SUPPLEMENTARY MATERIAL
Anti- skin aging activity of napthoquinones from Arnebia nobilis Reichb.f.
Satyabrata Mohapatraa*#, R. Sandeep Varmab#, L.M. Sharath Kumara, O.S. Thiyagarajanb,
M. Vijaykumara, K. Dilmela, M. Nishanta, U.V. Babua, and P.S. Patkic
aDepartment of Phytochemistry; bDepartment of Cell Biology; cMedical Services and Clinical Trials Research and Development, The Himalaya Drug Company, Bangalore-562 162, India
# Equal contribution
*Address for correspondence: Dr. Satyabrata Mohapatra, Research and Development, The Himalaya Drug Company, Bangalore 562 162, India. (T) +91 80 22169999. Extn: 9921 (F) +91 80 2371 4471.
Email:
Abstract
The present isolation and identification of napthoquinones from roots of Arnebia nobilis Reichb.f. can lead to the discovery of new anti-skin aging ingredient in colour cosmetics. Four compounds have been isolated and purified by rigorous column chromatography. The compounds are identified as β,β-dimethylacryl alkannin (AN-I), acetoxyisovaleryl alkannin (AAN-II), acetyl alkannin (AN-III) and alkannin (AN-IV) by interpretation of spectroscopic data. This study is the first to report the isolation of Acetoxyisovaleryl alkannin (AAN-II) from A. nobilis. The IC50 values of the compounds, determined in human skin cells (human dermal fibroblasts and human keratinocytes) and mouse embryonic fibroblasts (NIH3T3) varied significantly among the four alkannins. Among the four compounds, β-acetoxyisovaleryl alkannin (AAN-II) significantly inhibited hydrogen peroxide (H2O2) induced red blood corpuscle (RBC) haemolysis and cellular senescence in human dermal fibroblasts. Collagen-I, elastin and involucrin synthesis in human dermal fibroblasts or keratinocytes were up regulated by AAN-II. These results support the potential utility of alkannins as novel anti-aging ingredients.
Keywords: Arnebia nobilis, Alkannins, HDF cell, cellular senescence, skin barrier, colour cosmetics.
Experimental
Instruments
1H and 13C NMR spectra were recorded using a Bruker Avance 400 MHz NMR spectrometer with tetramethylsilane as an internal standard. LC-MS/MS spectra of four compounds have been recorded by API 2000 (Applied biosystem/MDS SCIEX, Canada) mass spectrometer coupled with ESI (Electron spray ionization) source and a chromatographic system of Shimadzu LC-20AD series pump and DUG-20A3 series shimadzu degasser. Luna C18 (250 X 4.6mm, 5um) phenomenex column have been used for chromatography. The mobile phase consisted of water (J. T. Baker) and methanol (J. T. Baker) in the ratio of 15: 85 v/v at a flow rate of 0.8 ml/min. The run time was about 30 min and peak elution was monitored in SPD-20 A detector at 525 nm. Batch acquisition and data processing was controlled by Analyst 1.5 version software. Intensity response was checked and found good in positive ionization mode.
Plant Material Collection
The commercially available roots of A. nobilis were purchased from a local vendor in Bangalore, India in the month February, 2012. The plant species was authenticated by Dr. Kannan, certified taxonomist and the voucher specimen (BG715/130102) is preserved in the R&D centre, The Himalaya Drug Company, Bangalore, India. The sample was washed with fresh water, shade dried and powdered in a pulveriser.
Extraction and Isolation
The dried and powdered material (1.0 kg) was extracted separately by stirring with 2 litres of n-hexane (Qualigen) at room temperature for 15 h (15 x 3 times). Concentration of the combined extract under vacuum yielded as 1.92%. Thin layer chromatography (TLC silica gel 60 F254, Merck) of the extract was performed with 15% ethyl acetate (Qualigen) in n-hexane to three well resolved red coloured spots. Second spot again resolved to two compounds by repeated chromatography (Fig. 1). The dark red colored extract (10 g) was adsorbed on silica gel (50 g, Acme Synthetic Chemicals) and chromatographed over a column (length: 150 mm; diameter: 50 mm) of silica gel (100-200 mesh) and eluted with solvent gradient form 100% n-hexane to 100% ethyl acetate and acetone (Qualigen). On repetitive silica gel column chromatographic separations of the fraction from the initial column, furnished four pure and single compounds. Further, AN-I and AN-IV have been crystallized in n-hexane. Interpretation of NMR and LC MS/MS spectra and comparison with their literature data, the compounds are characterized as AN-I (600 mg), AAN-II (12 mg), AN-III (10 mg) and AN-IV (08 mg) (Fig. 1).
