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Volume 3, No.1

Mexico / 2019january / april

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María de Lourdes Meza Jiménez, PhD.Universidad Popular Autónoma del Estado de Puebla, Puebla, Pue Sandra Victoria Ávila Reyes, PhD.Depto. Biotecnología, CeProBi, IPN, Yautepec, Mor María Ximena Quintanilla Carvajal, PhD.Facultad de Ingeniería, Universidad de la Sabana, Chía, Colombia

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Gerónimo Peña Climaco, PhD.Instituto Politécnico Nacional, México.

Daniel Alejandro Ramírez Villanueva, PhD.Universidad de la Sierra Juárez, Ixtlán de Juárez, Oaxaca.

Elizabeth González Terreros, PhD.Universidad de la Sierra Juárez, Ixtlán de Juárez, Oaxaca.

Fernando Uriel Rojas Rojas, PhD.Universidad del Valle de México.

Xariss Miryam Sanchez Chino, PhD. Catedra CONACyT ECOSUR Unidad Villahermosa L.

Luisa Ma Rodrigues Gouveia Da Silva, PhD. Laboratorio Nacional de Energía e Geología, Lisboa, Portugal.

Brenda Román Ponce, PhD. Universidad de Salamanca, España.

Aline Tezcucano, PhD. University Cres, Winnipeg, Canada.

Raúl Sánchez-Sánchez, PhD. Pohang University of Science and Technology, South Korea.

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JOURNAL OF BIOENGINEERING AND BIOMEDICINE RESEARCH, Año 3, Volumen 3, No. 1, enero - abril 2019, es una Publicación cuatrimestral editada por el Colegio Mexicano de Ingenieros Bioquímicos, A.C.,Calle Mar del Norte #5, Col. San Álvaro, Alcaldía Azcapotzalco, Ciudad de México, C.P. 02090, Tel. (55)2873 2956, www.cmibq.org.mx, [email protected], [email protected] Editor Responsable: Deilia Ahuatzi Chacón, Rosa María Ribas Aparicio.Reserva de derechos al uso exclusivo No. 04-2016-041313084800-203, ISSN: 2594-052X, ambos otorgados por el Instituto Nacional del Derecho de Autor. Responsable de la última actualización de este Número, José Alberto Romero León Prolongación de Carpio y Plan de Ayala s/n, Col. Santo Tomás,Alcaldía Miguel Hidalgo, C.P. 11340, Ciudad de México fecha de última modicación 25 de enero de 2019




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Volume 3, No.1

Mexico / 2019january / april

2019 - 2022

IBQ. Raúl Chávez AlvircioPresident

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INDEX

area authors pages

Food technology

Bioactive natural products

Environment and sustainability

1-5

6-11

12-19

20-27

Meza Márquez Ofelia Gabriela, Gallardo Velázquez Tzayhrí Guadalupe, Osorio Revilla Guillermo

Conrado Garcia Diana, Cano Sosa Julia, Noriega Trejo Rodolfo, Gongora Chin Ricardo, RamosDiaz Ana Luisa

Briceño Santiago Cinthia, Cano Sosa Julia, Andrada Ana, Ramos Díaz Ana Luisa

Rangel Mejía Virdiana, Rodríguez Casasola Felipe Neri, Leal Ríos Elvira, Rodríguez Casasola Ma. Teresa, Cruz Mondragón Carlos, Vázquez Ulloa Elenaé, Esparza García Fernando José

Copyright ©2019 COLEGIO MEXICANO DE INGENIEROS BIOQUIMICOS

JOURNAL OF BIOENGINEERING AND BIOMEDICINE RESEARCH (2019) Vol. 3 No. 1 1-5

1

Short communication

Determination of Clenbuterol Residues in Beef Meat, Liver and Kidney Marketed in Mexico by Gas ChromatographyMeza Márquez Ofelia Gabriela*1, Gallardo Velázquez Tzayhrí Guadalupe2, Osorio-Revilla Guillermo1

1 Departamento de Ingeniería Bioquímica, ENCB- Zacatenco, Instituto Politécnico Nacional. Unidad Profesional Adolfo López Mateos, Av. Wilfrido Massieu esq. Cda. Miguel Stampa S/N, 07738, Cd. de México. 2 Departamento de Biofísica, ENCB-Sto. Tomás, Instituto Politécnico Nacional. Prolongación de Carpio y Plan de Ayala, S/N. Col. Santo Tomás. 11340, Cd. de México.

*Corresponding author: [email protected]: 20 august 2018/ Accepted: 06 november 2018/ Published online: 25 january 2019

Abstract. Clenbuterol concentration ranged from 0.11 to 0.36 µg/kg in beef meat, from 0.15 to 7.29 µg/kg in liver and from 0.12 to 0.41 µg/kg in kidney. This procedure was validated obtaining recoveries larger than 90%. The results showed that clenbuterol was illegally used for some producers as bovine growth promoter.

Keywords. Bovine meat, quality control, clenbuterol.

Resumen. La concentración de clembuterol fue de 0.11-0,36 µg/kg en carne de res, 0.15-7.29 µg/kg en hígado y 0.12-0.41 µg/kg en riñón. Esta metodología fue validada obteniendo recuperaciones más altas que el 90%. Los resultados muestran que el clembuterol fue utilizado ilegalmente por algunos ganaderos como promotor del crecimiento bovino.

Palabras Clave. Carne de bovino, control de calidad, clenbuterol.

INTRODUCTION

Clenbuterol is a β2-adrenergic agonist used in veterinary medicine with therapeutic dose of 0.8 µg/kg body weight due to its bronchiolytic and tocolytic action. Clenbuterol may be added to feeds as a growth promoter due to its anabolic effect in dosages 5-10 times higher than the therapeutic one for economic benefit. However, the illegal use of the clenbuterol to increase the muscular mass of the bovine livestock is a problem of food safety, because the residues that are deposited in beef meat,

liver and kidney cause intoxications in people that consume them.1 The use of clenbuterol in livestock is prohibited in the European Union (EU), the United States of America (USA) and other countries.1 The residues of clenbuterol which accumulate in edible tissues especially liver, meat and kidney have a potential risk of adverse effects with signs of acute intoxication.2 Cases of clenbuterol food poisoning have been reported in several countries as Spain, France, Italy, China, Portugal, United States and México.2-8



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In Mexico, as well as in other parts of the world it is suspected the unlicensed use of clenbuterol in certain commercial cattle feedlots. 8 In 2001, allegations about the illegal use of clenbuterol among Mexican meat producers created a state of alert of the regulatory agencies.8 From 2002 to date, 2,780 cases of human intoxications with clenbuterol in Mexico were associated to the consumption of beef meat and bovine liver.9

Analytical methods for the determination of residues of clenbuterol in animal tissue have been described. These include Enzyme-Linked ImmunoSorbent Assay (ELISA) that is used as screening method10 and Gas Chromatography (GC) as confirmatory method.11-12 The aim of this study was to monitor the presence of clenbuterol residues in beef meat, liver and kidney samples marketed in Mexico by GC as confirmatory method.

METHODS, RESULTS AND DISCUSSION

Clenbuterol hydrochloride was obtained from Sigma (St. Louis, Mo., USA); Bis(trimethylsilyl)trifluoro-acetamide (BSTFA) was obtained from Supelco (Bellefonte, USA); ethyl acetate and methanol were from Merck (Darmstadt, Germany). All solvents were HPLC grade. All other reagents and solvents were analytical-reagent grade.

Stock solutions and standards: Clenbuterol hydrochloride was used to prepare a standard stock solution (10μg/50mL) in methanol. This solution was protected from light and stored at 4°C. Working standard solutions of different concentrations were prepared by dilution of the standard stock with methanol. 13

Samples of beef meat, liver and kidney: Sixty beef meat, liver and kidney samples (twenty of each one) were collected from different local butcheries and supermarkets in Mexico City.

