Chapter VI Bioactive guided fractionation and identification of active compounds from Gelidiella acerosa
CONTENTS 8.1. 8.2. Abstract Introduction 8.3. Materials and Methods 8.3.1. Preparation of seaweed extract 8.3.2. Fractionation of G. acerosa benzene extract using column chromatography 8.3.3. Evaluation of antioxidant potential by DPPH radical scavenging activity 8.3.4. Determination of cholinesterase inhibitory activity 8.3.5. LC MS analysis 8.3.6. Assessment of anti Alzheimer potential of the ligands through molecular docking 8.3.7. High performance Thin Layer Chromatography 8.3.8. Statistical analysis 8.4. Results and Discussion 8.4.1. Bioactive guided fractionation of G. acerosa benzene extract 8.4.2. Identification of active compounds from the pooled column fraction through LC MS analysis. 8.4.3. Assessment of antioxidant and anti cholinesterase potential of phytol 8.4.4. Investigation of binding mode of phytol in the binding site of AChE through docking studies 8.4.5. Quantitative High Performance Thin Layer Chromatography (HPTLC) for the analysis of pooled active fractions 8.5. Conclusion
Chapter VI 185 8.1. ABSTRACT The seaweed G. acerosa is a marine red macro alga with excellent therapeutic potentials. The results of the previous studies demonstrated that G. acerosa benzene extract possess noticeable neuroprotective activity, when evaluated through in vitro and in vivo systems. Hence, the bioactive compound which is responsible for the above mentioned protective effects was identified by bioactive-guided fractionation. The fractionation was done by column chromatography and the activity of the column fractions and the bioactive compound was evaluated by antioxidant and cholinesterase inhibitory assays. Identification of bioactive compound was done by LC-MS analysis. The results suggest that among all the fractions, fractions F9-F13 exhibited significant (P<.5) antioxidant and anti-cholinesterase activities. Hence these fractions were pooled together and verified for neuroprotective activity. The pooled fraction was subjected to LC-MS analysis, which yielded several compounds. Among all the compounds, phytol was previously reported to possess bioprotective activities and moreover it is a diterpene. Therefore, the antioxidant and anti-cholinesterase activity of phytol was evaluated and the results showed that phytol has significant (P<.5) antioxidant activities (25-125 µg/ml) and cholinesterase inhibitory potential (5-25 µg/ml). The mode of interaction through which phytol exerts its anti-cholinesterase effect was studied by docking analysis. The results suggested that phytol interacts with the enzyme through the latter s arginine residue. In addition, the amount of phytol present in the pooled column fraction was quantified by HPTLC. The results showed that the pooled fraction was found to contain about 6.266 µg of phytol per mg of pooled fraction. Overall, the study suggests that phytol might be the key compound responsible for the above mentioned neuroprotective potentials.
Chapter VI 186 8.2. INTRODUCTION The marine red alga Gelidiella acerosa has been employed as the principle agarophyte for the production of superior quality of agar. In recent years, the evaluation of pharmacological properties of the secondary metabolites present in G. acerosa has been greatly increased (Elsie et al., 11). The study led by Duraikannu et al. (14) reported that G. acerosa possess potential anti-cancer activity, when verified in Dalton s Ascitic Lymphoma (DAL) cells. Moreover the antioxidant capability of G. acerosa has also been demonstrated through in vitro antioxidant systems (Megha and Anjali, 13). The seaweed was also reported to have excellent anti-microbial activities against a wide range of bacterial and fungal species (Vivek et al., 11, Elsie and Dhanarajan, 1). Apart from these above mentioned therapeutic potentials, the results of the previous chapters suggest that G. acerosa possess potential antioxidant, anti-che and anti-amyloidogenic activities, when evaluated through various in vitro systems. The G. acerosa benzene extract was found to possess excellent neuroprotective potential against A 25-35 induced toxicities, when evaluated under in vitro and in vivo systems. Therefore, identification of the active compound, which is responsible for the neuroprotective activity, is crucial for the development of novel drug entities against AD. Among various methods, the phytochemical research based identification of natural compounds was considered as effective approach (Brusotti et al., 14). Hence in the present study, the G. acerosa benzene extract was subjected to bioactive-guided fractionation and the active compounds present in the fractions were also identified. The bioactive-guided fractionation involves step-wise separation of the extracted components, followed by the assessment of biological activity of the separated fractions. The active fractions were further analyzed to identify the active compounds responsible for the neuroprotective activity.