b,b-Dimethylacryl alkannin or Arnebin-1 (AN-I)
M.p. 116 oC; [α]D25 -50 (c = 0.02, CHCl3); 1H-NMR (CDCl3-400 MHz) δH: 6.97 (1H, s, H2), 7.17 (2H, s, H6, H7), 6.02 {1H, dd (7.2, 4.8), H11}, 2.49 (1H, m, H12), 2.62 (1H, m, H12), 5.14 (1H, t, 6.8, H13), 1.57 (1H, s, H15), 1.68 (1H, s, H16), 12.42 (1H, s, OH-5), 12.59 (1H, s, OH-8), Ester group: 5.78 (s), 2.15 (s), 1.94 (s); 13C-NMR (CDCl3-100 MHz) δC: 179.0(C1), 177.5(C4), 165.2(C5), 166.3(C8), 131.6(C2), 132.6, 132.7(C6, C7), 149.0(C3), 111.9(C10), 111.6 (C9), 135.9(C14), 32.9 (C12), 118.4(C13), 68.6(C11), 16.4(C15) , 17.9(C16), 166.8 (C1’), 132.4(C2’), 135.8 (C3’), 20.4 (C4’), 27.6 (C5’); LC-MS: 393.12, 293.02; MS/MS of 393.12: 271.01, 253.05, 229.04.
b-Acetoxyisovaleryl alkannin (AAN-II)
Deep red semisolid; [α]D25 -147 (c = 0.0013, CH2Cl2); 1H-NMR (CDCl3-400 MHz) δH: 12.57 (1H, s, H5), 12.40 (1H, s, H4), 7.18 (2H, s, H6,7), 7.01 (1H,d,H2), 6.04 (1H,ddd, H11), 5.12 (1H, t, H13), 2.95 (2H, dd, H2’), 2.47 (1H, m, H12), 2.61 (1H, m, H12), 2.00 (3H, s, H7’), 1.68 (3H, s, H16), 1.58 (3H, s, H15), 1.56 (3H, s, H5’), 1.54 (3H, s, H4’); LC-MS: 682.55, 352.70, 270.81; MS/MS of 372.70: 292.81, 270.80.
Acetyl Alkannin or Arnebin-3 (AN-III)
Deep red solid; M.p. 103 oC; [α]D25 -772 (c = 0.0013, CH2Cl2); 1H-NMR (CDCl3 - 400 MHz ) δH: 12.58 (1H, s,H5), 12.42(1H, s, H8), 7.18 (2H, s, H6,7), 6.99 (1H, s, H2), 6.03(1H, dd, H11), 5.12 (1H, t, H13), 2.62 & 2.47(2H, m, H12), 2.14(3H, s, H2’), 1.69 (3H, s,H16), 1.58 (3H,s,H15); 13C-NMR (CDCl3 – 100MHz) δC: 178.2(C4), 176.7(C1), 167.5(C8), 169.7(C1’), 166.9(C5), 148.4 (C3), 132.8(C7), 132.7(C6), 131.5 (C2), 136.1(C14), 111.8(C10), 111.6(C9), 32.8 (C12), 117.7(C13), 69.5(C11), 25.76(C15), 20.97(C2’), 17.97(C16); LC-MS: 883.35, 453.12, 293.18, 271.07; MS/MS of 453.12: 293.08, 229.20, 183.00.
