International Journal of Integrative Biology A journal for biology beyond borders ISSN

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1 Report International Journal of Integrative Biology A journal for biology beyond borders ISSN Free-radical scavenging activity and phytochemical analysis in the leaf and stem of Drymaria diandra Blume Palash Mandal 1,*, Tarun Kumar Misra 2, Mitali Ghosal 1 1 Department of Botany, North Bengal University, Darjeeling, WB, India 2 Institute of Plantation Science and Management, North Bengal University, Darjeeling, WB, India Submitted: 10 May. 2009; Revised: 2 Aug. 2009; Accepted: 12 Sep Abstract In different parts of plain land and hill area of Darjeeling district, leaves and stems of Drymaria diandra were evaluated for their phytochemical constituents like total phenol, ortho-dihydric phenol, flavonols, tannins and antioxidant activity against 2,2-diphenyl-2-picrylhydrazyl (DPPH), superoxide anion, hydroxyl radical, nitric oxide radical and anti-lipid peroxidation activity. Strong antioxidant scavenging activities were observed in both leaf and stem in different places. Anti-oxidative efficiency to inhibit anti-lipid peroxidation of these plant extracts in goat liver was investigated. Drymaria diandra stem showed moderate class of anti-lipid peroxidation against thioburbituric acid but the leaves have high anti-lipid peroxidation activity. The correlation was also drawn with antioxidants, its attributes and soil nutrients profile. This plant extract may be explored as therapeutic agent in future. Keywords: Drymaria diandra, free-radical scavenging, anti-lipid peroxidation, total phenols, flavonols. INTRODUCTION Reactive oxygen species (ROS), which consist of free radicals such as hydroxyl(oh - ), superoxide (O 2 - ), nitric oxide (NO), peroxyl (RO 2 - ), lipid peroxyl (LOO - ) radicals and non-free radical species such as hydrogen peroxide (H 2 O 2 ), singlet oxygen(o 2-1 ), ozone (O 3 ), lipid peroxide (LOOH), are different forms of activated oxygen (Helliwell et al., 1999; Yildirim et al., 2000; Gulcin et al., 2002a). ROS are produced by all aerobic organisms and can easily react with most biological molecules including proteins, lipids, lipoproteins and DNA. This ROS can generate oxidative stress and produce many pathophysiological disorders such as arthritis, diabetes, inflammation, cancer and genotoxicity (Kourounakis et al., 1999; Gulcin et al., 2002b). Antioxidants can terminate or retard the oxidation process by scavenging free radicals. These antioxidants are considered as possible protection agents for reducing oxidative damage of human body from ROS and retard the progress of many chronic diseases as well as lipid peroxidation (Peryor, 1991; Kinsella et al., * Corresponding author: Palash Mandal, Ph.D. Department of Botany, North Bengal University, Darjeeling, West Bengal, , India nbubotanypalash@rediffmail.com 1993; Lai et al., 2001). However, more recently the polyphenols have found to be beneficial as strong antioxidants (Vinson et al., 2002; Wang et al., 1997). Natural antioxidants are presumed to be safe since they occur in plants. Drymaria diandra is an herbaceous, annual plant, generally found as a weed throughout sub-himalayan part of West Bengal. This plant is commonly known as Abhijalo in Nepali language. The leaves and stems of this plant are traditionally used for curing asthma, snake bite and pneumonia diseases. A novel anti-hiv alkaloid drymaritin and new C- glycoside flavonoids, diandraflavone along with eight known compounds, torosaflavone A, isovitexin, spinasterol β-d-glycoside, p-hydroxybenzoic acid, p- hydroxybenzaldehyde, cis-p-coumarate, methyl 5- hydroxy-4-oxopetanoate and glycerol-α-lignocerate were present in D. diandra extracts (Hsieh et al., 2004). The aim of this work is to evaluate the free-radical scavenging properties like DPPH, nitric oxide radical, superoxide and anti-lipid peroxidation and to quantify phenolic constituents in methanol extracts from the leaves and stems of D. diandra plant in plain land and hilly area of Darjeeling. Antioxidants and its attributes were also correlated with the soil profile of that place for analyzing the location effect of naturally grown D. diandra. International Journal of Integrative Biology IJIB, 2009, Vol. 7, No. 2, 80