Determination of clenbuterol in beef meat, liver, kidney by GC: The extraction and clean-up procedures were those described by the ELISA kit manufacturer (R-biopharm, Darmstadt, Germany). Briefly, minced (beef meat, liver or kidney) samples (5 g) were homogenized for 20 min with 25 mL of 50 mM HCl and centrifuged (centrifuge, Cole-Parmer®), Colefor 15 min at 5600 rpm at 15 ºC. The supernatant was collected into a new centrifugal vial and mixed with 2 mL of 0.5 M NaOH for 10 min. 10 mL of 500 mM KH2PO4-buffer (pH 3) were added, mixed briefly and stored at 4 ºC for 1.5 h. The sample was centrifuged for 15 min at 5600 rpm at 15 ºC and finally, the entire supernatant was purified by RIDA® C18 column purification (solid-phase extraction).

The C18 column purification was first rinsed with 3 mL of methanol (100%) and then with 2 mL of 50 mM KH2PO4 buffer (pH 3). The sample was applied, and the column was rinsed again with 2 mL of 50 mM KH2PO4 buffer (pH 3). The fluid residues were removed by positive pressure for 2 min with nitrogen. The sample was eluted from the column with 1 mL of methanol (100 %) (Flow rate: 15 drops/min) and was evaporated to dryness under continuous flow of nitrogen at 45 to 50 ºC.13

The sample residue was derivatized as reported by Fente method.13 Briefly, 100 mL of BSTFA and 100 ml of ethyl acetate were added to the residue and heated at 70 ºC for 30 min. Once the derivatization process was completed, the solution was evaporated to dryness under continuous flow of nitrogen and reconstituted in 25 mL of ethyl acetate. Finally, 3 µL of this solution were injected into the gas chromatograph.

Chromatographic separation was analyzed on a Clarus 500 gas chromatograph (Perkin Elmer, Shelton, CT, USA) with a flame ionization detector (FID). Analyses were performed with a Heliflex® AT™-5MS capillary column (30 x



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0.25mm i.d x 0.25 µm of film thickness). Nitrogen (99.999%) was used as the carrier gas with at flow rate of 1.0 mL/min. The injector temperature was maintained at 280 °C and all injections were made in splitless mode. The GC oven temperature was held at 170 °C for 3 min, raised to 240 °C at 10 °C min-1, and then raised again to 280 °C at 30 °C min-1 and finally held at 280 °C for 3 min.8

Samples of the beef meat, liver and kidney free of clenbuterol (determined by ELISA) were spiked with clenbuterol at levels of 0.05, 1.0, 2.0, 3.5, 7.0 y 10 µg/kg, including one without any addition of clenbuterol (zero calibration). All spiked samples were processed according to the procedure described above. Peak retention times of clenbuterol were identified by injecting standards of clenbuterol.

Recovery (%R) and Precision (%CV) studies were performed at five levels (0.1, 1.5, 3.0, 6.5 y 10 µg/kg) by adding samples of beef meat, liver and kidney with clenbuterol and then quantifying the concentration by GC.8 Minitab Statistical Software version 16.1.0 (State College, PA, USA) was used for the analyses.

From sixty beef meat, liver and kidney samples (twenty of each) analyzed by the GC, clenbuterol concentration (µg/kg) ranged from 0.11 to 0.36 in beef meat, from 0.15 to 7.29 µg/kg in liver and from 0.12 to 0.41 µg/kg in kidney. No clenbuterol was detected in 40% of the samples (Table 1).

According to the Maximum Residue Limit (MRL) of 0.1 µg/kg in meat and 0.5 µg/kg in liver and kidney,14 fifty-two (52%) meat samples and seven (35%) liver samples would be considered positive samples (Table 1). However, Mexican regulations do not include MRL for clenbuterol residues in meat, liver and kidney, although unofficially, it is known that samples can be considered positive when presenting clenbuterol residues above 2 µg/kg.15

Table 1. Clenbuterol concentrations (µg/kg) in beef meat, liver and kidney samples determined by CG.

Clenbuterol concentration(µg/kg)

Sample Beef meat a Liver a Kidney a

M1 0.29 (± 0.03) 0.26 (± 0.07) < LOD b

M2 0.11 (± 0.01) 7.29 (± 0.02) < LOD b

M3 0.26 (± 0.04) < LOD b 0.14 (± 0.04)

M4 < LOD b 0.73 (± 0.03) < LOD b

M5 0.22 (± 0.02) 0.65 (± 0.01) 0.27 (± 0.01)

M6 0.35 (± 0.01) 0.28 (± 0.05) < LOD b

M7 < LOD b 6.54 (± 0.04) 0.18 (± 0.01)

M8 < LOD b 0.35 (± 0.02) < LOD b

M9 0.17 (± 0.08) 0.19 (± 0.01) < LOD b

M10 0.24 (± 0.06) < LOD b 0.35 (± 0.07)

M11 0.36 (± 0.05) 0.28 (± 0.02) 0.15 (± 0.06)

M12 0.28 (± 0.03) 0.34 (± 0.04) 0.41 (± 0.03)

M13 < LOD b 0.15 (± 0.01) < LOD b

M14 < LOD b < LOD b < LOD b

M15 < LOD b 0.64 (± 0.05) < LOD b

M16 0.34 (± 0.06) 0.39 (± 0.05) 0.12 (± 0.03)

M17 0.21 (± 0.01) < LOD b 0.26 (± 0.08)

M18 < LOD b 0.79 (± 0.02) < LOD b

M19 0.19 (± 0.06) 1.02 (± 0.04) < LOD b

M20 0.15 (± 0.07) < LOD b < LOD b

a Mean of three replicatesb LOD = Limit of detectionStandard deviation shown in brackets, preceded by the symbol ±

Clenbuterol peaks were observed at 9.65 ±0.09 min in beef meat sample (Figure 1). On the other hand, clenbuterol peaks were observed at 8.43 ±0.05 min in liver sample (Figure 1).

Fig 1. Chromatograms of beef meat sample. a) Clenbuterol standard at 10µg/L, b) blank beef meat sample, c) beef meat sample spiked at 3 µg/kg and d) contaminated beef meat sample (0.28 µg/kg).



4

Fig 2. Chromatograms of liver sample. a) Clenbuterol standard at 10µg/L, b) blank liver sample, c) liver sample spiked at 3 µg/kg and d) contaminated liver sample (6.54 µg/kg).

Method linearity was assessed by performing calibration curves using samples spiked with clenbuterol in the range from 0.05 to 10 µg/kg.

The response was linear in the range used with correlation coefficient of 0.971. Recovery studies were performed at five levels (0.1, 1.5, 3.0, 6.5 y 10 µg/kg), the results are summarized in Table 2. The average recoveries were in the range of 87.36-96.80%, 88.07-93.31% and 89.40-97.95% for beef meat, liver and kidney respectively. Average recovery thus indicated a reasonable parallelism and accuracy of the assay when applied to real samples.The variability expressed as a coefficient of variation (%CV) was in the range of 2.08-4.57%, 1.38-3.77% and 2.61-4.08% for beef meat, liver and kidney respectively (Table 2). The %CV values were below 5%, demonstrating an acceptable level of precision.

a R = (real concentration/added concentration) x 100b CV = (standard deviation/mean) x 100

CONCLUSIONS

The results show that clenbuterol was used illegally as a promoter of bovine growth by some farmers. The foregoing indicates that the authorities should carry out more vigilance to prevent it from being used illegally and to avoid intoxications by clenbuterol in people

FUNDING AND ACKNOWLEDGMENTS

Financial support from the Consejo Nacional de Ciencia y Tecnología (CONACyT) and the Secretaría de Investigación y Posgrado, Instituto Politécnico Nacional (SIP-IPN) is greatly appreciated.

REFERENCES

1) Council Directive 96/22/EC of 29 April 1996 concerning the prohibition on the use in stockfarming of certain substances having a hormonal or thyrostatic action and of ß-agonists, and repealing Directives 81/602/EEC, 88/146/EEC and 88/299/EEC. Official Journal of the European Communities, L125, 23/05/1996 P. 0003–0009. Last accessed: August 2018. https://eur-lex.europa.eu/legal-content/en/ALL/?uri=CELEX:31996L0022.

