Nothoscordum Bivalve (L.) Britton and Parthenium Incanum Kunth Extracts

Indian Journal of Traditional Knowledge Vol. 17(4), October 2018, pp. 699-706

Biological activity of Nothoscordum bivalve (L.) Britton and Parthenium incanum Kunth extracts

David Alejandro Hernández-Marín1,2, Fidel Guevara-Lara2, Catalina Rivas-Morales1, Jorge Armando Verduzco-Martínez3, Sergio Arturo Galindo-Rodriguez1 & Eduardo Sánchez-García1* 1Laboratorio de Química Analítica, Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, San Nicolás de los Garza, Nuevo León, México; 2Laboratorio de Biotecnología y Funcionalidad de Alimentos, Departamento de Química, Centro de Ciencias Básicas, Universidad Autónoma de Aguascalientes, Aguascalientes, Aguascalientes, México; 3Departamentode Botánica, Facultad de Ciencias Biológicas, Universidad Autónoma deNuevo León, San Nicolás de los Garza, Nuevo León, México E-mail: [email protected]

Received 04 January 2018, revised 6 July 2018

Medicinal properties of Nothoscordum bivalve (false garlic) have not been reported, while Parthenium incanum (mariola) is used to treat stomach and liver diseases. The aim of this work was to evaluate in vitro different biological activities of these plants to assess their pharmacological potential. Extracts were obtained by maceration or Soxhlet sequential solvent extraction. The extracts were tested by inhibiting the growth of Acinetobacter baumannii, besides for the techniques of Artemiasalina toxicity, cytotoxicity in human erythrocytes, cytoprotective, antioxidant capacity, quantification of soluble phenols and coagulation tests. MBC´s obtained were between 7.50±0.54 and 8.50±0.54 mg/mL. Toxicity assays showed LD50 from 380 to 1882 µg/mL. Cytotoxicity was between 0 and 92.98 %. Cytoprotective effect indicated 0 to 94.58 %. Antioxidant capacity levels of extracts were between 275.55±21 and 598.99±4 µmol TE/g. Soluble phenolics concentrations between 1.35±0.07 and 4.13±0.04 g GAE/100 g were observed. Regarding coagulation assays, only one P. incanum extract showed significant difference (p < 0.05) in the PT assay. Extracts from the sequential extraction showed less toxicity and cytotoxicity, and greater antioxidant and cytoprotective capacity; they also showed no significant prolongation in the extrinsic pathway of coagulation, and a significant (although small) prolongation in the intrinsic coagulation pathway.

Keywords: Parthenium incanum, Nothoscordum bivalve, Cytotoxicity, Antioxidant, Hemolysis, Coagulation

IPC Int. Cl.8: A61K 36/00, A01D 20/00, A62B, A61, C09K 15/00, B03D 3/02, B29C 67/06, C08C 1/14, C08F 6/22, A61K 38/36, C04B 103/60 The genus Parthenium contains approximately 16 worldwide6. N. bivalve (false garlic) is a perennial species of shrubs, herbaceous perennials, and plant up to 20 inches tall with white flowers. annuals1. P. incanum (Mariola) is a perennial plant 2- The species can be found in the eastern region of the 3 ft high; its leaves are grayish green and has white United States, Mexico, Argentina, Chile and small flowers. Its distribution covers from the South Uruguay7,8. There is not enough information on the western United States to the North of México. biological activity of the Nothoscordum genus to Mariola is a source of important food resources for establish its applications in traditional medicine, but grazing animals that are found in desert shrub lands2. antimicrobial activity and phytochemical screening The medicinal uses of this plant include treatment of has been reported for some species of the genus. With indigestion, sluggish liver and mild constipation3. respect to antimicrobial activity, the species N. gracile Various biological activities from related species and N. entrerianum have been studied; for the case of (P. hysterophorus) have already been reported such as N. gracile, phytochemical screening, FT infrared antimicrobial, antioxidant, antihemolytic, cytotoxicity spectroscopy and wavelength dispersive X-ray and lipid peroxidation inhibition4,5. fluorescence studies demonstrated the presence of On the other hand, the genus Nothoscordum secondary metabolites as well as inorganic elements comprises more than 30 species, distributed contained in their leaves and bulbs6,9.

