Vol. 38 No 3 2015

Foun ding Edi tor D. GAL, Hun ga ry

Edi tor-in-Chi ef S. K. IVA NOV, Bul ga ria

Edi tors M. I. BON EVA, Bul ga ria Zh. D. KA LITCHIN, Bul ga ria

Edi to ri al Bo ard R. L. AU GU STI NE, USA S. W. BEN SON, USA S. BOUR BI GOT, Fran ce D. BRA DLEY, UK A. M. BRAUN, Ger ma ny E. B. BUR LA KO VA, Rus sia J. DAHLMANN, Ger ma nyA. D’AMORE, Italy S. DOBE, Hun ga rySh. GAO, China N. GE TOFF, Au stria V. K. GUP TA, In dia J. HAP PEL, USA J. A. HOWARD, Can adaM. F. R. MUL CAHY, Au stra lia A. NE METH, Hun ga ry E. NI KI, Ja pan K. OHKU BO, Ja pan P. St. PETKOV, Bulgaria S. K. RAKOVSKY, Bulgaria R. A. SHEL DON, The Nether lands G. E. ZA I KOV, Rus sia J. J. ZIOL KOWSKI, Po land ZUWEI Xi, Chi na

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OXIDATION COMMUNICATIONS is the only scientific journal which, in its new form, is devoted to the global oxy-reduction interactions proceeding in nature.

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ISSN 0209-4541Printed in Bulgaria

Liquid oxidation processes – oxygen and hydrogen peroxide as oxidisers L. I. MATIENKO, L. A. MOSOLOVA, V. I. BINYUKOV, G. E. ZAIKOV Selective Ethylbenzene Oxidation with Dioxygen in the Presence of Binary and Triple Catalytic Systems Introduced Redox Inactive Metal Compound, LiSt, and Additives of Monodentate Ligands-modifiers: DMF, HMPA and PhOH. Kinetics and Mechanism 1169

M. KLOS, K. MALARCZYK, E. MILCHERT Influence of Technological Parameters on the Epoxidation of Linseed Oil with Peracetic Acid 1183 Ionic oxidation processes JINHUAN SHAN, QIANQIAN WANG Oxidation of Neopentyl Glycol and 1,3-Butanediol by Ditelluratocuprate(III) in Alkaline Medium. A Kinetic and Mechanistic Study 1195

A. S. VARALE Study of Thermodynamic Parameters for Oxidation of p-Hydroxy Benzoic Acid Hydrazide by Thallium(III) in Acidic Medium. A Kinetic and Mechanistic Approach 1204

A. S. VARALE Synthesis, Mechanistic Interpretation and Kinetic Approach of m-Toluic Acid Hydrazide by Thallium(III) in Acidic Medium 1213

D. PANDAY, S. KOTHARI Kinetics and Correlation Analysis of Reactivity in Oxidation of Organic Sulphides by Benzimidazolium Dichromate 1221 Inhibitors and antioxidants for foodstuffs and medicine SHENGJUAN JIANG, YUHAN MA, LIAN SONG, YUTING REN Effects of Extraction Conditions on Antioxidant Activities of Water and Ethanol Extractions from Lentinus edodes, Flammulina velutipes, Hericium erinaceus and Ganoderma lucidum 1233 D. FAZLI, GH. VAEZI, A. A. MALEKIRAD, M. ABDOLLAHI Antioxidative Protective Effects of Cinnamomum zeylanicum and Vaccinium myrtillus L. on DNA Damage and Myeloperoxidase Activity in Petrochemical Industry Workers 1244 K. H. SHANI, M. ZUBAIR, K. RIZWAN, N. RASOOL, S. ERCISLI, A. MAHMOOD, M. ZIA-UL-HAQ, L. DIMA, A. M. PASCU Compositional Studies of Oil and Antioxidant Capacity of Oil and Extracts of Diarthron vesiculosum 1252

Biological and biochemical oxidation processes Y. DOGAN Investigation of Micro and Macro Element Content of Wheat Varieties Grown Commonly in Turkey 1265

Y. DOGAN Amino Acid Profile, Nutrients Content and Yield of Chickpea (Cicer arietinum L.) Genotypes 1275

Y. DOGAN, N. TOGAY, Y. TOGAY Nutrient Status and Yield of Chickpea (Cicer arietinum L.) as Influence by Application of Different Harvest Timing and Sulphur Fertiliser

1286 A. OZDEMIR, G. KOCOGLU, N. UTKUALP Nutritional Supplement Use in High School Students 1296 TS. GANEVA, B. TSENOV, M. STEFANOVA, D. KOLEVA Assessment of Regenerative Potential of in vitro and ex vitro Leonurus cardiaca L. Plants by Anatomical Markers 1305 LIU DEJUN, SHANG ANQUAN, DONG TIELI Effect of a Neuroprotective Agent on Cerebral Ischemia Reperfusion Injury in Rat 1314 A. M. KUMLAY, B. A. YILDIRIM, K. EKICI, S. ERCISLI Screening Biological Activity of Essential Oils from Artemisia dracunculus L. 1320

