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: eduardo.sanchezgrc@uanl.edu.mx 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 species of shrubs, herbaceous perennials, and annuals1. P. incanum (Mariola) is a perennial plant 2- 3 ft high; its leaves are grayish green and has white small flowers. Its distribution covers from the South western United States to the North of México.
Mariola is a source of important food resources for grazing animals that are found in desert shrub lands2. The medicinal uses of this plant include treatment of indigestion, sluggish liver and mild constipation3. Various biological activities from related species (P. hysterophorus) have already been reported such as antimicrobial, antioxidant, antihemolytic, cytotoxicity and lipid peroxidation inhibition4,5.
On the other hand, the genus Nothoscordum comprises more than 30 species, distributed
worldwide6. N. bivalve (false garlic) is a perennial plant up to 20 inches tall with white flowers.
The species can be found in the eastern region of the United States, Mexico, Argentina, Chile and Uruguay7,8. There is not enough information on the biological activity of the Nothoscordum genus to establish its applications in traditional medicine, but antimicrobial activity and phytochemical screening has been reported for some species of the genus. With respect to antimicrobial activity, the species N. gracile and N. entrerianum have been studied; for the case of N. gracile, phytochemical screening, FT infrared spectroscopy and wavelength dispersive X-ray fluorescence studies demonstrated the presence of secondary metabolites as well as inorganic elements contained in their leaves and bulbs6,9.
The present investigation was focused on the in vitro biological activity of P. incanum and
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*Corresponding author
N. bivalve extracts, evaluating their antimicrobial activity against an isolated antibiotic-resistant strain of Acinetobacter baumannii, as well as their toxicity, cytotoxicity, antioxidant capacity, soluble phenolics content and their effects on blood coagulation to generate knowledge about their possible medicinal use.
Methodology Microorganism
Acinetobacter baumannii isolated strain was kindly donated by Dr. Elvira Garza-González, from Dr. José Eleuterio González University Hospital (Universidad Autónoma de Nuevo León) (UANL Hospital).
Preserved in Mueller-Hinton agar at 4 °C.
Plant material
Samples of aerial parts of N. bivalve (stem and flowers) and P. incanum (leaves and bark) were collected from the wild in the area around Santa Catarina, Nuevo León, México (25°38'50.78'' N, 100°43'24.19'' W). A voucher specimen of the plants was deposited in the herbarium of the Biological Science Faculty, Universidad Autónoma de Nuevo León, for correct identification. Collected plants were cut into small pieces and air-dried under shade at room temperature (25 °C ± 2 °C). Dried material was ground in a manual grain mill and stored in paper bags until use.
Extraction
Maceration extraction (ME): 100 g of P. incanum and 80 g of N. bivalve, were extracted with 600 mL and 500 mL of methanol (MeOH), respectively, for 24 h at room temperature in dark conditions, with occasional shaking. Then they were filtered through Whatman No. 1 filter paper; extraction was repeated 5 times on the same plant material10.
Soxhlet extraction (SE): 335 g of P. incanum and 280 g of N. bivalve, were placed in a Soxhlet equipment and extracted sequentially with 2 L of each of the following solvents: hexane, chloroform, and MeOH (in that order), on the same plant material, for 48 h each solvent11. All extracts (ME and SE) obtained, were concentrated at reduced pressure in a rotary evaporator (Yamato RE 200) until dryness.
Extracts were re-suspended in 15 mL of the solvent used for the primary extraction and stored at 4 °C in the dark until use.
Extraction yields of solids (%) = (mass of extracted solids/initial mass of the plant before extraction)
×100.
Drug resistance of A. baumannii
A sensitivity test for several antibiotics was carried out according to the methodology of Morales-Meza and Ruiz de Chávez-Ramos12 with slight modifications; the amount of microorganism was quantified by microdilution methods and was seeded at approximately 109 CFU/mL. The measurement and interpretation of inhibition halos were performed as indicated by the manufacturer.
Preliminary antimicrobial activity
The method reported by Sánchez et al.13 was used with slight modifications; 100 µL of bacterial suspension (approx 109 CFU/mL), previously activated in Mueller-Hinton broth, were homogeneously seeded onto Mueller-Hinton agar plates. MeOH extracts were evaluated by a well diffusion agar technique; briefly, wells (6 mm diameter) were cut out of the agar using a sterilized cup-borer, then wells were filled with 100 µL of the MeOH extracts. On the other hand, hexane and chloroform extracts were evaluated by a disc diffusion method in view of their insolubility in aqueous agar, in this case, 30 L of each extract were impregnated into sterile filter paper discs (6 mm in diameter); all discs were fully air-dried and were placed on the agar plate by use of a sterile needle.
