Anti-Oxiflammatory Profile of Date Extracts ( Phoenix sylvestris )

— Fruit of date palm ( Phoenix sylvestris L.) is edible and used as an anti-geriatric, anti-oxidant ethnomedi-cine. In this study, three different types ofdateextracts, methanolic, acidic ethanolic and basic ethanolicwere evaluated for their putative in vitro scavenging effects on reactive oxygen species (ROS), where scavenging of hydroxyl radicals (basic ethanolic>acidic ethanolic>methanolic), superoxide radicals (acidic ethanolic>basic ethanol-ic>methanolic), DPPH radicals (acidic ethanolic>methanolic>basic ethanolic), nitric oxide (NO) (methanolic>acidic ethanolic>basic ethanolic) and inhibition of lipid peroxidation (basic ethanolic>acidic ethanolic> methanolic) were found to occur in a dose dependent manner. Their flavonoid and phenolic contents proved to be the source of this potent free radical scavenging activity and indicated a direct correlation with their total anti-oxidant capacity. The date extracts have a positive therapeutic effect on bacterial lipopolysaccharide (LPS) - treated human embryonic kidney cell line (HEK) and on murine RAW macrophages. They significantly inhibit intracellular oxidative stress. This rein-statement of cellular homeostasis presumably occurs via mitochondrial pathways. activity (Hydroxyl radical scavenging assay shows that, in this system, the hydroxyl radical-scavenging activities of the three date palm extracts are in this order: basic ethanolic extracts>acidic ethanolic extract>methanolic extract). Various concentrations of date palm extracts, collected by three distinct methods, scavenge hydroxyl radical in a dose-dependent manner [r 2 =0.832 (p<0.01) for methanolic extract; r 2 = 0.852 (p<0.01) for acidic ethanolic extract; r 2 =0.882 (p<0.01) for basic ethanolic extract] As per IC 50 values basic ethanolic extract is more potent to scavenge hydroxyl radicals (IC 50 17.00 µg/ml) than the acidic ethanolic extracts (IC 50 17.30 µg/ml) and methanolic extracts (IC 50 18.20 µg/ml). Ascor- bic acid has been shown as positive control.


INTRODUCTION
One of the most primitive and yet effective mecha--nisms the mammalian physiology adopts in an exigent situation, which although predominantly non---specific is remarkably fast and conclusive for the next phases of immune response or structural tissue remodeling, is inflammation. Inflammatory response occurs in a phase---wise manner (Crunkhorn and Meacock, 1971). Inflammation is the principal and sometimes critical initiator of most disorders. A cross---talk between the structural and immune cells causes destruction on the one hand and healing or reconstruction on the other. In diseases such as asthma, rheumatoid arthritis, pso--riasis, multiple sclerosis, obesity and inflammatory bowel disease (Winter et al., 1962), the first phase of disease onset, followed by establishment, develop--ment, maintenance and exacerbations, leads to a com--pletion of various steps that are characteristic of the particular inflammatory disease. Although specific characteristics of inflammatory response in each dis--ease and their site ofoccurrence may vary, a universal feature governing this step---wise phenomenon is the complex interplay amongstthe various cell subsets of the inflammatory cascade and tissue resident cells, and the network of signaling governing the two (Vinegar et al., 1969;Winter et al., 1962), that affect various target tissues (Cortran, 2010).
Unmet needs in medicine and unknown phenomena prevailing in the mechanism of disease onset remain (Mohan, 2010). In traditional medicine, that haspro--vided solution to prevailing health issues at a global level, medicinal plants continue to provide valuable therapeutic agents. To avoid and mitigate various side effects and complications of modern medicine, and to address unmet needs of diseases, especially in the con--text of emerging complex etio---pathophysiological pathways, traditional medicine is gaining importance and is now being studied systematically using bio--technological tools, to find the scientific basis of their therapeutic actions.
Phytochemicals from fruits and other edible plant parts have been shown to possess significant antioxi--dant properties that may be associated with lower in--cidence and lower mortality rates of degenerative dis--eases in human. Different biological properties, anti--oxidant capacities and radical---scavenging activities of various herbal extracts have been widely demonstrat--ed, using in vitro techniques and in vivo models by different groups of researchers (Amann et al., 1995;Chatpalliwar VA, 2002). The anti---proliferative and anti---inflammatory activities of these herbal extracts have been documented in human oral, breast, colon, cervical, and prostate cancer cell lines as well as in preclinical animal models by attenuating certain inflammatory intermediates, including nitric oxide, NF---kB, and TNFα (Miles and Miles, 1952;Whittle, 1964).
This study was designed to identify anti---inflammatory potential in extracts from datewhich is a well--knownethno---medicine with high nutritive value. Among its contents, large amounts of several phenolic and non---phenolic compounds and other uncharacter--ized moieties may contribute to its use as a food sup--plement, a functional food or a nutraceutical sub--stance with prophylactic and therapeutic functions in oxidative inflammatory diseases. Anti---oxidative and anti---inflammatory activities of three types of date palm extracts, namely methanolic, basic ethanolic and acidic ethanolic, have been assessed, compared and validated, using both murine and human cell lines, in which inflammation has been induced by E. coli LPS, a potent pro---inflammatory agent. In addition, intracel--lular cell organelle specific targeting of this extract has also been assessed in order to seek information re--garding its mode of action in modulating the inflam--matory cascade in a biological system, which is the most critical part for any drug discovery programme addressing inflammatory disorders.

Extraction of date
Mesocarp tissue of date fruit was macerated and treat--ed with 50% water---methanol solution at 4 0 C. This was then divided into two parts: part A was evaporated to dryness, the remaining part was rinsed with water, dried at 50 0 C, alkaline hydrolyzed with sodium hy--droxide (0.1M) and filtered. The filtrate was acidified at pH 2.0, extracted with ethyl acetate and rinsed with 1% sodium bicarbonate and water. The organic layer was evaporated to dryness and then dissolved in wa--ter, to give part B. The residue after filtering out part B was alkaline hydrolyzed with 2M sodium hydroxide and filtered. The filtrate was further acidified at pH 2, extracted with ethyl acetate, rinsed with 1% sodium bicarbonate and water, and the organic layer evapo--rated to dryness (part C) (Fig.  1).

DPPH radical---scavenging activity
The antioxidant activity of the extracts was measured on the basis of the scavenging activity of the stable 1,1--diphenyl---2---picrylhydrazyl (DPPH) free radical (Braca et al., 2001). Aqueous extract was added to a 0.004% Methanol solution of DPPH on a 96 well ELISA plate. Absorbance at 517 nm was determined after 30 min, and the percent inhibition activity was calculated.

