Phytochemical composition, biological potential and enzyme inhibition activity of Scandix pecten-veneris L.

Phytochemical composition, biological potential and enzyme inhibition activity of Scandix pecten-veneris L. Scandix pecten-veneris L.的植物素、生物学潜力及酶抑制活性研究 1 first-author 2 3 4 2 5 2 6 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:hamayun84@yahoo.comimrancl@uop.edu.pk †E-mail:hamayun84@yahoo.comimrancl@uop.edu.pk hamayun84@yahoo.com 2 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:hamayun84@yahoo.comimrancl@uop.edu.pk †E-mail:hamayun84@yahoo.comimrancl@uop.edu.pk hamayun84@yahoo.com Department of Chemistry, Islamia College University, Peshawar-25120 KP, Pakistan Department of Chemistry, Islamia College University, Peshawar-25120 KP, Pakistan Biochemistry Section Institute of Chemical Sciences, University of Peshawar, Peshawar-25120 KP, Pakistan Biochemistry Section Institute of Chemical Sciences, University of Peshawar, Peshawar-25120 KP, Pakistan Department of Chemistry, University of Swabi, Anbar-23561 KP, Pakistan Department of Chemistry, University of Swabi, Anbar-23561 KP, Pakistan Department of Botany, Islamia College University, Peshawar-25120 KP, Pakistan Department of Botany, Islamia College University, Peshawar-25120 KP, Pakistan State Key Laboratory of Chemical Resource Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China State Key Laboratory of Chemical Resource Engineering, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China Interdisciplinary Research Center in Biomedical Materials, COMSATS Institute of Information Technology, Lahore-54000, Pakistan Interdisciplinary Research Center in Biomedical Materials, COMSATS Institute of Information Technology, Lahore-54000, Pakistan Objective: 2 Scandix pecten-veneris L. is a less studied wild edible herb and is considered an extinct plant species in many parts of the world. This study was designed to evaluate its phytochemical composition and biological potential of S. pecten-veneris L. Methods: 2 Phytochemicals including alkaloids, flavonoids, polyphenols, and tannins were determined in extracts of S. pecten-veneris . Antioxidant activity was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH), while reducing power was tested by ferric reducing/antioxidant power (FRAP) assay. Antimicrobial activity against seven bacterial and four fungal strains was evaluated using agar well diffusion assay. Enzymes inhibition study was performed for urease, phosphodiesterase-I, and catalase-II. Results: 2 S. pecten-veneris showed moderate antiradical activity and reducing potential of hydroxyl radicals to about 20% of the initial value. The antioxidant activity of various extracts of S. pecten-veneris showed a linear correlation with total phenolic contents in the order of water > n -butanol > chloroform > ethyl acetate > methanol extracts. S. pecten-veneris leaves showed the highest inhibitory activity against Staphylococcus aureus while the highest antifungal activity was observed against Candida albicans . The plant extract was most potent against urease enzymes but showed moderate activity against phosphodiestrase-I and carbonic anhydrase-II. Conclusions: 2 Our data demonstrate that in addition to its culinary uses, S. pecten-veneris has good medicinal potential and hence could be used for treating some specific health ailments. 目的 2 本研究旨在测定 Scandix pecten-veneris L.的植物素和评价其生物学潜力. 方法 2 测定 S. pecten-veneris 提取物中植物素的含量,包括生物碱、黄酮、多酚和单宁.使用2,2-二苯基-1-苦肼基(DPPH)测定抗氧化活性;同时通过铁还原/抗氧化能力(FRAP)测定还原能力;使用琼脂扩散测定法评价对七种细菌和四种真菌菌株的抗微生物活性.同时,对脲酶、磷酸二酯酶-I和过氧化氢酶-II进行酶抑制研究. 结论 2 沙门氏菌显示出适度的抗自由基活性;羟基自由基的潜能降至初始值的20%左右. S. pecten - veneris 多种提取物的抗氧化活性与总酚含量呈线性相关. S. pecten-veneris 叶对金黄色葡萄球菌表现出最高的抑制活性;对白色念珠菌表现出到最高的抗真菌活性.植物提取物对脲酶的活性最有效;对磷酸二酯酶-I和碳酸酐酶-II显示出中等活性.结果表明, S. pecten-veneris 具有良好的药用潜力,可用于治疗一些特定的疾病. Scandix pecten-veneris L. Phytochemical composition Antioxidant activity Antimicrobial potential Enzyme inhibition Scandix pecten-veneris L. 