s(br), H-6); galactosidemoiety: 5.67 (1H, d, J=8 Hz, H-I″), 4.3 (1H, t, J=8 Hz, H-2″),3.2–3.9 (m, galactose protons overlapped with hydroxyl andwater protons). 13C NMR data of 10: aglycone moiety: δ ppm156.8 (C-2), 133.1 (C-3), 178.0 (C-4), 160.5 (C-5), 98.2 (C-6),164.6 (C-7), 94.1 (C-8), 156.0 (C-9), 104.3 (C-10), 121.3 (C-1′),131.7 (C-2′ & C-6′), 161.8 (C-4′), 115.8 (C-3′ & C-5′); galacto-side moiety: 99.2 (C-1″), 77.6 (C-2″), 73.8 (C-3″), 67.7 (C-4″),76.2 (C-5″), 60.6 (C-6″).

2.4.2. Quercetin 3-O-β-(2″-sulphatogalactopyranoside)(11)A faint yellow amorphous powder, Rf-values: 0.91 (H2O),

0.85 (HOAc), 0.44 (BAW). Electrophoretic mobility: 2.6 cm, onWhatman no. 3 MM paper, buffer solution of pH 2, H2O–HCOOH–AcOH (89:8.5:2.5),1 1/2 h, 50 v/cm. UV λmax nm inMeOH: 255, 265 shoulder, 355; +NaOMe: 268, 327, 403;+NaOAc: 273, 323, 385; +NaOAc–H3BO3: 262, 300 shoulder,377; +AlCl3: 273, 295 shoulder, 330, 430; +AlCl3–HCl: 268,275 shoulder, 300 shoulder, 360, 398. ESIMS (negative mode):m/z=543 [M−Na]−,m/z=463 [M−SO3Na]−, ESIMS (positivemode): m/z=567 [M+H]+, 589 [M+Na]+; molecular formu-la: C21O15H20SNa. Normal acid hydrolysis gave galactose andquercetin (CoPC); the hydrolysate gave white BaSO4 precipitatewith BaCl2; flame atomic absorption of the hydrolysate: sodiumline at 589 nm. β-galactosidase enzymic hydrolysis: 11 recov-ered unchanged. Controlled acid hydrolysis, (10% AcOH,100 °C, 10 min): yielded quercetin 3-O-galactoside (CoPC,UV, 1H NMR and β-galactosidase enzymic hydrolysis). 1H NMRof 11: δ ppm: aglycone: 7.72 (1H, dd, J=8 and J=2Hz, H-6′),7.63 (1H, d, J=2 Hz, H-2′), 6.80 (1H, d, J=8 Hz, H-5′), 6.35(IH, s(br), H-8), 6.13 (IH, s(br), H-6); galactoside moiety:5.60 (1H, d, J=8 Hz, H-1″), 4.28 (1H, t, J=8Hz, H-2″), 3.2–3.9(m, galactose protons overlapped with hydroxyl and waterprotons). 13C NMR data of 11: aglycone moiety: δ ppm 158.5(C-2), 135.3 (C-3), 175.9 (C-4), 160.7 (C-5), 98.4 (C-6), 163.9(C-7), 93.5 (C-8), 156.2 (C-9), 103.1 (C-10), 122.3 (C-1′),115.3 (C-2′), 145.0 (C-3′), 147.6 (C-4′), 115.6 (C-5′), 120.0(C-6′); galactoside moiety: 99.3 (C-1″), 77.8 (C-2″), 73.8(C-3″), 68.0 (C-4″), 76.3 (C-5″), 60.7 (C-6″).

