Recently antioxidants have attained very important role because of their potential as therapeutic and prophylactic agents in various diseases. Traditional knowledge about medicinal plants has continuously direct the search for new cures. Supplementation of herbal antioxidants is necessary to suppress the oxidative stress in a healthier way. Use of man-made antioxidants like butylated hydroxy toluene (BHT) and butylated hydroxyl anisole (BHA) are restricted due to their side effects.1 Proanthocyanidins are flavonoids widely distributed in plants. Proanthocyanidins better known as condensed tannins are oligomeric and polymeric flavan-3-ols.2 Many plants produce proanthocyanidins in their fruits, bark, leaves and seeds. Proanthocyanidins are of great interest in nutrition and medicine because of its antioxidant, anti-allergic, anticancer, blood pressure and cholesterol lowering effects.3 Over the past years proanthocyanidins supplements have become popular in the form of oligomeric proanthocyanidin complexes (OPCs) for example grape seed extracts and maritime pine bark extracts.4
Cinnamomum zeylanicum bark (family: Lauraceae) is commonly called as cinnamon. C. zeylanicum is a small and evergreen tree, most well-known for its bark. Cinnamon is extensively used in commercially available products for its culinary value and it is almost entirely obtained from cultivated plants.5 It has been used as a spice and flavouring agent. A variety of pharmacological effects have also been observed with its use. The literature survey shows that bark of C. zeylanicum contains copious amounts of phenolic compounds which inhibit peroxidation reaction and therefore can be expected to prevent various chronic illnesses.6 So, in this study, methanolic extract of C.zeylanicum bark was analyzed for the presence of proanthocyanidins (polyphenolic compound).
The NF-κB family consists of a group of eukaryotic inducible transcription factors.7 NF-κB regulates the expression of genes that regulate inflammatory response, transformation, tumor promotion, tumor invasion, angiogenesis and metastasis. Activation of NF-κB is a tightly regulated event. In normal cells, NF-κB becomes activated only after the appropriate stimulation and then up regulates the transcription of its target genes. Thus, NF-κB activation is an inducible but transient event in normal cells.8 NF-κB family comprises of many homo- and heterodimers. A commonly known NF-κB consists of p50/p65 heterodimer. It is primarily composed of proteins with molecular masses of 50 KD (p50) and 65KD (p65) and is retained in the cytoplasm by its inhibitory subunit, IκBα.9 In response to a variety of stimuli including physical and chemical stresses, cytokines, reactive oxygen intermediates and ultraviolet light, the latent cytoplasmic NF-κB/IκBα complex is activated by the I-κB Kinase (IKK) complex.10 IKK is formed by three distinct subunits: IKKα, IKKβ and IKKγ. The activation of IKK complex leads to the phosphorylation by IKKβ of which targets IκB for ubiquitination and degradation by the 26S proteasome. The unmasked NF-κB can then enter the nucleus and binds to the DNA target elements present in the promoters of NF-κB regulated genes, as well as to co-activators of gene transcription to activate target gene expression.11 Deregulation and constitutive or aberrant activation of the NF-κB pathway has been observed in and attributed to the development of a variety of human ailments including cancer.12
Hence, search of natural C.zeylanicum bark derived proanthocyanidin which can inhibit NF-κB is the objective of this study.
MATERIAL AND METHODS
Collection of plant material
The bark of C. zeylanicum was collected from Anjarakandy Cinnamon Estate, Kannur district, Kerala and the methanolic extract was prepared.
The methanolic extract of C. zeylanicum bark was subjected to UV absorption in the range 190 – 400nm in a UV-visible spectrophotometer. Methanolic extract of C. zeylanicum bark was subjected to Reverse phase HPLC (Shimadzu chromatograph) analysis, a 250 mm reverse phase C-18 ODS column with UV visible detector was used. Methanolic extract of C. zeylanicum bark was dissolved in appropriate volume of HPLC grade methanol and 20μl was injected into the apparatus and detected at 280 nm.
Column and thin layer chromatographic separation and detection of proanthocyanidin rich fraction
The results of UV and HPLC analysis of methanolic extract of C. zeylanicum bark revealed that the bark is rich in flavonoids and phenolic compounds. Hence, an attempt was made to separate phenolics rich fraction from methanolic extract of C. zeylanicum bark using column chromatography.