Assay of superoxide radical O2 ---°s cavenging activity
The method used by Martinez et al. (Martinez et al., 2001) for determination of the superoxide dismutase was studied in the riboflavin---light--nitrobluetetrazolium (NBT) system (Beauchamp and Fridovich, 1971). Each 0.1 ml of reaction mixture con--tained 50 mM phosphate buffer (pH 7.8), 13 mM me--thionine, 2 µμM riboflavin, 100 µμM EDTA, NBT (75 µμM) and various doses of sample solution. The pro--duction of blue formazan was followed by monitoring the increase in absorbance at 560 nm after 15 min of illumination from a fluorescent lamp.

Assay of hydroxyl radical (---OH)---scavenging activity
The assay was based on the benzoic acid hydroxyla--tion method (Chung et al., 1997). Hydroxyl radicals were generated by direct addition of iron(II) salts to a reaction mixture containing phosphate buffer. In a 24 well plate, 0.15 ml of sodium benzoate (10 mM) and 0.15 ml of FeSO4.7H2O (10 mM) and EDTA (10 mM) were added. Then the sample solution and a phos--phate buffer (pH 7.4, 0.1 M) were added to give a total volume of 1.6 ml. Finally, 0.15 ml of an H2O2 solution (10 mM) was added. The reaction mixture was then incubated at 37 0 C for 2 h. After that, the fluorescence was measured at 407nm emission (Em) and excitation (Ex) at 305 nm. Measurement of spectrofluorometric changes has been used to detect the damage by the hydroxyl radical.

Lipid peroxidation assay
A modified thiobarbituric acidreactive species (TBARS) assay (Ohkawa et al., 1979) was used to measure the lipid peroxide formed, using egg yolk homogenates as lipid---rich media (Ruberto et al., 2000), where lipid peroxidation was induced by FeSO4.Malondialdehyde (MDA), produced by the oxi--dation of polyunsaturated fatty acids, reacts with two molecules of thiobarbituric acid (TBA), yielding a pinkish red chromogen with an absorbance maximum at 532 nm, which was measured using a 96 well ELISA plate reader. Percentage inhibition of lipid peroxida--tion by different concentrations of the extract was cal--culated.

Nitric oxide (NO) scavenging activity
Nitric oxide was generated from spontaneous decom--position of the Sodium nitropruside (20mM) in phos--phate buffer (pH 7.4) which interacts with oxygen molecule to produce nitrite ions, which can be meas--ured by the Griess reactions. The nitric oxide scaveng--ing activity of the date extracts, collected by three dif--ferent methods was determined by Shirwaikar et al (2006) with a slight modification. Briefly a stock solu--tion of each extract was prepared to contain 1 mg/ml. Different amounts of the stock solution were then transferred to different test tubes and volume was ad--justed to 1ml by adding double distilled water. 0.2 ml of sodium nitroprusside (20mM) in PBS (pH 7.4) and 1.8 ml of PBS solution was added and incubated at 37°C for 3 h. 1 ml of each solution was taken and di--luted with 1 ml of Griess reagent [1% sulfanilamide, 5% H3PO4and 0.1% N---(1---naphthyl)ethylenediamine]. Similarly, a blank was prepared containing the equivalent amount of reagents (only the sodium ni--troprusside and PBS), but without the extract. The ab--sorbances of these solutions were measured at 540 nm against the corresponding blank solution. Ascorbic acid (100 µμg/ml) was used as the positive control. The percentage inhibition of nitric oxide was calculated.

Determination of total antioxidant capacity
The assay is based on the reduction of Mo (VI) to Mo (V) by the extract and subsequent formation of a green phosphate/Mo(V) complex at acid pH (Prieto et al., 1999). Each well of a 96 well ELISA plate containing extract and reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molyb---date) were incubated at 95 0 C for 90 min. After the mix--ture had cooled to room temperature, the absorbance of each solution was measured at 695 nm against a blank. The antioxidant capacity was expressed as ascorbic acid equivalent (AAE).

Determination of reducing power
The reducing power of date extracts was determined according to the method (Salah et al., 1995) where dif--ferent concentrations of extracts were mixed with phosphate buffer and potassium ferricyanide. The mixture was incubated at 50°C for 20 min. 2.5 ml of trichloroacetic acid was added to the mixture. The up--per layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and FeCl3, and the absorbance was measured at 700 nm. Increased absorbance of the reac--tion mixture indicated increased reducing power. Ascorbic acid was used as positive control.

Determination of total flavonoid content
Total flavonoid content was determined using alumin--ium chloride (AlCl3) according to a known method, using Fisetin as a standard. The date extracts (0.1 ml) were added to 0.3 ml distilled water followed by 5% NaNO2 (0.03 ml). After 5 min at 25°C, AlCl3 (0.03 ml, 10%) was added. After further 5 min, the reaction mix--ture was treated with 0.2 ml of 1 mM NaOH. Finally, the reaction mixture was diluted to 1 ml with water and the absorbance was measured at 510 nm. The re--sults were expressed as mg Fisetin/g date extract.

Determination of total phenolic content
The total phenolic content of the date extracts was de--termined using the Folin---Ciocalteu reagent. The reac--tion mixture contained: 200 µμl of diluted extract, 800 µμl of freshly prepared diluted FolinCiocalteu reagent and 2 ml of 7.5% sodium carbonate. The final mixture was diluted to 7 ml with deionized water. Mixtures were kept in dark at ambient conditions for 2 h to complete the reaction. The absorbance at 765 nm was measured. Gallic acid was used as standard and the results were expressed as mg gallic acid (GAE)/g of the date extract.

UV based spectrophotometric Analysis:
The samples of various extracts was analysed using UV---Vis Spectrophotometer. About 40 mg of the air--dried samples was dissolved in 1 ml methanol, diluted to 1 mg/ml solution in different volumetric flasks and then applied on the spectrophotometer and scanned through the UV and visible region. The herbal func---tional groups were determined by analyzing the peaks.
Cell culture RAW 267.4 murine macrophage cell line and HEK 293 cell line were obtained from NCCS, Pune, India. The cells were grown in DMEM medium containing 5% inactivated fetal bovine serum, penicillin (100 U/mL), and streptomycin (20 µμg/mL) and kept at 37°C in a T---25 tissue culture flasks. Cell were grown to confluence in a humidified atmosphere containing 5% CO2.