2.4.3. 3-methoxyellagic acid 4-O-β-galactopyranoside (13)White amorphous powder, [α]D25 41.1o (c=0.071, MeOH),

Rf values: 0.13 (H2O), 0.28 (HOAc), 0.45 (BAW). UV λmax (nm)in MeOH: 236, 251, 352 sh., 361; in MeOH+NaOAc+H3BO3:254, 315 sh., 354. HRFTMS: m/z=477.210 [M−H]−,(C21H18O13). Normal acid hydrolysis gave galactose and 3-methoxyellagic acid (13a) (Co-PC). 3-Methoxyellagic acid(13a): Rf-values: 0.03 (H2O), 0.10 (HOAc), 0.76 (BAW). UVλmax (nm) in MeOH 251, 348 shoulder, 369; in MeOH+NaOAc+H3BO3: 257, 315 shoulder, 375. EIMS: [M]+ atm/z=316. 1H NMR data of 13: δ ppm: aglycone moiety:7.67 (1H, s, H-5), 7.52 (1H, s, H-5′), 4.0 (3 H, s, OMe-3); galacto-sidemoiety: 4.95 (1H, d, J=8 Hz, H-1″), 3.3–3.8 (m, other galac-toside protons overlapped by hydroxyl and water protons). 13CNMR data: 114.3 (C-1), 137.9 (C-2), 142.9 (C-3), 149.7 (C-4),111.1 (C-5), 111.1 (C-6), 159.7 (C-7), 112.9 (C-1′), 137.9(C-2′), 140.2 (C-3′), 152.8 (C-4′), 111.1 (C-5′), 105.8 (C-6′),160.3 (C-7′), 61.2 C-OMe); galactoside moiety: 102.0 (C-1″),70.3 (C-2″), 73.7 (C-3″), 69.8 (C- 4″), 77.0(C-5″), 61.3 (C-6″).

2.5. Biological methods

2.5.1. Determination of radical scavenging activity by DPPH assayRadical scavenging activity of plant extract against the stable

free radical DPPH (2,2-diphenyl-1-picrylhydrazyl, Sigma-AldrichChemie, Steinheim, Germany) was determined spectrophoto-metrically. When DPPH reacts with an antioxidant compound,which can donate hydrogen, it is reduced. The changes in color(from deep–violet to light–yellow) were measured at 517 nm.

Radical scavenging activity of the extract was measured byslightlymodifiedmethod of Brand-Williams, Cuvelier and Berset[12], where the extract solution was prepared by dissolving0.025 g of the dry extract in 10 ml of methanol. The solutionof DPPH inmethanol (6×10−5 M)was freshly prepared, beforeUVmeasurements. Threemilliliters of this solutionweremixedwith 9 different concentrations of the samples.

The resulting solutions were kept in the dark for 30 min atroom temperature and then the decrease in absorbance wasmeasured. Absorbance of blank sample containing the sameamount of methanol and DPPH solution was prepared andmeasured. The experimentwas carried out in triplicate. Radicalscavenging activity was calculated by the following formula:% Inhibition=[(AB−AA)/AB]×100, where:AB is the absorbanceof blank sample and AA is the absorbance of the tested samples.

IC50 value: the concentration of the substrate that causes50% loss of the DPPH activity (color), were calculated for thestandard and the extract from a graph plotted for the % inhi-bition against the concentration in μg/ml.

2.5.2. Determination of radical scavenging activity by ORACassay

ROS are generated by the thermal decomposition of [2,2″-azobis(2-amidino-propane) dihydrochloride (AAPH) and overtime quench the signal of the fluorescent probe fluorescein.The subsequent addition of antioxidants reduces the quenchingby preventing the oxidation of the fluorochrome. Briefly,6-Hydroxy-2,5,7,8-tetra-methylchroman-2-carboxylic acid(Trolox) and test compounds were dissolved/diluted in phos-phate buffered saline (10 mM , pH 7.4). In each well of a 96well plate 150 μl 10 nM fluorescein, 25 μl Trolox (0.2–3.13 μM)or 25 μl test compound were pipetted in quadruplicate. Plate

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was allowed to equilibrate at 37 °C for 30 min. After incubation,fluorescence measurements (Ex. 485 nm, Em. 520 nm) weretaken every 90 s to determine the background signal. After 3 cy-cles, 25 μl (240 mM) of AAPH was injected in each well. Mea-surements were continued for 90 min. Half life time offluorescein was determined using MS Excel software.