Tribological interactions M. ERDOGAN, R. TEKIN Oxidation of Artificially Aged AA 2014 Aluminum Alloy and Investigation of Its Mechanical Properties 1329 Synthesis of mesoporous nanostructured nickel electrode A. A. AL-OWAIS, I. S. EL-HALLAG Fabrication and Characterisation of Mesoporous Nanostructured Nickel Electrode 1342 Analytical section S. K. SHUKLA, A. PANDEY Quality Control Parameters of Sertraline Hydrochloride in Pure and Dosage Form Available in Indian Market 1349 K. STANCHEVA, S. PAVLOV, A. DAKASHEV Quantitative Chemical Analysis Based on Computer Processing Image of the Turbid Solution 1355 Ecologically friendly oxidative processes for the atmosphere, soils and water DAI XIU-LI, ZHU PEI-YU, ZHUANG YAN, DING JIA-BO, WANG YE Correlation Study of Tai Lake Conventional Water Quality 1364

WANG YANBO, LI QIONG Treatment of Petrochemical Wastewater via Supercritical Water Oxidation with Nano-Fe2O3 1373

LEI MA, XIE CHEN, MIN ZHAO Study on Supercritical Water Oxidation of Petrochemical Wastewater 1378 CAITING FENG, JUN WANG Treatment of Oily Wastewater via Supercritical Water Gasification (SCWG) 1384 FUQIANG WANG, SHIJIANG ZHU, XINGLONG GONG Gasification of Oily Sludge in Supercritical Water 1391 LUSI ZHANG, YAO FENG, GUANGHAO LI Oily Wastewater Treatment via Alkali Catalytic Gasification

1401 JING XU Treating Phenol Wastewater via Supercritical Water Oxidation with Ethylene Glycol

1409 MA GUANG-QIANG, ZOU MIN Alkali Catalytic Gasification of Phenolic Wastewater in Supercritical Water 1415 ZHANG CHUNYAN, YANG LIPING, LIU HUI Catalytic Gasification of Olive Oil Wastewater 1422 FU-YAN LUO, JING-XIA ZHANG Olive Oil Wastewater Treatment in Supercritical Water

1430 WANG GUANG PENG, ZHANG XUAN, SI GUI CAI Antibiotic Wastewater Treatment via Advanced Oxidation Process 1436 LI WEIDONG, JIANG XIA, YANG HUAYUN Gasification of Analgin Pharmaceutical Wastewater 1443 HAIYAN MAO, DINGGUO ZHOU, HENG CHEN, HAIYAN (HELENA) WANG Effective Degradation of Pharmaceutical Wastewater by Advanced Oxidation Process 1452

MIN ZHI HUA An Engineering Study on Gasification Treatment of Printing and Dyeing Wastewater 1458

SHUCONG ZHEN Supercritical Water Gasification (SCWG) of Coking Wastewater 1464

Ecology – remediation of water and soils ZHAN-DONG LI, PENG CHENG, HAI-XIANG ZHANG, CHU-HUI BAO, XIAO-MENG ZHU, BI-YING SHAO, JING MA Perspectives on Dye Wastewater Treatment 1470 F. T. KUKEYEVA, L. F. DELOVAROVA, T. A. ORMYSHEVA, K. N. SHAKIROV Sustainable Development and Water Management Issues: Transboundary Rivers Management Gaps between Kazakhstan and China (Case of Ili and Irtysh) 1480 E. SAKIN, A. SEYREK, E. D. SAKIN Comparison of Some Physicochemical Characteristics and Nutrient Element Status between Cultivated and Uncultivated Soils 1491

I. KIZILGOZ, E. SAKIN Effects of Increasing Soil Application of Nitrogen and Phosphorus on Dry Matter, Dry Weight, and Zinc and Boron Concentration in Wheat and Maize 1504 R. P. TIWARI, ARTI ALOK, R. P. SINGH

Selection of a Suitable Surfactant Based on Zeta Potential for Use as Purging Solution in Electrokinetic Remediation of Cadmium from Cadmium Contaminated Soil 1511

B. TAS Effect of Commercial and Organic Fertilisers on Some Heavy Metal Contents of Semolina 1521 H. AKGUL, C. C. ERGUL, D. YILMAZKAYA, I. AKATA, F. SELCUK, E. HUSEYIN Diversity of Microfungi on Fagaceae in Uludag Forests 1529 A. A. ZHAKUPOV, K. T. SAPAROV, O. B. MAZBAEV, G. M. DZHANALEEVA, M. N. MUSABAEVA, A. EGINBAEVA, E. ATASOY Fundamentals of Recreation-geographic Assessment for Tourism Development 1539

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* For correspondence.