植物素 抗氧化活性 抑菌能力 酶抑制 Introduction 1 Traditional plant medicines hold an important place in the treatment of many diseases, especially in developing countries (Kalemba and Kunicka, 2003 2003 ). Because of the presence of biologically active compounds, plant-based medicines have been recognized as an effective tool for the treatment of a wide range of human diseases (Newmann et al., 2003 2003 ; Bakkali et al., 2008 2008 ). The increasing resistance of pathogens to antibiotics and their associated side effects make plant-based therapy an attractive area of research. A number of plants have been recognized with multiple therapeutic potential. Scandix pecten-veneris L. commonly known as Shepherd’s needle, is a member of the Apiaceae or Umbelliferae family (Table 1 1 ), and is one of the species used against infectious diseases in traditional medicines. It is characterized by highly divided curly pinnate leaves and tiny white flowers arranged in clusters called umbels (Press and Gibbons, 1993 1993 ). S. pecten-veneris is a dicot, annual scapose herb (Aglaia, 2014 2014 ). The plant can grow up to 15–50 cm tall, and has long narrow and pointed fruit of 80 mm in length. It inhabits coastal sites, arable fields, and wasteland, and prefers heavy clay soils. The plant is native to Eurasia and present in Europe, reaching west to the UK and north to Denmark (Wildscreen Arkive, 2002 2002 ), and is also found in some parts of South Asia. S. pecten-veneris along with many other species is listed as an extinct or threatened weed species in many parts of the world (Pinke et al., 2011 2011 ). It was once widespread and abundant, but has declined considerably probably due to the destruction of field margin habitats and the application of chemical herbicides and fertilizers (Berry et al., 2002 2002 ). Also the seeds of S. pectin-veneris cannot stay dormant for long, increasing the risk of local extinction during times of improper habitat management (Wildscreen Arkive, 2002 2002 ). From Aglaia (2014) Taxa Name Kingdom Plantae Phylum Tracheophyta Class Magnoliopsida Order Apiales Family Umbelliferae Genus Scandix Species pecten-veneris L. Botanical name Scandix pecten-veneris L. English name Shepherds needle, Venus’s-comb S. pecten-veneris grows both in cultivated and waste places and prospers in loam, sandy, and heavy clay soils. It grows mainly during autumn and early winter. Seeds of the annual plant tend to germinate in October and early November (Wildscreen Arkive, 2002 2002 ), although they can also germinate in spring on arable land and flower between May and August (Press and Gibbons, 1993 1993 ). The leaves of S. pectin-veneris represent the main edible part of the plant and are consumed after being boiled and mixed with oil. They are also used in pies (Newman et al., 2003 2003 ; Algaia, 2014). Young stems of S. pecten-veneris are used as a pot-herb or in salads in both raw and cooked forms (Hedrick and Sturtevant, 1972 1972 ; Tanaka, 1976 1976 ). The plant has been used as remedy for toothache and other diseases. The important beneficial health effects include a reduction in the risk of chronic inflammatory diseases (Strzelecka et al., 2005 2005 ). Ethanolic extracts of S. pecten-veneris have been observed to decrease tumor necrosis factor α (TNFα) and nitric oxide synthesis in lipopolysaccharide (LPS)-activated monocytes, and significantly reduce the surface expression of VCAM1 on TNFα-stimulated endothelial cells. Additionally, they can decrease LPS or cytokine-stimulated iNOS mRNA levels (Strzelecka et al., 2005 2005 ). The consumption of wild plants in the Khyber Pakhtunkhwa regions of Pakistan has been documented for many years. The region is rich in wild flora and S. pecten-veneris is found growing there as a wild herb. Previously, we have determined