Chapter VI 187 8.3. MATERIALS AND METHODS 8.3.1. Preparation of seaweed extract The benzene extract of G. acerosa was prepared by the method as mentioned in the section 3.3.2. 8.3.2. Fractionation of G. acerosa benzene extract using column chromatography Silica-gel column (-1 mesh) was used for separation. The column bed (evenly set) was prepared using hexane. G. acerosa benzene extract (7 g) was impregnated with silica-gel and loaded on to the silica-gel column (-1 mesh). The solvents of varying ratio were introduced to the column and the fractions of double the bed volume were collected sequentially. The scheme of usage of solvent ratios has been described below: G. acerosa Benzene extract Column fractionation H:B 95:5 (F1) H:B 9:1 (F2) H:B 4:1 (F3) H:B 3:1 (F4) H:B 2:1 (F5) H:B 1:1 (F6) B:EA 19:1 (F7) B:EA 9:1 (F8) B:EA 4:1 (F9) B:EA 3:1 (F1) B:EA 2:1 (F11) B:EA 1:1 (F12) 1% EA (F13) EA:M 9:1 (F14) EA:M 3:1 (F15) EA:M 1:1 (F16) 1% M (F17) H- Hexane, B- Benzene, EA- Ethyl acetate, M- Methanol 8.3.3. Evaluation of antioxidant potential by DPPH radical scavenging activity (Shimada et al., 1992) The antioxidant potential of the separated fractions and the active component was evaluated by the method mentioned in the section 3.3.3.1.
8.3.4. Determination of cholinesterase inhibitory activity (Ellman et al., 1961; Ingkaninan et al., 8). The AChE and BuChE inhibitory activity of the separated fractions and the active component was evaluated by the method mentioned in the section 3.3.5. 8.3.5. LC MS analysis Chapter VI 188 The active fraction was subjected to LC-MS analysis using reverse phase C-18 column Shimadzu LC 1 ATVP with integrated library Metwin Version 2.. Electron spray ionization mode was employed (both positive and negative mode). 1 µl of sample was injected for analysis and the column temperature was maintained at 25 C. The m/z ratio range used was 5-1 for the detection of compounds. The mobile phase used was acetonitrile and methanol (5:5) and the flow rate maintained was 1.5 ml/min. The chemical components of the fraction were identified by comparing the compounds stored on the library Metwin version 2.. 8.3.6. Assessment of anti Alzheimer potential of the ligands through molecular docking The bioactive compound, which was found to be present in the active column fraction (through LC-MS analysis), was subjected to molecular docking. Since Donepezil is the currently used drug candidate for AD, it was used as positive control. Three dimensional (3D) structure of the active compound was retrieved from Pubchem database (http://pubchem.ncbi.nlm.nih.gov). The 3D structure of AChE enzyme (receptor protein) was obtained from Protein Data Bank (PDB code: 1B41). The binding pocket of the AChE protein was identified using Define and edit binding site protocol availed from Discovery Studio (Shafreen and Pandian, 13). The presence of water molecules in AChE was removed and was further subjected to docking analysis for the evaluation of the AChE inhibitory potential of the active compound. The active compound was docked into the rigid binding pocket of AChE. The molecular docking studies were performed using Ligand Fit module, which is available in Discovery Studio package, version 2.5. (Accelrys, Inc. USA). 8.3.7. High Performance Thin Layer Chromatography (HPTLC) The HPTLC analysis was performed to determine the amount of bioactive compound present in the pooled column fraction. The pooled column fraction was dissolved in methanol and the sample was loaded in 1 1 cm Silica gel F254 TLC