—————— The present investigation was focused on the *Corresponding author in vitro biological activity of P. incanum and 700 INDIAN J TRADIT KNOWLE, VOL. 17, NO. 4, OCTOBER 2018

N. bivalve extracts, evaluating their antimicrobial Drug resistance of A. baumannii activity against an isolated antibiotic-resistant strain A sensitivity test for several antibiotics was carried of Acinetobacter baumannii, as well as their toxicity, out according to the methodology of Morales-Meza cytotoxicity, antioxidant capacity, soluble phenolics and Ruiz de Chávez-Ramos12 with slight content and their effects on blood coagulation modifications; the amount of microorganism was to generate knowledge about their possible medicinal quantified by microdilution methods and was seeded use. at approximately 109 CFU/mL. The measurement and interpretation of inhibition halos were performed as Methodology indicated by the manufacturer. Microorganism Acinetobacter baumannii isolated strain was kindly Preliminary antimicrobial activity donated by Dr. Elvira Garza-González, from Dr. José The method reported by Sánchez et al.13 was used Eleuterio González University Hospital (Universidad with slight modifications; 100 µL of bacterial Autónoma de Nuevo León) (UANL Hospital). suspension (approx 109 CFU/mL), previously Preserved in Mueller-Hinton agar at 4 °C. activated in Mueller-Hinton broth, were homogeneously seeded onto Mueller-Hinton agar Plant material plates. MeOH extracts were evaluated by a well Samples of aerial parts of N. bivalve (stem and diffusion agar technique; briefly, wells (6 mm flowers) and P. incanum (leaves and bark) were diameter) were cut out of the agar using a sterilized collected from the wild in the area around Santa cup-borer, then wells were filled with 100 µL of Catarina, Nuevo León, México (25°38'50.78'' N, the MeOH extracts. On the other hand, hexane 100°43'24.19'' W). A voucher specimen of the plants and chloroform extracts were evaluated by a disc was deposited in the herbarium of the Biological diffusion method in view of their insolubility Science Faculty, Universidad Autónoma de Nuevo in aqueous agar, in this case, 30 L of each extract León, for correct identification. Collected plants were were impregnated into sterile filter paper discs (6 mm cut into small pieces and air-dried under shade at in diameter); all discs were fully air-dried and were room temperature (25 °C ± 2 °C). Dried material was placed on the agar plate by use of a sterile needle. ground in a manual grain mill and stored in paper Plates of both methods (well and disc diffusion) were bags until use. incubated at 37 °C for 24 h. Extraction solvents were

Extraction used as controls. Maceration extraction (ME): 100 g of P. incanum Minimal bactericidal concentration (MBC) and 80 g of N. bivalve, were extracted with 600 mL The MBC of the most active extracts was analyzed and 500 mL of methanol (MeOH), respectively, for 14 15 24 h at room temperature in dark conditions, with according to Sánchez et al. and Duarte et al. with occasional shaking. Then they were filtered through some modifications; extract concentrations between 0.5 and 15 mg/mL were evaluated with a bacterial Whatman No. 1 filter paper; extraction was repeated 5 9 times on the same plant material10. concentration of approximately 10 CFU/mL. After Soxhlet extraction (SE): 335 g of P. incanum and incubation (24 h at 37 °C), 20 µL were taken from the 280 g of N. bivalve, were placed in a Soxhlet wells where there was no apparent growth and were equipment and extracted sequentially with 2 L of each seeded by dripping in Mueller-Hinton agar, and of the following solvents: hexane, chloroform, and incubated for 24 h at 37 °C. MBC was defined as the MeOH (in that order), on the same plant material, for lowest concentration where no growth was recorded. 48 h each solvent11. All extracts (ME and SE) obtained, were concentrated at reduced pressure in a Toxicity in Artemiasalina rotary evaporator (Yamato RE 200) until dryness. Artificial sea water was prepared and 100 mg of Extracts were re-suspended in 15 mL of the solvent Artemiasalina eggs were added; after 48 h used for the primary extraction and stored at 4 °C in (maturation of nauplii) the extracts were evaluated in the dark until use. concentrations between 100 and 1000 µg/mL16,17. The Extraction yields of solids (%) = (mass of extracted lethal dose 50 (LD50) was calculated using the Probit solids/initial mass of the plant before extraction) method (SPSS ver. 23), according to the toxicity ×100. classification of Sánchez & Neira18. MARÍN et al.: BIOLOGICAL ACTIVITY OF N. BIVALVE AND P. INCANUM 701