2) Martínez-Navarro, JF. 1990. Food poisoning related to the consumption of illicit b-agonists in liver. The Lancet. 336:1311. doi: 10.1016/0140-6736(90)92990-Y

3) Pulce C, Lamaison D, Keck G, Bostvironnois C, Nicolas J, Descotes J. 1991. Collective human food poisonings by clenbuterol residues in veal liver. Vet. Human Toxicol. 33:480-481. Article ID: 1746141

4) Brambilla G, Loizzo A, Fontana L, Strozzi M, Guarino A, Sporano V. 1997. Food poisoning following consumption of clenbuterol-treated veal in Italy. J Amer Med Assoc. 278:635. doi: doi:10.1001/jama.1997.03550080045031

Tissue n Added concentration (µg/kg) Recovery%R a

Coefficient of Variation,

%CV b

Beef meat

3 0.1 96.80 3.433 1.5 87.74 3.653 3.0 92.34 4.023 6.5 87.36 4.573 10 90.35 2.08

Liver

3 0.1 88.07 2.873 1.5 92.73 1.783 3.0 93.31 3.773 6.5 90.47 2.193 10 92.92 1.383 0.1 89.40 2.613 1.5 90.15 2.83

Kidney 3 3.0 97.95 3.983 6.5 91.53 2.753 10 90.67 4.08

Table 2. Average Recovery (%R) and Coefficient of Variation (%CV) of clenbuterol by GC.



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5) Chan TYK. 1998. Food Poisoning Following the consumption of clenbuterol-treated livestock. Hong Kong Practitioner 20(7):366-369 Article ID: http://hkjo.lib.hku.hk/archive/files/50ff9e95f4cc7ddfaf08f60d54e1f7ba.pdf

6) Barbosa J, Cruz C, Martins J, Silva JM, Neves C, Alves C, Ramos F, da Silveira MIN. 2005. Food poisoning by clenbuterol in Portugal. Food Addit. Contaminants 22:563-566. doi: 10.1080/02652030500135102

7) Mitchell GA, Dunnavan G. 1998. Illegal use of b-adrenergic agonists in the United States. J Anim Sci 76:208-211. doi: 10.2527/1998.761208x

8) Estrada-Montoya, MC, González-Córdova AF, Torrescano G, Camou JP, Vallejo-Cordoba B. 2008. Screening and confirmatory determination of clenbuterol residues in bovine meat marketed in the Northwest of Mexico. CYTA-J Food, 6:130-136. doi:10.1080/11358120809487637

9) SAGARPA (2011). Programa de proveedor confiable (libre de clenbuterol). Segundo Foro Nacional de Rastros. SAGARPA. México. https://www.gob.mx/sagarpa Last accessed: August 2018.

10) Wang R, Zhang W, Wang P, Su X. 2018. A paper-based competitive lateral flow immunoassay for multi β-agonist residues by using a single monoclonal antibody labelled with red fluorescent nanoparticles. Mikrochim Acta. 185: 191. doi: 10.1007/s00604-018-2730-9

11) Wang L, Li Y-Q, Zhou Y-K, Yang Y. 2010. Determination of four β2-agonists in meat, liver and kidney by GC-MS with dual internal standards. Chromatographia 71:737-739. doi: 10.1365/s10337-010-1495-z

12) Ramos F, Cristino A, Carrola P, Eloy T, Silva JM, Castillo MC, Noronha da Silveira MI. 2003. Clenbuterol food poisoning diagnosis by gas chromatography–mass spectrometric serum analysis. Anal Chimica Acta. 483:207-

213. doi: 10.1016/S0003-2670(02)01020-613) Fente CA, Vázquez BI, Franco C, Cepeda

A, Gibosos PG. 1999. Determination of clenbuterol residues in bovine hair by using diphasic dialysis and gas chromatography-mass spectrometry. J. Chromatography B. 726:133-139. doi: 10.1016/S0378-4347(99)00014-6

14) Commission Regulation No. 2391/2000/EC of 27 October 2000, Official Journal of the European Communities, L276/5. Last accessed: August 2018. https://eur-lex.europa.eu

15) Norma Oficial Mexicana de Emergencia NOM-EM-015-ZOO-2002, Especificaciones técnicas para el control del uso de beta-agonistas en los animales. Diario Oficial de la Federación. 1 de Marzo de 2002, 57-64. Last accessed: August 2018. http://legismex.mty.itesm.mx/normas/zoo/zoo015em-02.pdf



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Research article

Establishment of a protocol of asymbiotic germination Bletia purpurea (Lam.) A. DC.Conrado-Garcia Diana1, Cano-Sosa Julia*1, Noriega-Trejo Rodolfo2, Gongora-Chin Ricardo2, Ramos-Diaz Ana Luisa1

1 Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A. C. Parque Científico Tecnológico de Yucatán, Tablaje Catastral 31264 km. 5.5 Carr. Sierra-Papacal-Chuburna Puerto. Yucatán, México. 2 Centro de estudios de Desarrollo Sustentable y aprovechamiento de la vida silvestre-Universidad Autónoma de Campeche. Avenida Héroe de Nacozari #480, C.P. 24079, San Francisco de Campeche, Campeche, México.

*Corresponding author: [email protected]

Received: 21 april 2018/ Accepted: 11 september 2018/ Published online: 25 january 2019

Abstract. Bletia purpurea is an orchid that, although it germinates easily compared to other species of orchids that require a long time for its germination, could be threatened by the destruction of its habitat, so it is important to have strategies for its reproduction and multiplication to be a plant with ornamental potential. Asymbiotic germination of seeds is a conservation tool that allows a rapid and large-scale propagation in orchid species. Disinfection of mature capsules was performed with Benzalkonium, Ethanol, followed by Sodium hypochlorite. For the asymbiotic germination were used the full strength Murashige & Skoog (MS) medium, MS at half-strength, Benzylaminopurine (BAP) (0.1, 0.5 mg / L1) and Activated Charcoal (AC) (2.2). % (p / v)). The disinfection showed 100% of the treatments free of contamination. Seed germination was observed in the MS medium in half-strength without BAP and AC, in the MS medium complete with BAP added at 0.5 mg / Li, and in the half-strength MS medium in with AC. The different concentrations of MS with or without BAP and CA allowed the germination of seeds and differences in the size of the leaves and roots of the seedlings are observed.

Keywords. Orchid, Bletia purpurea, Benzylaminopurine, Asymbiotic germination

Resumen. Bletia purpurea es una orquidea que, aunque germina facilmente en comparacion con otras especies de orquideas que requieren mucho tiempo para su germination, podria verse amenazada por la destruction de su habitat, por lo que es importante contar con estrategias para su reproduction y multiplication al ser una planta con potencial ornamental. La germinacion asimbiotica de semillas es una herramienta de conservation que permite una propagation rapida y a gran escala en especies de orquideas. La desinfeccion de capsulas maduras se realizo con Benzalconio, Etanol, seguido de Hipoclorito de sodio. Para los tratamientos de germinacion se uso el medio Murashige & Skoog (MS) de intensidad completa, MS a la mitad de su intensidad, Bencilaminopurina (BAP) (0.1, 0.5 mg / Li) y Carbon Activado (CA) (2.2). % (p / v)). La desinfeccion mostro el 100% de los tratamientos libres de contaminacion. Se observo germinacion de semillas en el medio MS a la mitad sin BAP y CA, en el medio MS completo con BAP adicionado a 0.5 mg / L i, y en el medio MS a la mitad con CA. Las diferentes concentraciones de MS con o sin BAP and CA permitieron la germinacion de semillas y se observan diferencias en cuanto al tamano de las hojas y raices de las plantulas.