Chapter VI 189 plate. The plate was kept in TLC twin trough developing chamber (after saturation) with the mobile phase of Benzene-Ethyl acetate (1:1). The developed plate was dried and scanned in CAMAG TLC scanner 3 at the wavelength of 254 nm. The data were recorded analyzed using wincats planar chromatography manager. 8.3.8. Statistical analysis Statistical analysis was performed using SPSS 17. software package. The results of all the experiments were represented as Mean S.D. Statistical analysis between the groups was performed by one-way ANOVA followed by Post- Hoc multiple comparisons. P- value <.5 were regarded as significant. IC5 values were calculated by probit analysis. 8.4. RESULTS AND DISCUSSION From the results of the previous chapters it is evident that the benzene extract of G. acerosa possess excellent neuroprotective potentials against AD. Hence to identify the active compounds responsible for the above mentioned activities, the G. acerosa benzene extract was subjected to separation through column chromatography. 8.4.1. Bioactive guided fractionation of G. acerosa benzene extract The bioactive-guided fractionation and separation of the extract was performed using a wide range of solvent ratios in the order of increasing polarity. The fractions eluted were evaluated for antioxidant and anti-cholinesterase activities using in vitro assays. The results of DPPH radical scavenging assay suggests that among all the fractions, F8-F14 exhibited significant (P<.5) antioxidant activity with the IC5 values of 36.13 ± 1.36 (F8), 38.8 ±.97 (F9),.55 ± 2.85 (F1),.9 ± 2.8 (F11), 41.66 ± 2.9 (F12), 41.99 ± 1.51 (F13), 39. ± 2.38 µg/ml (F14) respectively (Fig. 8.1). BHT, the standard antioxidant was used as positive control.
Percentage of inhibition 1 1 1 Chapter VI 19 5 1 Figure 8.1: Evaluation of DPPH radical scavenging activity of column fractions. The values are expressed as Mean±SD. P<.5 compared to control. The cholinesterase inhibitory activity of the column fractions separated from G. acerosa benzene extract was also evaluated. The results suggest that among all the fractions evaluated, fractions F9-F13 exhibited higher AChE activities at the concentration of µg/ml (Fig. 8.2). In the case of BuChE, fractions F9-F13 exhibited a significant (P<.5) inhibition against BuChE activity with the percentage of inhibition of 7.21 ± 5.86, 72.39 ± 1.5, 55.37 ±.45, 63.88 ± 3.81, 53.9 ± 2.31% for fractions F9, F1, F11, F12 and F13 respectively (Fig. 8.3). The IC5 values of the fractions were found to be 48.36 ± 1.38 (F9), 44. ± 2.2 (F1), 48.82 ± 6.91 (F11), 66.25 ± 12.89 (F12), 153.12 ± 16.79 µg/ml (F13) respectively for BuChE. Donepezil, the standard anti-cholinesterase drug was used as positive control. 1 Percentage of inhibition 1 5 µg/ml 1 µg/ml µg/ml Figure 8.2: Evaluation of AChE inhibitory activity of column fractions. The values are expressed as Mean±SD. P<.5 compared to control.
1 Chapter VI 191 Percentage of inhibition 1 1 5 µg/ml 1 µg/ml µg/ml Figure 8.3: Evaluation of BuChE inhibitory activity of column fractions. The values are expressed as Mean±SD. P<.5 compared to control. Since the fractions F9-F13 showed potent antioxidant and anti-cholinesterase activities, these fractions were pooled together and again verified for the antioxidant and anti-cholinesterase activities. The results of the assays demonstrated that the pooled fraction showed significant (P<.5) radical scavenging activity at the concentrations of 5, 1 and µg/ml with the IC5 value of 79.29 ± 18.65 µg/ml (Fig. 8.4). The cholinesterase inhibitory assays suggests that at the concentration of µg/ml, the pooled fraction also exhibited significant (P<.5) AChE (.38 ± 2.55%) and BuChE (54.15 ± 5.84%) inhibitory activities (Fig. 8.5 and 8.6). 1 Percentage of inhibition Control Pooled fraction BHT 5 µg/ml 1 µg/ml µg/ml Figure 8.4: Determination of DPPH radical scavenging activity of the pooled column fractions (F9 F13). The values are expressed as Mean ± S.D. P<.5 compared to control group.