Cytotoxicity (% hemolysis) of Trolox in 100 µL were prepared; for DPPH assay The methodology of Vinjamuri et al.19, was used each was allowed to react with 600 µL of 0.13 mM with some modifications; a suspension of 5 % human DPPH reagent (total reaction volume = 700 µL). erythrocytes was prepared with PBS buffer; extracts Likewise, 100 µL aliquots of plant extracts were were evaluated at concentrations between 100 and evaluated with the DPPH reagent. Reactions were 1000 µg/mL; distilled water and PBS buffer were kept in the dark for 20 min at room temperature23. used as positive and negative controls, respectively. Then the tubes were read at 515 nm24. For ABTS The final reaction volume was adjusted to 1.25 mL, assay, 100 µL of different Trolox concentrations (0-35 constituted by 250 µL of erythrocyte suspension, 980 nmol) were reacted with 1400 µL of ABTS reagent µL PBS, and 20 µL of the different extract (prepared using 7.4 mM ABTS and 2.6 mM concentrations. Then, the reaction mixture was potassium persulfate) for a total reaction volume of incubated for 30 min at 37±0.5 °C; samples were 1500 µL. Equally, 100 µL aliquots of extracts plants centrifuged and supernatants were read at 540 nm. were evaluated. Reactions were kept in the dark for Hemolysis was calculated with the following 30 min at room temperature. Then the tubes were read equation20: at 734 nm25. As Anc % Hemolysis = ×100 Soluble phenolics quantitation as gallic acid Apc Anc equivalents (GAE)

Where, As = Absorbance of the sample; The procedure was carried out in dim light. The Anc = Absorbance of the negative control; gallic acid curve (10-80 µg) was prepared in a final Apc = Absorbance of the positive control volume of 1 mL with 30 % MeOH. Similarly, different dilutions of the extracts were prepared. Cytoprotection (% inhibition of hemolysis) 125 µL of the 2 N Folin-Ciocalteu reagent was then A variant of the methodology of Sulaiman & added and the mixture was stirred and allowed to Hussain21and Chisté et al.22, was carried out. The final react for 6 min. Then 1.25 mL of 7 % sodium reaction volume of 1 mL was constituted by 375 µL carbonate was added, shaken and 625 µL of distilled of 150 mM AAPH in PBS, 250 µL of a 5 % water were added to give a final volume of 3 mL. erythrocyte suspension in PBS, 355 µL of PBS, and After stirring, the mixture was allowed to react for 90 20 µL of plant extract at variable concentrations (100 min in the dark at room temperature. After this time, without stirring the mixture, absorbance readings to 1000 µg/mL). The negative control was considered 24,26 as 250 µL erythrocyte suspension and 750 µL PBS; as were obtained at 757 nm in a spectrophotometer . a positive control, 250 µL erythrocyte suspension, 375 µL of 150 mM AAPH in PBS and 375 µL of PBS Coagulation tests (PT and aPTT) Blood samples (healthy patients) were obtained in (without extract). The reactions were maintained at 37 tubes with 3.2 % sodium citrate. They were ± 0.5 °C for 6 h in a shaking incubator (300 rpm); centrifuged at 13,000 rpm for 5 min to recover the afterwards, the samples were centrifuged and supernatants were read at 540 nm. The percentage of plasma. Then 1 mL of plasma was adjusted to the concentrations of 1000 and 500 µg/mL with the inhibition of hemolysis was obtained by using the extracts (980 µL plasma with 20 µL extract and 990 formula: µL plasma with 10 µL extract, respectively). For the As Anc  prothrombin time (PT), the reagent Trini CLOTPT % Inhibition of Hemolysis = 100-[  ×100] Excel (rabbit brain) and 100 µL of the treated plasma Apc Anc  were preheated separately (37 ± 0.5 °C) for 2 min in Where, As = Absorbance of the sample; the coagulometer, after which 200 µL of PT Excel Anc = Absorbance of the negative control; reagent were added to the plasma and the timer was Apc = Absorbance of the positive control activated. The coagulometer recorded the time to the formation of the clot. For activated partial Trolox equivalent antioxidant capacity (TEAC) thromboplastin time (aPTT), 100 µL of pre-heated using DPPH and ABTS reagents Trini CLOTaPTT-S (particles of phospholipids) All procedures were carried out in dim light; reagent was added to the treated plasma, the timer standard solutions containing between 0 and 35 nmol was activated and the reaction was incubated in the 702 INDIAN J TRADIT KNOWLE, VOL. 17, NO. 4, OCTOBER 2018