Cell viability using MTT assay
To test the cytotoxicity, 5×10 4 cells/well were seeded in a 96---well plate and incubated for 24 h with different concentrations of the date extract. The cells were washed, and each well was filled with 100 µμL of me--dium and 10 µμL of a tetrazolium salt, MTT. The plate was incubated for various time periods, and the ab--sorbance was measured at 540 nm. The percentage of viable cells was calculated using the absorbance of the control cells without extract as 100%. The assay was performed in triplicate twice.

HPLC analyses: Crude methanolic A, basic ethanolic B
& acidic ethanolic C were filtered by 0.22 µμm syringe filter (Milipore, Germany), and then the extracts were--analysed with RP---HPLC by using Waters 515 System with C---18 column as stationary phase. The mobile phase was Water (HPLC grade) and 90% acetonitrile. Sample volume was 100 µμl. Flow rate was 0.5ml/min. TLC analyses: Methanolic extract (A), Acidic Ethanolic extract (B) and Basic ethanolic extract (C) were applied on silica gel coated TLC plates (Milipore, Germany) by using capillary tubes and developed in a TLC chamber using mobile phase methanol:water:chloroform (2.5:0.2:7.3 v/v). The developed TLC plates were air dried and observed under ultra violet light UV at both 254 nm and 366 nm. TLC plates were sprayed with Libermann's solution, heated at 100-105°C and visual--ized under daylight. The movement of the analyte was expressed by its retention factor (Rf).

Methods
Taking 0% inhibition in the mixture without plant ex--tract, regression equations were prepared from the concentrations of the three date extracts which were collected by three different methods and percentage inhibition of free radical formation/prevention in dif--ferent systems of assay were calculated, viz. DPPH assay, superoxide radical---scavenging assay, hydroxyl radicals scavenging assay, lipid peroxidation assay and nitric oxide radicals scavenging assay. IC50 values (concentration of sample required to scavenge 50% of available free radicals or to prevent lipid peroxidation by 50%) were calculated from these regression equa--tions. IC50 value is inversely related to the activity of the extracts.

Assay of hydroxyl radical (OH)---scavenging activity
Superoxide radical was measured by the NBT reduc--tion assay. Each well of a 96---wells plate was seeded with RAW 264.7 macrophages suspension containing .5×10 4 cells/ml. The treatment of cells proceeded as described previously. After incubation, 40 µμL of a NBT solution at 1 mg/mL was added to the medium and incubated at 37°C, for 1 h. Then, the incubation medium was removed and cells were lysed with DMSO:2 M NaOH (1:1). The absorbance of reduced NBT, formazan, was measured at 620 nm, in a micro--plate reader (Multiskan ASCENT Thermo®).

NO estimation
In culture, the NO released by the macrophages into the medium is converted to several nitrogen deriva--tives, from which only nitrite is stable, being easily measured by Griess reagent (1.0% sulphanilamide and 0.1% N---(1)---naphthylethylenediamine in 5% phosphor--ic acid). After incubation, 100 µμL of culture medium supernatant was mixed with the same volume of Griess reagent, during 10 min, at room temperature. The nitrite produced was determined by measuring the optical density at 540 nm, in a microplate reader (Shimadzu).

ROS measurement
Intracellular formation of ROS was assessed by using oxidation sensitive dye DCFH---DA as a substrate (Wijesinghe et al., 2011). RAW macrophage cells were seeded in 24---well black plate at a concentration of 5x10 4 cells/ml. Cells were treated with 1 µμg/ml of E.coli LPS and then therapeutically various concentrations of date extracts were added on to it and incubated for 6 h. Negative control cells, i.e. only LPS treated as well as cells without any treatment were incubated for the same time period and then washed in PBS and after the addition of DCF---DA (5 µμg/mL) incubate for 30 min at 37 o C in dark. Non---fluorescent DCFH---DA dye, that freely penetrate into cells get hydrolyzed by intracel--lular esterase to 207---dichlorofluorescin (DCFH), and is trapped inside the cells. The formation of 207--dichlorofluorescin (DCF) due to oxidation of DCFH in the presence of ROS was read after 30 min at an excita--tion wavelength of 485 nm and emission wavelength of 525 nm using a spectrofluorometer. Percent scav--enging power of hydroxyl, superoxide and peroxide radicals was computed taking that by Ascorbic acid as 100%.

Measurement of mitochondrial membrane potential (MMP)
Mitochondrial membrane potential was monitored by the fluorescent dye, Rhodamine123. It is a cell perme--able cationic dye that preferentially enters into mito--chondria based on highly negative mitochondrial membrane potential (Wm). Depolarization of MMP results in the loss of Rhodamine 123 from the mito--chondria and a decrease in intracellular fluorescence intensity. After the addition of 1 µμg/ml E.coli LPS, var--ious concentrations of dateextracts were added to RAW macrophages cell line and incubated for 6 h in 37 o C. After incubation, the cells werewashed twice in cold PBS, then Rhodamine 123 (10 µμM) was added and incubated for 30 min at 37 o C in dark. Fluorescence was measured by spectrofluorometer with an excita--tion wavelength of 485 nm and emission wavelength of 525 nm.

TLC of Khejur Extracts
Methanolic extract (A), Acidic Ethanolic extract (B) and Basic ethanolic extract (C) were applied on silica gel coated TLC plates (Milipore, Germany) by using capillary tubes and developed in a TLC chamber using mobile phase methanol:water:chloroform(2.5: 0.2:7.3 v/v). The developed TLC plates were air dried and observed under ultra violet light UV at both 254 nm and 366 nm. TLC plates were sprayed with Liber--mann's solution, heated at 100-105°C and visualized under daylight. The movement of the analyte was ex--pressed by its retention factor (Rf).

HPLC Analysis
Crude methanolic A, basic ethanolic B & acidic etha--nolic C were filtered by 0.22 µμm syringe filter (Mili--pore, Germany), and then the extracts were analysed with RP---HPLC by using Waters 515 System with C---18 column as stationary phase. The mobile phase was Water (HPLC grade) and 90% acetonitrile. Sample volume was 100 µμl. Flow rate was 0.5 mL/min.

Statistical analysis
Statistical differences among samples were tested by Student t---test. A P---value less than 0.05 or 0.01 (as ap--plicable vis---à---vis the assay performed) was considered statistically significant.