2 MATERIALS AND METHODS Plant materials D. diandra (Abijal) was collected from three different places of plain land: NBU (26 º N, 88 º E, 443ft from amsl), NBU Gate No. 3 (26 º N, 88 º E, 440ft from amsl) and NBU Nibedeita (26 º N, 88 º E, 435ft from amsl) and two hill areas of Kurseong, Darjeeling district: Victoria I (26 º N, 88 º E, 5870ft from amsl) and Victoria II (26 º N, 88 º E, 5859ft from amsl), West Bengal, India. Leaves and stems were separated and kept for drying in oven for over night. The dried plant parts were separately crushed in mortar pestle to form fine powdered drug. Taxonomic position was authenticated by the Taxonomy and Environmental Biology Laboratory, Department of Botany, University of North Bengal. The material has been deposited in the NBU Herbarium recorded against the accession number 9482 dated Animal material Goat liver, used for anti-lipid peroxidation assay, were collected from slaughter house immediately after slay and experiment was conducted within one hour after collection. Chemicals Methanol (M), 2.2-diphenyl-1-picryl hydrazyl (DPPH), nitro blue tetrazolium (NBT), reduced nicotinamide adenine dinucleotide phosphate sodium salt monohydrate (NADPH), phenazine methosulphate (PMS), trichloro acetic acid (TCA), thiobarbituric acid (TBA), FeSO 4, 7 H 2 O, KOH, KH 2 PO 4, ethylenediamine tetra acetic acid (EDTA), ascorbic acid, vitamin-e, 2-deoxiribose, ferric chloride (Fecl 3 ), hydrogen peroxide (H 2 O 2 ), sodium nitroprusside, sodium carbonate (Na 2 CO 3 ) were either purchased from Sigma Chemicals (USA) or Himedia or Merck (Germany). Soil sampling and determination of physicochemical properties Soil samples were collected from five different naturally grown areas of D. diandra mentioned above (top and sub soil with 0-15 cm and cm depth respectively) and composite soil were prepared as per the method of Misra et al., The samples were processed for physicochemical analysis viz. ph, electrical conductivity, moisture contents, organic carbon, available form of nitrogen, potash as K 2 O, - phosphorus as P 2 O 5 and sulphur as SO 4 (Jackson, 1973). Extraction and determination of methanol extractive value Powdered drug of plant parts were separately extracted with methanol water in ratio 4:1, under Soxlet extractor for eight hours. The refluxed samples were dried in vacuo the extractive values were calculated on dry weight basis from the formula given below: Weight of dry extract % extractive value (yield %) = Weight taken for extraction DPPH based free radical scavenging activity. I The free radical scavenging activities of each fraction were assayed using a stable DPPH, following standard method (Blois, 1958). The reaction mixture contained 1.8ml of 0.1mM DPPH and 0.2ml of each serial dilution (0.5-2) of D. diandra methanol extracts. Simultaneously, a control was prepared without sample extracts. The reaction mixture was allowed to incubate for 5min at room temperature in the dark and scavenging activity of each fraction were quantified by decolourization at 515nm. Percentage of free radical scavenging activity was expressed as percent inhibition from the given formula: Abs.of control - Abs.of sample.. II % inhibition of DPPH radical = Abs.of control Superoxide radical scavenging assay Measurement of superoxide radical scavenging activity of D. diandra was done by using standard method (Nishikimi et al., 1972) followed by slight modification. The reaction mixture contained 1ml of NBT solution (312 μm prepared in phosphate buffer, ph-7.4), 1ml of NADH solution (936μM prepared in phosphate buffer, ph-7.4) and standardized 50 times methanol diluted different extracts of the sample were added. Finally, reaction were accelerated by adding 100μL PMS solution (120μM prepared in phosphate buffer, ph-7.4) to the mixture. The reaction mixture was incubated at 25 ο C for 5min and absorbance at 560nm was measured against methanol as control. Percentage inhibition was calculated (Eq. II). Hydroxyl radical scavenging assay Scavenging of the hydroxyl free radical was measured by the method of (Halliwell et al., 1989) with minor changes. All solutions were prepared freshly. 200μL of 2.8mM 2-deoxy-2-ribose, 5μL methanol extracts of D. diandra, 400μL of 200μM FeCl 3, 1.04mM EDTA (1:1 V/V), 200μL of H 2 O 2 (1.0mM) and 200μL ascorbic acid (1mM) was mixed to form a reaction mixture. After an incubation period of one hour at 37 o C the extent of deoxyribose degradation was measured by the International Journal of Integrative Biology IJIB, 2009, Vol. 7, No. 2, 81