coagulometer for 5 min at 37 ± 0.5 °C; after that, 100 in Table 1; the MeOH extracts presented the highest µL of pre-heated 0.025 M calcium chloride was activity. Minimal bactericidal concentrations (MBCs) added. The coagulometer recorded the time to the were obtained only for MeOH extracts because of formation of the clot. One-way analysis of variance their higher activity (Table 1), these results ranged (ANOVA) and Tukey’s test were used to find out from 7.50±0.54 to 8.50±0.54 mg/mL. Extracts from significant differences (p < 0.05)27. Parthenium species have been reported to show antimicrobial activity against Streptococcus mutans, Results and discussion Proteus vulgaris, and Salmonella typhi4. In the Extraction yields of solids obtained for P. incanum case of Nothoscordum, aqueous extracts from were: hexane (SE) 2.54 %, chloroform (SE) 2.51 % bulbs of three species showed activity against and MeOH (SE) 8.05 %, while yield of successive Candida albicans, Aspergillus fumigatus, and maceration only with MeOH was 14.49 %, it is Staphylococcus aureus9. On the other hand, important to mention that the cumulative yield Sánchez et al.14 reported that MeOH extracts from percentage of Soxhlet extraction was 13.1 %, slightly bulbs of N. bivalve had no antimicrobial activity lower than the aforementioned for successive against antibiotic-resistant strains of Klebsiella maceration (14.49 %). We found a similar behavior pneumoniae, Enterococcus faecalis, Escherichia coli, for N. bivalve, in this case, the obtained SE yields Stenotrophomonasmaltophilia, and Staphylococcus were: hexane 2.33 %, chloroform 1.57 % and MeOH aureus, all of them isolated at the UANL Hospital. 21.79 %, while successive maceration with MeOH MBCs ranged from 7.50 to 8.50 mg/mL; Raut & was 28.50 %, and similarly to the above, the Pukale31 reported various MBCs from polar extracts accumulated percentage of the Soxhlet extraction was of P. hysterophorus, being between 24 and 72 mg/mL 25.69 %. MeOH extractions for both plants showed for S. aureus, E. coli, and A. fumigatus. Nevertheless, the highest yields. It has been previously reported that for Nothoscordum, MBCs have not been previously MeOH is frequently used because it allows the reported for any microorganisms. extraction of a greater number of plant metabolites. In our work, maceration extraction (ME) was slightly Toxicity results of P. incanum and N. bivalve more efficient than Soxhlet extraction (SE); however, MeOH extracts against Artemiasalina showed previous studies also found that repeated extractions different degrees of toxicity (LD50 from 380 to 1882 on the same plant material showed higher extraction µg/mL); P. incanum ME 380 µg/mL (moderate yields, because a considerable amount of active toxicity), and P. incanum SE 1882 µg/mL (relatively principles of interest can remain in the plant after the innocuous); on the other hand, N. bivalve ME 916 first extraction10,28. µg/mL (slightly toxic) and N. bivalve SE 1023 µg/mL (practically non-toxic), according to the classification The A. baumanii strain was evaluated for resistance 18 to several antibiotics and proved to be resistant to of Sánchez & Neira . It is common to evaluate the amikacin (30 µg), ampicillin (10 µg), cephalotin (30 toxicity of natural products with biological activity using the A. salina test due to its ease of use, low cost, µg), cefepime (30 µg), cefotaxime (30 µg), 16,17 ceftriaxone (30 µg), chloramphenicol (30 µg), and reliability . gentamicin (10 µg), netilmicin (30 µg), nitrofurantoin Table 1 — Preliminary evaluation of antimicrobial activity and (30 µg) and sulfamethoxazole/trimethoprim (25 µg); MBC of P. incanum and N. bivalve extracts it was sensitive only to levofloxacin (5 µg). In recent Plants Extraction Preliminary MBC years, the pathogen A. baumannii has become activity(mm) (mg/mL) important at the nosocomial level around the world P. incanum SE Hexane 5.2±2.4 ND** due to the increase of its resistance to antibiotics and SE Chloroform NI* ND the prevalence in patients in intensive care units; it SE Methanol 9.4±0.8 8.50±0.54 belongs to the emergent pathogen group of ME Methanol 13.2±0.6 8.16±0.40 microorganisms known as ESKAPE. Regarding this, N. bivalve SE Hexane NI ND in the state of Nuevo León, México, A. baumannii SE Chloroform 6.5±1.6 ND was the most frequent isolate at the UANL Hospital SE Methanol 14.8±0.3 7.50±0.54 29,30 during 2011-2012 . ME Methanol 16.5±0.5 8.33±0.51 The results of preliminary evaluations of Values expressed as mean ± SE, n=6. *No inhibition. ** Not antimicrobial activity against A. baumannii are shown determined MARÍN et al.: BIOLOGICAL ACTIVITY OF N. BIVALVE AND P. INCANUM 703