RESULTS
Taking 0% inhibition in the mixture without plant ex--tract, regression equations were prepared from the concentrations of the three date extracts which were collected by three different methods and percentage inhibition of free radical formation/prevention in dif--ferent systems of assay were calculated, viz. DPPH assay, superoxide radical---scavenging assay, hydroxyl radicals scavenging assay, lipid peroxidation assay and nitric oxide radicals scavenging assay. IC50 values (concentration of sample required to scavenge 50% of available free radicals or to prevent lipid peroxidation by 50%) were calculated from these regression equa--tions. IC50 value is inversely related to the activityof the extracts (Fig.  2).
Various concentrations of date extracts, collected by three distinct methods, required to scavenge 50% free radical or to prevent lipid peroxidation by 50%. IC50 value is inversely related to the activity of the extracts. Both acidic and ethanolic date extracts are more active in scavenging the superoxide ions in anin vitro assay system than the methanolic date extracts, where as there is no significant variation in any of the three ex--tracts in scavenging the hydroxyl radical. The DPPH radical scavenging and nitric oxide scavenging activi--tiesare greater in acidic ethanolic extracts in compari--son to the methanolic extracts and basic ethanolic ex--tracts respectively. Interestingly prevention of lipid peroxidation activity is highest in basic ethanolic ex--tracts than the acidic ethanolic extracts and methanolic extracts. So, the ability to scavenge various free radi--cals or to prevent lipid peroxidation is different in all three extracts and it exerts the activity in a dose de--pendent manner. ods, required to scavenge 50% free radical or to prevent lipid peroxidation by 50%. IC 50 value is inversely related to the activity of the extracts where both acidic and ethanolic date palm extracts are more active to scavenge the superoxide ions in a in vitro assay system than the methanolic date palm extracts, where as there is no significant variation in all these three extracts to scavenge the hydroxyl radical but for the DPPH radical scavenging activity and Nitric oxide scavenging activity is greater in acidic ethanolic extracts in comparision to the methanolic extracts and basic ethanolic extracts respectively. Interestingly prevention of lipid peroxidation activity is highest in basic ethanolic extracts than the acidic ethanolic extracts and methanolic extracts respectively. So the scavenging power of various free radicals or to prevent the lipid peroxidation activities is different in all these three extracts and it exherts the activity in a dose dependent manner which has been shown in latter.

Met-OH Acidic-EtOH Basic-EtOH
Antioxidants, on interaction with DPPH transfer an electron (hydrogen atom) to DPPH, neutralizing its free radical character (Naik et al., 2003). The colour changes from purple to yellow and its absorbance at wavelength 517 decreases. Various concentrations of date extracts, collected by three distinct methods, quenched DPPH free radical in a dose---dependent manner [r 2 =0.909 (p<0.01) for methanolic extract; r 2 = 0.933 (p<0.001) for acidic ethanolic extract; r 2 =0.930 (p<0.001) for basic ethanolic extract]. IC50 values were 22.91 µμg/ml for methanolic extract, 14.61 µμg/ml for acidic ethanolic extract and 36.44 µμg/ml for basic etha--nolic extract. DPPH assay shows that, in this system, the radical---scavenging activities of the three varieties  . Hydroxyl radical (OH) scavenging activity (Hydroxyl radical scavenging assay shows that, in this system, the hydroxyl radical-scavenging activities of the three date palm extracts are in this order: basic ethanolic extracts>acidic ethanolic extract>methanolic extract). Various concentrations of date palm extracts, collected by three distinct methods, scavenge hydroxyl radical in a dose-dependent manner [r 2 =0.832 (p<0.01) for methanolic extract; r 2 = 0.852 (p<0.01) for acidic ethanolic extract; r 2 =0.882 (p<0.01) for basic ethanolic extract] As per IC 50 values basic ethanolic extract is more potent to scavenge hydroxyl radicals (IC 50 17.00 µg/ml) than the acidic ethanolic extracts (IC 50 17.30 µg/ml) and methanolic extracts (IC 50 18.20 µg/ml). Ascorbic acid has been shown as positive control.

Assay of hydroxyl radical (OH)---scavenging activity
By the addition of iron (II) salts to a phosphate buffer containing reaction mixture, hydroxyl radicals can be generated (Gutteridge, 1983). Benzoate, weakly fluo--rescent, after monohydroxylation forms highly fluo--rescent hydroxybenzoates (Gutteridge, 1987). Meas--urement of this spectrofluorometric change has been used to detect damage by hydroxyl radical. Date ex---tracts collected by three distinctly separate methods was found to be a powerful scavenger of hydroxyl radicals. There is a linear correlation between concen--tration of extract and OH---scavenging activity [r 2 =0.832 (p<0.01) for methanolic extracts of date; r 2 =0.835 (p< 0.01) for acidic ethanolic extracts of date; r 2 = 0.882 (p<0.01) for basic ethanolic extracts of date]. IC50 values are 18.20 µμg/ml for methanolic date extracts, 17.30 µμg/ml for acidic ethanolic date extracts and 17.00 µμg/ml for basic ethanolic extracts. Highest hydroxyl radical---scavenging activity was found in the date ex--tract collected by using basic ethanolic methods. The Figure 5. Assay of superoxide radical O 2 -°s cavenging activity (Superoxide radical scavenging assay shows that, in this system, the superoxide radical-scavenging activities of the three date palm extracts are in this order: acidic ethanolic extracts>basic ethanolic extract>methanolic extract). Various concentrations of date palm extracts, collected by three distinct methods, scavenge Superoxide radical in a dose-dependent manner [r 2 =0.809 (p<0.01) for methanolic extract; r 2 = 0.908 (p<0.01) for acidic ethanolic extract; r 2 =0.928 (p<0.01) for basic ethanolic extract] As per IC 50 values acidic ethanolic extract is more potent to superoxide radicals (IC 50 19.00 µg/ml) than the basic ethanolic extracts (IC 50 19.15 µg/ml) and methanolic extracts (IC 50 28.88 µg/ml). Figure 6. Nitric oxide (NO) scavenging activity (Nitric oxide scavenging assay shows that, in this system, the NO radical-scavenging activities of the three date palm extracts are in this order: methanolic extracts>acidic ethanolic extract>basic ethanolic extract). Various concentrations of date palm extracts, collected by three distinct methods, scavenge produced NO radicals, Sodium nitropruside used as substrate, in a dose-dependent manner [r 2 =0.852 (p<0.01) for methanolic extract; r 2 = 0.857 (p<0.01) for acidic ethanolic extract; r 2 =0.900 (p<0.01) for basic ethanolic extract] As per IC 50 values methanolic date palm extract is more potent to NO radicals (IC 50 5.505 µg/ml) than the acidic ethanolic extracts (IC 50 5.53 µg/ml) and basic ethanolic extracts (IC 50 5.60 µg/ml) . But there is no significant difference in all these three different extracts of date palm to scavenge the Nitric oxide in an in vitro system. 10 µ µg/ml 50 µ µg/ml 100 µ µg/ml 250 µ µg/ml 1 mg/ml NO scavenging %

Nitric oxide (NO) scavenging activity
The calculated IC50 values of all three different date extracts suggested that methanolic extract is the most   10µ µg/ml 50µ µg/ml 100µ µg/ml 250µ µg/ml 1mg/ml

Met-OH Acidic Et-OH Basic Et-OH
potent extract to scavenge the NO molecules followed by acidic ethanolic extracts and basic ethanolic ex--tracts. But surprisingly there is little or no significant variation of NO radical scavenging properties of all three dateextracts (Fig.  6).