25 grams of methanolic extract of C. zeylanicum was dissolved in 25mL of methanol and applied onto chromatography column (2.5×30 cm) packed with silica gel equilibrated with methanol. The column was developed using gradient solvent systems with mobile phase (eluent) of increasing polarity as follows:
Hexane (100%), hexane:chloroform (80%:20%), hexane:chloroform (70%:30%),hexane:chloroform 50%:50%, hexane: chloroform (30%:70%), chloroform (100%), chloroform: ethyl acetate (90%:10%), chloroform: ethylacetate(70%:30%) chloroform:ethylacetate (50%:50%), chloroform: ethylacetate (30%:70%), ethyl acetate 100%, ethyl acetate:ethanol (70%:30%), and ethyl acetate:ethanol (50%:50%). The fractions (7ml) were collected using a fraction collector. All the fractions were then successively concentrated in a rota evaporator. The concentrates were analysed for the presence of proanthocyanidins by Thin Layer Chromatography (TLC).13
Precoated TLC plates with silica of 100x200 meshes (Merck) were used for the analysis. The collected fractions were spotted on TLC plates and the plates were developed with 3 different solvent systems - Hexane, chloroform and toluene & ethyl acetate (1:1). After the run the plates were analyzed for the presence of coloured spots under UV light. The TLC plates which gave spots under UV light were then dipped in vanillin-HCl (10% vanillin in ethanol: concentrated HCl in 2:1 ratio) and heated to see the red spots in day light.14 Those fractions which gave similar spots and Rf value as standard procyanidin B2 were pooled together to get procyanidin B2rich fraction. The resultant procyanidin B2 rich fraction was subjected to GC-MS (Shimadzu) using procyanidin B2 as standard.
Molecular docking of procyanidin B2 against NF-κB
The latest version of Gold 3.0.1 program was used for molecular docking of procyanidin B2 against NF-κB.
The 3D structure of the target protein NF-κB (PDB id: 1NFI.pdb) was retrieved from Protein Data Bank (PDB). It is a complex of p50-p65 hetero dimer bound to IκBα inhibitory protein. p65 subunit contains 301 residues, p50 with 107 residues and IκBα with 213 residues (Figure 1A).
The first step in preparation of the target protein 1NFI.pdb was to remove all waters as well as other non-protein molecules. For this purpose, Deep view (Swiss-PDB viewer)15 was used. The inhibitor IκBα was then removed and the resulting *.pdb file was extracted and imported into MOE (Molecular Operation Environment, 2005). The “wash” function in MOE16 was used to add explicit hydrogen atoms and to set atom ionization states based on their formal charges. Finally, the protein structure was exported as *.pdb file for input into GOLD.17 The resulting *.pdb file was examined in order to identify a suitable binding site center atom for purpose of defining the binding site in the docking software, GOLD. The nitrogen atom in K 221 of p65 was selected.18 This residue is located deep in the pocket close to the center of the bound IκBα.
The structure of procyanidin B2 (Figure 1 B) was drawn using Chemdraw19 and exported to MOE as a *.mol file. Hydrogen atoms were added to the two dimensional structure and atom ionization set to formal charge using the “wash” function in MOE before conversion to three-dimensional structure. The structure was then exported in *.sdf format for input to GOLD.
The 3D structure of procyanidin B2 was docked into the binding site of prepared target protein, 1NFI. Docking experiments were performed using the default GOLD fitness function (VDW = 4.0, H-bonding = 2.5) and evolutionary parameters: population size = 100; selection pressure = 1.1; operations = 100,000; islands = 5; niche size = 2; migration = 10; mutation = 95; crossover = 95. The number of dockings to be performed on ligand was specified under GA runs: by default this value is 10.17 The nitrogen atom on K221 was selected as the binding site center for all calculations.
Ten docking runs were performed. If at any time 3 of the 10 poses were within 1.5 A° rmsd of each other, the docking run was terminated. All poses and the corresponding Gold scores were outputted into a single *.sdf file and text file respectively. The lowest-energy poses obtained from this stage were selected and were then rescored using the Gold Score function. Files containing best fitness function were saved.
RESULTS AND DISCUSSION
Figure 2 A shows the UV absorption spectrum of the methanolic extract of C. zeylanicum bark which revealed a peak at 220 nm, a small peak at 278 nm and a sharp peak at 300 nm. The peak at 278 nm in UV spectrum supports the presence of phenolic compound having absorbance at 278 nm in the bark extract.
The RP-HPLC spectrum at 280 nm of methanolic extract of C. zeylanicum bark is shown in Figure 2 B. Seven major peaks were observed.
Proestos et al.20 identified and quantified the phenolic compounds in Vitex agnus-castus, Origanum dictamnus, Teucrium polium, Lavandula vera and Lippia triphylla by RP-HPLC monitored at 280 nm and GC-MS by comparing them with spectrum of standards and reported the presence of caffeic acid (0.12-0.93 mg 100 g−1), ferulic acid (0.34-1.52 mg 100 g−1)and (+)-catechin (0.22-0.43 mg 100 g−1) in the dry samples of the selected plants. Basu and Maier21 studied the in vitro antioxidant activities of berry fruits and reported a positive correlations between IC50values for different radical scavenging activities and different polyphenolics. Red raspberry extract had the highest Pearson’s coefficient values (0.952–1) between total phenolics, flavonoids, and proanthocyanidins and DPPH and superoxide radical scavenging activities. Prabha et al22 reported the presence of monomeric flavonoids in Musa sapientum by using HPTLC techniques at 254 nm. In this study, the results of UV absorption and RP-HPLC monitored at 280 nm, showed the presence of phenolic compounds in methanolic extract of bark.