Extracts showed high Trolox equivalent increases. In contrast, P. incanum SE in all evaluated antioxidant capacities (TEAC) (> 200 µmol TE/g) by concentrations had between 0.07 and 1.43 % both methods (Table 2); it can be observed that SE hemolysis. N. bivalve ME at 1000 µg/mL showed extracts presented greater TEAC levels. Soluble 13.69 % hemolysis, whereas N. bivalve SE in all phenolics contents were calculated as gallic acid concentrations caused 0.16 to 2.39 % hemolysis. equivalents (GAE). P. incanum and N. bivalve Maceration extracts showed more hemolysis than extracts showed high levels of soluble phenolics those obtained by Soxhlet; this may be due to the ranging between 1.348±0.07 and 4.133±0.04 g extraction method applied on the plant material: It is GAE/100 g (Table 2). TEAC of extracts (275.55±21- known that many of the non-polar compounds present 598.99±4 µmol TE/g) were higher to those reported in in plant extracts, exhibit a strong cytotoxic activity35, foods like blue-kernel maize, nixtamalized this would explain the low cytotoxicity of the commercial blue corn flour, commercial blue methanolic extract obtained by soxhlet reflux, since baked tortilla chips and huitlacoche corn cobs during the soxhlet extraction, non-polar solvents (8.5-88.8 µmol TE/g)23; however, our values were (hexane and chloroform) were used before methanol, lower than those from aqueous and ethanol extracts removing the non-polar compounds of the plant, from plants of the genera Anacardium, Clausena, obtaining a methanolic extract which no longer Litsea, Ocinum, Piper, Pseuderanthenum, Spondias presents these kinds of compounds, while only MeOH and Syzygium (2.43 to 411.96 mmol TE/g)32. Soluble was used for maceration, which allows the extraction phenolics in extracts from both species (1.35±0.07- of different kinds of compounds even those with 4.13±0.04 g GAE/100 g) were similar or lower than cytotoxic activity; this explains why MeOH extracts those previously reported in aqueous and ethanol obtained after a polarity gradient (Soxhlet) will differ extracts from the genera Anacardium, Clausena, from those obtained by extraction without such a Litsea, Ocinum; Piper, Pseuderanthenum, Spondias gradient (maceration)10,27. It has been reported that and Syzygium (0.408-47.40 g GAE/100 g)32. Phenolic plant extracts may contain cytotoxic compounds, compounds are extracted with polar solvents such as which is why the hemolysis assay is used as a general MeOH; they possess various biological activities such indicator in the cytotoxicity study19,20,36. Our results as free radical uptake, antimicrobial activity and suggest the presence of cytotoxic (hemolytic) antiviral, among others. Therefore, it is always useful compounds in the non-MeOH Soxhlet extracts of both to correlate the soluble phenolics contents with the plants, a further investigation currently underway. antioxidant capacity of extracts32,33,34; Table 2 shows Likewise, a correlation can be observed between the that P. incanum and N. bivalve extracts do not show a A. salina and hemolysis tests; the ME of P. incanum correlation between TEAC and GAE. presented a lower LD50 and a high degree of Cytotoxicity evaluations of MeOH extracts of hemolysis, unlike the other extracts, which had higher P. incanum and N. bivalve are presented in Fig. 1, LD50 and lower percentages of hemolysis. where it can be clearly seen that the ME of Cytoprotective evaluations in Fig. 2 show that P. incanum exhibits approximately 92.98 % maceration extracts (ME) caused low or null hemolysis at 1000 µg/mL; as the concentration of extract increases, the percentage of hemolysis

Table 2 — Antioxidant capacity and soluble phenolics content of P. incanum and N. bivalve methanolic extracts Extracts TEAC TEAC Soluble ABTS assay DPPH assay phenolics (µmol TE/g) (µmol TE/g) content (g GAE/100 g) P. incanum ME 565.58±2.97* 433.33±11.43 1.348±0.07 P. incanum SE 598.99±3.47 459.11±22.55 4.133±0.04 N. bivalve ME 337.64±1.65 271.85±24.49 2.542±0.01 N. bivalve SE 362.35±2.17 275.55±21.05 3.119±0.02

Values expressed as mean ± SE., n=6. ME: Maceration extraction; Fig. 1 — Cytotoxicity (% hemolysis) of P. incanum and SE: Soxhlet extraction. N. bivalve methanolic extracts. 704 INDIAN J TRADIT KNOWLE, VOL. 17, NO. 4, OCTOBER 2018

Table 3 — Prothrombin time (PT) and activated partial thromboplastin time (aPTT) for P. incanum and N. bivalve methanolic Soxhlet extracts. Extract Plants PT(s) aPTT(s) Concentration P. incanum 500 µg/mL 13.06±0.15 35.33±1.51 1000 µg/mL 13.36±0.11 35.96±0.25 N. bivalve 500 µg/mL 12.36±0.15 33.86±2.55 1000 µg/mL 12.36±0.11 33.8±1.83 Solvent 10 µL 12.10±0.20 33.63±0.35 control 20 µL 12.16±0.11 33.36±0.95

