Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, like pathogens, damaged cells and irritants. It is a mechanism of innate immunity, and is characterized by pain, heat, redness, swelling and loss of function.

Betel vine (Piper betle L.), commonly called paan, and is the leaf of an evergreen, perennial vine of the piperaceae family. Betel leaves are consumed after heavy meals as a digestive stimulant and mouth freshener. Paan is said to have a number of medicinal properties, including anti-inflammatory and anti-oxidant activities.

Betel (Piper betle) belongs to the genus Piper of the family Piperaceae. The plant originated in central and eastern peninsular Malaysia, where it is locally called sirih [1,2]. It is distributed throughout east Africa and the tropical regions of Asia. It is a commercial crop that is widely cultivated in many parts of India and Sri Lanka [3]. The use of betel leaf can be traced as far back as two thousand years [4]. Fresh leaves are chewed with betel nut, Areca catechu (Arecaceae), and other adjuvants (betel quid) in most parts of India and the habit of chewing betel is so widely prevalent that it ranks next to consumption of alcohol, coffee and smoking [5,6]. Due to their strong pungent aromatic flavour, betel leaves are used as a masticatory in Asia. Its common names are betel (in English), paan (in Indian), phlu (in Thai) and sirih (in Bahasa Indonesian) [7]. Betel leaves are reported to contain an aromatic oil [8], minerals [9], glycosides [10], enzymes, vitamins, essential amino acids [11] and tannins [12].

It has been reported that betel leaf has a wide spectrum of therapeutic properties. The extract of betel leaves possesses antimutagenic, anticarcinogenic, antiplaque, antidiabetic, anti-inflammatory and antibacterial bioactivities [13-18]. Generally plants that have significant therapeutic properties have been found to be rich in phenolics, with high antioxidant properties [19]. This correlation has been confirmed with the antioxidant activity being detected in the extract of betel leaf [20]. The consumption of antioxidant-rich foods helps neutralize the free radicals in the body, thus preventing or delaying the oxidative damage of lipids, proteins and nucleic acids [21]. It has been shown that the antioxidants could reduce mortality rate of cardiovascular disease [22,23], and protect against cancer and other chronic diseases [24]. The aqueous extract of the inflorescence of Piper betle extract was effective in scavenging H₂O₂, superoxide radical and hydroxyl radical [25,26]. The extract also prevented hydroxyl radical-induced DNA strand breaks in the PUC18 plasmid [26]. Recently, Manigauha et al, [27] observed that methanolic extracts of the betel leaves possess reducing power, DPPH radical, superoxide anion scavenging and deoxyribose degradation activities [27,28]. Studies have also shown that the hydroalcoholic extract of the betel leaf possesses nitrogen oxide scavenging effect in vitro [26,28].

The betel leaf has long been used as a household remedy for the inflammation of the oral cavity [29]. Ethanolic extracts of betel leaf has been reported to possess anti- inflammatory activities at non- toxic concentrations, in the complete Freund’s adjuvant-induced model of arthritis in rats. Eugenol, one of the principal constituents of betel leaf, has been shown to possess anti-inflammatory effects in various animal models of studies with various inflammogens [30]. Betel leaf constituents, eugenol, hydroxychavicol and alpha-tocopherol,have also been shown to enhance the levels of GSH in mouse skin and liver [31,32].

Different species of betel leaves, for instance, from female (Bengaluru local) type, male (Madras) type and Meetha (sweet) type have been analysed for their anti-oxidant activity by FRAP (Ferric Reducing Anti-Oxidant Potential) Assay [1], their radical scavenging activity by DPPH (2,2-diphenyl-1-picrylhydrazyl) Assay [1], their anticancer activity by inhibiting proliferation of cells [2], their apoptosisinducing activity by inhibiting proliferation of cells and Sub G1 flow cytometry [2], and their cryoprotective actuighngnivity towards human fibroblast cells by MTT Assay [3]. The total phenol content and the flavonoid content of the betel leaves have been estimated, using Folin- Ciocalteau method and NaNO₂-AlCl3-NaOH respectively. Two of the constituent phenolic compounds have been identified as chevibetol and allyl pyrocatechol [4].

The anti-oxidant and anti-inflammatory properties of betel leaves have been attributed to various components of the leaves. Leaves with higher phenol and flavonoid content have been found to have higher anti-oxidant, radical scavenging and anti-cancer activities [1,2]. Quinic acid present in the betel quid may also play a role in health protection. [6].

Hydroxychavicol (HC) is a novel component of betel, which has been found to inhibit platelet aggregation. HC is a potent COX- 1/COX-2 inhibitor, ROS scavenger and inhibits platelet calcium signalling. HC could be used as a therapeutic agent for cardiovascular diseases through its anti-inflammatory effects, without any effect on haemostatic functions [7].

The anti-ulcerogenic activity has also been attributed to the high flavonoid content of betel leaves. It has been seen that betel extracts have the ability to heal gastric ulcers [8] and peptic ulcers [9].

The ethanolic extract of betel has been tested for radioprotective activity, and it has been found that it can prevent DNA-strand breaks induced by radiation. This property has been attributed to its radicalscavenging and lympho-proliferative activities [4].