Plates of both methods (well and disc diffusion) were incubated at 37 °C for 24 h. Extraction solvents were used as controls.
Minimal bactericidal concentration (MBC)
The MBC of the most active extracts was analyzed according to Sánchez et al.14and Duarte et al.15 with some modifications; extract concentrations between 0.5 and 15 mg/mL were evaluated with a bacterial concentration of approximately 109 CFU/mL. After incubation (24 h at 37 °C), 20 µL were taken from the wells where there was no apparent growth and were seeded by dripping in Mueller-Hinton agar, and incubated for 24 h at 37 °C. MBC was defined as the lowest concentration where no growth was recorded.
Toxicity in Artemiasalina
Artificial sea water was prepared and 100 mg of Artemiasalina eggs were added; after 48 h (maturation of nauplii) the extracts were evaluated in concentrations between 100 and 1000 µg/mL16,17. The lethal dose 50 (LD50) was calculated using the Probit method (SPSS ver. 23), according to the toxicity classification of Sánchez & Neira18.
Cytotoxicity (% hemolysis)
The methodology of Vinjamuri et al.19, was used with some modifications; a suspension of 5 % human erythrocytes was prepared with PBS buffer; extracts were evaluated at concentrations between 100 and 1000 µg/mL; distilled water and PBS buffer were used as positive and negative controls, respectively.
The final reaction volume was adjusted to 1.25 mL, constituted by 250 µL of erythrocyte suspension, 980 µL PBS, and 20 µL of the different extract concentrations. Then, the reaction mixture was incubated for 30 min at 37±0.5 °C; samples were centrifuged and supernatants were read at 540 nm.
Hemolysis was calculated with the following equation20:
% Hemolysis = As Anc Apc Anc
×100
Where, As = Absorbance of the sample;
Anc = Absorbance of the negative control;
Apc = Absorbance of the positive control Cytoprotection (% inhibition of hemolysis)
A variant of the methodology of Sulaiman &
Hussain21and Chisté et al.22, was carried out. The final reaction volume of 1 mL was constituted by 375 µL of 150 mM AAPH in PBS, 250 µL of a 5 % erythrocyte suspension in PBS, 355 µL of PBS, and 20 µL of plant extract at variable concentrations (100 to 1000 µg/mL). The negative control was considered as 250 µL erythrocyte suspension and 750 µL PBS; as a positive control, 250 µL erythrocyte suspension, 375 µL of 150 mM AAPH in PBS and 375 µL of PBS (without extract). The reactions were maintained at 37
± 0.5 °C for 6 h in a shaking incubator (300 rpm);
afterwards, the samples were centrifuged and supernatants were read at 540 nm. The percentage of inhibition of hemolysis was obtained by using the formula:
% Inhibition of Hemolysis =100-[ As Anc Apc Anc
×100]
Where, As = Absorbance of the sample;
Anc = Absorbance of the negative control;
Apc = Absorbance of the positive control
Trolox equivalent antioxidant capacity (TEAC) using DPPH and ABTS reagents
All procedures were carried out in dim light;
standard solutions containing between 0 and 35 nmol
of Trolox in 100 µL were prepared; for DPPH assay each was allowed to react with 600 µL of 0.13 mM DPPH reagent (total reaction volume = 700 µL).
Likewise, 100 µL aliquots of plant extracts were evaluated with the DPPH reagent. Reactions were kept in the dark for 20 min at room temperature23. Then the tubes were read at 515 nm24. For ABTS assay, 100 µL of different Trolox concentrations (0-35 nmol) were reacted with 1400 µL of ABTS reagent (prepared using 7.4 mM ABTS and 2.6 mM potassium persulfate) for a total reaction volume of 1500 µL. Equally, 100 µL aliquots of extracts plants were evaluated. Reactions were kept in the dark for 30 min at room temperature. Then the tubes were read at 734 nm25.
Soluble phenolics quantitation as gallic acid equivalents (GAE)
The procedure was carried out in dim light. The gallic acid curve (10-80 µg) was prepared in a final volume of 1 mL with 30 % MeOH. Similarly, different dilutions of the extracts were prepared.
125 µL of the 2 N Folin-Ciocalteu reagent was then added and the mixture was stirred and allowed to react for 6 min. Then 1.25 mL of 7 % sodium carbonate was added, shaken and 625 µL of distilled water were added to give a final volume of 3 mL.