Blank Untreated plasma 12.26±0.15 32.63±0.66 Fig. 2 — Cytoprotection activity (%) caused by P. incanum and Values expressed as mean ± SE, n=6. N. bivalve methanolic extracts cytoprotection (AAPH technique); for N. bivalve ME, assays were done with concentrations of 500 no cytoprotective activity was observed and for and 1000 µg/mL. Table 3 shows the PT and P. incanum at 500 µg/mL, about 41.92% efficacy was APTT results; for PT, P. incanum extracts at 500 present. However, when the extract concentration and 1000 µg/mL showed significant difference increased, the activity dropped to 12.91%. In the case (p < 0.05) with respect to the other means, including of Soxhlet extracts (SE), higher cytoprotective controls. On the other hand, in the APTT tests, activities were observed; for P. incanum extracts at no significant differences were found among the 500 and up to 1000 µg/mL, between 86.95 and means (p > 0.05), including the controls. With 94.58% cytoprotection was observed, while for N. respect to extract of N. bivalve did not cause bivalve, the cytoprotection was proportional to the a significant change in the coagulation times at increase in extract concentration, corresponding a 500 the evaluated concentrations. In recent years, µg/mL extract to 5.85 % cytoprotection, 750 µg/mL several studies of anticoagulant activities in to 39.24 % and 1000 µg/mL to 79.34 %. Low or null plant extracts have been carried out, as new cytoprotective activity in maceration extracts can be active principles are sought to treat thrombotic due to its hemolytic capacity at concentrations tested; diseases; it is known that the PT prolongation as shown in Fig. 1, as concentration increases, shows inhibition in the extrinsic coagulation pathway hemolysis increases. On the other hand, for the case and, on the other hand, the prolongation of APTT of the Soxhlet extracts, cytoprotection is observed at shows inhibition in the intrinsic pathway of 26,37 concentrations ≥ 500 µg/mL, the null or low coagulation . hemolysis at these concentrations can be clearly appreciated (Fig. 1). These differences between Conclusion maceration and Soxhlet extracts could be reflecting Extracts from P. incanum and N. bivalve the different extraction methods, as it was already showed various effects on biological activities such mentioned for hemolysis. The AAPH peroxide as antimicrobial, antioxidant, cytoprotective, toxicity, radicals attack the erythrocyte membrane; whereby cytotoxicity, and anticoagulant activity. Moreover, the plant extracts evaluated in this test could be an MeOH Soxhlet extracts were obtained from a auxiliary in the prevention or treatment of the more selective process in which low polarity oxidation provoked by free radicals. Previous studies compounds were first removed with hexane and have reported that plant extracts or their natural chloroform, which could explain their possible compounds counteract the action of radicals formed pharmaceutical potential, in view that they showed by AAPH21,22. inhibition of A. baumannii, have low toxicity, low Only MeOH extracts from the Soxhlet extraction cytotoxicity and cytoprotection of human were used for coagulation assays because they erythrocytes; they also showed no significant showed low toxicity, cytotoxicity, and significant prolongation in the extrinsic pathway of coagulation, cytoprotective effect. The PT (prothrombin time) and and a small but significant prolongation in the APTT (activated partial thromboplastin time) intrinsic coagulation pathway. MARÍN et al.: BIOLOGICAL ACTIVITY OF N. BIVALVE AND P. INCANUM 705