Oxidation Communications 38, No 3, 1252–1264 (2015)

Inhibitors and antioxidants for foodstuffs and medicine

COMPOSITIONAL STUDIES OF OIL AND ANTIOXIDANT CAPACITY OF OIL AND EXTRACTS OF Diarthron vesiculosum

K. H. SHANIa, M. ZUBAIRa, K. RIZWANa, N. RASOOLa, S. ERCISLIb, A. MAHMOODc, M. ZIA-UL-HAQd*, L. DIMAe*, A. M. PASCUe

aDepartment of Chemistry, Government College University, 38 000 Faisalabad, Pakistan bDepartment of Horticulture, Faculty of Agriculture, Ataturk University, Erzurum, Turkey cCollege of Pharmacy, Government College University, 38 000 Faisalabad, Pakistan dOffice of Research, Innovation and Commercialisation, Lahore College for Women University, Lahore, Pakistan eDepartment of Medicine, Transylvania University of Brasov, 500 036 Brasov, Romania E-mail: ahirzia@gmail.com; lorenadima@yahoo.com

ABSTRACT

The present study was carried out to examine the antioxidant, antimicrobial and haemolytic activities of different extracts, fractions and oils (essential and fixed) of Diarthron vesiculosum. GC–MS analysis revealed the presence of 18 and 16 com-pounds in the essential and fixed oils, respectively with 1,2-benzenedicarboxylic acid, diisooctyl ester (35.94 %), 1-(+)-ascorbic acid 2,6-dihexadecanoic (12.24%), Z,Z-3,13-octadecadien-1-ol (14.08 %), 9,12-octadecadienoic acid (Z,Z)-2,3-dihydroxypropyl ester (11.02%) as the major components. The extracts contained appreciable levels of total phenolic and total flavonoid contents and exhibited good DPPH radical scaveng-ing activity, (IC50 0.39–21.5 mg/ml ), and inhibition of peroxidation in linoleic acid system (65.8–89.3%). Moderate to high antimicrobial activity against selected strains of bacteria and fungi was observed. The extracts exhibited 2.84 to 13.15% haemolysis of red blood cells. The results of the present investigation demonstrated significant (p < 0.05) variations in the antioxidant, antimicrobial and haemolytic potential of the plant extracts and fractions.

Keywords: Diarthron vesiculosum, antioxidant activities, antimicrobial activities, oil.

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AIMS AND BACKGROUND

Diarthron vesiculosum F i s c h. & C. A. M e y. ex K a r. & K i r (Thymelaeaceae) is an annual herb native to Afghanistan, India, Pakistan, Kazakhstan and Russia1.The plant is well-known due to its cytotoxic, anthelmintic, insecticidal and antican-cer properties by local healers. Previously daphnane diterpene esters: vesiculosin, isovesiculosin, excoecariatoxin, simplexin and daphnoretin have been isolated and investigated from methanolic extract of seeds of D. vesiculosum. Daphnoretin found in Diarthron vesiculosum has been found to exhibit anti-tumor activity and effective cure to coronary diseases of heart2,3. The toxic potential of Diarthron vesiculosum is due to the presence of daphnane diterpenes. These compounds possess broad spectrum pharmacological activities such as abortionant, insect killing, anticancer, and cure for leukemia4. Despite its multipurpose usage, no detailed study exits on chemical and biological activities of this plant. As part of our studies on exploring medicinal flora of Pakistan for their compositional, nutritional and antioxidant potential5–28, we studied the plant D. vesiculosum to explore its antioxidant, antimicrobial, haemolytic potential and its essential and fixed oil composition.

EXPERIMENTAL

Extraction of plant material. Diarthron vesiculosum was collected from Quetta, Pa-kistan, identified by Dr. Rasool Bakhsh Tareen of Botany Department, University of Balochistan, Pakistan and a specimen (Voucher # DV-RBT-05) was deposited in same department. The whole plant was washed, shade dried and ground to a fine powder (500 g) which was extracted at room temperature for 7 days with methanol (500 ml × 3). The extract was filtered through Whatman No 1 filter paper and then concentrated at 40°C, using a rotary vacuum evaporator. The methanolic extract (80 g) was dissolved in distilled water and fractioned successively with n-hexane (13.39 g), chloroform (12.63 g), ethylacetate (10.86 g), and n-butanol (7.8 g). After drying the remaining crude aqueous extract (15.88 g) and obtained fractions were stored till further analysis.

Phytochemical screening. Phytochemical analysis was carried out by using the standard procedures as described by Edeoga et al.29

Essential oil extraction. Dried powdered plant (100 g) was subjected to hydro-dis-tillation for 5 h using a Clevenger-type apparatus for extraction of essential oil. The extracted essential oil was dried over anhydrous Na2SO4, filtered and stored in a vial at 4°C until further analysis.