Alkannin or Arnebin-4 (AN-IV)
Deep red amorphous powder; m.p. 145 oC; [α]D25 -164 (c = 0.015, CH2Cl2); 1H-NMR (CDCl3 - 400 MHz ) δH: 12.50 (2H, s, H4, 5),7-7.20 (Aromatic Hydrogen 3H, s, H6, 7, 3), 7.00(2H, s, H1, 8), 5.10 (1H, t, H10), 2.60 (2H, m, H11), 2.10 (2H, m, H2, 9), 1.68 (2H, s, H15, 13), 1.60 (2H, s, H16, 12). 13C-NMR (CDCl3–100MHz); LC-MS: 883.35, 453.12, 293.18, 271.07; MS/MS of 453.12: 293.08, 229.20, 183.00.
Cell culture
Human dermal fibroblasts (HDF), human keratinocytes (HaCaT) cells and mouse embryonic fibroblasts (NIH3T3) cells were obtained from National Centre for Cell Sciences, Pune, India. HDF cells were maintained in Minimum essential medium (MEM), whereas HaCaT and NIH3T3 cells were maintained in Ham’s F12 and Dulbecco’s minimum essential medium (DMEM) high glucose medium respectively with 10% fetal bovine serum and antibiotics (100 U of penicillin and 100 µg/ml of streptomycin) in a 95% air/5% CO2 humidified atmosphere incubator at 37˚C.
Cytotoxicity assay
In vitro toxicity of arnebins was checked in HDF, HaCaT and NIH3T3 cells. Briefly, different concentrations of arnebins (0-100µg/ml) was added to the cells and incubated for 24 h at 37°C. An MTT assay was performed to assess the basal cytotoxicity of arnebins. The resultant formazan dye was extracted with DMSO and spectrophotometric readings taken at 540 nm using a Synergy HT spectrophotometer (Bio-Tek, Winooski, VT). From the values obtained, the percentage toxicity (relative to survival of control cells) was calculated. Only non-toxic concentrations were used for further experiments.
RBC hemolysis
The volume of arnebin samples and control with erythrocyte suspensions were adjusted to 1 ml by adding saline (0.9%). Oxidative stress was then induced by adding 100 µM concentrations of hydrogen peroxide (H2O2) and incubated at 37°C for 3 h. After incubation, the mixture was centrifuged at 1200 rpm for 5 min, the supernatant were measured at 540 nm. RBC hemolysis by H2O2 was taken as 100% cell lysis and hemolysis of the treated and non treated erythrocytes was expressed as a percentage of this value. Ascorbic acid (100 µM) served as positive control. The experiment was repeated three times (n=3).
Cellular senescence assay
HDF cells were seeded in 40 mm petriplates at a uniform concentration of 1 x 105 cells/ml. The cells were treated with H2O2 (10 µM) and arnebins at two non toxic concentrations. H2O2 control and cell control was also maintained. Tannic acid (100 µg/ml) was used as positive control. The cells were incubated for 24 h at 37°C. The cellular senescence assay was carried out as per the instructions of the kit (Millipore, USA) and the stained cells were observed under light microscopy (10 x). The experiment was repeated three times (n=3).
ELISA for collagen-I and involucrin
ELISA kits were used for the quantitative measurements of collagen-I (BlueGene Biotech, China) and involucrin (Cloud-Clone Corp. USA) in cell supernatants or cell lysates. After treatment with arnebins, HDF or HaCaT cells were incubated for 96 h at 37°C. The cells were harvested and the cell supernatants or lysates prepared for ELISA. ELISA was carried out as per the instructions provided in the kit. Color developed in proportion to the amount of collagen or involucrin, which was measured at 450nm. Each sample was assayed with three replicates (n=3).
Collagen-I and elastin gene expression
Extraction of RNA from HDF was performed using TRI reagent, followed by DNase treatment to get rid of the contaminating DNA. Then, RNA was translated to complementary DNA using first strand cDNA synthesis kit (Fermentas, USA) according to the manufacturer's instructions. The GAPDH gene was used as internal control, and amplification for collagen-I and elastin was conducted using specific primers. Cycling conditions were 95°C for 10 min, followed by 35 cycles at 95°C for 15 s, at 60°C for 5 s, and at 72°C for 4–8 s. The products were detected by 1.5% agarose gel electrophoresis stained with ethidium bromide and photographed under UV. Densitometric analysis was carried out using the Image J software (Rasband, USA) and the band intensities were quantified and were normalized to those for GAPDH.