The aim of our study was to first detect potential anti-inflammatory and antioxidant activities of 9 varieties of Piper betle leaf extracts which are traditionally known as anti-geriatric compounds. High content of various phenolic and non-phenolic compounds and other uncharacterized moieties may contribute to its use, not only as a highly nutritive, edible plant part, but also as a nutraceutical substance, to be used prophylacticallyas well as therapeutically in oxidative inflammatory diseases.

Reagents

Chemicals, such as ethylenediamine tetra acetic acid (EDTA), trichloroacetic acid (TCA), butanol, ammonium molybdate, and sodium dodecyl sulphate, benzoic acid, sodium phosphate, and DMSO were purchased from E. Merck (India) Limited. 1,1 Diphenyl-2-picrylhydrazyl (DPPH), malondialdehyde, potassium ferricy anite, thiobarbituric acid (TBA) were procured from Sigma, USA. N-butanol, ferrous sulphate, ferric chloride, Folins reagent, Riboflavin, naphthylethylenediamine dihydrochloride (NED), and sulphanilamide in phosphoric acid, sodium bicarbonate, sodium hydroxide, and potassium hydroxide were purchased from Sisco Research Laboratories PVT Ltd India. Nitroblue tetrazolium (NBT), MTT reagent [(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], and DMEM media were purchased from Himedia, India. Fetal bovine serum (FBS) was purchased from Gibco. DCFH-DA and DHR 123 were purchased from Invitrogen. All other reagents were of analytical grade.

Preparation of plant extract

Fresh leaves were washed under distilled water and shade dried for 7 days and powdered. Then extraction was carried out by slightly modified method of Swati Dhote et al. [5]. Briefly, powered leaves were extracted with 80% methanol (1 g: 20 ml) with the help of cold maceration, at room temperature for about 24 hrs, shaking frequently. The process of extraction was repeated for three times. The solution was filtered using Whatman’s filter paper no1 and the solvent, allowed to evaporate completely to obtain the extract. The extract was stored in sterile glass vials at 4°C until use.

DPPH radical-scavenging activity

The antioxidant activity of the crude methanolic P.betle extracts was evaluated against 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals [33]. Various concentration of the methanolic extract was added to a 0.004% methanolic solution of DPPH on a 96 well ELISA plate. After 30 min incubation, Absorbance was determined at 517 nm and the percent inhibition activity was calculated.

Assay of superoxide radical O₂ ^-scavenging activity

Riboflavin-light-nitrobluetetrazolium (NBT) system [34] was used to determine the superoxide dismutase activity of the extract according to Martinez et al. [35]. Each 0.1 ml of reaction mixture contained 13 mM methionine, 50 mM phosphate buffer (pH 7.8), 100 μM EDTA, 2 μM riboflavin, NBT (75 μM) and various doses of sample solution. The production of blue formazan was followed by monitoring the increase in absorbance at 560 nm after 15 min of illumination from a fluorescent lamp.

Lipid peroxidation assay

Egg yolk homogenates as lipid-rich media [36] was used as lipid peroxide which was measured by modified thiobarbituric acid reactive species (TBARS) assay [36]. MDA which was produced by lipid peroxidation of polyunsaturated fatty acids by FeSO₄, reacts with two molecules of thiobarbituric acid (TBA) and pinkish red chromogen with an absorbance maximum at 532 nm, was measured using a 96 well ELISA plate reader. Percentage of inhibition of lipid peroxidation was calculated at various concentration of the crude methanolic P.betle extract [37].

Nitric oxide (NO) scavenging activity

Spontaneous decomposition of sodium nitroprusside (20 mM) in phosphate buffer (pH 7.4) generates nitric oxide which reacts with oxygen molecules to produce nitrite ions. Griess reaction was used to measure Nitrite ions. Slight modified method of Shirwaikar et al. [38] was used to determine the nitric oxide scavenging activity of the extracts 0.2 ml of sodium nitroprusside (20 mM) 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 different concentration of extract was taken and diluted with 1 ml of Griess reagent [1% sulfanilamide, 2% H3PO4 and 0.1% N-(1-naphthyl) ethylenediamine]. Similarly a blank was prepared containing the equivalent amount of reagents (only the sodium nitroprusside and PBS), but without the extract. The absorbance of these solutions was recorded at 540 nm against the corresponding blank solution. Ascorbic acid (100 μg/ml) was used as the reference. The percentage inhibition of nitric oxide was calculated as follows:

The NO scavenging activity(%) of methanolic extract of P.betle leaf =[(A_b-A_t)-A_b] X 100, where Ab is the absorbance of the blank and A_t is the absorbance in the presence of P.betle leaf extract or positive control.

Determination of total flavonoid content

Aluminium chloride (AlCl₃) was used to determine total flavonoid content of the extras Fisetin was used as a standard 0.1 ml methanolic extracts were added to 0.3 ml distilled water, followed by 0.03 ml, NaNO₂ (5 %) and after 5 min 0.03 ml AlCl₃ (10%) was added. After further 5 min, the reaction mixture 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. Fisetin was used as positive control.

Determination of total phenolic content

Folin-Ciocalteau reagent was used to determine total phenolic content of the P.betle leaf extract [39]. The reaction mixture contained 200 μl of diluted extract, 800 μl of freshly prepared diluted Folin Ciocalteau 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 was recorded at 765 nm. Gallic acid was used as standard and the results were expressed as mg Gallic acid (GAE)/10 g of the extract.