Fixed oil extraction. Briefly, 100 g of the powdered plant were extracted with 250 ml n-hexane (99.9% purity) by using a Soxhlet extractor for 6 h and pure fixed oil was kept at 4°C in the dark.

GC-MS analysis of essential and fixed oils. Essential and fixed oils were analysed by GC-MS (QP2010, Shimadzu, Japan) with following parameters (1 μl sample injected,

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split 1:50 column flow 1.0 ml /min., program temperature 200°C, rate 10°C/min) coupled with a quadrupolar MS 5973. GC was equipped with capillary column (30 m× 0.25 mm; film thickness 0.25 μm). Oven temperature was kept at 45°C first for 5 min, and then raised at 325°C at a 15°C/min for another 5 min. Helium gas was then employed at a flow rate of 1.1 ml /min (60 kPa pressure; 38.2 cm/s linear velocity). The identification of components was based on comparison of their mass spectra with those of NIST mass spectral library30.

Determination of total phenolic (TPC) and total flavonoid contents (TFC). The amount of TPC was determined using the Folin–Ciocalteu reagent and the total flavonoid contents (TFC) in plant extract and fractions were determined following the procedure as described by Dewanto et al.31

DPPH radical scavenging assay. Antioxidant activity of the fruit oils and extracts/fractions was assessed by their ability to scavenging DPPH stable radicals as reported earlier25–27. The samples (10 to 500 μg/ml ) were mixed with DPPH solution (1 ml; 90 μM) and then with methanol (95%) to a final volume of 4 ml. Synthetic antioxidant, butylated hydroxytoluene (BHT) was used as control. After 1 h incubation period at room temperature, the absorbance was recorded at 515 nm. Percent radical scaveng-ing concentration was calculated using the following formula:

radical scavenging (%) = 100 × (Ablank – Asample/Ablank),

where Ablank is the absorbance of the control and Asample – absorbance of the test samples.

Analysis of reducing power. The reducing power of the plant extracts, fractions and oils was determined according to the procedure22–25. The extracts at various concentra-tions (2.5–10 mg) were mixed with sodium phosphate buffer (pH 6.6; 5 ml; 0.2 M) and potassium ferricyanide (5 ml; 1.0%). The mixture was heated for 20 min at 50°C. Then trichloroacetic acid (5 ml; 10%) was added and centrifuged at 980 rpm at 5°C for 10 min. Further, its upper layer (5 ml) was dissolved in 5 ml distilled water and finally freshly prepared ferric chloride (1 ml; 0.1%) was added. The absorbance was calculated at 700 nm and a result for each sample was recorded in triplicate.

Inhibition of lipid peroxidation in linoleic acid system. The antioxidant activity of extracts as inhibition of peroxidation in linoleic acid system was evaluated by a re-ported method8–11. To each extract and fraction (5 mg) was taken in separate vials (50 ml) and linoleic acid (0.13 ml), 99.8% ethanol (10 ml) and sodium phosphate buffer (pH = 7; 10 ml) were added to each vial. Then mixture of each vial was diluted to 25 ml with distilled water and all these sample containing vials were incubated at 40oC. The inhibition of peroxidation was determined as: From each sample, 0.2 ml was taken into separate test tubes and added to each test tube 10 ml of 75% ethanol, 0.2 ml of 30% aqueous solution of ammonium thiocyanate and 0.2 ml of FeCl2. The mixture was stirred for 4 min and absorbance was noted at 500 nm. A blank sample was prepared by adding all the reagents to a test tube but without extracts and noted its

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absorbance at 500 nm. The positive control used was butylated hydroxytoluene (BHT) at a concentration of 2 mg/10 ml. The peroxidation level reached to its maximum value when observed after 15 days. The antioxidant activity in terms of inhibition of peroxidation was calculated by using the formula: % inhibition of peroxidation of linoleic acid = 100 – [(increase of absorbance of sample at

360 h/increase of absorbance of control at 360 h) × 100].

Ascorbic acid contents. Ascorbic acid contents of the extracts were determined by a reported procedure32. Dinitrophenyl hydrazine (1.0 g), thiourea (0.115 g) and CuSO4.5H2O (0.135 g) were dissolved in H2SO4 (50 ml; 5M). Aliquot (75 μl) of this solution was added to 500 μl reaction mixture containing 300 μl of extracts and frac-tions of D. vesiculosum, followed by addition of 100 μl of 13.3% trichloroacetic acid (TCA) and water, respectively and incubated the mixture at 37°C for 3 h. Finally, absorbance (520 nm) was observed.