A total of 96 fractions were collected from the column chromatography and all the fractions were analysed for the presence of procyanidin B2 by TLC and confirmed by dipping in vanillin-HCl Fractions in toluene:ethylacetate solvent gave positive result for the presence of procyanidin B2 (Figure 3). Fractions which showed similar spots and Retardation factor (Rf) values were pooled. Thus, procyanidin B2 rich fraction was got.
GC-MS analysis of procyanidin B2 rich fraction
From the results, it is revealed that the procyanidin B2 rich fraction contains five major compounds with RT values of 11.30, 11.66, 12.23, 12.52 and 12.65. The peak at RT 12.23 for procyanidin B2 rich fraction was comparable with the peak at RT 12.25 obtained for standard procyanidin B2.The results in this study, confirms the presence of dimeric proanthocyanidins in the bark extract.
Molecular docking of procyanidin B2 against NF-κB.
The transcription factor NF-κB is an interesting target molecule for the design of lead compounds of anti-tumor, anti-inflammatory and pro-apoptotic drugs.10 The best ligand docking gold score value was 55.03 with lowest binding energy of -6.03 (kcal/mol) and formed H-bonding with six amino acid residues (E 282, P283, V244, K221, H 181, I196) of p65 subunit of NF-κB (Figure 5 and Table 1).
In a mini review survey by Patrick,18 showed the list of amino acid residues involved in interactions between IκBα and p50/ p65.The amino acids in p65subunit, Y20, E 22, E49, H181, K 221, S 238, F 239, S240, Q241, A 242, D 243, R 246, D 291, I295, K 298 involved in binding with IκBα.
Thavamani et al 23 carried out molecular docking for 12 active compounds present in Cocculus hirsutus against hepato cellular carcinoma targets such as Aurora kinase, c-Kit, fibroblast growth factor, nuclear factor kappa B (NF-kB), B-cell lymphoma-extra-large, and vascular endothelial growth factor (VEGF) with the software Grid-Based Ligand. The results indicated that coclaurine, haiderine, and hisutine had good inhibition on NF-κB signaling pathway.
The present study shows the procyanidin B2 formed H-bonding with H 181 and K 221 of p65 sub unit of target protein. Hence, it supported the fact that the ligand is located in IκBα binding site of target protein. This shows that procyanidin B2 could have the potency to mimic the action of IκBα.
|Ligand name||Protein name||Atom in ligand||Atom in protein||Hydrogen bond distance||Score|
Piccagli et al.10 studied the amino acids of p50/p65 heterodimer involved in DNA binding and revealed that 13 p50 residues (C59, K144, Y57, Q274, Q306, R305, K145, K272, S63, G65, G66, N136, K77) and twelve p65 amino acids (C38, K122, Y36, K123, Q220, Q247, R246, K221, A 43, S42, S45, K56) made several contacts in DNA backbone.
Chen et al.24 studied the crystal structure of p50/p65 heterodimer of transcription factor NF-κB bound to DNA and showed that G65, G 66, G 88, H64, N54, N56, E 60, T67,K 241,C59, Y 57, K 144, K145, Q274, K 275, R 305 are the residues in p50 of p50/p65hetrodimer bind with DNA. Similarly R246, K 221, K218, E 220, E247, R246, Y36, C38,K 122, K 123 R 33, R 35, R187, and E 39 are the amino acid residues in p65 subunit of p50/p65 hetreodimer bind with DNA.
In the present findings also, K221 formed H-bonding with procyanidin B2 thus could interfere with binding of p50/p65 heterodimer to κB DNA binding sequence. This proved that the ligand is positioned in a cleft surrounded by active site amino acids of NF-κB. The hydrogen bonding plays the most important role in determining the specificity of intermolecular interactions. These findings supported that procyanidin B2 by binding to active site of NF-κB could inhibit the nuclear translocation and DNA binding of p50/p65 heterodimer to κBDNA sequences. Thus, procyanidin B2 can act as the inhibitor for transcription factor NF-κB.
Natural products play an important role in the novel drug discovery for the treatment of human diseases including cancer.25 Chemical compounds derived from plants inhibit carcinogenesis through many mechanisms which include antioxidant activity, changes in carcinogen metabolism, modulation of cell cycle, alternations in their intracellular signaling and inhibition of angiogenesis.26 So, procyanidin B2 present in C. zeylanicum bark can be used as a potential lead compound for drug development against cancer and other oxidative stress disorders.
The bark of Cinnamomum zeylanicum is rich in medicinally essential phytoconstituents especially phenolic compounds.
The methanolic extract is found to be rich in procyanidin B2
In silico Molecular drug docking of procyanidin B2 against NF-κB by Gold
Gold score value was 55.03 with lowest binding energy of -6.03 (kcal/mol)
Formed H-bonding with six amino acid residues (E 282, P283, V244, K221, H 181, I196) of p65 subunit of NF-κB
Thus, procyanidin B2 can act as the inhibitor for transcription factor NF-κB, can be used as a potential lead compound for drug development against cancer and other oxidative stress disorders.