Anti-HIV-1 Activity of Ellagic acid Isolated from Terminalia paniculata

Objective: The present investigation evaluates the potential Anti-HIV-1 and antioxidant activity of the methanol extract of the leaves of Terminalia paniculata. This search for inhibitors of Anti-HIV-1 Integrase and Protease from medicinal plants of Western Ghats, India led to the isolation of Ellagic acid from the methanol extract of T. paniculata leaves. Methodology: The in vitro antioxidant activity was determined by different standard methods such as 1,1-diphenyl-2-picryl hydrazyl radical (DPPH), metal chelation, superoxide radical scavenging and lipid peroxidation assays. The in vitro antiHIV-1 enzyme inhibition assays were carried out by standard protocols using Multiplate integration assay. The structure of Ellagic acid was elucidated on the basis of NMR, LC-MS and FTIR studies. Results: Ellagic acid has shown excellent in vitro anti-HIV-1 activity in the enzyme inhibition assays carried out with an IC50 value of 8.7 μm and 70.4 μm against HIV-1 Integrase and HIV-1 Protease enzymes respectively. For the antioxidant assays carried out Ellagic acid has shown scavenging potential of 92.2 ± 0.03% of DPPH radical, 88.1 ± 0.41% chelation of metal ions, 68.02 ± 0.12% scavenging of superoxide anion radical and 85.52 ± 0.06% inhibition of lipid peroxidation all at a concentration of 40 ppm. Conclusion: The isolated bioactive molecule, Ellagic acid has shown highly efficient in vitro anti-HIV and antioxidant activity. The present study happens to be the first report on the HIV-1 inhibition ability of T. paniculata.


INTRODUCTION
Extracts of natural materials have always proved to be a vital source in the characterization and developing lead molecules having various biological activities.By thorough screening of extracts of natural products it becomes possible to discover a multitude of structurally diverse drugs and enzyme inhibitors.This kind of diversity cannot be achieved by screening of synthetic entities.Till date, a large number of molecules derived from natural products have been discovered and added to the list of anti-HIV agents. 1 Medicinal plants have proven to be a rich source of potential and new natural bioactive molecule having high therapeutic value.One of the first inhibitor of the enzyme integrase was a phenethyl derivative of caffeic acid (CAPE), which is a plant derived product.This molecule was reported along with Doxorubicin. 2 In the life cycle of the deadly Human Immunodeficiency virus, there are several crucial steps which are targeted for therapeutic intervention.HIV-1 Protease is the enzyme which is essential to cleave the newly synthesized polyprotein components at specific sites in order to create mature protein components.These mature protein units in turn aid in forming an infectious HIV virion. 3Without the action of HIV-1 Protease, it is impossible for the HIV virions to attain infectivity. 4,5HIV-1 Integrase is the key viral enzyme which is responsible for catalyzing the integration of DNA.In vitro assays for 3 ' -end processing and strand transfer have proven to be invaluable in determining the mechanisms which control integration. 6,7The in vitro anti-HIV assays have been extremely helpful and have facilitated the discovery and characterization of new HIV-1 inhibitors with proven therapeutic value.
The discovery of more number of inhibitors of HIV would definitely go a long way in improving the existing treatment paradigm for people suffering from HIV. Working towards this, we have successfully isolated and characterized a bioactive molecule namely Gallogen, also known as Ellagic acid from the leaves of Terminalia paniculata.
Of late, there has been a tremendous rise in interest towards isolating natural antioxidants.This is owing to the mounting evidence everyday on how these antioxidants can go a long way in protecting the human body against the action of free radicals thereby retarding the progression of several chronic diseases. 8Phenolic compounds, nitrogen compounds, carotenoids belong to the family of natural antioxidants. 9rminalia paniculata is a medicinal tree native to the Western Ghats, India.Other species of Terminalia have been widely researched and have shown several therapeutic properties.But, T.paniculata still remains to be unexplored on a larger scale for its possible therapeutic activity and this was the reason to choose this medicinal tree for the present study.
T.paniculata has shown to have hepatoprotective activity 10 and anti-diabetic activity. 11Isolation of Ellagic acid having potent anti-HIV-1 activity coupled with strong antioxidant activity from T.paniculata shows a new promising source of natural anti-HIV agent.