Lipid peroxidation inhibition assay
Egg yolk lipids undergo rapid non---enzymatic peroxi--dation when incubated in the presence of ferrous sul--phate. Lipid peroxides are likely involved in numer---  where as the total Phenolic content was determined using the Folin-Ciocalteu reagent. (n=5) results were expressed as mg of Flavonoid contents in fisetin/g of extracts and mg of gallic acid/ 10 mg of the extract, taking ±SD. Total Flavonoid and Phenol contents show a direct corelation with the total anti oxidant capacity of the tested compounds. Acidic ethanolic extract is rich in both Phenolic and Flavonoid contents, and it can be correlated to it's radical scavenging activities and total antioxidant properties as the data has shown, than the other two tested extracts of date palm. Figure 11. UV based spectrophotometric Analysis. UV-VIS based spectrophotometric analysis reveal that acidic and basic ethanolic extracts contain maximum amounts of phenolic compounds which comes under the UV zone where the methanolic extracts shows a very poor quantities of these compounds in this area under study. Significantly the acidic ethanolic extracts indicates that it contain more phenolic and flavonoid contents than the standard control ascorbic acid solution which has been correlated to the radical scavenging activities, lipid peroxidation inhibitory potential, total phenolic and flavonoid contents and the total anti oxidant properties also.
ous pathological events, including inflammation, met--abolic disorders and cellular aging (Ames et al., 1993;Wiseman et al., 1996) suggested that consumption of date may afford a cy--toprotective effect by lowering the lipid peroxidation level (Fig.  7).

Figure 12. Cell viability determination using MTT assay. (A) RAW 264.7 Cells were treated with 1µg/ml of E.coli LPS and then methanolic date palm extracts
were added into it therapeutically and incubated for two different time periods, 1hour and 24 hour. Taking control as 100% viability data was calculated (p<0.01). Dexamethasone (9nM) was used as positive control (a= 250,b=100,c=50,d=10 µg/ml). (B) RAW 264.7 Cells were treated with 1µg/ml of E.coli LPS and then acidic ethanolic date palm extracts were added into it therapeutically and incubated for two different time periods, 1hour and 24 hour. Taking control as 100% viability data was calculated (p<0.01). Dexamethasone (9nM) was used as positive control. (a= 250,b=100,c=50,d=10 µg/ml). (C) RAW 264.7 Cells were treated with 1µg/ml of E.coli LPS and then basic ethanolic date palm extracts were added into it therapeutically and incubated for two different time periods, 1hour and 24 hour. Taking control as 100% viability data was calculated (p<0.01). Dexamethasone (9nM) was used as positive control. (a= 250,b=100,c=50,d=10 µg/ml) (* for 1 hour group, # for 24 hour group in comparission to the untreated control group).

Figure 13. Super oxide scavenging assay, following NBT inhibition method, using three different date palm extracts show that for twenty four hour LPS induced model, both acidic ethanolic extract and methanolic extract is more capable to inhibit LPS induced inflammatory oxidative burst than the basic ethanolic tested extracts in a dose dependent manner. In lower doses basic ethanolic extract shows proinflammatory activities by inducin oxidaive sress on murine RAW macrophages. (A) Super oxide Ion scavenging activities of methanolic date palm
extracts on LPS (1µg/ml) induced RAW macrophages, after 24 hour incubation, were determined by using NBT inhibition methods, taking control group as 100% inhibition (p<0.05 in comparission to the untreated control group). (B) Super oxide Ion scavenging activities of acidic ethanolic date palm extracts on LPS (1µg/ml) induced RAW macrophages, after 24 hour incubation, were determined by using NBT inhibition methods, taking control group as 100% inhibition (p<0.05 in comparission to the untreated control group).

Determination of reducing power
The reducing power of different date extract samples using the potassium ferricyanide reduction method was evaluated. It has been investigated from the Fe 3+ -Fe 2+ transformation in the presence of three different extract samples. Highest activity was found in basic ethanolic extracts, followed by acidic ethanolic ex--tracts of date and the lowest activity was found in methanolic extract of date (Fig.  8).

Total antioxidant capacity (equivalent to ASA/mg of plant material)
Total antioxidant capacity of date extract is expressed as the number of equivalents of ascorbic acid. The as--say is based on the reduction of Mo(VI) to Mo(V) by the extract and subsequent formation of a green phos--phate/Mo(V) complex at acid pH. The phosphomo--lybdenum method is quantitative since the antioxi--dant activity is expressed as the number of equiva--lents of ascorbic acid (Prieto et al., 1999). Acidic etha--nolic extract had a higher capacity than the other two varieties. The results from various free radical--scavenging system revealed that the three date extract samples collected by three different methods had sig--nificant antioxidant activity. The extracts were found to have different levels of antioxidant activity in the systems tested. The antioxidant activities of the three varieties were in the order acidic ethanolic ex--- Figure 16. Intracellular ROS measurement in RAW macrophages (all three date palm extracts are capable to inhibit LPS induced oxidative stress in RAW macrophages in a dose dependent manner). Intracellular formation of ROS was assessed by using oxidation sensitive dye DCFH-DA as a substrate where non-fluorescent DCFH-DA dye, that is freely penetrate into cells get hydrolyzed by intracellular esterase to 207dichlorofluorescin (DCFH), and traps inside the cells. The formation of 207-dichlorofluorescin (DCF) due to oxidation of DCFH in the presence of ROS was read after 30 min at an excitation wavelength of 485 nm and emission wavelength of 525 nm using a spectrofluorometer. Intracellular ROS scavenging activities of three date palm extract followed a dose dependend manner where at 250 μg/ml, 100 μg/ml and 50 μg/l doses all three exracts are capable to inhibit LPS induced oxidative stress in RAW macrophages significantlly, but at lower dose, i.e. 10 μg/ml only methanolic and acidic ethanolic extracts are capable to reduce oxidative burst significantly. (*p <0.05 in comparission to the untreated control group) From this data, it can be concluded that all three date palm extracts are capable to inhibit LPS induced oxidative stress in RAW macrophages in a dose dependent manner.