Cell culture

HEK 293 cell line and RAW 267.4 murine macrophage cell line was obtained from NCCS, Pune, India. The cells were grown in DMEM medium containing 5% inactivated fetal bovine serum, streptomycin (20 μg/mL) ,penicillin (100 U/mL), and kept at 37°C in T-25 tissue culture flasks at CO₂ incubator. Cell was grown to confluence in a humidified atmosphere containing 5% CO₂.

Cell viability using MTT assay

Cytotoxicity of betel leaf extract was measured according to Das et al. [40-42] by seeding , 5 × 10⁴ cells/well in a 96-well plate and incubated for 24 h with different concentrations of the betel leaf extract . Each well was filled with 100 μL of medium and 10 μL of a tetrazolium salt, MTT after washing the cell properly. The plate was incubated for various time periods and then absorbance was measured at 540. The percentage of viable cells of control (without extract) considered as 100%. The assay was performed in triplicate twice.

HPLC analysis of the P.betle extracts:

Crude methanolic extract of P.betle leaf was filtered by 0.22 μm syringe filter (Milipore, Germany) and 100 μl of filtered was administered for HPLC analysis. The HPLC analysis was carried out using Waters 2695 System Controller coupled with 2487 dual absorbance detector and Waters 515 pump reversed-phase C18 columns was used as stationary phase. Acetonitrile/Water (1:1, flow rate 0.5 mL/min) was used as and peaks were detected at 254 nm [41].

IC₅₀ (half maximal inhibitory concentration) is a quantitative measure of how much of a drug is required to inhibit a biological process or its component by half. Lower the IC₅₀, the more efficient is the drug.

DPPH radical scavenging activity gives an idea of the anti- oxidant activity of the compound, and is expressed as the concentration of sample needed for 50% reduction of DPPH colour intensity. IC₅₀ of lipid peroxidation assay, NO assay and superoxide radical assay are the concentrations needed to prevent lipid peroxidation, NO production and superoxide production, respectively, by 50%.

The lowest IC₅₀ values are of varieties C and A, for DPPH radical scavenging activity, which are found to be close (1.02 fold higher and 1.09 fold lower, respectively) to the IC₅₀ of ascorbic acid, a standard anti- oxidant, whereas that of variety D is very high (7.30 fold). The IC₅₀ of varieties D, G and H are lower (1.13 fold, 1.16 fold and 1.15 fold respectively) than ascorbic acid, for lipid peroxidation assay. The NO scavenging activity of variety D is lowest among all the compounds (8.45 μg/ml), but it is 2.16 fold higher than ascorbic acid. Superoxide radical scavenging activity of varieties H and G are highest (IC₅₀ 0.27 and 0.77, respectively), which are 11.56 and 4.05 folds lower than ascorbic acid.

Inhibition of DPPH radical, or DPPH radical scavenging activity, is seen in all 9 varieties of betel leaves, at a concentration of 1 mg/ml. Maximum inhibition is seen in variety C (81.03%), whereas minimum inhibition is seen in variety D (10%). The inhibitory activity of the compounds decrease in the order C>A>B>F>G>I>E>H>D.

The ability to prevent lipid peroxidation is seen in all 9 varieties of betel leaves, at a concentration of 1 mg/ml. Maximum inhibition is seen in variety H (77.5 %), whereas minimum inhibition is seen in variety C (22.2 %). The inhibitory activity of the compounds decrease in the order H>G>F>E>D>I>A>B>C.

Nitric oxide scavenging activity is seen in all 9 varieties of betel leaves, at a concentration of 1 mg/ml. Maximum inhibition is seen in variety D (41.8%), whereas minimum inhibition is seen in variety G (18.7%). The inhibitory activity of the compounds decrease in the order D>C>A>E>I>F>B>H>G.

Total phenol content of the P.betle leaves was assessed using the Folin-Ciocalteau method, using gallic acid as a standard. Results are expressed as mg gallic acid per 10 gms of extract. It is seen that variety A has the highest phenolic content (1.12 mg GAE/10 gm extract), whereas variety E has the lowest (0.76 mg GAE/10 gm extract). The phenol content of the samples decrease in the order A>B>C>H>I>D>G>F>E.

Total flavonoid content of the P.betle leaves was assessed using the aluminium chloride (AlCl3) method, using fisetin as a standard. Results are expressed as mg fisetin per gm of extract. It is seen that variety B has the highest phenolic content (499.15 mg fisetin/gm extract), whereas variety D has the lowest (46.01 mg fisetin/gm extract). The flavonoid content of the samples decrease in the order B>F>H>E>A>C>I>G>D.

Cell viability of RAW 264.7 cells was assessed using MTT assay. Viability of untreated control cells was taken as 100%, and the viabilities of cells treated with extracts were calculated accordingly. Dexamethasone (dexa) was taken as positive control, and cells treated with only LPS as negative control. With LPS treatment, there is a 1.54 fold decrease in viability, which is restored by dexa treatment. However, the viability of cells treated with sample A has not increased, but decreased from LPS. The decrease is seen at all the concentrations, with the maximum decrease at 10 μg/ml. This shows that sample A is not very effective in combating the inflammation caused by LPS.