Plant Material
The fresh leaves of T. paniculata were collected from Mijar, Moodabidri, Dakshina Kannada District, Karnataka.

Herbaria of the plant was deposited in Herbarium Collection
Centre at the Department of Studies in Microbiology, University of Mysore, Mysore.The accession number being T. paniculata (MGMB/003/2011-12).The leaf material was air dried an ground into a fine powder.100 g of the leaf powder was then extracted with 500 ml of methanol.It was then filtered, squeezed and evaporated under reduced pressure to obtain the MeOH extract.

Extraction and Isolation
Briefly, the leaf powder of T. paniculata was extracted with MeOH at room temperature and the solvent was evaporated under reduced pressure to obtain the MeOH extract (570.12 mg).A part of the MeOH extract (250 mg) was then used to carry out column chromatography on silica gel by eluting with Hexane / Ethyl acetate (9.1 : 0.9, 9.05 : 0.95, 9 : 1, 8.95 : 1.05) to yield four fractions (Fraction F1-Fraction F4).Based on the comparison of Rf value, fractions F1 to F4 were pooled to obtain fraction F5A.Fraction F5A was again subjected to column chromatography using Hexane and ethyl acetate as elution solvents.This in turn gave four sub-fractions F5a, F5b, F5c, F5d.Sub-fraction F5d was purified by preparative TLC with Hexane Ethyl acetate (2 : 1) to obtain the purified compound Ellagic acid (8.4

UV-255 nm
Copies of original spectra (NMR, LC-MS and FTIR) are available from the corresponding author.

Enzymes and Chemicals
Recombinant HIV-1 Protease, substrate peptides and acetyl pepstatin were purchased from Sigma Chemical Co., St.Louis, USA.Recombinant HIV-1 Integrase was expressed in Escherichia coli, purified and stored at -80 o c until use.

Assay of HIV-1 Protease Inhibitory activity
This assay was carried out according to the protocol reported by Min et al, 12 with minor modifications.The recombinant HIV-1 Protease enzyme solution was diluted with a buffer consisting of 50 mM sodium acetate, 1 mM ethylene diamine disodium and 2 mM 2 -Mercaptoethanol.The above mentioned reagents were mixed with glycerol in the ratio 3:1.The sequence of substrate peptide was Arg-Val-Nle-(pNO 2 -Phe) Glu-Ala-NH 2. This substrate peptide was diluted with 50 mM sodium acetate buffer.2 µl of the plant extract and 4 µl of HIV-1 Protease solution were then added to the solution containing 2 µl of 50 mM buffer solution and 2 µl of substrate solution.The reaction mixture was incubated for a period of 1 hour at 37 o c.A control reaction was carried out under similar conditions but without the plant extract.The reaction was brought to a halt by heating the reaction mixture at a temperature of 900 o c for 1 minute.Subsequently, 20 µl of sterile water was added.An aliquot of 10 ml was then analyzed by HPLC (RP-18 column, 4.6 X 150 mm ID, Supelco 516 C-18, USA). 10 µl of the reaction mixture was injected to the column and gradiently eluted with acetonitrile (10-40%) and 0.2% tifluroacetic acid in water.The elution profile was carried out at 280 nm.The inhibitory activity of HIV-1 Protease was calculated as follows:

% inhibition = (A control-A sample) X 100 /A of control
Where, A is the relative peak of the product hydrolysate.Acetyl pepstatin was used as a positive control.

Multiplate Integration Assay
This assay has been carried out according to the method described by Tewtrakul et al., 13 A mixture consisting of 12 µl Integrase buffer, 75 mM Mncl 2 , 5 mM dithiothritol, 25% glycerol and 500 µg/ml bovine serum albumin along with 1 µl of 5 pmol/ml digoxigenin -labeled target DNA and 32 µl of sterilized water were added into each well of a 96 -well microtiter plate.To this reaction mixture, 6 µl of plant extract and 9 µl of 1/5 dilution of Integrase enzyme was added.Following this, the microtiter plate was incubated at a temperature of 37 0 c for 80 minutes.After this, wells were washed with Phosphate Buffered Saline four times.Following this, 100 µl of 500 mU/ml alkaline phosphatase labeled anti-digoxigenin antibody was added and again incubated for 1 hour at 37 0 c.To this, 150 µl of Alkaline phosphatase buffer was added to each of the well and incubated for 1 hour at 37 \0 c.The absorbance of the plate was measured at 405 nm using a microplate reader.A control reaction was carried out consisting of a reaction mixture of 60% DMSO and Integrase enzyme.Sumarin, which is a polyanionic inhibitor of integrase enzyme was used as a positive control.% inhibition against HIV-1 Integrase was calculated as follows:

Free radical scavenging activity DPPH radical scavenging activity
1,1 -diphenyl-2-picryl hydrazyl (DPPH) is a stable free radical which is extensively used to determine the free radical scavenging ability of various compounds.It is known to have maximum absorbance at 515 nm.The absorbance decreases when antioxidants donate protons to DPPH.The DPPH radical scavenging activity was assayed according to the protocol of Sultanova et al. 14 The reaction mixture consisted of 300 µm DPPH solution in methanol dissolved in different concentrations of the plant extract.The absorbance was detected at 515 nm after incubation for 20 minutes at room temperature.The radical scavenging activity was calculated as a % of the reduction in radicals.The assay was carried out in triplicates.Ascorbic acid was used as reference compound.The % of radicals scavenged was calculated using the formula:

Metal Chelation assay
The chelating potential of the plant extract for metal ions such as ferrous ions (Fe 2+ ) was tested by adding 100 µl of different concentrations of the plant extract to 135 µl of distilled water.To this, 2 mM Fecl 2 , 5 mM ferrozine was also added.The reaction mixture was incubated for 10 minutes and absorbance measured at 562 nm.EDTA was used as control.The % chelation of the plant extract for Fe 2+ was calculated as:

% chelation = (A 0 -A 1 )/A 0 X 100
Where A 0 is the absorbance of the control and A 1 is the detected absorbance in presence of the plant extract. 15

Superoxide radical scavenging assay
To check the ability of the plant extract to scavenge superoxide anion radicals, an in vitro non -enzymatic phenazine methosulphate-nicotinamide adenine dinucleotide (PMS -NADH) system was generated.This system consisted of a reaction mixture of PMS, NADH and oxygen.It was assayed by the reduction of nitroblue tetrazolium (NBT).The absorbance was measured at 560 nm.BHA was used as control. 16,17e % scavenging of superoxide anion radical was calculated as follows:

Lipid peroxidation assay
Freshly excised goat liver was processed to obtain 10% homogenate in cold phosphate buffered saline using a homogenizer.It was then filtered to obtain a clear homogenate.The degree of lipid peroxidation was assayed by the TBARS. 18The standard method of Ohkawa et al., 19 with minor modifications was followed.Different concentrations of the plant extract (10-40 ppm) in water were added to the liver homogenate.The reaction was initiated by the addition of 100 µl of 15 mM FeSO 4 solution to 3 ml of liver tissue homogenate.After 30 minutes, 100 µl of this reaction mixture was transferred into a test tube containing 1.5 ml TBA in 50% acetic acid.After this, the reaction mixture was heated for 30 seconds on a boiling water bath.A pink colored complex was formed, the absorbance of which was measured at 535 nm.% of inhibition of lipid peroxidation was calculated as follows:

Total flavonoid content
The content of total flavonoid was determined according to the protocol of Sudha et al. 20 The absorbance was measured against the blank at 510 nm.Quercitin was used as reference standard.The results were expressed as mg of Quercitin equivalent per gram of dry weight.

Total flavonol content
The content of total flavonol was assessed as described by Miliauskas et al. 21Absorbance was measured at 510 nm using a UV-Visible Spectrophotometer.The total flavonol content was expressed as mg of Quercitin equivalent per gram of plant extract.

Total phenolic content
The total phenolic content was determined by using Folin-Ciocalteu reagent.In the procedure followed, 200 µl of the plant extract was mixed with 1.5 ml of Folin-Ciocalteu reagent.After 5 minutes, 1 ml of saturated Na 2 CO 3 was added to the mixture.The mixture was then incubated for 90 minutes at room temperature in dark.The absorbance was measured at 725 nm against the blank. 22The content of total phenolics was expressed as mg of Gallic acid equivalent (GAE) per gram of dry weight.