The primer sequences used for gene amplification were Elastin: Forward 5’- GGTGCGGTGGTTCCTCAGCCTGG- 3’ and Reverse 5’- GGGCCTTGAGATACCCCAGTG-3’. Collagen-I: Forward primer 5’-TCTTGGTCGGTGGGTGACTCT-3’ and reverse primer 5’- CCCCCTCCCCAGCCACAAAG-3’. The primer sequence used for GAPDH was Forward primer: 5’-ATGAAGATCCTGACCGAGCGT-3’ and Reverse primer: 5’-AACGCAGCTCAGTAACAGTCCG-3’.
Statistical Analysis
All results were expressed as mean ± standard deviation. Statistical analysis of the data was determined by Student’s unpaired t-test using GraphPad Prism-4.0 software (La Jolla, CA).
Table S1: Cytotoxicity of alkannins on HDF, HaCaT and NIH3T3 cells.
Sl. No / Sample / IC50 Value in µg/mlHDF / HaCaT / NIH3T3
1 / AN-I / 22.31 ± 0.7 / 23.85 ± 0.4 / 23.77 ± 0.2
2 / AAN-II / 916.63 ± 3.2 / 966.20 ± 4.7 / 973.67 ± 3.9
3 / AN-III / 21.31 ± 0.9 / 45.30 ± 0.7 / 24.58 ± 0.3
4 / AN-IV / 1.16 ± 0.1 / 1.21 ± 0.3 / 1.18 ± 0.2
Table S2: Effect of alkannins on H2O2 induced RBC hemolysis.
Sl.No / Samples / Concentration tested (µg/ml) / % inhibition of RBC hemolysis1 / AN-I / 10
5 / 56.12 ± 1.2
32.15 ± 1.8
2 / AAN-II / 10
5 / 61.40 ± 1.0
33.86 ± 1.3
3 / AN-III / 10
5 / 22.50 ± 1.0
15.93 ± 0.7
4 / AN-IV / 0.5
0.25 / 9.21 ± 0.5
3.08 ± 0.4
5 / Ascorbic Acid
(AA) / 50 / 65.96 ± 0.8
Fig.S1
Figure S1. Effect of alkannins on cellular senescence in HDF cells. The cells were treated with arnebins and H2O2 (10 µM), incubated for 1 h at 37°C. The cells were stained, further incubated for 24h at 37°C. The cells were counted under inverted microscope (10 x). (A) Cell control (B) H2O2 treated cells (C) Cells treated with AN-I and H2O2 (D) Cells treated with AN-III and H2O2 (E) Cells treated with AAN-II (50µg) and H2O2. (F) Cells treated with AN-IV (50µg) and H2O2. (G) Cells treated with Tannic Acid (100µg) (H) Percentage reduction in SA-β-gal cells, expressed as mean ± SEM (n = 3). (P < 0.05), TA denotes Tannic acid (standard).
Fig. S2
Figure S2. (A) Effect of alkannins on Collagen I production in HDF cells. Mean ± SEM (n = 2); (P < 0.01). (B) Effect of alkannins on Collagen I gene expression in HDF cells. The gene expression was quantified and normalized to GAPDH and the data represented as mean ± SD (n = 2). The image shown is a representative from among two replicates. (C) Densitometric analysis of collagen-I gene transcripts. Values depict arbitrary units. AA denotes Ascorbic acid (standard). (D) Effect of alkannins on involucrin production in HaCaT cells. Involucrin content in cell supernatants was estimated by ELISA and results expressed as percentage of control. Mean ± SEM (n = 2); (P < 0.01). KN denotes kinetin (standard). (E) Effect of alkannins on elastin gene expression in HDF cells. The gene expression was quantified and normalized to GAPDH and the data represented as mean ± SD (n = 2). The image shown is a representative from among two replicates. (F) Densitometric analysis of elastin gene transcripts. Values depict arbitrary units.
Fig.S3
Figure S3. Major mass fragment of compounds AN-I - AN-IV in LC-MS2 spectra.