2.5.3. Cell culture and assay conditionsCell culture and assay conditions: The spontaneously trans-

formed non-tumorigenic human keratinocyte cell line HaCaT(kindly provided by Prof. Fusenig of the German Cancer Re-search Centre, Heidelberg, Germany) was cultured in growthmedium at 37 °C with 5% CO2 in a humidified atmosphere.Growth medium (RPMI 1640) was supplemented with 8% heatinactivated fetal calf serum (FCS, Sigma, Taufkirchen, D) and an-tibiotics (penicillin 100 units ml, streptomycin 100 μg/ml). Me-dium was changed every three days. Cells were subculturedroutinely using EDTA (0.05% in phosphate buffered saline, PBS)and trypsin/EDTA (0.05%/0.02% in PBS). For the experiments,growth medium was replaced by RPMI 1640 containing 0.01%bovine serumalbumine (BSA, Sigma Taufkirchen, D) andpenicil-lin/streptomycin (BSA medium). Cell culture plastics and medi-um supplements were obtained from Biochrom AG (Berlin, D),except otherwise stated. Cell viability determination: All assayswere conducted between passages 50 and 70. HaCaT cells wereplated in 96 well plates in growth medium at a density of2×104 cells per well. After 24 h, medium was replaced by BSAmedium and cells were incubated with different concentrationsofH. crude extract (CE) and pure compound 10 for 72 h at 37 °C.As positive control, 100 μg/ml Aloe vera extract [Unformulated,freeze-dried Aloe vera inner gel powder (AV, supplied by AloeVera of America Inc., USA or Rainbow Naturprodukte GmBH,Germany)] [19] was used. After incubation, the cells were ob-served under the microscope for cell integrity and were treatedwith MTT solution (BSA medium, final concentration0.5 mgmL−1) for 3 h at 37 °C. Formazan crystals were dissolved(DMSO) and optical density (O.D.) was measured at 550 nmusing a multi well plate reader (BMG Omega, BMG Labtech,Offenburg, D). Cell viability was expressed as a percentage of ve-hicle control. Interleukin assay and UVB irradiation Cells wereseeded in growth medium into 60 mm dishes and left undis-turbed for 24 h. Medium was removed and cells were washedtwice with Hank's balanced salt solution (HBSS, PAA, Pasing,D). Cell layerwas then coveredwith 1.5 mlHBSS anddishes irra-diated using Philips PLS 9WTL12 UVB broadband emitting fluo-rescence bulbs with a mean radiation power of 1.4 mW/cm2.After 5 min, HBSS was replaced by 4 ml growth medium con-taining test samples or controls (1 μM caffeic acid). After 24 h,cell culture supernatant was retrieved, centrifuged, aliquotedand stored at −70 °C until analysis. Two independent experi-ments were performed. Il-6 and Il-8 were measured using a cy-tokine human 10plex bead based assay from Life Technologies(Invitrogen, Karlsruhe, D) according to the manufacturer's pro-tocol. Data were obtained by measurement in a Luminex 200system and finalized using MS Excel.

3. Results and discussion

Following column chromatographic fractionation of theaqueous alcohol extract obtained by extraction of the leavesof H. caffrum by aqueous alcohol (75%), 21 compounds (1–21)

were isolated. Conventional and spectral analysis mainly byNMR spectroscopy and by mass spectrometry indicated thatthree of these compounds are new natural products (10, 11and 13).