the nutritional composition of S. pecten-veneris (Imran et al., 2007 2007 ), but the in vitro and in vivo biological activity of this species is largely unknown. Therefore, this study was performed to determine the phytochemical composition and biological activity, including antioxidant, antimicrobial, and enzyme inhibition activity of S. pecten-veneris . Materials and methods 1 Chemicals 2 The solvents (chloroform, ethyl acetate, methanol, n -butanol) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich (USA). Iron (III) chloride (FeCl 3 ·6H2O) and sodium acetate (CH 3 COONa·3H2O) were purchased from Merck (Germany). Enzymes including phosphodiesterase-I and carbonic anhydrase-II were purchased from Sigma-Aldrich (USA) and urease from Sigma-Aldrich (Germany). Sample collection 2 S. pecten-veneris leaves were collected from Tarnab Farm Agricultural area, Peshawar, Khyber Pakhtunkhwa of Pakistan during spring (March to April) 2016. The plant was identified at the Botany Department, Peshawar University, Pakistan. Before analysis any visible dirt or insect parts were removed from the plant samples. Fresh and healthy leaves were dried in the shade for at least two weeks, then crushed and stored in the dark. Extraction and phytochemical screening 2 Extraction was carried out by soaking dry powdered leaves (about 2 g) of S. pecten-veneris in methanol (30 ml) for 3 d. The extract was filtered, dried, and subjected to phytochemical screening (Ayoola, et al., 2008 2008 ; Wadood et al., 2013 2013 ). Results for the phytochemicals screening were expressed as “+” or “−” for the presence or absence of the phytochemicals, respectively. Estimation of phytochemicals 2 The total phenolic content was quantified using the Folin-Ciocalteu method (Ozyigit et al., 2007 2007 ). Briefly, a mixture containing 0.5 ml of extract in methanol (1 mg/ml), 2.5 ml of Folin-Ciocalteu’s reagent (10% (v/v) dissolved in water), and 2.5 ml NaHCO 3 (7.5% (75 mg/ml)) was incubated at 45 °C for 45 min. A blank was concomitantly prepared by mixing 0.5 ml methanol, 2.5 ml Folin-Ciocalteu’s reagent, and 2.5 ml NaHCO 3 . Absorbance was measured at 765 nm using spectrophotometer (SP-3000 plus Optima, Japan). Gallic acid (GA) was used as a standard and total phenol was expressed in terms of gallic acid equivalents (mg of GA/g of extract). Flavonoid content was determined by the aluminum chloride colorimetric method (Lin and Tang, 2007 2007 ). Briefly, 1 ml each of AlCl 3 (2% (0.02 g/ml) solution in methanol) and methanolic plant extract (1 mg/ml) was mixed and incubated for 1 h at room temperature. Absorbance was measured using the spectrophotometer (SP-3000 plus Optima, Japan) at 415 nm. The same procedure was repeated for standard solutions of rutin (RU). Flavonoid content was determined and expressed in terms of rutin equivalents (mg of RU/g of extract). The total alkaloid content was estimated gravimetrically using aluminum hydroxide precipitation (Gracelin et al., 2013 2013 ). For alkaloid determination, 1 g of the ground plant material was suspended in 200 ml acetic acid (10% (v/v) in ethanol) for 4 h. The filtrate was concentrated in a water bath to one-fourth of the original volume, followed by the drop wise addition of concentrated ammonium hydroxide until precipitation of the alkaloid was complete. The precipitated alkaloid was washed with dilute ammonium hydroxide, dried, and weighed. Total tannin was quantified spectrophotometrically (Gracelin et al., 2013 2013 ). Briefly, 0.5 g of sample was suspended in 50 ml of distilled water and shaken for 1 h. The mixture was filtered into a 50-ml volumetric flask and made up to the mark. Then 5 ml of the filtrate was mixed with 2 ml of 0.1 mol/L FeCl 3 in 0.1 mol/L HCl and 0.008 mol/L potassium ferrocyanide. The mixture was incubated for 10 min and its absorbance was measured at 120 nm. This procedure