Palabras clave. Orquídea, Bletia purpurea, Benzylaminopurine, Germinación asbiótica



7

INTRODUCTION

Bletia is a genus comprising about 40 species of Neotropical terrestrial orchids.1,2 One of the species that is distributed from Florida, Mexico, Central America to South America and the West of the Indians, is Bletia purpurea (Lam.) A. DC.3 This orchid is characterized by the color of its flowers, which are purple pink.4 Currently their populations have decline to the North of America by the loss of habitat caused by the urbanization, the previous thing has suggested the need to establish protocols of germination of seeds that allow the propagation of this species.3 The first step for the establishment of seedlings from orchid seeds, is the germination of the seeds.5 The seed of orchids are small that not contain endosperm,6 thus require the establishment of a symbiotic relationship with a compatible fungus for the development of the protocormo and the growth of the future seedling.7 One of the tools for the production of orchids is the tissue culture from seeds.8 A form of propagation is asymbiotic seed germination, wich the association with a fungus is not required for the development of protocormo.9 In seed planting orchid asymbiotic, disinfection is important to maintain the in vitro culture under aseptic conditions.7,9 The establishment of orchid asymbiotic seed germination requires to determine the nutritional requirements, which involves modifications of organic salt concentrations and the addition of organic compounds. A The use of growth regulators is to stimulate germination of seeds and further development, should be studied in the orchid asymbiotic germination.11 Studies on growth regulators in relation to asymbiotic germination are scarce and the role of auxins and cytokinins during development and germination is unclear.12

Particularly, the information on the protocols on the germination requirements in seeds of B. purpurea is scarce.3 The objective of this study

is to establish a disinfection and germination protocol in B. purpurea with the interaction of factors such as the basal medium Murashige and Skoog (MS),13 Benzylaminopurine (BAP) and Activated Charcoal (CA) in the medium of culture.

MATERIALS AND METHODS

Sourse capsule and disinfection

The mature capsules come from plants collected in the state of Campeche, Mexico. The capsules were stored under refrigeration at 4 °C in darkness for their conservation until the time of planting. Seed were disinfection under aseptic conditions as the first step, the capsule was submerged in 5% Benzalkonium chloride solution for 20 minutes and rinsed with distilled water. Then it was immersed in 96% ethanol for one minute and rinsed 3 times with distilled water. Finally, it was treated at a concentration of 5% with sodium hypochlorite solution (commercial bleach) for 20 minutes and rinsed as many times as necessary to eliminate the smell of sodium hypochlorite.

The culture medium was sterilized in an autoclave at 121 °C and was distributed in 30 ml the glass flasks. The disinfection and seeding of the seeds was carried out in a laminar flow chamber. After the disinfection, the capsule was passed near the flame and in a petri dish and with a scalpel they were extracted the seeds from capsule and placed in solution of 30 ml of sterilized distilled water. To homogenize the seed volume by each flasks with medium, a sterile 3 ml syringe was used. For this, aliquot of 0.3 ml of seeds was taken and immediately inoculated into the culture medium. Immediately each flask was covered and covered with PVC film and were incubated under controlled conditions (25 °C, sixteen hours light photoperiod and eight hours dark).



8

Experimental design

All the treatments contained the MS medium, 2.2 (w/v) gelrite and 3% sucrose. The following three components were evaluated: full intensity Murashige and Skoog11 medium (MS), half-strength MS medium (/ MS) and concentration of Benzylaminopurine (BAP 0.1, 0.5 mg, L1),

Activated charcoal (AC 2.2% (w / v)). The MS medium was used as a control, with 100% concentration and /, having a total of 12 treatments (Table 1). Three replicates were used for each germination treatment, each replica was represented by a culture flask, for a total of 48.

Table 1. Effect of the concentration of BAP, the MS medium and AC in the germination of B. purpurea seeds.

After four months, the development effect on germinated seeds was evaluated. The measurement was carried out qualitatively using a scale of response phases (Table 2) since, when extracted from the culture medium, they are vulnerable to the contamination of microorganisms.

Table 2. Scale used to evaluate the response phases of seed development of B. purpurea (adapted by Stewart and Zettler).19

RESULTS

Effect of the method of disinfection in treatments.

No contamination was registered in the planting medium in any of the disinfection treatments or in seeds.

Effect of MS basal medium, BAP and AC in the seed germination.

The effect of the basal medium of MS, BAP and AC medium was different in the germination treatments. Germination was observed 19 days after planting in treatments 2, 11 and 12. The other treatments showed stage 0, that is, with seeds of ungerminated embryos. The size of the seeds in B. purpurea is approximately 1 pm (Figure 1).

Development after germination asymbiotic of seed

Differences were observed in the three treatments in relation to the development of the seedling after germination. This result is evident in comparing leaf and root development in Figure 2. The control treatment 12 (Figure 2a) showed a development phase 5 under the concentration of MS in the middle without growth regulator and AC. The development of a seedling with more than two leaves and eight long and thin roots was observed. Treatment 2 (Figure 2b) that contained a concentration of BAP 0.5 mg. L_1 and complete MS without AC showed the development of leaf shoots without roots. The treatment 11 (Figure 2c) showed the development of a seedling in phase 5, with the leaves rolled down and with necrotic parts, as well as four short and thin roots showed the development of leaf shoots without roots. The treatment 11 (Figure 2) showed the development of a seedling in phase 5, with the leaves rolled down and with necrotic parts, as well as four short and thin roots.

Treatments BAP mg/L MS (%) AC (%)1 0.5 100 2.22 100 0.03 50 2.24 50 0.05 0.1 100 2.26 100 0.07 50 2.28 50 0.09 0.0 100 2.210 100 0.011 50 2.212 50 0.0

Phase Description0 Seeds with ungerminated embryo1 Embryo expansion, rupture of the testa (= germination)2 Appearance of protocormo and rhizoids3 Emergency and appearance of the first leaf4 A leaf and appearance of roots5 Presence of two or more leaves, root present (= seedling)



9

Figure 1. Seeds of B. purpurea. (a) Seed seen on the stereoscope (bar scale 1 pm). (b) Seed seen under a 10 X optical microscope. The left arrow indicates seed coat (b); The right arrow indicates seed embryo (b).

Figure 2. Response of treatments in the development of B. purpurea seeds. (a) Seedling developed in 4 MS in which the growth of more than two leaves and several long roots is shown. (b) Foliar sprouts without root in full strength MS medium| added with 0.5 mg. L BAP. (c) Seedling developed with leaves and root in 4 MS added with AC; Arrow indicates multiplication of leaves (b); necrotic parts of the leaves (c).

DISCUSSION

The disinfection method provided pathogen-free seed culture media. Another successful method of disinfection in mature capsules of B. purpurea has been in the solution containing 100% ethanol: 6.0% NaOCl: sterile deionized distilled (dd) water (5: 5: 90) for 1 min.3 Although reports of disinfection methods in this orchid are few, mature capsules have not shown difficulty in decontamination. The difference of the method of disinfection reported and the method used in this work, is that the benzalkonium solution was used first, different concentrations of each solution were used and the exposure time was higher because the capsule used was already open and therefore, it is more exposed to pathogen contamination.

The result in seed germination response was phase 0 in the rest treatments at four months after planting. Unlike what was reported by Dutra and collaborators,3 in which a rapid germination was obtained in two week in the MS medium at half of its concentration with 0.1% of AC and without growth regulators. Other species such as Bletia catenulata germinated successfully 45 days after planting in medium Knudson supplemented with vitamins and AC.14 The ½ MS of treatment control was the first to germinate and develop a seedling with complete foliar and roots. Previous studies have shown that seeds of B. purpurea germinate easily in the medium ½ MS even without sucrose % This is the first report where the BAP was added to the medium for the germination of B. purpurea seeds and had a different effect to that of the other two treatments that germinated. Although there are few studies of this cytokinin in seed germination, it has been reported that the high concentrations of BAP have stimulated the asymbiotic germination in the orchid Ophrys apifera in comparison with other phytoregulators.11

The use of other growth regulators in vitro germination have been documented in Cymbidium dayanum Reichb,16 and in the Calanthe genus.5 Regarding the treatment with AC, a positive germination result was obtained. The positive effect of CA on the in vitro germination of terrestrial orchids of the genus Disa.17 This is attributed to the fact that the addition of the AC can inhibit or absorb substances from the culture medium and in some cases the stimulation in the germination of orchid seeds % After the germination it was observed that the leaves were rolled and with necrotic parts, and the roots and that this could be caused by the activated charcoal that may be retaining some nutrient for the normal development of the leaves.