Chapter VI 192 1 Percentage of inhibition 1 Control Pooled fraction Donepezil 5 µg/ml 1 µg/ml µg/ml Figure 8.5: Determination of AChE inhibitory activity of pooled column fractions. The values are expressed as Mean±SD. P<.5 compared to control. 1 Percentage of inhibition 1 Control Pooled fraction Donepezil 5 µg/ml 1 µg/ml µg/ml Figure 8.6: Determination of BuChE inhibitory activity of pooled column fractions. The values are expressed as Mean±SD. P<.5 compared to control. 8.4.2. Identification of active compounds from the pooled column fraction through LC MS analysis. A total of 13 compounds were obtained through LC-MS analysis of the pooled column fraction (Table 8.1). The major compounds identified in the pooled fractions
Chapter VI 193 include coumaryl alcohol, sinapyl alcohol, citramalic acid and phytol. Of the major compounds identified, Phytol has been reported to possess neuroprotective effect in response to pilocarpine induced seizures in mice (Costa et al., 12). Phytol is an acyclic monounsaturated diterpene alcohol terpenoid and a part of chlorophyll molecule. It is also present in vitamin K, vitamin E and other tocopherols. The compound phytol has also been demonstrated to exhibit anti-schistosomal activity, when evaluated in the parasite Schistosoma mansoni (Moraes et al., 14). In addition to that phytol also exhibited excellent anti-bacterial activity against Staphylococcus aureus at its least concentration (Inoue et al., 5). Apart from these potentials, more importantly, this diterpene was reported to possess higher antioxidant activity as it scavenges hydroxyl and nitric oxide radical and also reduces the peroxidation of lipids. Moreover, the anti-nociceptive activity of phytol has also been revealed, by verifying the effect in mice. (Santos et al. 13). Based on this background information on phytol, in the present study, the antioxidant and anti-cholinesterase activity was evaluated. Table 8.1: Identification of active components present in the pooled column fractions of G. acerosa benzene extract S. NO COMPOUND IDENTIFIED MOLECULAR MASS 1. Phytol 296.54 2. Pentadecane 212.42 3. Nonadecane 268.53 4. Acetophenone 1.15 5. Hydroxy phenone 15.14 6. Citramalic acid 148.11 7. Coumaryl alcohol 15.18 8. Undecalactone 184.28 9. Linalyl acetate 196.29 1. Sinapyl alcohol 21.33 11. Alanine 89.1 12. Valeric acid 12.14 13. Malonic acid 14.7
8.4.3. Assessment of antioxidant and anti cholinesterase potential of phytol Chapter VI 194 The antioxidant potential of phytol was evaluated through DPPH radical scavenging assay and the results suggests that phytol possess significant (P<.5) scavenging activity at the concentration of 1 µg/ml with the percentage of inhibition of 52.63 ± 2.67% (Fig. 8.7). The IC5 value for phytol was found to be 95.27 ± 1.65 µg/ml. BHT, the standard antioxidant was used as positive control. 1 Percentage of inhibition 1 Control Phytol BHT 25 µg/ml 5 µg/ml 75 µg/ml 1 µg/ml 125 µg/ml Figure 8.7: Evaluation of radical scavenging effect of various concentrations (25 125 µg/ml) of phytol. The values are expressed as Mean±SD. P<.5 compared to control. The cholinesterase inhibitory potential of phytol was evaluated through AChE and BuChE inhibition assay. The results of the experiment demonstrated that phytol was found to possess significant AChE inhibitory activity (at the concentration of 5 µg/ml) with the percentage of inhibition of 92.41 ± 5.83% (Fig. 8.8). Similarly, phytol was also able to inhibit BuChE significantly (P<.5) at the concentration of 1 µg/ml with the percentage of inhibition of 74.21 ± 3.57% (Fig. 8.9). The IC5 values of phytol against AChE and BuChE was found to be 2.74 ±.7 and 5.798 ±.72 µg/ml respectively.