Acknowledgment 12 Morales-Meza MG & Ruiz de Chávez-Ramos CG, The authors are thankful to the Chemical Analitical Diferencias en la resistencia a los antimicrobianos de cepas de Staphylococcus aureus obtenidas de diversas fuentes de Laboratory, Facultad de Ciencias Biológicas, aislamiento, Universidad La Salle, Rev Cent de Investig, 25 Universiadad Autónoma de Nuevo León for providing (7) (2006) 45-64. the facilities for conducting the research study. And to 13 Sánchez E, Heredia N, Camacho-Corona MR & García S, the Departments of Microbiology and Chemistry of Isolation, characterization and mode of antimicrobial action Universidad Autónoma de Aguascalientes. Technical against Vibrio cholera of methyl gallate isolated from Acacia farnesiana, J Appl Microbiol, 115 (2013) 1307-1316. support from Ma. Lorena Sandoval-Cardoso, José 14 Sánchez E, Rivas-Morales C, Castillo S, Leos-Rivas C, Luis Carrasco-Rosales and José Luis Moreno- García-Becerra L, et al., Antibacterial and antibiofilm Hernández-Duque. Support and doctorate grant from activity of methanolic plant extracts against nosocomial CONACYT to DAHM (556587). microorganisms, Evid-Based Compl Alt, 2016, Article ID 1572697, 8 pages. 15 Duarte AF, Ferreira S, Oliveira R & Domingues FC, Effect References of coriander oil (Coriandrum sativum) on planktonic and 1 Ebadi M, Pharmacodynamicbasis of herbal medicine, biofilm cells of Acinetobacter baumannii, Nat Prod nd Chapter 25, 2 edn, (CRC Press, Boca Raton, Florida), 2006, Commun, 8 (5) (2013) 673-678. 285. 16 Solís PN, Wright CW, Anderson MM, Gupta MP & 2 Villalobos C, Estimating aboveground biomass of Mariola Phillipson JD, A microwell cytotoxicity assay using (Parthenium incanum) from plant dimensions, In: Artemiasalina (brine shrimp), Planta Med, 59 (1993) 250- Proceedings: Shrubland dynamics—fire and water; 2004 252. August 10-12, edited by Sosebee RE, Wester DB, Britton 17 Peréz-Hernández RA, Leos-Rivas C, Oranday-Cárdenas A, CM, McArthur ED, Kitchen SG, (Lubbock, TX. Proceedings Hernández Luna CE, Sánchez-García E, et al., Effecto in RMRS-P-47. Fort Collins, CO: U.S. Department of vitro en la inhibición del proceso de nucleación en litiasis Agriculture, Forest Service, Rocky Mountain Research renal, capacidad de captura de radicales libres, actividad Station), 2007, 132-135. antimicrobiana y tóxica del extracto metanólico de Berberis 3 Kane CW, Herbal medicine of the American Southwest: A trifoliata, Rev Mex Cienc Farm, 46 (1) (2015) 70-76. guide to the identification, collection, preparation and use of 18 Sánchez L & Neira A, Bioensayo general de letalidad en medicinal and edible plants of the Southwestern United Artemia salina, a las fracciones del extracto etanólico de States, (Lincoln Town Press, Tucson, USA), 2006, 163-164. Psidium guajava L. y Psidium guineense. SW, C Cient, 4 Kumar S, Mishra A & Pandey AK, Antioxidant mediated 3 (2005) 40-45. protective effect of Parthenium hysterophorus against 19 Vinjamuri S, Shanker D, Ramesh RS & Nagarajan S, In vitro oxidative damage using in vitro models, BMC Comple Altern evaluation of hemolytic activity and cell viability assay of Med, 13 (120) (2013) 1-9. hexanoic extracts of Bridelia ferruginea Benth, World J 5 Kumar S, Pandey S & Pandey AK, In vitro antibacterial, Pharm Pharm Sci, 4 (7) (2015) 1263-1268. antioxidant and cytotoxic activities of Parthenium 20 Farooq S & Sehgal A, Evaluation of antioxidant and hysterophorus and characterization of extracts by LC-MS antigenotoxic effects of kahwa, Indian J Tradit Knowle, analysis, Bio Med Res Int, Vol. 2014, Article ID 495154, 10 16 (2) (2017) 277-283. pages. 21 Sulaiman AA & Hussain SA, Concentration and time 6 Sidhu MC & Thakur S, Phytochemical and elemental dependent cytoprotective effects of anthocyanins against exploration of Nothoscordum gracile (Aiton) Stearn for its oxidative hemolysis induced by water and lipid soluble free medicinal potential, J Chem Pharm Sci, 9 (4) (2016) 2627- radical initiators: an in vitro study, Oxid Antioxid Med Sci, 2631. 1(2) (2012) 133-140. st 7 Howard TM, Bulbs for warm climates, 1 edn, (University of 22 Chisté RC, Freitas M, Mercadante AZ & Fernandes E, Texas, Austin), 2001, 35-36. Carotenoids are effective inhibitors of in vitro hemolysis of 8 Richardson A & King K, Plants of deep South Texas: A field human erythrocytes, as determined by a practical and guide to the woody and flowering species, 1stedn, (Texas optimized cellular antioxidant assay, J Food Sci, 79 (9) A&M University Press), 2011, 24. (2014) 1841-1847. 9 Lazo W & Ravenna P, Antimicrobial activity of three species 23 Saha MR, Kar P & Sen A, Assessment of phytochemical, of the genus Nothoscordum (Alliaceae), Bol Micol, 4 (2) antioxidant and genetic diversities among selected medicinal (1989) 91-92. plant species of Mimosoideae (Mimosaceae), Indian J Tradit 10 Castillo SL, Heredia N, Contreras JF & García S, Extracts of Knowle, 17(1) (2018) 132-140. edible and medicinal plants in inhibition of growth, 24 Amador-Rodríguez KY, Martínez-Bustos F, Pérez-Cabrera adherence, and cytotoxin production of Campylobacter jejuni LE, Posadas-del-Río FA, Chávez-Vela NA, Sandoval- and Campylobacter coli, J Food Sci, 76 (6) (2011) 421-426. Cardoso ML & Guevara-Lara F, Effect of huitlacoche 11 Murugan R & Parimelazhagan T, Comparative evaluation of (Ustilago maydis DC Corda) paste addition on functional, different extraction methods for antioxidant and anti- chemical and textural properties of tortilla chips, Food Sci inflammatory properties from Osbeckia parvifolia Arn.-An Technol, 35 (2015) 452-459. in vitro approach, J King Saud Univ Sci, 26 (4) (2014) 267- 25 TaskinT, Sadikoglu N, Birteksoz-Tan S & Bitis L, In vitro 275. screeninig for antioxidant and antimicrobial properties of 706 INDIAN J TRADIT KNOWLE, VOL. 17, NO. 4, OCTOBER 2018