10

CONCLUSIONS

The use of 5% of benzalkonium, 96% of ethanol and 5% of commercial sodium hypochlorite allowed maintaining 0% contamination in all treatments, which is an alternative to a method of disinfecting mature capsules in this species. Factors such as MS in different concentrations, either alone or with the addition of BAP and AC have positive effects on seed germination and post-germination on the formation of leaves and the root system of the seedlings. The use of BAP at 0.5 mg. L stimulated the multiplication of leaf shoots and was documented for the first time in seeds of B. purpurea. The half MS at its concentration without organic supplements had the best effect on the germination response and on the development of a complete seedling, being an adequate culture medium to propagate plants in vitro and contribute to the conservation of the germplasm of this threatened species.


The financial support to Consejo Nacional de Ciencia y Tecnología for the scholarship No.612022 and Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C with the project RED INTERNA REMMBEVA.

REFERENCES

1) Sosa, V. 2007. A Molecular and Morphological Phylogenetic Study of S u b t r i b e Bletiinae (Epidendreae, Orchidaceae). Systematic botany 32 (1): 34-42. doi: 10.1600/036364407780360175

2) Nino, M. S., & Losada, M. S. 2011. A new natural hybrid, Bletia xekmanii (Orchidaceae), from Cuba. Willdenowia 41 (1): 107-11. doi: 10.3372/wi.41.41112

3) Dutra, D., Johnson, T. R., Kauth, P. J., Stewart, S L., Kane, M. E., & Richardson, L. 2008.

Asymbiotic seed germination, in vitro seedling development, and greenhouse acclimatization of the threatened. Plant Cell Tiss Organ Cult. 94:11-21. doi:10.1007/s11240-008-9382-0

4) Palestina, R. A. y Sosa, V. 2012. Morphological Variation in Populations of Bletia purpurea (Orchidaceae) and Description of the New Species B. riparia Sosa. Springer on Behalf of the New York Botanical Garden 54: 99-11. doi: 10.1663/0007- 196X(2002)05410099: MVIPOB12.0.CO:2

5) Godo, T., Komori, M., Nakaoki, E., Yukawa, T., In, S., Cellular, V., June, N. M. 2010. Germination of mature seeds of Calanthe incannata Lindi. an endangered terrestrial orchid, by asymbiotic culture in vitro. In Vitro Cell Dev Biol Plant. 46: 323-328. doi: 10.1007/s11627-009-9271-1

6) Rasmussen, H. N., Dixon, K. W., Jersakova, J., & Tesitelova, T. 2015. Germination and seedling establishment in orchids: A complex of requirements.Ann Bot 116 (3):391-402. doi: 10.1093/aob/mcv087

7) Vasudevan, R., & Van Staden, J. 2010. In vitro asymbiotic seed germination and seedling growth of Ansellia africana Lindl. Sci Hortic (Amsterdam) 123 (4):496-504. doi: 10.1016/i.scienta.2009.11.010

8) Sasamori, M. H., Endres Junior, D., & Droste, A. 2015. Asymbiotic culture of Cattleya intermedia Graham (Orchidaceae): the influence of macronutrient salts and sucrose concentrations on survival and development of plantlets. Acta Bot Brasilica 29(3): 2 9 2 - 2 9 8 . doi:10.1590/0102- 33062014abb0054.

9) Billard, C. E., Dalzotto, C. A., & Lallana, V. H. 2014. Desinfeccion y siembra asimbiotica de semillas de dos especies y una variedad de orquideas del genero Oncidium. Polibotanica 38: 145-157. ISSN: 1405-2768

10) Nongdam, P., & Tikendra, L. 2014. Establishment of an efficient in vitro regeneration protocol for rapid and mass propagation of Dendrobium chrysotoxum lindl. Using seed



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culture. ScientificWorldJournal 2014:1- 8. doi: 10.1155/2014/740150

11) Pierce, S., Guidi, V., Ferrario, A., Ceriani, R. M., Labra, M., Vagge, I., & Cerabolini, B. E. L. 2015. Pea seed extracts stimulate germination of the terrestrial orchid Ophrys apifera Huds. during a habitat restoration project. Plant Biosystems, 149(1): 54-60. doi:10.1080/11263504.2013.809814

12) Yeung, E. C. 2017. A perspective on orchid seed and protocorm development. Botanical Studies, 58(33): 2-14. doi.10.1186/s40529-017-0188-4

13) Murashige, T., y F. Skoog. 1962. “A revised medium for rapid growth and bioassays with tobacco tissue culture”. Physiol Plant. 15: 473-497. doi: 10.1111/i.1399-3054.1962.tb08052.x

14) Borges, V., Neto, D. P., Paula, A., Correa, M., Roxana, D., Padilha, C., Zuffo, R. 2015. The Bletia catenulata ornamental orchid is self-compatible but pollinator-dependent for reproduction. Pesqui Agropecu Trop 45 (4): 473-479. ISSN: 1517-6398

15) Johnson, T. R., Kane, M. E., & Perez, H. E. 2011. Examining the interaction of light, nutrients and carbohydrates on seed germination and early seedling development of Bletia purpurea (Orchidaceae). Plant Growth Regul 63 (1): 89-99. doi:10.1007/s10725-010-9516-3

16) Chongtham, P. N. and N. 2012. In vitro Seed Germination and Mass Propagation of Cymbidium dayanum Reichb.: An Important Ornamental Orchid of North-East India. Trends in Horticultural Research 2: 28-37. doi: 10.3923/thr.2012.28.37

17) Thompson, D. I., Edwards, T. J., & Van Staden, J. 2007. A novel dual-phase culture medium promotes germination and seedling establishment from immature embryos in South African Disa (Orchidaceae) species. Plant Growth Regulation, 53(3):163-171. doi.10.1007/s10725-007-9215-x

18) Thomas, T. D. 2008. The role of activated charcoal in plant tissue culture. Biotechnol Adv 26 (6):618-

63. doi:10.1016/i.biotechadv.2008.08.00319) Stewart, S. L., & Zettler, L. W. 2002. Symbiotic

germination of three semi-aquatic rein orchids (Habenaria repens, H. quinquiseta, H. macroceratitis) from Florida. Aquatic Botany, 72(1): 25-35. doi. 10.1016/S0304-3770(01)00214-5


Copyright ©2019 COLEGIO MEXICANO DE INGENIEROS BIOQUIMICOS 12

Research article

Pollen description of melliferous flora with ornamental and medicinal potential in YucatanBriceño-Santiago Cinthia1, Cano-Sosa Julia*1, Andrada Ana 2, Ramos-Díaz Ana Luisa1, Noriega-Trejo Rodolfo3

1 Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C (CIATEJ SURESTE). Km 5.5 Carretera Sierra Papacal-Chuburná Puerto, Sierra Papacal Mérida, Yucatán, México.2 Departamento de Agronomía, Universidad Nacional del Sur. Altos del Palihue, (8000) Bahía Blanca, Argentina. 3 Centro de estudios de Desarrollo Sustentable y aprovechamiento de la vida silvestre-Universidad Autónoma de Campeche. Avenida Héroe de Nacozari #480, C.P. 24079, San Francisco de Campeche, Campeche, México.

*Corresponding author: [email protected]

Abstract. The physicochemical and sensorial characteristics of honey depend on the flora that bees visit, that is why it is necessary to generate all possible knowledge about the flora of each region. In the present study, a collection of plant material was made in the surroundings of apiaries visited in localities of the state of Yucatan, Mexico. Afterwards, a palynological analysis was carried out, the pollen morphology of the species was described, and the pollen material was included in a palinotheque that will be housed in the southeast unit of CIATEJ. Twelve plants were identified at the species level, each with a melliferous, ornamental and medicinal value. Four species were endemic, two native and six species among cultivated, introduced and wild. The families that were identified in the study were Asteraceae, Bignoniaceae, Boraginaceae, Fabaceae, Cactacea, Convolvulaceae, Malpighiaceae, Rutaceae, Urticaceae and Verbenaceae. Based on the aperture of the pollen, five pollen types and seven types of ornamentation were observed. The representative species of the study was Piscidia piscipula, it is native to the Yucatan Peninsula and is important in beekeeping in the region. The results obtained in this research are part of the characterization of the collected plant material and will allow associating it with honeys produced in Yucatan and its main medicinal properties.

Keywords. Melliferous flora, palynological analysis, pollen morphology.