was repeated for tannic acid as a standard. Total tannin content was expressed in terms of tannic acid equivalent (mg of tannic acid/g of extract). Antioxidant activity 2 2.5.1 DPPH radical scavenging activity The total antioxidant activity was measured using 2,2-diphenyl-1-picrylhydrazyl (DPPH), according to the previously reported procedure (Dorman et al., 2004 2004 ; Huang et al., 2012 2012 ). Leaves of S. pecten-veneris were subjected to methanolic extraction (3 d) and fractionated using chloroform, ethyl acetate, n -butanol, and water. DPPH (2 ml, 0.1 mmol/L in methanol) and plant extract in various solvents (1 ml, 1.0 mg/ml) were mixed thoroughly, and incubated at room temperature for 30 min. The optical densities of the samples were measured (at 517 nm) using the spectrophotometer (SP-3000 plus Optima, Japan). Vitamin C (ascorbic acid) was used as the positive control. The following equation was used for calculation of DPPH radical scavenging potential: DPPH scavenging effect (%)=(OD 1 –OD 0 )/OD 0 ×100%, where OD 0 is the optical density of the negative control and OD 1 is the optical density of the sample or the positive control. 2.5.2 Ferric reducing power assay Ferric reducing/antioxidant power (FRAP) assay was carried out using the method reported by Iamsaard et al. ( 2014 2014 ). Plant extracts (1 ml, 1 and 3 mg/ml) were mixed with phosphate buffer (2.5 ml, pH 6.6) and potassium ferricyanide (2.5 ml, 1% (0.01 g/ml)), incubated for 20 min at 50 °C, and centrifuged after the addition of trichloroacetic acid (2.5 ml, 10% (0.1 g/ml)). The upper layer was separated and mixed with ferric chloride (0.5 ml, 0.1% (1 g/L)) and distilled water (2.5 ml). The optical density (at 700 nm) was measured using the spectrophotometer (SP-3000 plus Optima, Japan). Vitamin C was used as the positive control. Antibacterial and antifungal assays 2 The antimicrobial effects of S. pecten-veneris were evaluated against 11 strains of microorganisms including bacteria and fungi. Antimicrobial activity was determined by the agar well diffusion method. Seven bacterial species were tested, selected as representatives of both Gram-positive and -negative classes: Escherichia coli , Salmonella typhi , Streptococcus pneumoniae , Proteus vulgaris , Pseudomonas aeruginosa , Staphylococcus aureus , and Bacillus subtilis . Fungal species tested included Aspergillus flavus , Aspergillus niger , Aspergillus parasiticus , and Candida albicans . Ten microliters of bacterial and fungal cultures were added to different flasks containing pre-melted and cooled (45 °C) nutrient agar (1.5% (15 g/L) agar, 0.5% (5 g/L) peptone, and 0.3% (3 g/L) beef extract in distilled water) and potato dextrose agar (0.2% (2 g/L) agar, 0.15% (1.5 g/L) peptone, and 0.04% (0.4 g/L) potato starch in distilled water), respectively. The mixtures were then transferred to different culture dishes, solidified, and using sterile steel cork borer, wells of uniform diameter (6 mm) were made on the solidified agar. One hundred microliters of plant extract (5 mg/ml) were added to wells and incubated at 37 °C for 24 h. Standard drugs and dimethyl sulphoxide (DMSO) were used as the positive and negative controls, respectively. Zones of inhibition were measured in mm (Stepanovie et al., 2003 2003 ). Enzyme inhibition 2 2.7.1 Urease inhibition assay Urease inhibition activity was determined using the phenol red method (Amin et al., 2013 2013 ). Briefly, reaction mixtures comprising 55 μl of phosphate buffer (pH 6.8), 25 μl of Jack bean ( Canavalia ensiformis ) urease, 5 μl of plant extract (0.2 mg/ml) in DMSO and 100 mmol/L of urea were incubated at 30 °C for 15 min in 96-well plates followed by the addition of 45 μl of phenol reagent (0.005% (0.05 g/L) sodium nitroprusside and 1% (0.01 g/ml) phenol), and 70 μl of alkali reagent (0.1% (1 g/L) NaOCl and 0.5% (5 g/L) NaOH). Optical density was measured at 630 nm in a microplate reader (Molecular Devices, CA, USA). Thiourea was used as the standard inhibitor. Percent urease inhibition was calculated as follows: inhibition (%)=1−(OD sample /OD control )×100%, (1) where OD sample is the optical density of the sample and OD control is the optical density of the control. 