Figure 17. Mitochondrial ROS measurement in RAW macrophages (all three date palm extracts are capable to inhibit LPS induced mitochondrial oxidative stress in RAW macrophages in a dose dependent manner).
Mitochondrial ROS production was monitored by the fluorescent dye, Rhodamine123 where due to oxiudative stress depolarization of mitochondrial membrane potential results in the loss of Rhodamine123 from the mitochondria and a produce the fluorescence intensity. Mitochondrial ROS scavenging activities of three date palm extract followed a dose dependent manner where at 250 μg/ml, 100 μg/ml and 50 μg/l doses all three exracts are capable to inhibit LPS induced Mitochndrial oxidative stress in RAW macrophages significantlly, but at lower dose, i.e. 10 μg/ml though they reduce mitochondria oxidative burst in comparision t the LPS treated group, but as the values are so much higher than the control group, so the values are not counted (*p <0.05 in comparission to the untreated control group). From this data, it can be concluded that all three date palm extracts are capable to inhibit LPS induced mitochondrial oxidative stress in RAW macrophages in a dose dependent manner.

Total Flavonoid and phenol content.
Total

Figure 15. Cell viability assay using three different date palm extracts show that for one hour LPS induced model, there is no significant variation to maintain cell viability of all three extracts where as in 24 hour model, both acidic and basic ethanolic extract is more capable to inhibit LPS induced inflammation than the methanolic extract in a dose dependent manner on human embryonic kidney cell lines. (A)
Cell viability determination using MTT assay. HEK 293 Cells were treated with 1µg/ml of E.coli LPS and then methanolic date palm extracts were added into it therapeutically and incubated for two different time periods, 1hour and 24 hour. Taking control as 100% viability data was calculated (p<0.01). (B) Cell viability determination using MTT assay. HEK 293 Cells were treated with 1µg/ml of E.coli LPS and then acidic ethanolic date palm extracts were added into it therapeutically and incubated for two different time periods, 1hour and 24 hour. Taking control as 100% viability data was calculated (p<0.01). (C) Cell viability determination using MTT assay. HEK 293 Cells were treated with 1µg/ml of E.coli LPS and then basic ethanolic date palm extracts were added into it therapeutically and incubated for two different time periods, 1hour and 24 hour. Taking control as 100% viability data was calculated (p<0.01). Dexamethasone (9nM) was used as positive control. (a= 250,b=100,c=50,d=10 µg/ml) (* for 1 hour group, # for 24 hour group).

UV based spectrophotometric Analysis:
When different extracts of date was analysed in a UV---VIS spectrophotometer to assess their solubility in dif--ferent solvents to determine actual active compounds remain in that solution, we got a striking result where date extracts in basic ethanolic solvents yielded best solubility in comparison to their acidic ethanolic sol--vent, whereas the methanolic extracts failed to show any such significant peak, indicating very low or no solubility of the date extract into it which states that ethanolic solvent is the best to exhibit the potent anti--inflammatory biological activities of the date which correlates with the cellular analysis further (Fig  11).

Cell viability of RAW macrophages:
In order to ascertain the anti---inflammatory activities of the date extracts, collected by three distinct meth--odologies, LPS induced preclinical in vitro models, using both RAW macrophage like murine cell lines and HEK, human embryonic kidney cell lines were used. Cells were seeded into the 96 well plate and in--cubated for 12 hours, then 1 µμg/ml LPS was added into it following the various doses of the extracts and incubated for one hour and twenty four hour time periods respectively. In both the models using RAW macrophages, three extracts possess their anti---in--flammatory activities by regulating the cell viability in a dose dependent manner where methanolic extracts are not potent to maintain cell viability in 24 hour in--flammatory models in comparison to the other two extracts where acidic ethanolic extracts retain the cell viability significantly in its lower doses, i.e. 100 µμg/ml, 50 µμg/ml, 10 µμg/ml and 1 µμg/ml respectively in both one hour and 24 hour models (Figure 12a, 12b), but the basic ethanolic extracts maintain the cell viability only in higher doses, i.e. 250 µμg/ml, 100 µμg/ml and 50 µμg/ml, where in lower doses (1 µμg/ml) it acts as pro--inflammatory causing the cell death (Fig 12c). So all these extract may act as either anti---inflammatory or pro---inflammatory depending on their doses and na--ture of extraction.

Assay of hydroxyl radical (OH)---scavenging activity
As the in vitro study suggested that all three date crude extracts have potent hydroxyl radical and su--peroxide ion scavenging activities, the ex vivo result, using RAW macrophages, also advocated the same where acidic ethanolic extract has been shown to have strongest hydroxyl radical scavenging power, >50% inhibition (Fig.  13b), followed by basic ethanolic (Fig.  13c) and methanolic extracts (Fig.  13a). This mayhave been accomplishedby modulating the mitochondria associated NADPH oxidase activity.

NO estimation
None of the tested compounds induced changes in NO basal levels, when incubated without LPS (data not shown). To evaluate the nitrosative stress, Griess reagent was used. After 24 h LPS treatment, cellular NO production increases nearly two folds (Figure  14a) in comparison to the untreated control samples, whereas the three date extracts ameliorate the LPS induced NO production to the basal level like untreat--ed group in a dose dependent manner which strongly showing there NO scavenging capabilities which is already been established by the in vitro assays. Here dexamethasone has been taken as positive control (Fig.  14a,b,c).

Cell viability of HEK 293 cells
To investigate the cytotoxicity of the three date ex--tracts MTT assay was performed as MTT is reduced bymitochondrial dehydrogenase to form formazan, an insoluble purple compound and one can measure the cytotoxicity in terms of the intensity of the purple compound. Whereas on the other hand, dead cells do not form any purple formazan because the enzyme is degraded and lack regular function. Among the three extracts of date, acidic ethanolic extracts ameliorated the LPS induced cell death to retain the cell viability in both one hour and twenty four hour inflammatory models (Fig. 15b) followed by basic ethanolic (Fig.  15c) and methanolic extracts (Fig. 15a) in a dose de--pendent manner. In lower doses all these three ex--tracts fail to inhibit LPS induced cell death where as they are active only in the higher concentrations, i.e. in 250 µμg/ml and 100 µμg/ml doses which is similar to the LPS induced inflammatory models applied on murine macrophage cell line. This data suggests that acidic ethanolic extracts contain potent anti---inflammatory compound(s) which is/are able to inhibit the LPS in--duced cytotoxicity in a dose dependent manner. Dex--amethasone was used as positive control in the entire study.