Resumen. Las características fisicoquímicas y sensoriales de la miel dependen de la flora que las abejas visitan, por lo cual es necesario generar todo el conocimiento posible sobre la flora de cada región. En el presente estudio se realizó colecta de material vegetal, en los alrededores de apiarios visitados en las localidades del estado de Yucatán, México. Posteriormente se realizó análisis palinológico, se describió la morfologia del polen de las especies, y el material polínico fue incluido en una palinoteca que estará albergada en la unidad sureste del CIATEJ. Se identificaron doce plantas a nivel de especie, cada una con un valor melífero, ornamental y medicinal. Cuatro especies fueron endémicas, una nativa y seis especies entre cultivadas, introducidas y silvestres. Las familias que se identificaron en el estudio fueron, Asteraceae, Bignoniaceae, Boraginaceae, Fabaceae, Cactacea, Convolvulaceae, Malpighiaceae, Rutaceae, Urticaceae y Verbenaceae. Con base en las aberturas del polen se observaron cinco tipos polínicos, y siete tipos de ornamentación. La especie representativa del estudio fue Piscidia piscipula, es nativa de la Península de Yucatán e importante en la apicultura de la región. Los resultados obtenidos en esta investigación forman parte de la caracterización realizada al material vegetal colectado y permitirán asociarlo a mieles producidas en Yucatán y a sus principales propiedades medicinales.

Palabras clave. Flora melífera, análisis palinológico, morfología del polen.

Received: 03 may 2018/ Accepted: 07 january 2019/ Published online: 25 january 2019



Received: 03 may 2018/ Accepted: 07 january 2019/ Published online: 25 january 2019

INTRODUCTION

Angiosperms, flowering plants, the most diverse group of the Yucatan Peninsula, is represented by 2, 262 taxa in species or lower levels distributed in 922 genera and 144 families of which 119 endemic child species.1 This heterogeneity is manifested in the detailed Mayan knowledge of plants, and that man has adopted for different uses (medicine, food, apiculture activity and construction).2,3 Melliferous plants are important for beekeeping and contribute to the physicochemical and sensory characteristics of honey.4 Until now, around 600 species that visit the bees have been reported for the collection of pollen and / or nectar, necessary for their feeding. However, ethnobotanical studies report some 1800 species of which are considered of great interest for the production of honey.5 Within this variety of species, some are of ornamental use for their striking colors or aromas that attract bees, and others contribute to the honey’s phenolic compounds, among which are the flavonoids that are of interest for human health.6

Considering that one of the regions with the highest honey production is the state of Yucatan, contributing in the year 2017 a volume of 7,490 tons per year, it is important to have accurate information about the melliferous flora that can contribute with better management of the apiaries.7 Currently, the science of palynology is used as a tool to know the relationship between bees and the flora of the region. This discipline is based on the recognition of pollen through its morphology (shape, size, symmetry, polarity, germinal openings and sporodermis, characteristics that allow to identify its botanical origin and thus attribute it to a family, gender or species. Subsequently they are annexed to a reference palinoteca, which is used as a consultation to determine the pollen present in honey.8 The aim of the present study is to describe the pollen of the honey flora with ornamental and medicinal potential in the apiaries of the state of Yucatan.


Fieldwork

The selection of specimens was made in the surroundings of the apiaries of three localities belonging to the municipality of Mérida, Yucatán (Xcunyá, Xmatkuil, and Tamanché) in the months of may, jun and july corresponding to the rainy season of the year 2017. It was observed that the plants were in the flowering stage and specimens with closed flowers were collected. The flower buds were put in paper envelopes and taken to the facilities of CIATEJ SOUTHEAST for analysis in the laboratory; all collections were duly labeled with date, number, location, and name of the species. Photographs were taken of the plants with a NIKON camera model P900. For the selection of plants that could have a melliferous, medicinal and/or ornamental use, the available bibliography was consulted and the popular local knowledge of the chosen species was considered.

Laboratory work

The sheltered plants were analyzed by a microscope to identify the parts of the anthers, which is where the pollen is found. All the anthers of the buds were collected in their entirety, and deposited in Eppendorf tubes previously labeled with the collection number and each one is added five drops of glacial acetic acid to dehydrate the sample.9 The material was squeezed so that the pollen is exposed, and then filtering was carried out by placing the pollen residue in graduated 10 ml conical glass tubes gauging up to 10 ml with acetic acid. Subsequently, the steps were followed according to the methodology of the acetolysis technique (Erdtman, 1992 10) to eliminate the cellular content of pollen and intine allowing a detailed analysis of the sporodermis. For the assembly of pollen part of the residue was taken with a dissection needle in which previously a small piece was pricked of



glycerin-gelatin and placed on a slide. It warmed gently on the flame and was homogenized with a needle, then place the paraffin in the four corners and support the cover-objects. It was heated again over the flames to seal the preparation. Finally, the assemblies were labeled, for which three plates were prepared per species: two were sheltered for the reference palinotheque in the CIATEJ SOUTHEAST and a plaque was incorporated into the LabEA, Department of Agronomy - UNS. They observed and took pictures of the pollen grains with a Microscope Leica DM 500 with built-in camera (ICC50E), with software LEICA Application Suite (LAS) EZ; they were compared and corroborated the morphological description of pollen with bibliography corresponding to other authors.11, 12, 13

RESULTS

During the visits to the apiaries, 12 specimens belonging to 10 families were collected. All species presented were identified as important for their medicinal, ornamental, melliferous value, and their distribution (Table 1 and 2).

Table 1. List of species identified in the three apiaries (Xcunyá, Xmatkuil, and Tamanché) visited in the months of may, jun and july 2017 respectively.

*Distribution (Dtb): C=Cultivated; N=Native; E=Endemic;

I=Introduced; W= Wild.

Table 2. Medicinal properties of the species identified in the three apiaries (Xcunyá, Xmatkuil and Tamanché) visited in the months of may, june and july 2017 respectively.

The image recording determined the morphological variation of the twelve pollen types (Figure 1). According to the size, 41.7% of the species presented a large pollen, 33.3% a small pollen and 25% were known medium. Five types of aperture were observed: tricolporated, tricolpate, periporate, pantoporated, and diporate. The most variable pollen characteristic is the ornamentation, seven types were registered: equine and granulated, reticulated, microreticulated, subequine, striate, scrabate, and verrucate.25 In Table 3, the pollen morphology of each species is presented.

Family Species *Dtb

Asteraceae Tithonia diversifolia (Hemsl.) A. Gray C

Bignoniaceae Tecoma stans (L.) Juss. Ex Kunth N

Boraginaceae Cordia dodecandra A. DC. N

Cactaceae Nopalea gaumeri Britton & Rose E

Convolvulaceae Ipomoea carnea Jacq. I

Leguminosae

(Fabaceae)

Bauhinia divaricata L. N

Cassia fistula L. I

Piscidia piscipula (L.) Sarg N

Malpighiaceae Byrsonimia crassifolia (L.) Kunth C

Rutaceae Murraya paniculata (L) Jack I

Urticaceae Cecropia peltata L. W

Verbenaceae Stachytarpheta jamaicensis (L.) Vahl I

Species Medicinal properties

T.diversifoliaAntidiabetic -Anti-inflammatory

Analgesic-Anticarcinogenic14, 15

T. stans

Diuretic -Antiepasmolytic

Anthelmintic-Anti-inflammatory

Antidiabetic .16, 17

C. dodecandra Respiratory diseases -Antibacterial. 18

I. carneaAnti-inflammatory -Antioxidant

Antidiabetic -Antimicrobial 19

B. divaricataRespiratory diseases -Disinfectants

Anti-inflammatory-Kidney problems.20

C. fistula

Antitussive-Anti-inflammatory

Antitussive-Antifungal

Antibacterial activity.21

P. piscipulaAnalgesic-Antispasmodic,

Antitussive 22

B. crassifolia

Antipyretic -Stringent properties,

Kidney diseases

Anti-inflammatory of the ovaries.18

M. paniculataAstringent-Analgesic,

Anti-inflammatory properties. 23

C. peltataAntidiabetic-bladder inflammation,

Bone fractures 18

S. jamaicensis

Antiacid –Analgesic-Anti-helmithicAnti-inflammatory-Diuretic-Hypotensive-Laxa-tive, Purgative-Diseases respiratory 24



Figure 1. Registration of pollen images of melliferous species with ornamental and medicinal use. a) Polar view of T. diversifolia; b) Equatorial view of T. diversifolia; c) Polar view of T. stans; d) Equatorial view of T, stans; e) Polar view of C, dodecandra; f) Equatorial view of C, dodecandra; g) Equatorial view of N. gaumeri; h) Polar view of I. carnea; i) Equatorial view of B. divaricata; j) Polar view of B. divaricata; k) Polar view of C. fistula; l) Equatorial view of C. fistula; m) Polar view of P. piscipula; n) Equatorial view of P. piscipula; ñ) Polar view of B. crassifolia; o) Equatorial view of B. crassifolia; p) Polar view of M. paniculata: q) Equatorial view of M. paniculata; r) Polar view of C. peltata; s) Polar view of S. jamaicensis.