Cell viability of RAW 264.7 cells was assessed using MTT assay. Viability of untreated control cells was taken as 100%, and the viabilities of cells treated with extracts were calculated accordingly. Dexamethasone (dexa) was taken as positive control, and cells treated with only LPS as negative control. With LPS treatment, there is a 1.54 fold decrease in viability, which is restored by dexa treatment. With addition of extract B, the viability has increased 1.12 fold with 250 μg/ml, and has increased very slightly with 100 μg/ml. However, the viability of cells has decreased with 50 μg/ml and 10 μg/ml. This shows that sample B has an anti-inflammaotyr effect only at high concentrations.

Cell viability of RAW 264.7 cells was assessed using MTT assay. Viability of untreated control cells was taken as 100%, and the viabilities of cells treated with extracts were calculated accordingly. Dexamethasone (dexa) was taken as positive control, and cells treated with only LPS as negative control. With LPS treatment, there is a 1.54 fold decrease in viability, which is restored by dexa treatment. With sample C, the viability has increased 1.36 fold with 250 μg/ml, and has increased very slightly with 100 μg/ml. However, the viability of cells has decreased with 50 μg/ml. This shows that sample C has an antiinflammatory effect at higher concentrations.

Cell viability of RAW 264.7 cells was assessed using MTT assay. Viability of untreated control cells was taken as 100%, and the viabilities of cells treated with extracts were calculated accordingly. Dexamethasone (dexa) was taken as positive control, and cells treated with only LPS as negative control. With LPS treatment, there is a 1.54 fold decrease in viability, which is restored by dexa treatment. With addition of sample D, the viability has increased 1.10 fold with 250 μg/ ml.It has increased very slightly with 100 μg/ml (1.01 fold) and with 50 μg/ml (1.01 fold). However, the viability of cells has decreased with 10 μg/ml (1.51 fold). This indicates that sample D has an antiinflammatory effect at comparatively high concentrations, since the viability does not increase till 250 μg/ml.

Cell viability of RAW 264.7 cells was assessed using MTT assay. Viability of untreated control cells was taken as 100%, and the viabilities of cells treated with extracts were calculated accordingly. Dexamethasone (dexa) was taken as positive control, and cells treated with only LPS as negative control. With LPS treatment, there is a 1.54 fold decrease in viability, which is restored by dexa treatment. With treatment with sample E, the viability has increased 1.10 fold with 250 μg/ml, 1.11 fold with 100 μg/ml, 1.29 fold with 50 μg/ml and 1.42 fold with 10 μg/ml. This indicates that sample E has an anti-inflammatory effect at a concentration as low as 10 μg/ml. It is effective even at high concentrations, without being detrimental.

Cell viability of RAW 264.7 cells was assessed using MTT assay. Viability of untreated control cells was taken as 100%, and the viabilities of cells treated with extracts were calculated accordingly. Dexamethasone (dexa) was taken as positive control, and cells treated with only LPS as negative control. With LPS treatment, there is a 1.54 fold decrease in viability, which is restored by dexa treatment. The viability has increased 1.52 fold with 250 μg/ml, 1.36 fold with 100 μg/ml, and 1.33 fold with 50 μg/ml, with sample F, but has decreased 1.11 fold with 10 μg/ml. This indicates that sample F has an antiinflammatory effect at concentrationsof 50 μg/ml and above, but not at 10 μg/ml.

Cell viability of RAW 264.7 cells was assessed using MTT assay. Viability of untreated control cells was taken as 100%, and the viabilities of cells treated with extracts were calculated accordingly. Dexamethasone (dexa) was taken as positive control, and cells treated with only LPS as negative control. With LPS treatment, there is a 1.54 fold decrease in viability, which is restored by dexa treatment. With addition of sample G, the viability has increased 1.21 fold with 100 μg/ml, 1.35 fold with 50 μg/ml, and 1.14 fold with 10 μg/ml, but has decreased 1.03 fold with 250 μg/ml. This indicates that sample G has an anti-inflammatory effect at lower concentrations, but may be toxic at higher concentrations.

Cell viability of RAW 264.7 cells was assessed using MTT assay. Viability of untreated control cells was taken as 100%, and the viabilities of cells treated with extracts were calculated accordingly. Dexamethasone (dexa) was taken as positive control, and cells treated with only LPS as negative control. With LPS treatment, there is a 1.54 fold decrease in viability, which is restored by dexa treatment. It is seen that with compound H, the viability has not increased even at low concentrations, indicating that sample H has a pro-inflammatory activity, rather than anti- inflammatory activity.

Cell viability of RAW 264.7 cells was assessed using MTT assay. Viability of untreated control cells was taken as 100%, and the viabilities of cells treated with extracts were calculated accordingly. Dexamethasone (dexa) was taken as positive control, and cells treated with only LPS as negative control. With LPS treatment, there is a 1.54 fold decrease in viability, which is restored by dexa treatment. With treatment with sample I, the viability has increased 1.35 fold with 250 μg/ml, 1.26 fold with 100 μg/ml, 1.16 fold with 50 μg/ml and 1.10 fold with 10 μg/ml. This indicates that sample I has an antiinflammatory effect at concentrations as low as 10 μg/ml, and even at high concentrations without being toxic to the cells.