2.7.2 Phosphodiesterase-I inhibition assay Phosphodiesterase-I activity (snake venom) was determined as described previously (Rauf et al., 2015 2015 ). Tris-HCl (33 mmol/L), magnesium acetate (30 mmol/L) buffer (pH 8.8), plant extract and enzyme (0.742 mU/well) were incubated at 37 °C for 30 min followed by the addition of 0.33 mmol/L bis-( p -nitropheny1) phosphate. The optical density was measured at 410 nm in a plate reader (Molecular Device, CA, USA). Ethylene diamine tetra acetate (EDTA) was used as the positive control. The percent phosphodiesterase inhibition activity was calculated by Eq. (1). 2.7.3 Carbonic anhydrase-II inhibition assay Carbonic anhydrase-II (CA-II) inhibition assay was performed as reported previously (Iyer et al., 2006 2006 ), with slight modification. Briefly, the HEPES-Tris solution (20 mmol/L, 140 µl, pH 7.2–7.9) was mixed with 20 µl of bovine erythrocyte CA-II (0.1 mg/ml in deionized water), 20 µl of plant extract and 4-nitrophenylacetate (4-NPA) at a concentration of 0.8 mmol/L. The mixture was incubated for 15 min and the optical density was monitored at 1 min intervals for 30 min in a plate reader (Molecular Device, CA, USA) at 400 nm. Acetazolamide was used as the positive control, CA-II inhibitor. The percent carbonic anhydrase-II inhibition activity of S. pectin-veneris was calculated by Eq. (1). Statistical analysis 2 All experiments were performed in triplicate and results were expressed as mean±standard deviation (SD). Student’s t -test was used to test differences between the means and a P- value of < 0.05 was regarded as statistically significant. Results and discussion 1 Phytochemical composition 2 Phytochemical screening of S. pecten-veneris revealed the presence of flavonoids, phenols, tannins, reducing sugars, cardiac glycosides, etc. However, saponins, pholobatanins, or anthraquinones were not detected in plant leaves (Table 2 2 ). A quantitative analysis was carried out for alkaloids, flavonoids, phenols, and tannins (Table 3 3 ). The methanolic extract was found to contain (48.90±3.93), (98.00±6.78), (48.00±4.45), and (65.00±3.93) mg/g (on dry weight basis) alkaloids, flavonoids, tannins, and phenols, respectively. Flavonoids have been shown to have broad biological activity including antiviral, anti-inflammatory, antibacterial, vasodilatory, antineoplastic, antithrombotic, and antiallergic properties (Miller, 1996 1996 ). To determine the total phenolic contents, the methanolic extract of S. pecten-veneris was further fractionated using various organic solvents including, ethyl acetate, chloroform, n -butanol, and water (Table 3 3 ). High phenolic content was observed in the water fraction ((100.00±2.15) mg/100 g), while the values for ethyl acetate, chloroform, and n -butanol were (10.00±0.72), (8.00±0.40), and (12.00±0.89) mg/g, respectively. However, the values in all cases were lower than those reported by Simopoulos ( 2004 2004 ). These differences could be due to geographical location, plants age, or soil composition as these factors have a great impact on the nutrient contents, phytochemical composition, antioxidant activity, and biological potential of plants (Pinos-Rodríguez et al., 2008 2008 ; Khattak and Rahman, 2015 2015 ). No. Phytochemicals Inference 1   Flavonoids + 2   Tannins + 3   Terpenoids + 4   Steroids + 5   Caumarins + 6   Emodins + 7   Alkaloids + 8   Reducing sugar + 9   Phenols + 12   Saponins − 13   Pholobatanins − 14   Anthraquinones − Rutin equivalent (mg of rutin/g of extract); Tannic acid