After stirring, the mixture was allowed to react for 90 min in the dark at room temperature. After this time, without stirring the mixture, absorbance readings were obtained at 757 nm in a spectrophotometer24,26. Coagulation tests (PT and aPTT)
Blood samples (healthy patients) were obtained in tubes with 3.2 % sodium citrate. They were centrifuged at 13,000 rpm for 5 min to recover the plasma. Then 1 mL of plasma was adjusted to the concentrations of 1000 and 500 µg/mL with the extracts (980 µL plasma with 20 µL extract and 990 µL plasma with 10 µL extract, respectively). For the prothrombin time (PT), the reagent Trini CLOTPT Excel (rabbit brain) and 100 µL of the treated plasma were preheated separately (37 ± 0.5 °C) for 2 min in the coagulometer, after which 200 µL of PT Excel reagent were added to the plasma and the timer was activated. The coagulometer recorded the time to the formation of the clot. For activated partial thromboplastin time (aPTT), 100 µL of pre-heated Trini CLOTaPTT-S (particles of phospholipids) reagent was added to the treated plasma, the timer was activated and the reaction was incubated in the
coagulometer for 5 min at 37 ± 0.5 °C; after that, 100 µL of pre-heated 0.025 M calcium chloride was added. The coagulometer recorded the time to the formation of the clot. One-way analysis of variance (ANOVA) and Tukey’s test were used to find out significant differences (p < 0.05)27.
Results and discussion
Extraction yields of solids obtained for P. incanum were: hexane (SE) 2.54 %, chloroform (SE) 2.51 % and MeOH (SE) 8.05 %, while yield of successive maceration only with MeOH was 14.49 %, it is important to mention that the cumulative yield percentage of Soxhlet extraction was 13.1 %, slightly lower than the aforementioned for successive maceration (14.49 %). We found a similar behavior for N. bivalve, in this case, the obtained SE yields were: hexane 2.33 %, chloroform 1.57 % and MeOH 21.79 %, while successive maceration with MeOH was 28.50 %, and similarly to the above, the accumulated percentage of the Soxhlet extraction was 25.69 %. MeOH extractions for both plants showed the highest yields. It has been previously reported that MeOH is frequently used because it allows the extraction of a greater number of plant metabolites. In our work, maceration extraction (ME) was slightly more efficient than Soxhlet extraction (SE); however, previous studies also found that repeated extractions on the same plant material showed higher extraction yields, because a considerable amount of active principles of interest can remain in the plant after the first extraction10,28.
The A. baumanii strain was evaluated for resistance to several antibiotics and proved to be resistant to amikacin (30 µg), ampicillin (10 µg), cephalotin (30 µg), cefepime (30 µg), cefotaxime (30 µg), ceftriaxone (30 µg), chloramphenicol (30 µg), gentamicin (10 µg), netilmicin (30 µg), nitrofurantoin (30 µg) and sulfamethoxazole/trimethoprim (25 µg);
it was sensitive only to levofloxacin (5 µg). In recent years, the pathogen A. baumannii has become important at the nosocomial level around the world due to the increase of its resistance to antibiotics and the prevalence in patients in intensive care units; it belongs to the emergent pathogen group of microorganisms known as ESKAPE. Regarding this, in the state of Nuevo León, México, A. baumannii was the most frequent isolate at the UANL Hospital during 2011-201229,30.
The results of preliminary evaluations of antimicrobial activity against A. baumannii are shown
in Table 1; the MeOH extracts presented the highest activity. Minimal bactericidal concentrations (MBCs) were obtained only for MeOH extracts because of their higher activity (Table 1), these results ranged from 7.50±0.54 to 8.50±0.54 mg/mL. Extracts from Parthenium species have been reported to show antimicrobial activity against Streptococcus mutans, Proteus vulgaris, and Salmonella typhi4. In the case of Nothoscordum, aqueous extracts from bulbs of three species showed activity against Candida albicans, Aspergillus fumigatus, and Staphylococcus aureus9. On the other hand, Sánchez et al.14 reported that MeOH extracts from bulbs of N. bivalve had no antimicrobial activity against antibiotic-resistant strains of Klebsiella pneumoniae, Enterococcus faecalis, Escherichia coli, Stenotrophomonasmaltophilia, and Staphylococcus aureus, all of them isolated at the UANL Hospital.
MBCs ranged from 7.50 to 8.50 mg/mL; Raut &
Pukale31 reported various MBCs from polar extracts of P. hysterophorus, being between 24 and 72 mg/mL for S. aureus, E. coli, and A. fumigatus. Nevertheless, for Nothoscordum, MBCs have not been previously reported for any microorganisms.