Natural products are in great demand owing to their extensive biological properties and bioactive components which have proved to be useful against a large number of diseases. It has been proved that present extracts of Piper betleleaves show a wide array of activities like anti-inflammatory, antioxidant and also pro-inflammatory.

Various reactive oxygen species (ROS) can be formed in cells by transition metal {especially Fe (II)} mediated reactions along with oxygen metabolism [42], and radiation exposure [43], leading to deleterious effects on membrane lipids and DNA. For the present work, the antioxidant activity of the methanolic extracts of nine varieties of P. betle leaves were screened by the DPPH radical scavenging and reducing power assays. Antioxidants, on interaction with DPPH, either transfer an electron or hydrogen atom to DPPH, thus neutralising its free radical character [44,45]. The colour changes from purple to yellow and its absorbance at wavelength 517 decreases. Methanolic extract of P. betle leaf extract quenched DPPH free radical (Table 1). DPPH assay shows that, in this system, the radical-scavenging activities of the various Piper leaf extracts are in this order: C>A>B>F>G>I>E>H>D (Table 1). Methanolic extract of C variety P.betle leaf exhibited the highest antioxidant activity by strongly inhibiting 50% of DPPH radicals at a lower concentration (4.38 μg/ml) where ascorbic acid showed 50% inhibition at 4.78 μg/ml (Figure 1). Among 9 varieties of P.betle leaf, D variety was very less active in scavenging free radicals. IC₅₀ value of D variety of P.betle leaf was 34.9 μg/ml (Figure 1).

Table 1: DPPH Scavenging Activities of P.betle leaf.

Figure 1: IC₅₀values were calculated from these regression equations. IC₅₀ values of DPPH radical scavenging activity lipid peroxidation, NO radical scavenging activities and superoxide radical scavenging activity of nine different varieties of P.betle leaves (A,B,C,D,E,F,G,H & I) were compared with the IC₅₀ value of Ascorbic Acid in each system to assess the antioxidant property of P.betle leaves.

All of the extracts demonstrated highest inhibition of lipid peroxidation at concentration of 1mg/ml (Table 2). The percentage of lipid peroxidation inhibition, IC₅₀ values (concentration of sample required to scavenge 50% free radical or to prevent lipid peroxidation by 50%) was calculated from regression equations. IC₅₀ values of the nine varieties of P. betle leaves were compared with the IC₅₀ value of ASA in each system to assess the antioxidant property of P. betle leaves (Figure 1). IC₅₀ value is inversely related to the activity. The percentage of lipid peroxide inhibition can be described in the following order: G>H> D>E>F>I>A>C>B. Among the nine varieties of betle leaf extract, G,H, D and E varieties showed more inhibition of lipid peroxidation than ascorbic acid. G variety of betle leaf has the highest activity which was about 1.16 times more than that of ASA.IC₅₀ value of lipid peroxidation of F variety P.betle leaf extract was same as ASA, i.e. 5.57 μg/ml (Figure 1). But A,B,C& I varieties of P.betle leaf extract were less effective than ASA in inhibiting lipid peroxidation. The results suggest that consumption of G,H,D, and E varieties of P. betle leaf may afford a cytoprotective effect.

Table 2: Prevention of lipid peroxidation by P.betle leaf.

Nitric oxide (NO) is a potent pleiotropic mediator in physiological processes and a diffusible free radical too in the pathological conditions. It reacts with superoxide anion and form a potentially cytotoxic molecule, the ‘peroxynitrite (ONOO-)’. Its protonated form, peroxynitrous acid (ONOOH), is a very strong oxidant [46,47] . The main route of damage is the nitration or hydroxyllation of aromatic compounds, particularly tyrosine. Under physiological conditions, peroxynitrite also forms an adduct with carbon dioxide dissolved in body fluid and responsible for oxidative damage of proteins in living systems [48,49]. Methanolic extract of 9 varieties of P. betle leaf were examined for their possible regulatory effect on nitric oxide (NO) levels using sodium nitroprusside as an NO donor in vitro. All of the P. betle leaf extracts demonstrated direct scavenging of NO and exhibited significant activity. The potency of scavenging activity was in the following order: D>C>A>E>I>F>B>H>G (Table 3). IC₅₀ value of the D variety of P. betle leaf extract was 8.45μg/ml, whereas IC₅₀ value of ascorbic acid was 3.92 μg/ml (Figure 1) [50].

Table 2: Prevention of lipid peroxidation by P.betle leaf.

Phenols are the largest group of secondary metabolites of plant. Plant phenolic compounds have various biological effects, including antioxidant (1,49,50,51,52).Plant phenolic compounds are highly effective free radical scavengers and antioxidants. Consequently, the antioxidant activities of plant/herb extracts are often explained by their total phenolic content with good correlation. We also observed similar correlations in the present study [51].

Figure 2 demonstrated the total phenolic content (TPC) of nine varieties of methanolic extracts of P.betle leaf. It was found that the A variety of P.betle leaf extract has the highest TPC, i.e. 1.121 ± 0.019 mg gallic acid/10 gm of P.betle leaf extract [52].

Figure 2: Bar graph showing comparative phenol content of 9 varieties of P.betle leaves. Results expressed as gallic acid equivalent (mg gallic acid/10gm) ± SEM of P. betle leaf extract, in duplicate.