Compound 10 is a light yellow amorphous powder that ex-hibits chromatographic properties, anionic character on elec-trophoretic analysis and UV maxima in MeOH and afteraddition of diagnostic shift reagents [20,21], (see Experimental)which suggested it to be an anionic flavonol derivative, mostprobably a 3-substituted kaempferol. On controlled acid hydro-lysis (10% aqueous AcOH, 10 min, 100 °C), compound 10yielded an intermediate 10a, separated by prep. PC and identi-fied to be kaempferol 3-O-β -galactoside [CoPC, UV, 1H NMRand β-galactosidase enzymic hydrolysis (β-galactosidase fromyeast, lactase Y:3.2.1.23, lyophilized powder: activity approx.1 EU/mg, BDH)], [22]. 10 was recovered unchanged afterbeing treated for 24 h with β-galactosidase, but it was hydro-lysed when refluxed with 2 N aqueous HCl at 100 °C for15 min to yield kaempferol and galactose (CoPC). The hydroly-sate gave a precipitate with aqueous BaCl2. Dry 10 gave the typ-ical sodium flame test (golden color). The presence of sodiumwas confirmed by flame atomic absorption analysis, thus prov-ing that the sulfate increment(s) in this molecule exists as sodi-um sulfate. On negative FTESIMS 10 exhibited a molecular ion[M−Na]− at m/z=527, and an ion at m/z=447 attributed to[M−SO3Na]−, while on positive ESIMS analysis, it exhibited amolecular ion [M+H]+ at m/z=551, together with an [M+Na]+ ion at m/z=573. These MS data revealed the molecularformula C21O14H20SNa. The above given analytical data sug-gested a kaempferol 3-O-sulphatogalactoside structure forcompound 10. From the 13CNMR spectra, thepresence of galac-tosemoiety followed from the anomeric carbon signal at δ ppm99.2. That the sulphatosugar moiety must be attached to posi-tions 3 of kaempferol followed from the upfield shift of the fla-vonol carbon signal, C-3 while the corresponding ortho andpara-carbon signals were shifted downfield, all in comparisonwith the corresponding carbon signals in the spectrum of theaglycone (see Experimental) [23]. Similar shifts are well-knownfrom the work of Markham et al., 1978. The β-configuration ofthe galactose moiety was derived from the C-l chemical shiftat 99. Attachment of the sulfate radical to C-2 of galactosewas indicated by the shift of the sugar C-l signal to δ 99.2(α-upfield shift) and of its C-2 signal to 77.6 ppm (downfieldshift caused by sulfate substituent), [24]. The chemical shiftvalues of all galactose carbons confirmed its pyranose form[25]. In the 1HNMR spectrumof 10, a triplet sugar signal locateddownfield at δ ppm 4.3 was recognized. This signal wasassigned to the galactose carbon C-2 bearing the sodium sulfatesubstituent, an assignment which was then confirmed by1H–1H COSY experiment, whereby a cross peak correlatingthe anomeric galactose doublet signal at δ ppm 5.67 to thattriplet signal was recognized in the received spectrum.2DHSQC experiment of 10 allowed the unambiguous assign-ments of all sugar carbons and finally confirmed its structureto be kaempferol 3-O-(2″-sulphatogalactopyranoside). Thisis a flavonoid structure which has not been reported in naturebefore. It should be noted however that, Kaempferol sulphato-glycosides are of rare occurrence. They include kaempferol3-O-β-(3″-sulphatoglucoside) and kaempferol 3-O-β-(6″-sulphatoglucoside) from Cystopterris fragilis, kaempferol 3-O-α-(6″-sulphatoglucoside) from Asplenium filix-foemina

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and kaempferol 3-O-α-(6″-sulphatorhamnoside) from Dar-idsomia pruriens [18].

Compound 10: R=HCompound 11: R=OH

Compound 11was isolated as faint yellow amorphous pow-der which showed anionic property on electrophorotic analy-sis. On normal hydrolysis 11 gave quercetin, galactose (CoPC)and sodium sulfate (white precipitate with BaCl2 and goldenyellow flame test). On negative ESIMS, 11 exhibited a molecu-lar ion, [M−Na]− atm/z=543, and an ion atm/z=463 attrib-uted to [M−SO3Na]−, while on positive ESIMS analysis, itexhibited a molecular ion [M+H]+ at m/z=567, togetherwith an [M+Na]+ ion at m/z=589. These MS data revealedthe formula C21O15H20SNa. These data togetherwith theUV ab-sorption spectral data (see Experimental) indicated that 11 isthe quercetin analog of 10. 1H and 13C (see Experimental) spec-troscopic analysis of 11 confirmed its structure to be quercetin3-O-(2″-sulphatogalactopyranoside) which has not beenreported before as a natural product.