The morphology description of the pollen (Table 3) of the species analyzed in the present work was similar to that reported by other authors.11, 12, 13

Table 3. Description of the pollen morphology of the collected species of melliferous, ornamental and medicinal use according to the microscopy analysis

Orn: Ornamentation; M: medium; L: large; S: small; TCPD: tricolporate; TCPT: tricolpate; PRPT: periporate; PNPD: pantoporated; DIPT: diporate; SPHE: spheroidal; SUBP: subprolate; PROL: prolate; EQUI: equine; GRAN: granulated; RETI: reticulated; MRET: microreticulate; SUBE: subequine; STRI: striate; SCRA: scrabate; VERT: verrucate.

DISCUSSION

In the present work, some of the collected species in the apiaries of the state of Yucatán are identified as honey species; it was corroborated with other authors.26,27,28,11,12,13 An exception was the species Nopalea gaumeri and Stachytarpheta jamaicensis, which were not reported as meliferas at least in the Yucatan Peninsula. However, it has been reported with ornamental and medicinal use, respectively.24, 29

This information is important because it is the first work that studies the flora related to the apiaries of the state of Yucatan, and its contribution either a mellifera species or species with an ornamental potential and medicinal use that can be used in the apiaries. Asteraceae is a family that predominates in the botanical classifications of honeys from the Yucatan Peninsula, providing nectar and pollen. The species T. diversifolia belonging to the family Asteraceae has been reported as a

Species Size Aperture Form Orn

T. diversifolia M TCPD SPHEEQUI

GRAN

T. stans M TCPT SUBP RETIC. dodecandra L TCPD SPHE RETIN. gaumeri L PRPT SPHE MRETI. carnea L PNPD SPHE SUBEB. divaricata M TCPD SPHE STRIC. fistula L TCPD SUBP MRETP. piscipula S TCPD SUBP SCRAB. crassifolia S TCPD PROL MRETM. paniculata S TCPD PROL MRETC. peltata S DIPT PROL SCRAS. jamaicensis L TCPT PROL VERT



honeybee 30 (however in the melisopalinological analysis of the state of Yucatan this species has not been reported.11, 12, 28

The family of the Fabaceae has been reported as important in the production of honey because it provides both nectar and pollen to bees. Two of these species are C. fistula and P. piscipula 12. In the monofloral honeys of the Yucatan peninsula, Piscidia piscipula pollen has been found with high percentages.28 The T. stans species belongs to the Bignoniaceae family that has been reported to receive visits from the bees for their collection of nectar and pollen.31 Within the Boraginaceas is found in the species C. dodecandra reported in the samples of honey in the Yucatan peninsula with a frequency of 0.55%.28 The family Convolvulaceae have been reported of minor importance in apiculture; however, in the Yucatán Peninsula presents flowering throughout the year that provides pollen and nectar bees, as well as the maintenance of hives throughout the cycle of the apicola, providing pollen when not much available flora is found.32,12 Another species from the family Malphighiaceae is B. crassifolia and from the family Rutaceae the species M. paniculata whose contribution to the feeding of bees are nectar and pollen.11 In the sample of honey collected in the Yucatan Peninsula, the species Cecropia peltata presents high percentages, which is why it is considered a species with pollinifera value.28 Some species of meliferous use are considered ornamental due to certain characteristics that attract bees, generally due to their striking colors, aromas or the vailability of nectar and pollen in the plant. Some analyzed species such as C. dodecandra, C. peltata, B. crassifolia, T. stans and M. paniculata, in addition to be feeding option for bees, there are ornamental trees that are recommended in the cities to decorate streets and sidewalks.33 Within the Convolvulaceae family the Ipomea genus are used as an ornamental

for their striking flower colors.34 Some species grow wild but have been adapted as ornamental plants within them is the T. diversifolia species belonging to the family Asteraceae. S. jamaicensis has been mentioned as having an ornamental utility in parks and gardens.35 It has been found that some species of meliferous importance have phenolic compounds such as flavonoids that have great antioxidant activity which is of interest in human health. There is one study that evaluated the flavonoids of Viguiera dentata and Gymnopodium floribundum, which are the most influential species in the production of honey in Yucatan. However, the information available for Yucatan species is scarce, and there is a variety of melliferous plants.6 In this work, it was identified that melliferous plants from apiaries of three localities belonging to Yucatán have a contribution to traditional medicine has been reported for respiratory diseases such as bronchitis, cough and asthma to the species T. diversifolia, B. divaricata and P. piscipula. The species T. stans, I. carnea and C. peltata have been reported for diabetes. The species N. gaumeri, S. jamaicensis and B. crassifolia have been used for diseases of the digestive system, while M. paniculata has been used for disorders of the nervous system.36, 30

The description of the morphology coincides with that reported by other authors.11,12,13 It is worth mentioning that the results presented in this work was limited to qualitative descriptors.

CONCLUSIONS

Twelve species represented by the family were identified Asteraceae, Bignoniaceae, Boraginaceae, Fabaceae, Cactaceae, Convolvulaceae, Malpighiaceae, Rutaceae, Urticaceae and Verbenaceae. Two species Nopalea gaumeri and Stachytarpheta jamaicensis they were collected in the apiaries



and have not been reported as melifera species. All species had an ornamental value, while only eleven were species reported as medicinal. Four of which are native to the region T. stans, C. dodecandra, B. divaricata, P. piscipula. The representative species of the study was Piscidia piscipula, native to the Peninsula, important in beekeeping in the region. It was determined that the pollen of the species was very varied in size and morphology. The twelve species described were annexed to the CIATEJ palinoteca. All the species independently that were reported as meliferous and medicinal, have an ornamental use. This information is considered relevant to emphasize the importance of the species in the ornamental production sector.


Consejo Nacional de Ciencia y Tecnología (CONACYT) for the grant awarded N° 453588Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco A.C (CIATEJ).Department of Agronomy, National University of the South, Bahia Blanca, Argentina Project Organic HoneyCompany Plenumsoft

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23) Casado Martín C. M, Gutiérrez Gaitén Y. I, Rodríguez Amado E. 2011. Acercamiento al género Murraya (Rutaceae) y a la especie Murraya paniculata (L.) Jack. Revista Cubana de Plantas Medicinales, 16(4), 408-418. ISSN 1028-4796.

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25) Saenz de Rivas, C. 1978. Polen y esporas: Introducción a la palinología y vocabulario palinológico (No. Sirsi) a97207).

26) Villanueva G. R. 1999. Pollen resources used by European and Africanized honey bees in the Yucatán Peninsula, Mexico. Journal of apicultural research, 38(1-2) ,105-111. https://doi.org/10.1080/00218839.1999.11101001.