Antimicrobial assay. Bacillus subtilis ATCC 14579, Escherichia coli ATCC 25922, Pasturella multocida locally isolated, Staphylococcus aureus API Staph tac 6736153 were used as bacterial strains and Aspergillus niger ATCC 10595, Aspergillus flavus ATCC 32612, Rhizophus solani ATCC 10231 as fungal strains. The pure bacterial and fungal strains were obtained from the Department of Veterinary Microbiology, University of Agriculture, Faisalabad, Pakistan. The bacterial strains were cultured overnight at 37°C in nutrient agar (Oxoid, UK) while fungal strains were cultured overnight at 28°C using potato dextrose agar (Oxoid, UK).

Antimicrobial disc susceptibility test. Antimicrobial activity of the plant oils and extracts/fractions was determined by using the disc susceptibility test as used by our research group previously22–28 and the inhibition zones were measured. The positive antimicrobial activity was read based on growth inhibition zone.

Minimum inhibitory concentration (MIC). The MIC of the plant oils and extracts was estimated following resazurin microtitre plate assay reported by Sarker et al.33

Hemolytic activity. The hemolytic activity against human red blood cells (RBCs) using Triton X-100 as positive control was estimated by following already reported method34.

RESULTS AND DISCUSSION

Phytochemical constituents. In the present study, efforts were made to qualitatively estimate the various medicinally active constituents such as flavonoids, saponins, tannins, steroids, alkaloids and terpenoids present in D. vesiculosum in dry plant. These constituents were present in the plant, with the exception of anthraquinones and steroids. Powell et al.34 reported the presence of terpenoids in D. vesiculosum. The findings showed that the highest components were flavonoids (4.05 ± 0.01%) in ethylacetate fraction and the lowest were saponins (0.66 ± 0.02%) in absolute methanol

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extract. Tanins were only found in absolute methanol extract (1.21 ± 0.02%) and were absent in other fractions. The percentages of other constituents alkaloids, phenolics, flavonoids, tannins and saponins were found to be significant in all fractions (Table 1).

Table 1. Quantitative phytochemical analysis of extractsExtracts/fractions Alkaloids

(mg/g)Flavonoids

(mg/g)Tannins(mg/g)

Phenolics(mg/g)

Saponins(mg/g)

Absolute methanol 1.65±0.02 0.88±0.01 1.21±0.02 3.53±0.03 0.66±0.0280% Methanol 1.27±0.01 1.52±0.02 – 1.94±0.02 0.92±0.01Crude aqueous – 2.54±0.01 – 1.05±0.05 –n-Butanol 0.86±0.02 1.64±0.03 – 1.57±0.02 –n-Hexane – 1.63±0.03 – 7.93±0.02 –Chloroform 2.24±0.02 2.24±0.02 – 3.88±0.01 –Ethylacetate 1.13±0.02 4.05±0.01 – 1.61±0.01 1.27±0.01Results are the mean of triplicate experiments ± SD.

Percentage yield, total phenolic and total flavonoid contents. The percentage yield of extracts and fractions of D. vesiculosum varied from 1.17–16.14 g/100g of dry powder. The methanol extract of the plant showed maximum yield whereas the yield of aqueous crude extract was minimum (Table 2).The various extracts and fractions of the whole plant D. vesiculosum in the decreasing order of their %age yield are as follows: absolute methanol > ethylacetate > n-hexane > 80% methanol > chloroform > n-butanol > crude aqueous.

Total phenolic contents (TPC) of extracts and fractions ranged from 79.13 mg/100g (n-hexane) to 381.77 mg/100 g (absolute methanol). Decreasing order of total phenolics of extracts and fractions was as follows: absolute methanol > chloroform > 80% methanol > ethylacetate > n-butanol > crude aqueous > n-hexane.

The total flavonoid contents ranged from 99.51 mg/100 g (n-hexane) to 423.8 mg/100 g (absolute methanol extract). The decreasing order of the total flavonoid contents was found to be: absolute methanol > ethylacetate > n-butanol > crude aqueous > chloroform > 80% methanol > n-hexane. The absolute methanol and n-hexane fraction of extract showed highest and lowest amount of total phenolic and total flavonoid contents, respectively (Table 2).

Antioxidant activity. An inverse relationship was found between % free radical (DPPH) scavenging activity and IC50 values. The IC50 values of extracts and fractions in the decreasing order are as follows: n-hexane > chloroform > 80% methanol > crude aqueous > ethylacetate > n-butanol > absolute methanol. The absolute methanolic extract showed highest DPPH radical scavenging activity while n-hexane showed least. All extracts and fractions showed less DPPH radical scavenging as compared to standard BHT (Table 2).