3 TBA reaction. 1.5ml of 2.8% TCA was added in the reaction mixture and kept for 20 min. at 100 o C taking Vitamin E as positive control. Percentage inhibition was calculated (Eq. II). Nitric oxide radical scavenging assay Nitric oxide was generated from sodium nitroprusside and measured by the Greiss reaction. This assay was done by the method of (Marcocci et al., 1994). 320μL methanol extract, 360μL sodium nitroprusside, 216μL Greiss reagent (1% sulfanilamide, 2% H 3 PO 4 and 0.1% napthylethylenediamine dihydrochloride) was mixed and incubated at 25 o C for one hour. Lastly 2ml water was added and absorbance was taken at 546nm. Percentage inhibition was calculated (Eq. II). Anti-lipid peroxidation (ALP) assay The anti-lipid peroxidation assay in the goat liver homogenate was measured by the standard method (Dhalwal et al., 2005) followed by slight modification. 2.8ml of 10% goat liver homogenate, 0.1ml of 50mM FeSO 4 and 0.1ml extract was mixed. The reaction mixture was incubated for 30min. at 37 o C. 1ml reaction mixture was taken with 2ml 10%TCA-0.67%TBA in acetic acid (50%) for stopped the reaction. Then the mixture was boiled for 1hour at 100 o C and centrifuged at 10,000rpm for 5min.. Supernatant was taken for absorbance at 535nm against a blank. This contained all reagents except liver homogenate and extract. Identical experiments were performed to determine the control (without extract and FeSO 4 ) and induced (without extract) Vitamin E was used for standard. ALP percentage was calculated using the following formula. IC 50 values of all experiment were calculated using different concentration of extract. Abs. of Fe2 + induced peroxidation abs. of sample... III %ALP = Abs. of Fe2 + induced peroxidation abs. of control Estimation of total phenol content Total phenolic compounds were determined according to the protocol described as (Slinkerd et al., 1977). 40 μl of methanolic extract of leaf and stem of D. diandra plant were mixed separately with 1ml Folin-Ciocalteu reagent and 2ml of 20% Na2CO3. It was mixed, boiled for one hour, cooled, and centrifuged at 10,000rpm for five minutes. Absorption was recorded at 575nm in spectrophotometer. Estimation of ortho-dihydric phenol content The estimation of ortho-dihydro phenol was based on the method of Arnow s (Kim et al., 2003). Made the volume of 0.2ml of extracts upto1ml.1ml of 0.05N HCl, 1ml of Arnow s reagent, 10ml water and 2ml of 1N NaOH were mixed thoroughly with the extract. Maintain the reagent blank similarly with out the extract. Absorbance was measured at 515nm in spectrophotometer. Estimation of flavonols content The amount of total flavonoids content for each extract was determined by the method of (Kim et al., 2003). To 1ml sample: water (50:50, v/v) or standard solutions quercetin (0-500 mg L -1 ) was added to 4ml H 2 O in a 10 ml volumetric flask. At zero time, 0.3 ml of 50g L -1 NaNO 2 was added to the flask. After 5min., 0.3ml AlCl 3 (100gL -1 ) was added. At 6min, 2ml 1mol L -1 NaOH were added to the mixture and immediately diluted with 204ml of water. Absorbance of the mixture was read at 510nm vs. water blank. Estimation of tannins content Tannin content was measured by Folin-Denis method (Oyaizu, 1986). 