Statistics
Statistical analysis for the results of HIV-1 Protease inhibitory activity was expressed as mean ± S.D. of three determinants.Whereas, the results of HIV-1 integrase inhibitory activity were expressed as mean ± S.D. of four determinants.For the antioxidant assays, the statistical comparison among the groups were performed with one way ANOVA followed by Tukey's HSD as Post Hoc Test (Whenever F value was significant) using statistical presentation system software version. 16.

RESULTS AND DISCUSSION
From the methanol extract of the leaves of T.paniculata, a polyphenol compound Ellagic acid was isolated.Ellagic acid isolated from Tristaniopsis species has shown to possess antiplasmodial activity, 23 antioxidant activity. 24Derivatives of Ellagic acid isolated from Terminalia chebula Retz has shown to downregulate the expression of quorum sensing genes to attenuate Pseudomonas aeruginosa. 25Ellagic acid is a polyphenolic compound.It is a dimeric derivative of Gallic acid.In the combined form it exists with its precursor hexahydroxydiphenic acid or in the bound form as Ellagitannins.Ellagic acid has been known to possess antimutagenic, antiviral, skin whitening and antioxidative properties. 26It is of high importance to note that Ellagic acid has been approved for use as a food additive in Japan for its functioning as an antioxidative agent. 27It can hence be said that Ellagic acid is completely safe for human consumption.Ellagic acid is effective against several carcinogens such as nitrosamines, azoxymethane, mycotoxins and polycyclic aromatic hydrocarbons.
Table 1 shows the antioxidant activites of the methanol extract versus Ellagic acid and the reference compound used for the study.Ellagic acid isolated from T. paniculata leaves was a potent DPPH radical scavenger.The radical quenching ability of the methanol extract against DPPH radical at 40 ppm was found to be 92.2 ± 0.03% (Figure 3) whereas that of Ellagic acid was 88.3 ± 0.36%.
Metal ion chelating activity of an antioxidant molecule prevents the generation of oxy radicals and its consequent oxidative damage.Chelation of metal ion decreases the concentration of the catalyzing transition metal in lipid peroxidation. 28The metal ion chelating potential (%) of the methanol extract, Ellagic acid and EDTA at 40 ppm were 97.1 ± 0.12, 88.1 ± 0.41 and 91.2 ± 0.06 respectively (Figure 4).
Superoxide is the most important radical produced in vivo in the mitochondria.During electron transfer chain, it regularly leaks out of mitochondria.The superoxide anion scavenging potential (%) of methanol extract, Ellagic acid and BHA at 40 ppm were found to be 78.1 ± 0.09, 68.02 ± 0.12 and 91.03 ± 0.03 respectively (Figure 5).Though, superoxide anion is a weak oxidant, it is capable of giving rise to potentially dangerous hydroxyl radicals and singlet oxygen.Both of these are known to trigger oxidative stress.Hence, the scavenging of superoxide radical by antioxidants has physiological implications. 29,30e oxidation of lipids or lipid peroxidation is a crucial step in the pathogenesis of several diseases as has been evident by several studies in recent years.Lipid peroxidation is a     naturally occurring process happening in small amounts in the body.It mainly occurs by the effect of several reactive oxygen species.These reactive oxygen species act by readily attacking the polyunsaturated fatty acids in the fatty acid membrane.This initiates a self-propogating chain reaction.The destruction of membrane lipids coupled with the end products of such lipid peroxidation reactions are especially dangerous for the viability of cells and tissues (Figure 6).
The methanol extract was evaluated for the concentration of total phenolics, total flavonoids and flavonols.The content of total phenolics was found to be 59.5 mg GAE/g extract.The flavonoid and flavonol content was found to be 27.5 mg and 13.2 mg GAE/g extract respectively.
The results of the present investigation clearly indicate that the isolated compound is most potent against HIV-1 Integrase with an IC 50 value of 8.7 µm/ml and against HIV-1 Protease with an IC 50 value of 70.4 µm/ml respectively (Figure 1 and Figure 2).In our previous study, the anti-HIV-1 activity of several Indian medicinal plants has been reported, 31 but the active fractions were not characterized.In this regard, the present study was designed to isolate the most potent active component from the numerous plants which we screened for anti-HIV-1 activity.

Figure 4 :
Figure 4: Metal ion chelating activity of Ellagic acid Each value is expressed as mean ± S.D. (n=3).