equivalent (mg of tannic acid/g of extract). Data are expressed as mean±SD No. Phytochemicals Quantity (mg/g dry weight) 1   Alkaloids 48.90±3.93 2   Flavonoids * * 98.00±4.78 3   Tannins ** ** 48.00±3.45 Plant phenols have an excellent capability to inhibit enzymes that cause inflammation, act as antioxidants, and modify the prostaglandin pathways thereby protecting platelets from clumping. Nowadays, the search for natural antioxidants is growing for three main reasons: (1) the consumption of fruits and vegetables rich in antioxidants is associated with decreased risks of developing chronic diseases including diabetes, cancer, and cardiovascular disorders; (2) safety concerns regarding the potential harmful effects of the chronic intake of synthetic antioxidants, such as butylhydroxytoluene and butylhydroxyanisole, in beverages and foods; and (3) the public’s perception that synthetic antioxidants are more harmful than their natural analogues (Dastmalchi et al., 2007 2007 ). Antioxidant activity 2 Free radicals have been found to be a cause of some major diseases including heart diseases, diabetes, cancer, and aging. Reduction of these radicals by antioxidants is crucial to overcome these health risks. Polyphenolic compounds and flavonoids present in plants act as antioxidants and show remarkable biological potential against these life threatening diseases (Lobo et al., 2010 2010 ). Both 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and DPPH radicals are used to assess the free radical scavenging abilities of plant extracts and pure compounds. The antioxidant activity of leaf extracts of S. pecten-veneris was determined on the basis of their DPPH radical scavenging potential and compared with vitamin E, which was used as a standard. The DPPH radical scavenging activity of different extracts (crude methanolic extract and ethyl acetate, chloroform, n -butanol, and water fractions) of S. pecten-veneris leaves is given in Fig. 1 1 . All these extracts were used at a concentration of 1.0 mg/ml. A crude methanolic extract of S. pecten-veneris showed 40% scavenging potential against DPPH (Fig. 2 2 ). The aqueous fraction showed the highest activity ( P < 0.001 vs. other fractions) against DPPH, followed by n -butanol, chloroform, and ethyl acetate fractions. This means that non-polar antioxidants contribute to the antioxidant activity. Vitamin C which was used as a positive control exhibited the highest DPPH scavenging potential ( P < 0.01 vs. water fraction and P < 0.000 vs. other fractions). The antioxidant potential of S. pecten-veneris could be attributed to the synergistic effect of various phytochemicals including steroids, flavonoids, and phenolic compounds. The amounts of various phytochemicals obtained in the current study are lower than those reported by Simopoulos ( 2004 2004 ), but are in agreement to those of Pieroni et al. ( 2002 2002 ). Data are expressed as mean±SD Data are expressed as mean±SD The reducing power of an extract or a pure compound represents its ability to act as antioxidant. The FRAP assay was used to determine the total antioxidant potential of S . pecten-veneris . The FRAP test is considered an accurate indicator of total antioxidant activity (Tezcan et al., 2009 2009 ). The S. pecten-veneris reducing power assay was performed using different extracts at different concentrations (1 and 3 mg/ml) of methanol, n -butanol, ethyl acetate, water, and chloroform with vitamin C as a standard (Fig. 3 3 ). Among these extracts, the reducing potential of the water fraction was significantly higher than that of other fractions ( P < 0.000), while the reducing potential of the ethyl acetate fraction was the lowest. Crude methanolic extract was more potent than non-polar fractions in reducing free radicals. The reducing ability of vitamin C was higher than that of the plant extracts. This high