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Table 2. Percentage yield, total phenolic and total flavonoid contents and antioxidant activitya

Extracts/frac-tions

Yield(%)

TFCb (mg/100 g)

TPCc (mg/100 g)

% Inhibition of linoleic

acid (% per-oxidation)

DPPH scav-enging IC 50

(mg/ml)

Ascorbic acid con-

tents (mg/g)

Absolute methanol

16±0.200 423.8±7.48 381.7±7.52 89.3±1.22 0.390±0.007 3.11±0.10

80% Methanol 3.54±0.004 152.7±0.66 192.1±0.53 67.1±1.09 3.16±0.001 1.17±0.01Crude aqueous 1.17±0.002 253.2±0.85 102.0±0.39 69.7±1.04 2.261±0.001 1.07±0.55 n-Butanol 1.98±0.052 324.9±0.96 156.1±0.48 75.2±1.38 0.741±0.001 2.69±0.07n-Hexane 4.09±0.005 99.5±0.53 79.1±0.34 65.8±1.20 21.5±0.006 2.28±0.05Chloroform 3.13±0.011 206.5±1.17 350.1±10.5 78.1±1.47 4.56±0.001 1.99±0.03Ethylacetate 5.27±0.093 405.6±1.08 159.9±0.49 82.7±1.23 0.932±0.001 2.47±0.13BHT – – – 92.6±1.10 0.164±0.001 –aValues are mean ± SD of three separate experiments; btotal phenolic contents calculated in mg/100 g of dry plant material are expressed as gallic acid equivalent; ctotal flavonoid contents calculated in mg/100 g of dry plant material are expressed as catechin equivalent.

The extracts and fractions of D. vesiculosum exhibited % inhibition of peroxi-dation in linoleic acid system ranging from 65.84% (n-hexane fraction) to 89.36% (absolute methanol extract). Percentage inhibition of peroxidation of extracts and fractions was as follows: absolute methanol > ethylacetate > chloroform > n-butanol > crude aqueous > 80% methanol > n-hexane. The methanolic extract, ethyl acetate fraction, n-butanol fraction, showed significant % inhibition of peroxidation of linoleic acid. BHT showed highest inhibition of peroxidation (92.36%) slightly higher than that of methanolic extract (89.362%) (Table 2).

Antioxidants activity was also determined by the measurement of reducing power of extracts/fractions. The reducing potential (Fig. 1) at concentrations of 2.5–10 mg/ml was noted to be in the range of 0.237–1.82 nm. The maximum absorbance (1.821 nm) was recorded for ethylacetate fraction, while the minimum for n-hexane (0.237 nm) fraction.

The decreasing order of reducing power of extracts and fractions was as follows: ethylacetate > absolute methanol > n-butanol > crude aqueous > chloroform > 80% methanol > n-hexane. The ethylacetate fraction and absolute methanolic extract, n-butanol fraction showed excellent reducing power whereas 80% methanolic extract, crude aqueous and chloroform fractions exhibited better reducing powers as compared to n-hexane fraction (Fig. 1).

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Fig. 1. Reducing power activity of extracts and fractions of D. vesiculosum

Total ascorbic acid contents. High amount of total ascorbic acid content was observed in absolute methanolic extract of plant and lower values were observed in crude aqueous extract (Table 2). While other fractions n-butanol, ethylacetate, chloroform showed significant contents of ascorbic acid. Results of the present study showed that among all the extracts and fractions, absolute methanolic extract of plant extracted the highest amount of TPC and TFC, which also demonstrated the highest antioxidant activity as measured by DPPH radical scavenging, inhibition of linoleic acid oxidation and reducing power assay. This may be due to the high polarity of methanol, whereas, n-hexane fraction demonstrated the least antioxidant activity probably because of its low polarity. Previous reports26 also revealed that the methanolic extracts of plant materials offer more effective antioxidants.

Antimicrobial activity. The antimicrobial activity of extracts and fractions was ex-pressed as inhibition zone (IZ, Table 3) and minimum inhibitory concentrations (MICs, Table 4). The absolute methanol extract showed potent antibacterial activity against Nitrospora (IZ = 22.2 mm; MIC = 0.92 mg/ml) whereas n-hexane fraction did not showed any activity against Nitrospora. n-butanol (IZ = 14.7 mm; MIC = 1.29 mg/ml), crude aqueous (14.7 mm; MIC = 1.16 mg/ml) and chloroform (IZ = 13.2 mm; MIC = 1.19 mg/ml) fractions showed potent activity against E. coli. n-butanol (IZ = 15.2 mm; MIC = 1.33 mg/ml ) and ethylacetate (IZ = 12.1 mm; MIC = 1.66 mg/ml) fractions showed potent activity against B. cerus while n-hexane fraction exhibited no activity against B. cerus.