Compound 13 was obtained as a white amorphous pow-der. The molecular ion peak at m/z 477.210 [M−H]− (calc.:477.209) and the 1H and 13C NMR data suggested the molec-ular formula C21H18O13. The characteristic chromatographicproperties (weak mauve spot on PC under UV light) and UVabsorption maxima in methanol suggested that 13 is an ella-gic acid derivative. The pronounced red shift of the absorp-tion maxima at 250 and 272 (shoulder) nm of the aromaticchromophors in the molecule of (13), observed on additionof NaOAc+H3BO3 (see Experimental) might be attributed tothe presence of free diorthohydroxyl group(s) in the aromaticring(s). Normal acid hydrolysis of 13 yielded galactose(Co-PC), and compound13a. The latterwas also released on in-cubating of 13 at 37 °C for 24 h, together with β-galactosidase.Compound 13awas extracted by EtOAc from the 2 N acidic hy-drolysate and was found to possess a molecular weight of 316as established by EIMS ([M]+ at m/z=316), corresponding toa molecular formula of C15H8O8. Chromatographic properties,UV absorption maxima and EIMS data suggested that 13a is amonomethoxyellagic acid. The 1H and 13C NMR data of 13aconfirmed its structure to be 3-methoxyellagic acid [26–28].Consequently, the parent compound 13 is 3-methoxyellagicacid mono-β-O-galactopyranoside. Comparison of the 1D

NMR data of 13 and 13a proved that the former contained a ga-lactoside moiety which revealed its anomeric proton as a dou-blet at δ ppm 4.95, (J=8 Hz). This finding, together with theresult of hydrolysis with β-galactosidase enzyme proved theβ-configuration of the existing galactose moiety. In the 13CNMR spectrum of 13, the β-configuration was derived fromthe δ values of the recorded sugar carbon resonances (seeExperimental) [29]. In the HMBC spectrum, a 3J correlation ofthe anomeric galactose proton H-1″ (δ=4.95) to the aromaticcarbon C-4 (δ=145.30) allowed positioning of this moiety atthis carbon. The recognizable 2J correlation of the downfield ar-omatic proton (δ=7.67) to the same C-4 carbon was in accor-dance with this conclusion. Correlations of the methoxylprotons (δ=4.00) to C-3 (δ 140.24) and of the same downfieldaromatic proton (δ=7.67) to the same C-3 carbon confirmedthat the site of attachment of the galactosyl moiety is at the C-4 position of the methoxy ellagic acid moiety. The completestructure of compound 13 was therefore determined to be 3-methoxyellagic acid 4-O-β-galactopyranoside,which representsto the best of our knowledge a new natural product.

Compound 13

In addition, the known compounds, 3-methoxy gallic acid5-sodium sulfate (1), 1,3-di-O-galloyl glucose (2), 2,3-di-O-galloyl glucose (3), gallic acid (4), gentesic acid 2-O-glucoside(5), gentesic acid 5-O-glucoside (6), protocatechuic acid (7),p-hydroxybenzoic acid (8), 3-methoxy gallic acid (9), 3,3″-dimethoxy ellagic acid 4-O-glucoside (12), quercetin 3-O-arabinopyranoside (14), kaempferol 3-O-rhamnoside (15),quercetin 3-O-rhamnoside (16), kaempferol 3-O-galactoside(17), quercetin 3-O-galactoside (18), 3,3′,4-trimethoxyellagicacid (19), kaempferol (20), quercetin (21), were also isolatedand identified by applying the conventional and spectralmethods of analysis. It should be noted however that, com-pounds (4), (7), (15), (16), (20) and (21) were isolated, previ-ously from the same plant, but were identified on the basis ofthe conventional methods of analysis only [11]. On the otherhand, the rest of the isolated known compounds were isolatedand identified here for the first time from H. caffrum.