Toxicity results of P. incanum and N. bivalve MeOH extracts against Artemiasalina showed different degrees of toxicity (LD50 from 380 to 1882 µg/mL); P. incanum ME 380 µg/mL (moderate toxicity), and P. incanum SE 1882 µg/mL (relatively innocuous); on the other hand, N. bivalve ME 916 µg/mL (slightly toxic) and N. bivalve SE 1023 µg/mL (practically non-toxic), according to the classification of Sánchez & Neira18. It is common to evaluate the toxicity of natural products with biological activity using the A. salina test due to its ease of use, low cost, and reliability16,17.
Table 1 — Preliminary evaluation of antimicrobial activity and MBC of P. incanum and N. bivalve extracts
Plants Extraction Preliminary activity(mm)
MBC (mg/mL) P. incanum SE Hexane 5.2±2.4 ND**
SE Chloroform NI* ND
SE Methanol 9.4±0.8 8.50±0.54 ME Methanol 13.2±0.6 8.16±0.40
N. bivalve SE Hexane NI ND
SE Chloroform 6.5±1.6 ND
SE Methanol 14.8±0.3 7.50±0.54 ME Methanol 16.5±0.5 8.33±0.51 Values expressed as mean ± SE, n=6. *No inhibition. ** Not determined
Extracts showed high Trolox equivalent antioxidant capacities (TEAC) (> 200 µmol TE/g) by both methods (Table 2); it can be observed that SE extracts presented greater TEAC levels. Soluble phenolics contents were calculated as gallic acid equivalents (GAE). P. incanum and N. bivalve extracts showed high levels of soluble phenolics ranging between 1.348±0.07 and 4.133±0.04 g GAE/100 g (Table 2). TEAC of extracts (275.55±21- 598.99±4 µmol TE/g) were higher to those reported in foods like blue-kernel maize, nixtamalized commercial blue corn flour, commercial blue baked tortilla chips and huitlacoche corn cobs (8.5-88.8 µmol TE/g)23; however, our values were lower than those from aqueous and ethanol extracts from plants of the genera Anacardium, Clausena, Litsea, Ocinum, Piper, Pseuderanthenum, Spondias and Syzygium (2.43 to 411.96 mmol TE/g)32. Soluble phenolics in extracts from both species (1.35±0.07- 4.13±0.04 g GAE/100 g) were similar or lower than those previously reported in aqueous and ethanol extracts from the genera Anacardium, Clausena, Litsea, Ocinum; Piper, Pseuderanthenum, Spondias and Syzygium (0.408-47.40 g GAE/100 g)32. Phenolic compounds are extracted with polar solvents such as MeOH; they possess various biological activities such as free radical uptake, antimicrobial activity and antiviral, among others. Therefore, it is always useful to correlate the soluble phenolics contents with the antioxidant capacity of extracts32,33,34; Table 2 shows that P. incanum and N. bivalve extracts do not show a correlation between TEAC and GAE.
Cytotoxicity evaluations of MeOH extracts of P. incanum and N. bivalve are presented in Fig. 1, where it can be clearly seen that the ME of P. incanum exhibits approximately 92.98 % hemolysis at 1000 µg/mL; as the concentration of extract increases, the percentage of hemolysis
increases. In contrast, P. incanum SE in all evaluated concentrations had between 0.07 and 1.43 % hemolysis. N. bivalve ME at 1000 µg/mL showed 13.69 % hemolysis, whereas N. bivalve SE in all concentrations caused 0.16 to 2.39 % hemolysis.
Maceration extracts showed more hemolysis than those obtained by Soxhlet; this may be due to the extraction method applied on the plant material: It is known that many of the non-polar compounds present in plant extracts, exhibit a strong cytotoxic activity35, this would explain the low cytotoxicity of the methanolic extract obtained by soxhlet reflux, since during the soxhlet extraction, non-polar solvents (hexane and chloroform) were used before methanol, removing the non-polar compounds of the plant, obtaining a methanolic extract which no longer presents these kinds of compounds, while only MeOH was used for maceration, which allows the extraction of different kinds of compounds even those with cytotoxic activity; this explains why MeOH extracts obtained after a polarity gradient (Soxhlet) will differ from those obtained by extraction without such a gradient (maceration)10,27. It has been reported that plant extracts may contain cytotoxic compounds, which is why the hemolysis assay is used as a general indicator in the cytotoxicity study19,20,36. Our results suggest the presence of cytotoxic (hemolytic) compounds in the non-MeOH Soxhlet extracts of both plants, a further investigation currently underway.
Likewise, a correlation can be observed between the A. salina and hemolysis tests; the ME of P. incanum presented a lower LD50 and a high degree of hemolysis, unlike the other extracts, which had higher LD50 and lower percentages of hemolysis.