50μL of extract was made upto 7.5ml by adding double distilled water. Then 0.5ml Folin- Denis reagent and 1ml of Na 2 CO 3 were mixed with it. Again Volume was made upto 10ml by double distilled water. Absorption was recorded at 700nm. Statistical analysis The data was pooled in triplicate and subjected to analysis of correlation co-efficient matrix using SPSS (Version 10.00) for drawing the relation between soil physicochemical properties and antioxidant attributes and MS Excel was used for comparing the antioxidant attributes of the parts of plant collected from five different places. Smith s Statistical Package (Version 2.5) was used for determining the IC 50 values of antioxidants and their standard error of estimates (SEE). RESULTS AND DISCUSSION Fig.1 [Supplementary data] shows that the methanol extracts of D. diandra has antiradical activity by inhibiting DPPH radical with the IC 50 value of (0.47 to 3.28µg/ml) which was comparable with quercetin standard. IC 50 value is the effective concentration at which the antioxidant activity is 50%. DPPH is usually used as a substrate to evaluate anti-oxidative activity of antioxidant. The method is based on the reduction of methanol DPPH solution in the presence of a hydrogen donating antioxidant due to formation of the non radical form DPPH-H by the reaction. The extract was able to reduce the stable radical DPPH to the yellow colored diphenyl picrylhydrazine. It has been found that cysteine, glutathione, ascorbic acid, tocopherol, poly-hydroxy aromatic compounds (hydroquinone, pyrogallol, gallic acid, etc.) reduce and decolorize DPPH by their hydrogen donating ability (Blois, 1958). International Journal of Integrative Biology IJIB, 2009, Vol. 7, No. 2, 82

4 It appears that extracts of D. diandra possess hydrogen donating abilities to act as an antioxidant. Fig.1 shows the highest DPPH radical scavenging effect (IC 50 value 0.47µg/ml) present in the leaf extracts of hilly area (Kurseong Victoria I and Victoria II) than the samples of plain land (NBU Basketball Court, Gate No. 3 and Nibedeita Hostel). The data were even superior to standard antioxidant BHT (IC 50 value: 20.15µg/ml.). In the PMS/NADH-NBT system, superoxide anion derived from dissolved oxygen by PMS / NADH coupling reaction reduces NBT. The decreases of absorbance at 560nm with antioxidants thus indicate the consumption of superoxide anion in the reaction mixture. Addition of various extracts of D. diandra in above coupling reaction showed decrease in absorbance (Fig. 2 [Supplementary data]). Maximum inhibition (IC 50 value53.85 µg/ml) was found in leaf sample of hilly area (Kurseong Victoria I). Hsieh et al. (2004) observed that the C-glycoside flavonoids present in the D. diandra extract have significantly selective inhibition on superoxide anion generation from human neutrophils stimulated by fmlp/cb with an IC 50 value of 10.0µg/ml. The extracts with liver homogenate undergo rapid peroxidation when incubated with FeSO 4 and produce peroxide (Aruma, 1996) and they attack the biological material. This leads to the formation of MDA (malonodialdehyde) and other aldehydes, which form a pink chromogen with TBA, absorbing at 535nm (Kosugi et al., 1987). It was observed (Fig. 3 [Supplementary data]) that methanol extract of D. diandra have high anti-lipid peroxidation effect against goat liver in all samples collected from different places of plains and hills. Addition of Fe 2+ /ascorbate to the liver, cause increase lipid peroxidation. The extracts showed inhibition of peroxidation effect in all concentration. The highest anti-lipid peroxidation activity was found (IC 50 value µg/ml) in leaf sample of hill area (Kurseong Victoria I). In extracts inhibition value was found to be lesser than the standard, Vitamin E (IC 50 value 14.75µg/ml). The extract was examined for its ability to act as OH. radical scavenging agent. Ferric EDTA was incubated with H 2 O 2 and ascorbic acid at ph -7.4; hydroxyl radicals were formed in free solution and were detected by their ability to degrade 2-deoxy-2-ribose into fragments that on heating with TBA and low ph form a pink chromogen (Halliwell et al., 1987; Aruoma et al., 1989). The extract and vitamin E exhibited strong scavenging effect of hydroxyl radical which could inhibit lipid damage in different extracts of leaf and stems of D. diandra collected by different places of Darjeeling. When the extracts and vitamin E were added to the reaction