Anti-inflammatory versus pro- inflammatory effect

The effect of methanolic extract of 9 varieties of P.betle leaf on the viability of RAW 264.7 cells was evaluated therapeutically in a dose dependent manner (Figure 3). RAW 264.7 cells were treated with 1μg/ml of E.coli LPS and then various concentrations of methanolic Piper leaf extracts (250 μg/ml, 100 μg/ml, 50 μg/ml and 100 μg/ml) were added into it therapeutically and incubated for 24 h. Sample A did not exhibit any anti-inflammatory effect on LPS induced inflamed Raw 264.7 cells (Figure 4). Sample B (250 μg/ml) showed very less antiinflammatory activity and it retained 72.69% viability, only 7.69% more than LPS induced RAW264.7 cells, which showed 65% viability (Figure 5). 250 μg/ml of sample C showed significant anti-inflammatory effect and it retained 88.4%viability, but the other three concentrations did not show effective viability on LPS treated RAW264.7 cells (Figure 6). Sample D retained 71.79% viability at a concentration of 250 μg/ml, and it is 6.79% more than negative control (Figure 7). In sample E, we found significant anti- inflammatory effect at every concentration and cell viability was retained 91.22%, 71.98 %, 84.01% and 92.09% at 250 μg/ml, 100 μg/ml, 50 μg/ml and 10 μg/m respectively on LPS treated RAW cells. Maximum anti- inflammatory activity was seen at a concentration of 250 μg/ml and 10 μg/ml, which is close to positive control i.e. Dexamethasone (Figure 8). Among the 9 varieties of P.betle leaf, the F variety was most effective concentration of 250 μg/ml and it retained cell viability 99%, very close to control and positive control. 2other concentrations of sample F, 100 μg/ml and 50 μg/ml, also showed significant viability, 88.48% and 86.49% respectively, under same experimental conditions. However, at a concentration of 10 μg/ml, there is no anti-inflammatory activity (Figure 9). Methanolic extract of sample G showed significant anti-inflammatory activity at a concentration of 50 μg/ml with respect to cell viability. At this concentration, it retained cell viability by 88.06 %, which is 23.06 % more than cell viability of negative control, i.e. 65% (Figure 10). 2 other concentrations of this sample, 100 μg/ml and 10 μg/ml, also showed anti-inflammatory activity, and retained cell viability by 78.86% and 73.9% respectively (Figure 10). But at the highest concentration (250 μg/ml), this variety of P.betle leaf showed lowest activity, but the other 7 varieties of P.betle leaf (A,B,C,D,E,F, and I) showed highest activity at concentration of 250 μg/ml. Variety I of P.betle leaf showed antiinflammatory activity at 4 different concentrations. At a concentration of 250 μg/ml, it showed maximum viability of 87.74%, 22.74% more than negative control (Figure 11). Interestingly, sample H showed pro-inflammatory activity, whereas the other 8 varieties of P.betle leaf showed anti- inflammatory activity. Cell viability was retained 36.35%,39.61%,39.13% and 39.35% at concentrations of 250 μg/ml, 100 μg/ml, 50 μg/ml and 10 μg/ml of methanolic extract of sample H respectively,where LPS treated RAW cells showed 65% cell viability (Figure 12).

Figure 3: Bar graph showing comparative flavonoid content of 9 varieties of P.betle leaves. Results expressed as fisetin equivalent (mg fisetin/gm) ± SEM of P.betle leaf extract, in duplicate.

Figure 4: Bar graph showing effect of various concentrations of P.betle leaf extract (A) on viability of LPS induced inflamed RAW 264.7 cell line. Viability of Control (untreated) is considered as 100%, +Dexa (Dexamethasone) is used as positive control and +LPS as negative control, to determine antiinflammatory property P.betle leaf extract of sample A by MTT assay.

Figure 5: Bar graph showing effect of various concentrations of P.betle leaf extract (B) on viability of LPS induced inflamed RAW 264.7 cell line. Viability of Control (untreated) is considered as 100%, +Dexa (Dexamethasone) is used as positive control and +LPS as negative control.

Figure 6: Bar graph showing effect of various concentrations of P.betle leaf extract (C) on viability of LPS induced inflamed RAW 264.7 cell line. Viability of Control (untreated) is considered as 100%, +Dexa (Dexamethasone) is used as positive control and +LPS as negative control.

Figure 7: Bar graph showing effect of various concentrations of P.betle leaf extract (D) on viability of LPS induced inflamed RAW 264.7 cell line. Viability of Control (untreated) is considered as 100%, +Dexa (Dexamethasone) is used as positive control and +LPS as negative control.

Figure 8: Bar graph showing effect of various concentrations of P.betle leaf extract (E) on viability of LPS induced inflamed RAW 264.7 cell line. Viability of Control (untreated) is considered as 100%, +Dexa (Dexamethasone) is used as positive control and +LPS as negative control.

Figure 9: Bar graph showing effect of various concentrations of P.betle leaf extract (F) on viability of LPS induced inflamed RAW 264.7 cell line. Viability of Control (untreated) is considered as 100%, +Dexa (Dexamethasone) is used as positive control and +LPS as negative control.