reducing power indicates that the compounds present in the extracts are electron donors and can reduce the oxidized intermediates of protein and lipid per oxidation processes, and can serve as primary and secondary antioxidants. Data are expressed as mean±SD Antibacterial and antifungal activity 2 The increasing number of infectious diseases poses a major threat to human health globally. The growing resistance of pathogens to antibiotics is exacerbating this problem (Alli et al., 2011 2011 ). The isolation and synthesis of new compounds with good antimicrobial activity have thus become imperative. The methanolic extract of S . pecten-veneris showed variable degrees of antimicrobial activity both for bacteria and fungi. Table 4 4 shows the inhibitory activity identified in the plant extracts against both Gram-negative and -positive bacterial strains, which are known to cause gastrointestinal, urinary, and respiratory disorders in humans. Streptomycin and gentamycin were used as standard drugs for comparison. Most of the tested bacteria showed an appreciable sensitivity towards the plant extract. The plant extract showed good activity against P . vulgaris , B . subtilis , and S . pneumoniae , but was most active against E. coli , S . aureus and S . typhi . The increasing order of the size of the zone of inhibition of different bacteria was P . aeruginosa<P . vulgaris<B. subtilis=S. pneumoniae<E. coli<S. typhi<S. aureus . S. aureus showed the largest zone of inhibition ((22.00±1.17) mm), while P . aeruginosa was not sensitive to the S. pecten-veneris extract. The antibacterial activity of the plant extract was compared with that of streptomycin and gentamycin as standard antibiotics. The results showed that standard antibiotics have higher activity than the crude extract. Standard drugs. NT: not tested; NS: not sensitive. Data are expressed as mean±SD 2 No. 2 Bacterial strain 3 Zone of inhibition (mm) Extract Streptomycin * * Gentamycin * * 1 E . coli 18.00±1.02 25.00±1.34 NT 2 S . typhi 20.00±1.14 NT 28.00±1.21 3 S . pneumoniae 16.00±0.94 35.00±1.18 NT 4 P . vulgaris 15.00±0.75 33.00±1.07 NT 5 P . aeruginosa NS 28.00±0.91 NT 6 S . aureus 22.00±1.17 NT 28.00±1.32 7 B . subtilis 16.00±0.92 NT 26.00±1.19 The potential antifungal activity was evaluated using four fungal strains as tester microorganisms, selected from among mycotoxin producers, food contaminators, and food spoilage pathogens including A . flavus , A . niger , A . parasiticus , and a clinical oral isolate C . albicans . Results of the antifungal potential of S. pecten-veneris are given in Table 5 5 . S. pecten-veneris showed the highest activity against C . albicans with a zone of inhibition of (30.00±1.25) mm and the lowest activity against A . parasiticus ((12.00±1.11) mm). The plant extract produced zones of inhibition with (21.00±2.45) and (23.00±1.78) mm against A . flavus and A . niger , respectively. Clotrimazole, used as a standard drug against A . flavus and C . albicans produced (28.00±1.36) and (35.00±1.22) mm zones of inhibition, respectively, while the standard drug fluconazole showed (29.00±1.02) and (30.00±1.10) mm inhibitory activity against A . niger and A . parasiticus , respectively. The antifungal property of S . pecten-veneris extract was lower than that of the standard drugs, but demonstrated a promising effect on the growth of C . albicans . The antimicrobial potential of plant species depends on their chemical composition. The phenolic compounds and flavonoids found in S . pecten-veneris could be responsible for its antimicrobial potential against the tested microorganisms. Standard drugs. NT: not tested. Data are expressed as mean±SD 2 No. 2 Fungal strain 3 Zone of inhibition (mm) Extract Clotrimazole * Fluconazole * 1 A . flavus 21.00±2.45 28.00±1.36 NT 2 A . niger 23.00±1.78 NT 29.00±1.02 3 A . parasiticus 12.00±1.11 NT 30.00±1.10 