3.1. DPPH assay

During evaluation of the biological activity the aqueousmethanol extract of H. caffrum showed high values for

Table 1Antioxidant activity of the aqueous methanolic extract of H. caffrum.

μl of the methanolicsolution of the substanceadded to 3 ml DPPH

Conc inμg/ml

% inhibition⁎

Ascorbic acid H. caffrum extract

0 0 0 01.75 1.4 39.5±0.35 8.9±0.422.3 1.9 53.2±0.54 13.9±0.133.5 2.9 74.3±0.42 17.8±0.337 5.8 88.6±0.32 34.5±0.1610 8.3 94.4±0.51 44.3±0.2114 11.6 97.8±0.39 65.7±0.5819 15.8 98.1±0.32 76.5±0.3738 31.6 98.2±0.32 92.6±0.3477 64.1 98.3±0.15 94.2±0.24

⁎ Values are the average of triplicate experiments and represented asmean±standard deviation.

Fig. 1. Antioxidant activity of H. caffrum samples measured in the ORACassay: half life time of fluorescein after protection with 12.5 μg/ml test sam-ple. Positive control Trolox (3.13 μM). 10=pure compound 10, CE=H. caf-frum crude extract, H1–H7 fractions of CE. Experiments carried out induplicate with 6 replicates each.

1270 M. Nawwar et al. / Fitoterapia 82 (2011) 1265–1271

Fig. 2. Influence of H. caffrum samples on the interleukin 6 and 8 productionof keratinocytes after 20 mJ UVB radiation. 10=pure compound 10,CE=Harpephyllum crude extract. Experiments carried out in triplicatewith 3 replicates each.

absorbance inhibition at the different concentrations used,the extract completely inhibited DPPH absorbance at a con-centration of 77 μl (Table 1), the percentage obtained(94.2%) can be considered as a full absorbance inhibition ofDPPH, because after completing the reaction the final solu-tion always possesses some yellowish color and thereforeits absorbance inhibition compared to colorless methanol so-lution cannot reach 100%. Permanent residual absorbance re-sults in up to 7% of the total absorbance inhibition.

Also, the IC50 (the concentration that inhibits 50% of the ab-sorbance of DPPH), was determined from the graph plotted forthe % inhibition against concentration in μg/ml for ascorbic acidand H. caffrum extract which found to be 1.83±0.34 μg/ml and8.4±0.23 μg/ml respectively. Values are the average of triplicateexperiments and represented as mean±standard deviation.

It could be concluded from the obtained results that the H.caffrum extract shows a very high antioxidant capacity whichis very close to the value of the reference standard used.

3.2. ORAC assay

TheORAC assay demonstrated a distinguishable anti-oxidantcapacity of the crude extract and some fractions (Fig. 1). Themost active fractions (H3, H4) were used to isolate the majorcompound 10. While compound 10 showed the overall highestactivity in this assay, probably due to its good solubility in aque-ous systems.

3.3. Cell viability, interleukin assay and UVB irradiation

In another set of experiments keratinocytes were irradiatedwith UV-B broadband (20 mJ/cm2). This irradiation dose hasbeen found to be well suitable for the detection of possibleUV protecting effects before [30]. Irradiation induces a clear in-crease in the production of the proinflammatory cytokine IL-6by the exposed cells. The level of IL-6 and IL-8 can bemeasuredin the cell culture medium. Control (caffeic acid) or Harpephyl-lum extract was added immediately after irradiation. 24 h laterthe IL-6 and IL-8 content of themediumwasmeasured bymul-tiplex bead based assay. Higher doses of the Harpephyllum ex-tract inhibited the IL-6 production in the UV-exposed cells.40 μg/mL decreased IL-6 production to about 50% of irradiatedvehicle control whereas 2 μg/mL did not change IL-6 levels.