Figure 10: Bar graph showing effect of various concentrations of P.betle leaf extract (G) on viability of LPS induced inflamed RAW 264.7 cell line. Viability of Control (untreated) is considered as 100%, +Dexa (Dexamethasone) is used as positive control and +LPS as negative control.

Figure 11: Bar graph showing effect of various concentrations of P.betle leaf extract (H) on viability of LPS induced inflamed RAW 264.7 cell line. Viability of Control (untreated) is considered as 100%, +Dexa (Dexamethasone) is used as positive control and +LPS as negative control.

Figure 12: Bar graph showing effect of various concentrations of P.betle leaf extract (I) on viability of LPS induced inflamed RAW 264.7 cell line. Viability of Control (untreated) is considered as 100%, +Dexa (Dexamethasone) is used as positive control and +LPS as negative control.

HPLC profile

Figures 13-21 demonstrated the HPLC fingerprint of 9 crude extract of methanolic P. betle leaf. Among 9 varieties, B,D and F varieties of the P.betle leaf extracts showed very sharp and single pick at 254 nm (Figures 14,16 & 18) and F varieties showed highest antiinflammatory activity .But Other six varieties of P. betle leaf extract showed multiple picks. Further analysis like MS, NMR etc. has to be done for identification of the pure bioactive compounds which have potent biological antioxidant and anti-inflammatory activities.

Figure 13: HPLC profile of a methanolic extract of variety A of Piper betle. Chromatogram of crude Sample A prepared from methanolic extract of P.betle L. leaf, on analytical RP-HPLC in isocratic mode using acetonitrile and water (1:1) as mobile phase and C18 column as stationary phase, at a flow rate of 0.5 ml/min. Three major peaks were identified at 254 nm and their retention times were 5.25, 7.36 and 9.33 min respectively.

Figure 14: HPLC profile of a methanolic extract of variety B of Piper betle. Chromatogram of crude Sample B prepared from methanolic extract of P. betle L. leaf, on analytical RP-HPLC in isocratic mode using acetonitrile and water (1:1) as mobile phase and C18 column as stationary phase, at a flow rate of 0.5 ml/min. Only one peak was found at 254 nm and its retention time was 6.53 min.

Figure 15: HPLC profile of a methanolic extract of variety C of Piper betle. Chromatogram of crude Sample C prepared from methanolic extract of P. betle L. leaf, on analytical RP-HPLC in isocratic mode using acetonitrile and water (1:1) as mobile phase and C18 column as stationary phase, at a flow rate of 0.5ml/min. Six peaks were identified at 254nm and their retention times were 5.21, 6.08, 6.42, 7.01, 7.44 and 7.97 min respectively.

Figure 16: HPLC profile of a methanolic extract of variety D of Piper betle. Chromatogram of crude Sample D prepared from methanolic extract of P.betle L. leaf, on analytical RP-HPLC in isocratic mode using acetonitrile and water (1:1) as mobile phase and C18 column as stationary phase, at a flow rate of 0.5 ml/min. Only one peak was found at 254 nm and its retention time was 6.54 min.

Figure 17: HPLC profile of a methanolic extract of variety E of Piper betle. Chromatogram of crude Sample E prepared from methanolic extract of P. betle L. leaf, on analytical RP-HPLC in isocratic mode using acetonitrile and water (1:1) as mobile phase and C18 column as stationary phase, at a flow rate of 0.5 ml/min. Only two peaks were found at 254 nm and their retention times were 6.73 and 7.16 min respectively.

Figure 18: HPLC profile of a methanolic extract of variety F of Piper betle. Chromatogram of crude Sample F prepared from methanolic extract of P. betle L. leaf, on analytical RP-HPLC in isocratic mode using acetonitrile and water (1:1) as mobile phase and C18 column as stationary phase, at a flow rate of 0.5 ml/min. Only one peak was found at 254 nm and its retention time was 3.01 min.

Figure 19: HPLC profile of a methanolic extract of variety G of Piper betle. Chromatogram of crude Sample G prepared from methanolic extract of P. betle L. leaf, on analytical RP-HPLC in isocratic mode using acetonitrile and water (1:1) as mobile phase and C18 column as stationary phase, at a flow rate of 0.5 ml/min. Four peaks were identified at 254 nm and their retention times were 15.04, 16.19, 17.79 and 18.79 min respectively.

Figure 20: HPLC profile of a methanolic extract of variety H of Piper betle. Chromatogram of crude Sample H prepared from methanolic extract of P. betle L. leaf, on analytical RP-HPLC in isocratic mode using acetonitrile and water (1:1) as mobile phase and C18 column as stationary phase, at a flow rate of 0.5 ml/min. Three peaks were identified at 254 nm and their retention times were 6.40, 6.70 and 9.42 min respectively.

Figure 21: HPLC profile of a methanolic extract of variety I of Piper betle. Chromatogram of crude Sample I prepared from methanolic extract of P. betle L. leaf, on analytical RP-HPLC in isocratic mode using acetonitrile and water (1:1) as mobile phase and C18 column as stationary phase, at a flow rate of 0.5 ml/min. Four peaks were identified at 254 nm and their retention times were 13.82, 14.36, 15.28 and 17.66 min respectively.

Piper betle L. (locally known as Paan) is a widely distributed plant in the tropical and subtropical regions, its leaves being largely consumed               as a masticator and mouth freshener. It is valued as a mild stimulant             and also has its use in Ayurvedic medicines.