4 C . albicans 30.00±1.25 35.00±1.22 NT Enzyme inhibition 2 Natural products from medicinal plants are widely used in the treatment of many diseases as they inhibit many pathogenic enzymes. Secondary metabolites provide a good pharmacophore template for new drugs. The methanolic extract of S . pecten-veneris was assessed for in vitro enzyme inhibitory potential against urease, phosphodiestrase-I, and carbonic anhydrase-II. The extract was found to be significantly more active against urease than standard thiourea but showed low activity against phosphodiestrase-I and carbonic anhydrase-II (Table 6 6 ). Phosphodiesterases (PDEs) control cellular concentrations of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) by hydrolyzing them to 5'-GMP and 5'-AMP, respectively. Inhibitors of PDEs are being used in many pharmacological applications for their vasodilatory, cardiotonic, antidepressant, smooth muscle relaxant, anti-inflammatory, and bronchodilator effects. Many plant metabolites have been shown to be active PDE inhibitors (Rahimia et al., 2010 2010 ). Mammalian α-carbonic anhydrases have important physiological functions in respiratory homeostasis, pH balance, regulation of intracellular osmotic pressure, and gluconeogenesis. Consequently, a number of carbonic anhydrase isoenzymes are the intended targets of drugs or inhibitors. These inhibitors are commonly used for the treatment of gastric and duodenal ulcers, hypertension, certain types of cancers, epilepsy, and as antiglaucoma drugs (Winum et al., 2006 2006 ; Supuran, 2008 2008 ). Furthermore, carbonic anhydrase isoenzymes present in prokaryotic mammalian pathogens including Neisseria gonorrhoeae , Helicobacter pylori , and the eukaryotic malarial parasite Plasmodium falciparum could also be targeted by these inhibitors. Urease catalyzes the hydrolysis of urea to ammonia, which is important for growth and survival of H. pylori in the acidic environment of the stomach and as a result causes various gastrointestinal disorders (Hiyama and Wynder, 1986 1986 ; Devesa et al., 1998 1998 ; Shabnam et al., 2015 2015 ). Many medicinal plants have long been applied as remedies to cure such diseases. Euphorbia decipiens (Ahmad et al., 2003 2003 ) and sulfated polysaccharide of brown seaweed are examples of natural substances reported to have urease inhibition activity (Limuro and Wakabayashi, 2003 2003 ). The data on enzyme inhibitory activity clearly show the potential of S . pecten-veneris as an active inhibitor of urease and hence suggest that it could be effective in the treatment of gastrointestinal disorders. However, further work is required on the isolation and correct identification of the active constituents of the extract. Data are expressed as mean±SD 2 Group 3 Inhibitory activity (%) Urease Phosphodiestrase-I Carbonic anhydrase-II Extract 72.80±5.25 33.11±3.98 44.90±3.32 Standard 98.20±4.72 (Thiourea) 80.10±5.25 (EDTA) 89.00±2.25 (Acetazolamide) Conclusions 1 S . pecten-veneris L. showed inhibitory activity against a wide range of Gram-negative and -positive bacteria and fungi, as well as good free radicals scavenging potential. A crude methanolic extract of S. pecten-veneris exhibited excellent inhibitory activity against urease, suggesting its possible use in treating gastrointestinal disorders. 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This article does not contain any studies with human or animal subjects performed by any of the authors. 10.1631/jzus.B1600443 Project supported by the Directorate of Science and Technology (DoST), Khyber Pakhtunkhwa, Pakistan, under Research Project Developmental Scheme (Bio-Tech Sector, Phase-1, A03959 Bio-Tech) 2016-10-05 2017-05-04 2022-01-05 Copyright © Zhejiang University and Springer-Verlag GmbH Germany, part of Springer Nature 2018 2018 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 02 19