Caffeic acid (1 μM) reduced IL-6 production to 72% of control.However, the isolated compound 10 did not interact withUVB triggered IL-6 production ofHaCaT keratinocytes. IL-8 con-centrations were only slightly affected by either treatment(Fig. 2). Clearly, Harpephyllum crude extract diminishes UVphototoxic reaction of keratinocytes. This effect might bedue to radical scavenging or protection of oxygen sensitiveenzymes like dihydropteridine reductase and remains to beclarified in future. The remarkable radical scavenging effectand the inhibition of UV induced IL-6 production of humankeratinocytes that is produced by the leaf aqueous alcohol ex-tract could be due to the existing combination of constituentsor it could be attributed to one or more of the phenolic com-pound(s). Although compound 10 showed impressive antiox-idant effects it failed to reduce the IL-6 production. Ascytotoxicity is also neglible (Fig. 3), a minimal uptake of thishighly polar substance into the eukaryotic cells may be a rea-son for this observation. Further studies are needed to identifythe effective compounds within the crude extract.

Fig. 3. a- Viability of keratinocytes after treatment with crude leaf extract ofH. caffrum, measured in the MTT assay. Experiments carried out in triplicatewith 6 replicates each; b- Viability of keratinocytes after treatment withcompound 10, measured in the MTT assay Viability of keratinocytes aftertreatment with crude extract of Harpephyllum crude extract, measured inthe MTT assay. Experiments carried out in triplicate with 6 replicates each.

1271M. Nawwar et al. / Fitoterapia 82 (2011) 1265–1271

Acknowledgment

We are indebted to AvH (Alexander von Humboldt) foun-dation for the donation of a Schimazu UV-Visible-1601 spec-trophotometer and an 8001-Kruess polarimeter to MahmoudNawwar. We thank the DAAD and the STDF, Cairo, Egypt forfinancing the stay of Reham El-Sharawy and MahmoudNawwar in Germany through a the GESP project. ID: 1399.

References

[1] Tebbe B. Relevance of oral supplementation with antioxidant for pre-vention and treatment of skin disorders. Skin Pharmacol Appl Skin Phy-siol 2001;14:296–302.

[2] Afaq F, Mukhtar H. Effects of solar radiation on cutaneous detoxificationPathways. J Photochem Photobiol B 2001;63:61–9.

[3] Goihman-Yahr M. Skin aging and photoaging: an outlook. Clin Dermatol1996;14:153–60.

[4] Afaq F, Mukhtar H. Photochemoprevention by botanical antioxidants.Appl Skin Physiol 2002;15:297–306.

[5] Svobodová A, Psotová J, Walterová D. Natural phenolics in the preven-tion of UV-induced skin damage. A review. Biomed Pap 2003;147:137–45.

[6] Van Wyk E, Van Oudtshoorn B, Gericke N. Plants of South Africa.Pretoria, South Africa: Briza Publications; 1997.

[7] Buwa J, Van Staden J. Antibacterial and antifungal activity of traditionalmedicinal plants used against venereal diseases in South Africa. Ethno-pharmacol 2006;103:139–42.

[8] Ojewol J. Hypoglycemic and hypotensive effects of H caffarum. Cardio-vasc J S Afr 2006;17:67–72.

[9] Jiger A, Hutchings A, Van Stadten J. Screening of Zulu medicinal plantsfor prostaglandin-sythesis inhibitors. J Ethnopharmacol 1996;52:95–100.

[10] Moyo M, Ndhlala A, Finnie J, Van Staden J. Phenolic composition, anti-oxidant and acetylcholinesterase inhibitory activities of Sclerocarya bir-rea and H. caffrum (Anacardiaceae) extracts. Food Chem 2010;123:63–8.