The present study investigates on the free radical scavenging potential               as well as total phenolic and aflavanoid contents of methanolic               extract of 9 different varieties of Piper betle leaves (A,B,C,D,E,F,G,H               & I). These extracts from diverse localities and varying on treatment,               maturation at plucking etc. were used to evaluate antioxidant and               anti-inflammatory activities. The antioxidant potential of all 9               varieties of methanolic leaf extracts were evaluated by six methods:               1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging               activity, superoxide radical O₂ - scavenging activity by the riboflavinlight-               nitroblue tetrazolium (NBT) system, Lipid peroxidation assay               was done by A modified thiobarbituric acid reactive species (TBARS)               assay, Nitric oxide (NO) scavenging activity. By the Griess reaction               and determination of total phenolic content was done by the Folin-               Ciocalteau reagent. Gallic acid was used as standard to determine               total phenolic content. Total flavonoid content was determined using               aluminium chloride (AlCl3) and the results were expressed as mg               Fiseting P. betle leaf extract. P. betle leaves. MTT assay was performed               to evaluate the anti-inflammatory effect of the extract on RAW 264.7               cell line in dose dependent manner. RP-HPLC analyses of 9 varieties of               methanolic crude extract, was done in isocratic mode using acetonitrile               and water (1:1) as mobile phase and C18 column as stationary phase               and pick were monitored at 254 nm. All of the 9 extracts demonstrated               highest antioxidant activities at concentration of 1mg/ml. C variety               P.betle leaf exhibited the highest DPPH radicals scavenging activity               (IC50-4.38 μg/ml) which was more than Ascorbic acid (IC₅₀-4.78 μg/               ml) where D variety was less effective(IC₅₀ :34.9 μg/ml). G variety of               betle leaf extract was most effective to inhibit peroxidation of lipid,               about 1.16 times more effective than Ascorbic acid. In superoxide and               nitric oxide radical scavenging assays, among 9 varieties, D showed               highest NO nitric oxide radical scavenging activity (IC₅₀ 8 μg/ml) and               H variety demonstrated highest superoxide radical O₂ - scavenging               (IC₅₀- 0.77 μg/ml) when compared to the reference compounds. . It               was found that B variety showed the highest phenolic content (499.15               mg fisetin/ gm extract), whereas variety D had the lowest (46.01 mg               fisetin/ gm extract). Among the 9 varieties of P. betle leaf, the F variety               was most effective at a concentration of 250 μg/ml and it retained cell               viability 99%, very close to control and positive control but H variety of               P.betle leaf extract showed pro-inflammatory activity and cell viability               decreased by 29% than LPS induced RAW 264.7 cell. The results of               the experiments suggest that selected variety of P. betle may be used as               natural antioxidant as well as an alternative or supplementary herbal               remedy for the treatment of inflammatory disease [53]. This is the               first communication that betel leaves cannot be generalized in terms               of their potential medicinal properties or toxic traits. It is a very fine               environmental issue. The betel leaves used in the study have been               collected from various geographical localities; have different farming               profiles including various environmental traits and anthropogenic               factors that lead to variations in their functional profiles. The details of               the environmental and anthropogenic factors require detailed analyses               and are beyond the scope of this paper. But this is a unique finding and               needs to be brought to the attention of food scientists, drug hunters,               ecologists, economists and social scientists because of the far reaching               consequences of taking on leaf versus another and their long term               health consequences as functional food. While some varieties show               powerful anti-oxidant and anti-inflammatory activities, others show               antagonistic functions which are potentially dangerous and long term             use may lead to serious health conditions. These need to be avoided but common people generally believe paan to be of only one variety and             do not consider the micro-nutrients aspects that must be factored in.             Additionally biodiversity conservation also should be a major thrust             area for the conservation /preservation of each variety which are by             no means uniform and farming techniques and human interference             and handling also must be thoroughly studies to understand the             implication of each and every factor contributing to its ultimate             functional phenotype and implications in terms of its transcriptomic             components that in turn influence the metabolomics of the human             being taking them as recreational food. Thus, the present study             warrants further investigation involving components of P. betle for             possible development of new class of anti-inflammatory drugs as well             as focussed biodiversity conservation policies.

RD has performed all assays, analysed data and drafted this manuscript. SM has analysed data and made significant intellectual contribution to this work. KM had performed some preliminary assays. UPS has mentored all the proteomic analyses pertaining to this project and helped in the acquiring of data and all final analyses. ERB has conceptualized the project, planned all experiments, analysed all data and wrote this manuscript.

The authors would like to acknowledge University of Calcutta and Center for Research in Nanoscience and Nanotechnology for all infrastructure related funding for setting up the lab, University Grants Commission for providing funds for initiation of viable research projects in the ERB lab, WB DST for paying RD his fellowship, WB DBT for paying KM his fellowship, UGC for paying SM her fellowship (she is a UGC-NET fellow) and Indian Institute of Chemical Biology for providing UPS his infrastructural requirements. We would also like to acknowledge Prof. Bratati De and her fellow for providing the paan extracts. ERB is the PI on all funded intramural and extramural projects that have directly or indirectly funded the research embodied in this manuscript.

The authors have declared that no conflict of interest exists.