mol # 79020molpharm.aspetjournals.org/content/suppl/2012/06/... · k.varghese, geetha b. kumar,...

MOL # 79020

Athira Omanakuttan, Jyotsna Nambiar, Rodney M. Harris, Chinchu Bose, N. Pandurangan, Rebu

K.Varghese, Geetha B. Kumar, John A. Tainer, Asoke Banerji, J. Jefferson P. Perry and Bipin G. Nair,

Anacardic acid inhibits the catalytic activity of matrix metalloproteinase-2 and matrix

metalloproteinase-9, Journal of Molecular Pharmacology.

SUPPLEMENTARY FIGURE 1: HPLC profiles of CNSE (A), Cardol-Cardanol mixture (B) and

Anacardic acid mixture (C) using C-18 , reverse phase column, PDA and acetonitrile/ H2O / acetic acid

(72:18:10) as mobile phase at 245 nm. Anacardic acid mixture shows 3 major peaks for the unsaturated

forms of the material and a 4th near negligible peak for the saturated form.

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SUPPLEMENTARY FIGURE 2: Mass Spectrometry profiles. The masses (m/e) of the three major

HPLC molecular peaks, MH+ corresponds to 343 for the triene (A), 345 for the diene (B) and 347 for the

monoene (C) forms of anacardic acid.

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SUPPLEMENTARY FIGURE 3A: 1H NMR spectra for anacardic acid (triene), recorded on a Bruker

(Wissembourg, France) AM 500 spectrometer (500 MHz, for 1H NMR) using tetramethylsilane (TMS) as

a standard. Deuterated trichloromethane (CDCl3) solvent was used for all the samples. Chemical shift and

J are given in δ and Hz, respectively. The following NMR shifts corresponds to: 7.3 (C4, t, J=7.9Hz, 1H)-

aromatic proton, 6.87 (C5, d, J=8.3 Hz, 1H)-aromatic proton, 6.72 (C3, d, J=7.4, 1H)-aromatic proton,

4.97-5.7 (C15, C14, C12, C11, C10, C9, C8, m, 7H)-alkene protons, 2.98 (C7, t, 2H)-methylene protons

near alkene, 2.8 (C10, C13, q, 4H)-Skip methylene,2.0 (C1, q, 2H)- Benzyl proton, 1.3 (C6, C5, C4,

C3,C2, m )-methylene protons.

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SUPPLEMENTARY FIGURE 3B: 1H NMR spectra for anacardic acid (diene). The following NMR

shifts corresponds to: 7.3 (C4, t, J=7.9Hz, 1H)-aromatic proton, 6.87 (C5, d, J=8.3 Hz, 1H)-aromatic

proton, 6.72 (C3, d, J=7.4, 1H)-aromatic proton, 5.3 (C12, C11, C9, C8, m, 4H)-alkene protons, 3.0 (C7,

t, 2H)-methylene proton near alkene, 2.77 (C10, q, 2H)-Skip methylene, 1.6 (C1, t, 2H)- Benzyl proton,

1.3 (C6, C5, C4, C3, C2, m)-methylene protons, 0.9 (C15, t, 3H)- methyl protons.

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SUPPLEMENTARY FIGURE 3C: 1H NMR spectra for anacardic acid (monoene). The following

NMR shifts corresponds to: 7.3 (C4, t, J=7.9Hz, 1H)-aromatic proton, 6.87 (C5, d, J=8.3 Hz, 1H)-

aromatic proton, 6.72 (C3, d, J=7.4, 1H)-aromatic proton, 5.3 (C9, C8, t, 2H)-alkene protons, 3.0 (C7, t,

2H)-methylene proton near alkene, 1.6 (C1, t, 2H)- Benzyl protons, 1.2 (C2, C3, C4, C5, C6, C11, C12,

C13, m)-methylene protons, 2.0 (C15, t, 3H)-methyl protons, 0.89 ( C14 t, 2H)- methylene protons near

to methyl group.

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SUPPLEMENTARY FIGURE 4: Cell viability using MTT cytotoxicity assay was performed for the

cells treated with anacardic acid isolated from CNSE (at a concentration range of 1-12.5µM) in serum

containing DMEM. Each bar represents the mean ± SE of triplicate determinations from three

independent experiments. **P < 0.01, ***P < 0.001 (one-way ANOVA with Dunnett's multiple-

comparison post-test).

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SUPPLEMENTARY FIGURE 5. Plot showing relative fluorescence units (RFU) in the absence of (A)

or presence of (B) MMP-2 catalytic core domain.

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SUPPLEMENTARY FIGURE 6. Fluorescence-based studies on MMP-2 inhibition by

commercially available anacardic acid. Plot showing comparison of % inhibition of MMP-2 catalytic

core domain in the presence of different concentrations of commercially available saturated anacardic

acid and anacardic acid isolated from CNSE. ***P < 0.001 (one-way ANOVA with Dunnett's multiple-

comparison post-test).

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SUPPLEMENTARY FIGURE 7. Confirmation of MMP-2 in the conditioned media. (A) Zymogram

showing molecular weight marker (lane 1-molecular weight marker, lane 2-MMP-2). (B) Zymogram

incubated in developing buffer in the absence of 20mM EDTA (lane 1) or presence of EDTA (lane 2).

(C) Zymogram of conditioned media prepared in SDS-PAGE sample buffer in the absence of 5mM DTT

(lane 1) or in the presence of 5mM DTT (lane 2).

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SUPPLEMENTARY FIGURE 8. Effect of different modulators on MMP-2 enzymatic activity.

Representative plot (of the zymogram) showing % MMP-2 inhibition by Anacardic Acid (AA), Salicylic

Acid (SA) and Aspirin. Each bar represents the mean ± SE of triplicate determinations from three

independent experiments. ***P < 0.001 (one-way ANOVA with Tukey’s multiple comparison test).

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SUPPLEMENTARY FIGURE 9. HPLC profiles of cardanol (A) Mass Spectrometry profiles of

cardanol showing the masses (m/e) of the three major HPLC molecular peaks, MH+ corresponds to 299

for the triene (1), 301 for the diene (2) and 303 for the monoene (3) forms of cardanol (B) IR spectra of

cardanol and anacardic acid recorded on a Shimadzu IR Affinity -1 in Film(C).

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SUPPLEMENTARY FIGURE 10. Fluorescence-based studies on MMP-2 inhibition by cardanol.

Plot showing comparison of % inhibition of MMP-2 catalytic core domain in the presence of different

concentrations of cardanol and anacardic acid isolated from CNSE. ***P < 0.001 (Student’s t-test).

mol # 79020molpharm.aspetjournals.org/content/suppl/2012/06/... · k.varghese, geetha b. kumar,...

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