[11] El-Sherbeiny A, El-Ansari M. The polyphenolics and Flavonoids of H.caffrum. Plant Med 1976;29:129–32.

[12] Brand-Williams W, Cuvelier M. Use of a free radical method to evaluateantioxidant activity. Food sci Technol 1995;28:25–30.

[13] Ou B, Hampsch-Woodill M, Prior R. Development and validation of animproved oxygen radical absorbance capacity assay using fluoresceinas the fluorescent probe. J Agric Food Chem 2001;49:4619–926.

[14] Boukamp P, Petrussevska R. Normal keratinization in a spontaneouslyimmortalized aneuploid human keratinocyte cell line. J Cell Biol1988;106:77761–71.

[15] Urbanski A, Schwarz T, Neuner P, Krutmann J, Kirnbauer R, Köck A, et al.Ultraviolet light induces increased circulating interleukin-6 in human.J Invest Dermatol 1990;94:808–11.

[16] Petit-Frére C, Clingen P, Grewe M, Krutmann J, Roza L, Arlett C, et al. In-duction of interleukin-6 production by ultraviolet radiation in normalhuman epidermal keratinocytes and in a human keratinocyte cell lineis mediated by DNA damage. J Invest Dermatol 1998;111:354–9.

[17] Williams C, Harborne J, Greenham J, Briggs B, Johnson L. Flavonoid pat-terns and the revised classification of Australian Restionaceae. Phyto-chemistry 1998;49:529–52.

[18] BarronD, Varin L, IbrahimR,Harborne J,Williams C. Sulphated flavonoids—an update. Phytochemistry 1988;27:2375–95.

[19] Habeeb F, Stables G, Bradbury F, Nong S, Cameron P, Plevin R, et al. Theinner gel component of Aloe vera suppresses bacterial-induced pro-inflammatory cytokines from human immune cells. Methods 2007;42:388–93.

[20] Harborne J, Williams C. The Flavonoids. In: Harborne J, Mabry J, MabryH, editors. London: Chapman & Hall; 1975. p. 37–441.

[21] Mabry J, Markham R, Thomas B. The Systematic Identification of Flavo-noids. Heidelberg: Springer; 1970.

[22] Miller K, Guyon V, Evans J, Shuttleworth W, Taylor L. Purification, clon-ing, and heterologous expression of a catalytically efficient flavonol 3-O-galactosyltransferase expressed in the male gametophyte of Petuniahybrida. J Biol Chem 1999;274:34011–9.

[23] Markham K, Ternai B, Stanley R, Geiger H, Mabry T. Carbon-13 NMRstudies of flavonoids-III. Tetrahedron 1978;34:1389–97.

[24] Nawwar M, Buddrus J. A gossypetin glucuronide sulphate fromn theleaves of Malva sylvestris. Phytochemistry 1981;20:2446–8.

[25] Breitmaier E, Voelter W. 13C NMR Spectroscopy. New York: VCH; 1978.[26] Nawwar M, Hussein S, Merfort I. NMR spectral analysis of polyphenols

from Punica granatum. Phytochemistry 1994;36:793–8.[27] Nawwar M, Buddrus J, Bauer H. Dimeric phenolic constituents from the

roots of Tamarix nilotica. Phytochemistry 1982;21:1755–8.[28] Sato T. Spectral differentiation of 3, 3″ di-O-methylellagic acid from

4,4″-di-Omethylellagic acid. Phytochemistry 1987;26:2124–5.[29] Kalinowski H, Berger S, Braun S. 13C NMR Spektroskopie. Stuttgart:

George Thieme; 1984.[30] Lindequist U, Wende K, Harms M, Witt S, Poerksen J, Jülich W. Biolog-

ical and chemical investigations of Ganoderma pfeifferi and possibleapplication of this new medicinal mushroom in cosmetics ConferenceProceedings, 5. InternationalMedicinalMushroom Conference, Nantong,China, September; 2009. p. 615–22.