Chapter VI 195 1 Percentage of inhibition 1 Control Phytol Donepezil 5 µg/ml 1 µg/ml 15 µg/ml µg/ml 25 µg/ml Figure 8.8: Evaluation of AChE inhibitory activity of various concentrations (5 25 µg/ml) of phytol. The values are expressed as Mean±SD. P<.5 compared to control. 1 Percentage of inhibition 1 Control Phytol Donepezil 5 µg/ml 1 µg/ml 15 µg/ml µg/ml 25 µg/ml Figure 8.9: Evaluation of BuChE inhibitory activity of various concentrations (5 25 µg/ml) of phytol. The values are expressed as Mean±SD. P<.5 compared to control. 8.4.4. Investigation of binding mode of phytol in the binding site of AChE through docking studies The most challenging task in evaluating the therapeutic potentials of active compounds is to identify its affinity and selectivity towards their respective targets.
Chapter VI 196 Molecular docking has been regarded as the simple and rapid computational method, which is used for the assessment of the binding affinity of the ligands over the receptor (Wichapong et al., 14). Hence in the present study, the ability of phytol to interact with the binding pocket of the enzyme AChE in all the possible poses was studied. About 23 binding sites were predicted in AChE enzyme (the receptor molecule) through the binding pocket analysis using Ligand Fit module. Among these 23 binding sites, site 17 of AChE was found to have maximum binding capability to bind with that of the control ligands (Rivastigmine, Donepezil, Galantamine). Table 8.2 illustrates the binding mode of the control ligands and the test ligand (phytol). ARG177 has been identified as the active site residue in AChE, which is essential for the establishment of strong interaction with the control ligands. Interestingly, the test ligand showed a similar kind of interaction with the arginine residue (ARG177) with a dock score of 27.372. The interaction of the control and test ligands with the AChE enzyme has been illustrated in Fig. 8.1. Table 8.2: Comparison of docking score and interactions of phytol with standard anti AD drugs. S. No Compound Pubchem ID Docking score Interaction Distance 1. Donepezil 3152 34.16 ARG177 (NH1):O2 ARG177 (NH1):O3 2.694 2.526 2. Galantamine 9651 43.27 ARG177 (NH1):O2 2.345 3. Rivastigmine 77991 25.161 ARG177 (NH1):O2 3.154 4. Phytol 5366244 27.372 ARG177 (NH1):O1 3.145
Chapter VI 197 Figure 8.1: Molecular interaction of Donepezil (A), Galantamine (B), Rivastigmine (C) and Phytol (D) with the enzyme acetylcholinesterase.
Chapter VI 198 8.4.5. Quantitative High Performance Thin Layer Chromatography (HPTLC) for the analysis of pooled active fractions HPTLC is a simple and rapid technique for the qualitative and quantitative analysis of the natural compounds. This technique is a modern adaptation of TLC with advanced separation efficiency and detection limits (Shivatare et al., 13). Since phytol possesses excellent antioxidant and anti-cholinesterase activities, the amount of phytol present in the pooled active column fraction was quantified by HPTLC analysis. Fig. 8.11 illustrates the HPTLC chromatogram of pooled column fraction in comparison with phytol. The results suggests that the pooled fraction of G. acerosa contains 6.266 µg/mg of phytol (Rf value.79). (A) (B) Figure 8.11: HPTLC chromatogram of pooled column fraction (A) in comparison with the standard phytol (B).
Chapter VI 199 8.5. CONCLUSION In the present study, the bioactive compound, which is responsible for the observed neuroprotective effects, was identified through the bioactive-guided fractionation. The fractions separated from G. acerosa benzene extract were assessed for antioxidant and anti-cholinesterase activities. The active fractions were pooled together and again evaluated for the antioxidant and anti-cholinesterase activities and then subjected to LC-MS analysis. The pooled fraction showed the presence of active compound phytol, a diterpene with potent bioprotective potentials. Hence the antioxidant and anti-cholinesterase activity of phytol was evaluated through in vitro assays and the results demonstrated that phytol possess excellent antioxidant and anticholinesterase activities. In addition to that, the molecular docking studies suggest that phytol interacts with AChE through the arginine residue and thereby it might exhibit its inhibitory effect. Hence the outcome of the study suggests that phytol might be the active compound responsible for the above mentioned neuroprotective effects.