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Table 3. Antimicrobial activities of various extracts and fractions of D. vesiculosumExtracts/fractions Inhibition zone (IZ) (mm)

B. cereus E. coli Nitrospira A. flavus C. albicans A. nigerAbsolute methanol 10.3±0.15 10.1±0.15 22.2±0.25 12.0±0.02 11.1±0.11 –80% Methanol 11.1±0.10 12.3±0.35 13.1±0.21 21.9±0.10 16.2±0.20 –Crude aqueous 9.13±0.06 14.7±0.25 12.1±0.15 20.8±0.26 14.2±0.25 –n-Butanol 15.2±0.10 14.7±0.35 8.27±0.25 15.2±0.07 18.8±0.12 –n-Hexane – 8.83±0.15 – 25.7±0.45 14.1±0.10 –Chloroform 10.1±0.06 13.2±0.26 14.7±0.15 – 13.2±0.20 –Ethylacetate 12.1±0.15 9.33±0.31 10.1±0.10 15.7±0.40 – –Standard 26.04±0.11 26.5±0.22 22.4±0.24 27.9±0.3 25.9±0.28 22.3±0.23Results are the mean of triplicate experiments±SD, P < 0.05; Rifampicin and fluconazole were used as standard drugs (control) for bacteria and fungus, respectively.

Table 4. Minimum inhibitory concentration (MIC) of D. vesiculosum extracts and fractionsExtracts/fractions MIC (mg/ml )

B. cereus E. coli Nitrospira A. flavus C. albicans A. nigerAbsolute methanol 1.91±0.01 1.47±0.01 0.92±0.01 1.73±0.01 1.79±0.01 –80% Methanol 1.78±0.01 1.29±0.01 1.51±0.01 0.85±0.02 1.41±0.01 –n-Butanol 1.33±0.01 1.07±0.01 2.08±0.01 1.54±0.01 1.12±0.01 –Ethyl acetate 1.66±0.01 1.66±0.01 1.92±0.01 1.49±0.01 – –Chloroform 1.96±0.01 1.19±0.01 1.22±0.01 – 1.64±0.01 –n-Hexane – 1.79±0.01 – 0.79±0.01 1.59±0.01 –Crude aqueous 2.18±0.01 1.16±0.01 1.83±0.01 0.97±0.01 1.51±0.01 –Standard fungone

for fungus and ciprofloxin for bacteria

0.74±0.01 1.02±0.01 0.75±0.01 0.67±0.01 0.72±0.01 0.81±0.01

Results are the mean of triplicate experiments ± SD, P < 0.05; Rifampicin and fluconazole were used as standard drugs (control) for bacteria and fungus, respectively.

Among tested fungal strains the plant showed no activity against A. niger, whereas n-hexane fraction (IZ = 25.7 mm; MIC = 0.79 mg/ml) and 80% methanol extract (IZ = 21.9 mm; MIC = 0.85 mg/ml) showed potent activity against A. flavus. All extracts and fractions showed significant antifungal activity against C. albicans except ethyl-acetate fraction. Overall the results indicated that the all tested extracts and fractions of the plant showed significant activity against tested microbial strains. The secondary metabolites identified during the phytochemical assay in the plant saponins, flavo-noids, alkaloids and terpenoids are responsible for this activity as shown in Table 4.

Haemolytic activity. The extracts and fractions expressed as percentage haemolysis of red blood cells ranged from 2.84% (crude aqueous fraction) to 13.15% (80% methanol extract). The extract which exhibits haemolysis of RBCs less than 10% is safe to use

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in herbal medicines whereas those with 10% or higher haemolysis of RBCs might be used as anticancer or antitumor medicine. Methanolic (80%) extract exhibited 13.15% haemolysis of RBCs whereas all the other extracts or fractions showed less than 10% RBCs haemolysis.

Fig. 2. Percentage haemolysis of RBC by extracts and fractions of D. vesiculosum

The higher value of % haemolysis of RBCs might be due to the presence of dit-erpenes whose presence have already been reported in D.vesiculosum2 which possess anticancer or antitumor activities (Fig. 2). The mechanical stability of the membrane of red blood cells (RBCs) is a good indicator to evaluate in vitro the effects of various compounds when screening for cytotoxicity. Treating cells with a cytotoxic compound can cause different problems to human beings. The cells may undergo a loss of mem-brane integrity and die rapidly as a result of cell haemolysis.

GC-MS analysis of D. vesiculosum oils. The GC-MS analysis identified 18 and 16 compounds in the essential and fixed oils of D. vesiculosum plant respectively (Figs 3 and 4, Table 5). The major components found in essential oil were 1,2-benzenedicar-boxylic acid di-isooctyl ester (35.94%) which is a fatty acid ester and 1-(+)-ascorbic acid 2,6-dihexadecanoate (12.24%) which is a vitamin. While in fixed oil the major components were alcohol namely Z,Z-3,13-octadecadien-1-ol (14.08%); fatty acid ester, i.e. 9,12-octadecadienoic acid (Z,Z)-2,3-dihydroxypropyl ester (11.02%) and sesquiterpene namely verrucarin A,7′-deoxo-7′-(l-hydroxyethyl)- (9.45%). Hydrocar-bons present in essential oil were 2,2-dimethoxybutane, 3,3-dimethoxy-2-butanone, undecane, nonane, 5-(l-methylpropyl)-, while hydrocarbons present in essential oil were tridecane, 1-pentadecyne, heptadecane and 2,6,10,15-tetramethyl-ester.