ROS measurement
The evidence presented above suggests that date ex--tracts mediates its effects through the anti---oxidant pathway. We used a DCF---DA probe to examine whether this mechanism can scavenge ROS inside the RAW macrophage cells. Cells were treated with E.coli LPS and then various concentrations of date extracts were added into it therapeutically and incubated for 6 h, and analyzed by florescent spectrophotometer. Date extracts ameliorate LPS induced ROS levels signifi--cantly over the control in a dose dependent manner. This effect was observed best in acidic ethanolic ex--tracts where it inhibited the ROS generation in higher doses, i.e. in 250 µμg/ml and 100 µμg/ml and increased ROS levels slightly thereafter. ROS levels decreased significantly in methanolic and basic ethanolic extracts also but increased significantly in its lower doses compared to control cells (Fig.  16).

Measurement of mitochondrial membrane potential (MMP)
After the assessment of total cellular anti---oxidant property, in the next phase we targeted mitochondria, the most important site inside the cell for ROS genera--tion. Here we have found that all three extracts of date is capable of reducing LPS induced oxidative stress in murine macrophage like cells significantly, in compar--ison to the control group in a dose dependent manner, which advocates that the date extracts ameliorate the LPS induced oxidative stress by targeting the mito--chondria in a dose dependent manner and thus main--tain the cell viability (Fig.  17).  Figure  18.However only two peaks show appreciably high absorbance when observed with 254 nm wavelength. The peak at 23.5 min is the major peak while that at 34.0 min is a minor peak. The peak at 23.5 min appears about 20 fold attenuated when observed with 370 nm wavelength while the peak at 34.0 min is not observed at this wavelength.

TLC analyses
Sample B: Extract B shows four closely spaced peaks between 6.0 - 9.0 min appearing at 6.7, 7.3, 7.8 and 8.4 min with major peak being at 7.3 min when observed with 254 nm wavelength. These peaks were also ob--served with 370 nm wavelength but with about 4 fold attenuation in peak absorbance values.
Sample C: The extract C showed roughly three distinct peaks. They were at 5.1---5.2 min, 6.3 min and 7.9 min using 254 nm wavelength. The first two peaks are ma--jor peaks but are not well separated. The third peak at 7.9 min is a minor peak in this sample. There is anoth--er minor peak at about 6.8 min observed with 254 nm wavelength which may be a spurious peak as it lies on the shoulder of the 6.3min peak and didn't appear in repeat scan. The absorbance maxima of these peak positions remained similar when seen in 370nm wave--length although their intensities were attenuated by about 10 fold. Comparing the absolute absorbance values of peak maxima of sample C with others sam--ples (A &B), it indicates that sample C contains much higher concentration of compound(s) as similar values of samples (100---200 µl) were injected for HPLC analy--sis in each case.   69, 6.16, 7.23, 8.27, 24.88, 26.42, 29.01, & 33.01 min, respectively.   Their HPLC retention times were 5.14,6.30,6.77,7.94,23.61,33.89 min, respectively.