Cytoprotective evaluations in Fig. 2 show that maceration extracts (ME) caused low or null
Table 2 — Antioxidant capacity and soluble phenolics content of P. incanum and N. bivalve methanolic extracts Extracts TEAC
ABTS assay (µmol TE/g)
TEAC DPPH assay (µmol TE/g)
Soluble phenolics
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;
SE: Soxhlet extraction. Fig. 1 — Cytotoxicity (% hemolysis) of P. incanum and
N. bivalve methanolic extracts.
cytoprotection (AAPH technique); for N. bivalve ME, no cytoprotective activity was observed and for P. incanum at 500 µg/mL, about 41.92% efficacy was present. However, when the extract concentration increased, the activity dropped to 12.91%. In the case of Soxhlet extracts (SE), higher cytoprotective activities were observed; for P. incanum extracts at 500 and up to 1000 µg/mL, between 86.95 and 94.58% cytoprotection was observed, while for N.
bivalve, the cytoprotection was proportional to the increase in extract concentration, corresponding a 500 µg/mL extract to 5.85 % cytoprotection, 750 µg/mL to 39.24 % and 1000 µg/mL to 79.34 %. Low or null cytoprotective activity in maceration extracts can be due to its hemolytic capacity at concentrations tested;
as shown in Fig. 1, as concentration increases, hemolysis increases. On the other hand, for the case of the Soxhlet extracts, cytoprotection is observed at concentrations ≥ 500 µg/mL, the null or low hemolysis at these concentrations can be clearly appreciated (Fig. 1). These differences between maceration and Soxhlet extracts could be reflecting the different extraction methods, as it was already mentioned for hemolysis. The AAPH peroxide radicals attack the erythrocyte membrane; whereby the plant extracts evaluated in this test could be an auxiliary in the prevention or treatment of the oxidation provoked by free radicals. Previous studies have reported that plant extracts or their natural compounds counteract the action of radicals formed by AAPH21,22.
Only MeOH extracts from the Soxhlet extraction were used for coagulation assays because they showed low toxicity, cytotoxicity, and significant cytoprotective effect. The PT (prothrombin time) and APTT (activated partial thromboplastin time)
assays were done with concentrations of 500 and 1000 µg/mL. Table 3 shows the PT and APTT results; for PT, P. incanum extracts at 500 and 1000 µg/mL showed significant difference (p < 0.05) with respect to the other means, including controls. On the other hand, in the APTT tests, no significant differences were found among the means (p > 0.05), including the controls. With respect to extract of N. bivalve did not cause a significant change in the coagulation times at the evaluated concentrations. In recent years, several studies of anticoagulant activities in plant extracts have been carried out, as new active principles are sought to treat thrombotic diseases; it is known that the PT prolongation shows inhibition in the extrinsic coagulation pathway and, on the other hand, the prolongation of APTT shows inhibition in the intrinsic pathway of coagulation26,37.
Conclusion
Extracts from P. incanum and N. bivalve showed various effects on biological activities such as antimicrobial, antioxidant, cytoprotective, toxicity, cytotoxicity, and anticoagulant activity. Moreover, MeOH Soxhlet extracts were obtained from a more selective process in which low polarity compounds were first removed with hexane and chloroform, which could explain their possible pharmaceutical potential, in view that they showed inhibition of A. baumannii, have low toxicity, low cytotoxicity and cytoprotection of human erythrocytes; they also showed no significant prolongation in the extrinsic pathway of coagulation, and a small but significant prolongation in the intrinsic coagulation pathway.
Fig. 2 — Cytoprotection activity (%) caused by P. incanum and N. bivalve methanolic extracts
Table 3 — Prothrombin time (PT) and activated partial thromboplastin time (aPTT) for P. incanum and N. bivalve
methanolic Soxhlet extracts.
Plants Extract
Concentration PT(s) aPTT(s) 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
control
10 µL 12.10±0.20 33.63±0.35 20 µL 12.16±0.11 33.36±0.95 Blank Untreated plasma 12.26±0.15 32.63±0.66 Values expressed as mean ± SE, n=6.
Acknowledgment
The authors are thankful to the Chemical Analitical Laboratory, Facultad de Ciencias Biológicas, Universiadad Autónoma de Nuevo León for providing the facilities for conducting the research study. And to the Departments of Microbiology and Chemistry of Universidad Autónoma de Aguascalientes. Technical support from Ma. Lorena Sandoval-Cardoso, José Luis Carrasco-Rosales and José Luis Moreno- Hernández-Duque. Support and doctorate grant from CONACYT to DAHM (556587).
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