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Fig. 3. GC-MS spectrum of essential oil of D. vesiculosum

Fig. 4. GC-MS spectrum of fixed oil of D. vesiculosum

Fatty acid esters recorded were 9,12-octadecadienoic acid (Z,Z)-,2,3-dihydroxy-propyl ester, octadecanoic acid, methyl ester, hexadecanoic acid, butyl ester, hexa-decanoic acid, 1-(hydroxymethyl)-1,2-ethandiyl ester, hexadecanoic acid and butyl ester. Major fatty acid was 1-beta-d-ribofuranosyl-1,2,4-triazole-3-carboxylic acid while main alcohol was Z,Z-3,13-octadecadien-1-ol, ethanol, 2-(9-octadecenyloxy)-, (Z)-. The chemical constituents found in oil are responsible for biological activities and pharmacological properties exhibited by oil. 1, 2-benzenedicarboxylic acid di-isooctyl ester has antimicrobial properties. 1-(+)-ascorbic acid 2,6-dihexadecanote has antioxidant, anti-inflammatory, anti-nociceptive and anti-bacterial properties35,36.

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Table 5. GC-MS product distribution and relative proportion (wt.% of total compounds) of essential oil and fixed oil from D. vesiculosum (mg/g oil)Retention time (min)

Chemical constituents Composition (%)essential

oilfixed oil

3.40 2,2-dimethoxybutane 10.07 7.614.857 3,3-dimethoxy-2-butanone 2.02 –4.974 acetaldehyde, di-sec-butyl acetal 0.82 –8.693 undecane 1.85 –8.695 tridecane – 1.4816.402 octadecanoic acid, methyl ester 0.74 –16.641 1-(+)-ascorbic acid 2,6-dihexadecanoate 12.24 –16.641 n-hexadecanoic acid – 6.3517.6 1-pentadecyne – 1.1617.881 Z,Z-3,13-octadecadien-1-ol – 14.0817.908 ethanol,2-(9-octadecenyloxy)-,(Z)- 3.81 –18.026 hexadecanoic acid, 1-(hydroxymethyl)-1,2-ethandiyl ester – 3.6318.033 1-beta-d-ribofuranosyl-1,2,4-triazole-3-carboxylic acid 1.61 –18.176 hexadecanoic acid, butyl ester – 3.3419.808 unidentified 1.04 –19.254 9,12-octadecadienoic acid (Z,Z)-,2,3-dihydroxypropyl ester – 11.1220.07 heptadecane,2, 6, 10, 15-tetramethyl- – 1.5120.072 nonane,5-(l-methylpropyl)- 1.79 –20.301 1,2-benzenedicarboxylic acid,diisooctyl ester 35.94 5.6220.775 d-glucopyranose, 4,6-o-nonylidene- – 2.0521.008 1, 2, 4, 6-o-dibenzylidene-d-glucose – 2.0021.162 erythro-9,10-dibromopentacosane 3.09 –21.165 N,N-dimethylcholestan-6-amine – 2.8921.317 5-(p-methoxyphenyl)-3-(6-methyl-3-pyridyl)-2-pyrazoline 0.67 –21.358 t* 0.98 –21.835 ergostane-5,25-diol,3,6,12-tris[(trimethylsilyl)oxy]-,25-

????acetate,(3beta,5alpha,6beta,12beta)- 1.30 –

21.859 cholestano[3,2-c]isoquinolin-1′ (2’H)-one, 3′,4′-dihydro-6′,7′-dimethoxy-

– 2.04

22.008 verrucarin A,7′-deoxo-7′-(l-hydroxyethyl)- 2.25 9.4522.108 5-tridecaflourohexyl-uridine – 4.6122.307 hexadecane,8-hexyl-8-pentyl- 1.58 –23.392 11-nor-delta-8-tetra-hydrocannabinol-9-carboxylic acid

di-TMS 1.33 –

t* – traces; compounds identification was carried on the basis of retention time and EI/MS.

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CONCLUSIONS

The extract and fractions of the plant exhibited good antioxidant and antimicrobial activities but methanolic extract was more potent as antioxidant agent and it possessed highest amount of total phenolic and total flavonoid contents. More research is needed to isolate the constituents responsible for the biological actions.

ACKNOWLEDGEMENTS

We are highly thankful to the Higher Education Commission Islamabad, Pakistan for providing funds through the project (Project No 20-1563/R&D/09/1582) for the purchase of chemicals and other research-related materials.

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Received 29 January 2015 Revised 25 February 2015