DISCUSSION
In the present study to evaluate anti---inflammatory potential of the extracts of date collected by three dif--ferent extraction procedures (Fig. 1) where one or more fractions of the mesocarp tissue is likely to be included and the rest excluded, various inflammatory markers were used against a powerful and well char--acterized pro---inflammatory stimulus---LPS, a major component of the outer membrane of Gram---negative bacteria. This molecule shows potent pro inflammato--ry action on various cell types, including macrophag--es, endothelial cells, and fibroblasts (Allaith, 2008) in--duced inflammation in RAW 264.7 and HEK 291 cell lines by interacting with TLR4, a trans---membrane re--ceptor on macrophages, which recognizes molecules derived from gram negative bacteria activating the downstream NF---κB (nuclear factor kappa) signaling pathway leading to increased production of cytokines, and/or ROS/RNS generation following the activation of mitogen---activated protein kinases (MAPK) and NF---κB mediated signaling pathway which ultimately drive the tissue degeneration.
In the present study, datefruit was extracted using various organic solvents, and their anti---oxidative and anti---inflammatory activities were evaluated. Phenolic compounds as well as Flavonoids, which are a group of poly phenolic compounds possessing free radical scavenging properties, potential inhibitory actions of hydrolytic and oxidative enzymes as well as anti--inflammatory action. Obtained data have clearly demonstrated that date extracts, depending on their mode of collectionpossess potent anti---oxidative prop--erties indicated by significant inhibition of lipid perox--idation (Fig. 7), superoxide radical scavenging, hy--droxyl radical scavenging, DPPH radical scavenging and nitric oxide (NO) scavenging activities. The calcu--lated IC50 values of dateindicate strong anti---oxidative properties of these crude extracts.
Data obtained from studies using these date extracts, collected by three different methods indicate potent anti---oxidative properties by showing significant inhi--bition of lipid peroxidation, superoxide radical scav--enging, hydroxyl radical scavenging, DPPH radical scavenging and nitric oxide (NO) scavenging activi--ties. The calculated IC50 valuesof these three extracts of datehaving revealedstrong anti---oxidative properties (Fig. 9, Table 7), their anti---inflammatory potencies warranted confirmation through additional studies on cells. Both human and murine cell lines were assessed. Oxidative stress being the main driving force in any inflammatory cascade, studies weredesigned to evalu--ate theirameliorative effect during and post---oxidative stress induced inflammation.
As we have identified the anti---oxidative capacity ( Fig.  9) of dietary date extracts, collected by three different extraction procedures, i.e. methanolic, basic ethanolic and acidic ethanolic methods, (Fig.  1) as an interesting lead that can stabilize the intracellular reactive oxygen and nitrogen intermediates in both murine and hu--man cell lines in vitro at non cytotoxic concentrations, we were therefore interested to evaluate the in vivo anti---inflammatory activities of these compounds in preclinical models, but before this cytotoxic and anti--inflammatory properties of these three extracts have been evaluated thoroughly, targeting the mitochon--drial reactive oxygen and nitrogen intermediates (Fig.  17).
The acidic ethanolic extracts of date extract amelio--rates the LPS induced oxidative stress by inhibiting the NO production (Fig.  6) and subsequently protect--ing the mitochondrial respiratory chain complex func--tion, thus maintaining the cellular homeostasis or cell viability per se. As we have mentioned earlier that oxidative stress and mitochondrial dysfunction are key features for any inflammatory cascade and here the date extracts are capable of inhibiting this, thus, these crude extracts must have potent anti--inflammatory power. Another important point to be noted here is that the acidic ethanolic extract of the date has shown a more potent anti---inflammatory properties in murine RAW macrophages (Fig.  12b) in comparison to other two extracts (Fig. 12a, 12c), whereas in case of LPS induced human HEK cells, the methanolic extract (Fig.  15a) is better at inhibiting the LPS induced oxidative stress and in maintaining the cellular homeostasis, than either acidic or basic etha--nolic extracts. From studies done by various research--ers as stated and referred to in the preceding para--graphs, we now know that plants have various phe--nolic compounds and flavonoids, which are basically secondary metabolites of the pentose phosphate, shi--kimate, and phenylpropanoid pathways, exhibit strong anti---oxidative redox properties allowing them to act as reducing agents, hydrogen donors, and sin--glet oxygen quenchers. Also, many of them have been shown to exhibit the antagonism for the classical NF---κB signaling pathways and for this reason in past few years there has been an upsurge of interest in the ther--apeutic potentials of medicinal plants as antioxidants in reducing such free radical induced tissue injury. Although several phytochemicals have been shown to possess pharmacological properties of potential inter--est anti---inflammatory properties and/or therapy, their activity in the in vitro and in vivo preclinical model organisms is still not well understood due to varia--tions of their extraction and collection procedures, heterogeneous solubility in different solvents and most importantly, route of administration of these compounds in a dose dependent manner in different disease models. The mechanism by which various date extracts mediates its antioxidant effects remains unclear.
Mitochondria are the major source for ROS generation in the cell. Evidence from our studies suggests the role of mitochondria in date induced ROS scavenging pathways (Fig.  17). It is possible that date extracts in--hibit the mitochondrial enzymes that lead to produc--tion of ROS and thus maintain the oxidative stress induced cell damage (Fig. 16). The inhibition of ROS by date extracts could occur through its interaction with thioredoxin reductase thus changing its activity to NADPH oxidase, which could then lead to the scavenging of ROS. It is not clear yet which structural group or compound of date extracts is responsible for inhibiting the ROS production. Present extracts of date also has shown a high level of phenol (Fig. 10b) and flavonoid (Fig. 10a) content and this may be the rea--son behind their anti---inflammatory activities.
We can conclude that the inhibitory effect of date ex--tracts on LPS induced inflammation in RAW macro--phages is via the downstreaming of the classical TLR4 mediated signaling cascades scavenging the mito--chondrial ROS production (Fig.  17). Until phytochem--ical screening of these extracts is carried out, this will be very early to conclude. However, it is true that the compounds present in these extracts can inhibit the endotoxin induced oxidative stress (Fig. 16) in both murine and human cell lines in a dose dependent manner. The phytochemical screening and the struc--ture activity relationship of these extracts are under investigation now along with the detailed anti--inflammatorysignaling pathways. But the question is, what compound (s) are exactly present in these ex--tracts that are truly exhibiting the anti---inflammatory properties and the signaling pathways that they fol--low, is still not clear yet. For this HPLC and mass spectrometry of the samples are being carried out and from database analysis individual eluents shall be used in anti---inflammatory and anti---oxidative assays to assess their oxi---flammatory potential. This is ongoing work in the lab.
Acting as second messengers, transient free radicals, synthesized during regular metabolismoften trigger further downstream sequence of activation networks. But produced in unregulated catabolic cycles, they demonstrate deleterious compounds such as toxins and wastes which further contribute to the disease etiology. Studies on intermediate free radicals, espe--cially reactive oxygen species (ROS) and their action on cellular physiology, have shown that they play a pivotal role in causing secondary tissue degeneration in various inflammatory diseases, such as rheumatoid arthritis (Filippin et al., 2008), multiple sclerosis (Offen et al., 2004), thyroiditis (Burek and Rose, 2008), and type 1 diabetes (Chen et al., 2008).
ROS are known to perform essential roles in immune response to pathogens, including bacterial killing via induction of superoxide anion during respiratory burst in activated macrophages and neutrophils (Kanayama and Miyamoto, 2007;Lambeth, 2004). Fur--ther studies on patients with chronic granulomatous disease (CGD) or genetically engineered mice lacking components of the NADPH oxidase enzyme (NOX) (Morgenstern, 1997;Shiloh et al., 1999) provide cor--roborative data.
Natural products have yielded as many as 70% of the drugs used today for inflammation and degeneration (Newman et al., 2002). Consumption of large portions of fruits and vegetables have been recommended for reduction of risk of cardiovascular diseases, autoim--mune diseases, cancer, and various other chronic ill--nesses (Neuhouser, 2004) although little is known about the actual active compounds or moieties which are beneficial. It is an important project to identify and validate direct action and participation of such active ingredients in modulating disease.
Figures 18---20 show data on proteomic analyses by TLC, HPLC and MS. On preliminary investigation TLC data revealsthat extract A may not contain im--portant compounds although anti---inflammatory activ--ity was seen in the other two extracts. Clear peaks were not obtained in scan data of the other two which necessitates further purification steps and MS analyses of the individual peaks and NMR. This work is ongo--ing and is beyond the scope of this work.

CONCLUSION
In this study, three different types of date extracts, methanolic, acidic ethanolic and basic ethanolic pro--duce effects on reactiveoxygen species where scaveng--ing of hydroxyl radicals, superoxide radicals, nitric oxide scavenging activity, and inhibition of lipid pe--roxidation were found to occur in a dose dependent manner. Moreover, they have a significant inhibition of anti---inflammatory activities. Invitro experiments prove that the effects of the extracts islikely mediated through inhibition of mitochondria derived (ROS) scavenging pathways. Thisis an important finding as it shows not only the extracts' pathway of action but also provesthe direct involvement of a sub---cellular signaling network that can be interfered with, using anatural products without the possibility of drastic side effects. Ongoing proteomic analysis has already revealed scan data using various sensitive chromato--graphic techniques of certain specific peaks. Further analyses are needed to conclusively identify the peaks to reveal identity of one or more compounds for de--tailed characterization and development into novel drug entities.