mixture they removed hydroxyl radical and prevented the degradation of 2-deoxy-2- ribose. The maximum inhibition (IC 50 value 24.97µg/ml) was found in leaf samples of hill area (Kurseong Victoria II). Fig. 4 [Supplementary data] shows that the plants of hill area have more H 2 O 2 scavenging activity than in plain land. In addition to reactive oxygen species, nitric oxide is also implicated in inflammation, cancer and other pathological conditions (Moncada et al., 1991).The plant or plant products may have the property to counteract the formation of nitric oxide radicals and in turn may be of considerable interest in preventing the ill effects of excessive nitric oxide generation in the human body. But the extracts from this plant of both hill and plain areas executed very less scavenging power against NO as documented from their higher IC 50 values [12, ,127.33mg/ml] (Fig. 5 [Supplementary data]). Still this scavenging activity may be considered important because the plant is consumed as green vegetables in large quantity by local tribes. Among these, the highest nitric oxide scavenging activity was again showed by the samples of hilly areas (Kurseong Victoria I). A number of studies have focused on the biological activities of phenolic compounds, which are potential antioxidants and free radical scavengers (Marja et al., 1999; Sugihara et al., 1999). Their was a wide variation in the amount of total polyphenols in different extracts of D. diandra tested. Among these extracts, the highest phenol content was found (Fig. 6 [Supplementary data]) in leaf sample of hill area (Kurseong Victoria I). From the results, flavonol (Fig. 7 [Supplementary data]), tannin (Fig. 8 [Supplementary data]) and ortho-dihydric phenol content (Fig. 9 [Supplementary data]) was found more in leaf sample of plain land. Although all samples have these phytochemicals in high amount. A new Anti-HIV C- glycoside flavonoids-diandraflavone have been recently isolated from D. diandra. It is well known that flavonoids possess a wide range of antioxidant activities. These antioxidant properties are based on their phenolic structures. Phenolic compounds are also thought to be capable of regenerating endogenous α- tocopherol, in the phospholipids bi-layer or lipoproteins particles, back to its active antioxidant form. They are also known to inhibit various types of oxidizing enzyme. So this flavonoids-diandraflavone acts as antioxidant in D. diandra. These potential mechanisms of antioxidant action make the diverse groups of phenolic compounds an interesting target in the search for health beneficial phytochemicals. The methanol: water leaf extractive value (percentage of yield) was highest (35.00%) in sample collected from NBU Nibedeita Hostel and lowest (7.09 %) in Victoria I (Fig. 10 [Supplementary data]). Mineral nutritional status and physical properties generally ph and EC of soil greatly influence the phytochemicals constituents present in the different parts of the plant. It is also reported that International Journal of Integrative Biology IJIB, 2009, Vol. 7, No. 2, 83

5 phytochemicals constituents directly influences the antioxidant properties of the plant extract (Misra et al., 2008). Correlation coefficient matrix analysis in Table1 [Supplementary data] showed that antioxidant properties, phytochemicals constituents and physicochemical properties of soil significantly correlated with each other at P< 0.05% level. Therefore, marked difference of antioxidant activities as well as phytochemicals were observed in the leaf and stem part of extract of D. diandra collected from different places of North Bengal. CONCLUSION This study suggests that the D. diandra Blume plant (leaf and stem) extract has antioxidant activity, which might be helpful in preventing or slowing the progress of various oxidative stress-induced diseases. The results of the present study also indicate that the plant parts possess many phytochemicals which could be beneficial for human health. References Aruma OI (1996) Characterization of drugs as antioxidant prophylactics. 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