five commercial Origanum species from Turkey, Indian J ethanol crude extracts of 13 Thai traditional plants, Int J Tradit Knowle, 16 (4) (2017) 568-575. Curr Microbiol Appl Sci, 3 (1) (2014) 549-558. 26 Tatiya A, Kalaskar M, Patil Y & Surana S, Chemical 33 Piluzza G & Bullitta S, Correlations between phenolic analysis, nutritional content and antioxidant property of content and antioxidant properties in twenty-four plant Eulophiaherbacea Lindl. tubers: a medicinally versatile species of traditional ethnoveterinary uses in the Indian tribal nutritional food supplement, Indian J Tradit Mediterranean area, Pharm Biol, 49 (2011) 240-247. Knowle, 17(1) (2018) 141-147. 34 Do QD, Angkawijaya AE, Tran-Nguyen PL, Huynh LH, 27 Karim MAA, Noor SM, Seman Z, Tohit ERM & Idris F, Soetaredjo FE, et al, Effect of extraction solvent on total Evaluation of anticoagulant property of aqueous and phenol content, total flavonoid content, and antioxidant ethanolic extracts of Morinda citrifolia, Int J Trop Med, 8 (1) activity of Limnophila aromatic, J Food Drug Anal, 22 (3) (2013) 1-5. (2014) 296-302. 28 Pandey A & Tripathi S, Concept of standardization, extraction and pre-phytochemical screening strategies for 35 Taşkın T, Özakpınar ÖB, Gürbüz, B, Uras F, Gürer ÜS & herbal drug, J Pharmacog Phytochem, 2 (5) (2014) 115-119. Bitiş L, Identification of phenolic compounds and evaluation 29 Llaca-Díaz JM, Mendoza-Olazarán S, Camacho-Ortíz A, of antioxidant, antimicrobial and cytotoxic effects of the Flores S & Garza-González E, One-year surveillance of endemic Achillea multifidi, Indian J Tradit Knowle, 15 (4) ESKAPE pathogens in an intensive care unit of Monterrey, (2016) 594-603. México, Chemotherapy, 58 (6) (2012) 475-481. 36 Araújo de Oliveira VM, Basilio-Carneiro AL, de Barros- 30 Gonzalez-Villoria AM & Valverde-Garduno V, Antibiotic- Cauper GS & Pohlit AM, In vitro screening of Amazonian resistant Acinetobacter baumannii increasing success plants for hemolytic activity and inhibition of platelet remains a challenge as a nosocomial pathogen, J Pathog, 6 aggregation in human blood, Acta Amazon, 39(4) (2009) (2016) 1-10. 973-980. 31 Raut SV & Pukale PS, Study on antimicrobials principles of 37 Félix-Silva J, Souza T, Gomes-Camara RB, Cabral B, Silva- Parthenium hysterophorus and its application as a Júnior AA, et al., In vitro anticoagulant and antioxidant disinfectant, Plant Arch, 10 (2) (2010) 919-926. activities of Jatropha gossypiifolia L. (Euphorbiaceae) leaves 32 Chanudom L, Bhoopong P, Khwanchuea R & Tangpong J, aiming therapeutical applications, BMC Comple Altern Med, Antioxidant and antimicrobial activities of aqueous & 14 (405) (2014) 1-13.