27) Porter Bolland .2003. La apicultura y el paisaje maya. Estudio sobre la fenología de floración de las especies melíferas y su relación con el ciclo apícola en La Montaña, Campeche, México. Mexican Studies, 19(2), 303-330. DOI: 10.1525/msem.2003.19.2.303

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Research article

Biotransformation of progesterone by Cunninghamella elegans H260 as an effect of detoxification

Rangel Mejía Virdiana1,2, Rodríguez Casasola Felipe Neri3, Leal Ríos Elvira1, Rodríguez Casasola Ma. Teresa1, Cruz Mondragón Carlos1, Vázquez Ulloa Elenaé2*, Esparza García Fernando José1**

1 Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional (CINVESTAV). Av. Instituto Politécnico Nacional 2508, Gustavo A. Madero, San Pedro Zacatenco, 07360 Ciudad de México. 2 Tecnológico Nacional de México, Instituto Tecnológico de Gustavo A. Madero. Av. 608 y Av. 412 No. 300 Col. San Juan de Aragón. Delegación Gustavo A. Madero, 07470, Ciudad de México.3 Instituto Politécnico Nacional, Escuela Nacional de Ciencias Biológicas. Prolongación de Carpio y Plan de Ayala s/n, Miguel Hidalgo, Santo Tomás, 11340 Ciudad de México, CDMX.

*Corresponding author:*[email protected], **[email protected]

Received: 25 april 2018/ Accepted: 14 january 2019/ Published online: 25 january 2019

Abstract. Emerging contaminants are compounds of different origin and chemical nature, including steroidal hormones, which represent a problem in wastewater treatment. Bioremediation seems to be a good option for elimination of these contaminants. In the present work we propose the use of zygomycete fungus Cunninghamella elegans to treat these hazardous waste. Progesterone inhibits the growth (radial and dry weight) of Cunninghamella elegans, but even though the fungus is capable of biotransform the progesterone in by-products with greater polarity as we demonstrated in thin-layer chromatography (TLC). The biotransformation occurs in the first 8 hours of fermentation obtaining good yields in the treatment of this pollutant.

Keywords. Emerging contaminants, Steroid hormones, Zygomycete fungi.

Resumen. Los contaminantes emergentes son compuestos de diferente origen y naturaleza química, incluidas las hormonas esteroides, que representan un problema en el tratamiento de aguas residuales. La biorremediación se presenta como una buena opción para la eliminación de estos contaminantes. En el presente trabajo, se propone el uso del hongo zigomiceto Cunninghamella elegans para tratar estos residuos peligrosos. La progesterona inhibe el crecimiento tanto radial como por peso seco de Cunninghamella elegans, sin embargo, el hongo es capaz de biotransformar a la progesterona en subproductos de mayor polaridad y menos tóxicos, tal como lo demuestran los resultados de cromatografía en capa fina. La biotransformación ocurre a las 8 horas tras empezar la fermentación con lo que se obtiene buen rendimiento en el tratamiento de este contaminante.

Palabras Clave. Contaminantes emergentes, hormonas esteroideas, hongos cigomicetos.



INTRODUCTION

Emerging contaminants are a group of natural or synthetic compounds,1 that can enter to the environment through sources such as wastewaters, such as domestic or industrial, waste from treatment plants, hospital effluents, agricultural, livestock activities and septic tanks, beside others.2

A wide range of pharmaceutical products has been detected in surface and groundwater, associated with the disposal of wastewater.2 These pharmaceutical waste are transported to the water cycle through different routes: the wastewater treatment plants act as a gateway for these products to water bodies, but sometimes these compounds are not really retained in their processes.3

The presence of pharmaceutical chemicals in the aquatic environment has been recognized as a concern.4 The main entrance of pharmaceutical products to the environment are through human excretion, because the elimination of unused products.5

This emerging pollutants lack of control in the environment and legal regulation, so they have an unknown impact on ecosystems and human health.4

Emerging contaminants include steroid hormones such as progesterone.1, 2 The presence of hormones, particularly progesterone, in wastewater has been associated with feminization and masculinization of aquatic organisms,7 alteration of circadian rhythms in fish6 and with impact on fertility and as endocrine disruptors.5 The elimination of this compound is difficult since it persists in conventional wastewater treatments,6 so new technologies are being investigated.

The biological treatment of wastewater seems to be an ecological alternative for the degradation of hormones in non-toxic byproducts.3 The zygomycete fungi of the Cunninghamella genus, from which seven species has been recognized, are filamentous microorganisms isolated from soil, animal and plant materials. Especially in Mediterranean and subtropical zones, it is a common opportunistic contaminant fungus.5

Cunninghamella have been used as a study model for drug metabolism since they are capable of mimicking the toxic biotransformation in mammals. It has been shown that Cunninghamella has enzymes involved in xenobiotic biotransformation.8 In fact, the monooxygenase activity, capable of oxidizing aromatic compounds by incorporating oxygen atoms in the aromatic rings, has already been demonstrated for some species of Cunninghamella.9 This activity has been used in the synthesis of various products, for example quinolone derivatives, used for malaria, tuberculosis and pain relief; can be hydroxylated by Cunninghamella spp.10 This fungal genera, specifically C. elegans have also been used for the study of the metabolism of the antidepressant drug mirtazapine,11 the respiratory mucus-normalizer bromhexine,12 among other therapeutic compounds.

In the present work, we used the Cunninghamella elegans fungus for the biotransformation of progesterone in more polar by-products, presumably hidroxilated. The results presented here, support the use of this kind of organisms for wastewater treatment.


Strain

The strain of Cunninghamella elegans H260 was obtained from the bank of strains of the Centro de Investigacion y de Estudios Avanzados del Instituto Politecnico Nacional (CINVESTAV) of the Microbial Collection Carlos Casas Campillo C.C.B.B.



Stock of spores

The stock of spores was obtained cultivating the fungi and scrapping the plate surface with 15ml of distillated water with the help of a scraper. The stock was preserved at 4°C until its use.

Spores counting

The number of spores was determined by Neubauer chamber (Improve brand) counting. Each experiment was carried out with approximately 2.75x10-6 esp/ml.

Progesterone resuspension

The concentration of progesterone used in all the experiments was 80mg/L in ethanol.15

Colony Morphology evaluation

Fungus spores were seeded in YPG medium, ethanol and progesterone. After 48 h of incubation, colony morphology was described taking in consideration color, shape and texture of each colony.

Cellular Morphology evaluation

Fungus spores were seeded in YPG, ethanol and progesterone culture. After 48 h of incubation, a steril sample was took and reseeded in a micro-culture for morphology observation. Briefly, a square of 1cm per side of solid media was cut and place on a glass triangle, a sample fungus from the original culture was inoculated in the square and a coverslip was placed on the top. The culture was leaved until the fungus was visible. The coverslip was detached and stained with lactophenol cotton blue staining and watched under the microscope. The ethanol and progesterone treatment were added in to the culture.

Growth kinetics

For the evaluation of radial growth, spores were seeded in the center of the culture by puncture in the solid medium. After 24 h of incubation at 30°C, the ethanol or ethanol-progesterone mixture were added and incubated under the same conditions. Radial growth, the size of the fungus from the center to the borders of the dish, was measured at 24, 48 and 72 h and reported in cm.

Biotransformation kinetics

0.25 ml of the spore stock was inoculated into 125 ml flasks with modified YPG liquid medium and incubated for 24 hours at 30°C under stirring (150 rpm). After 24 hours, ethanol or ethanol-progesterone mixture was added to reach a concentration of 0.08 mgml-1 and the medium was incubated for another 24 hours. The samples were taken at 8, 24 and 48 hours.

Dry weight biomass determination

Progesterone was extracted by solid-liquid extraction. The biomass was dry crushed in a mortar and the extraction was carried out by micro soxhlet, using 99.9% pure chloroform as solvent. The residual progesterone and by-products were collected in vials.

A further liquid-liquid extraction was carried out in a 250 ml separating funnel, placing the supernatant of the filtration and 50 ml of 99.9% pure chloroform. Again, the residual progesterone and by-products were collected in vials. Subsequently, a total evaporation of the samples obtained from the extractions was carried out and the dry residue was dissolve in 5 mL of HPLC methanol.

By-products analysis

The by-products were analyzed by thin layer paper chromatography (TLC). An ethyl acetate/



chloroform (2:1) solution was used as mobile phase.13 The chromatogram was revealed with UV light at 254 and 364 nm wavelength.17

Densitometric image analysis

Intensity of bands were evaluated with the image J software to determine pixel bands intensity. Results from three replicates were averaged and reported as fold change intensity from the control.

Statistical analyses

In order to determine the differences in the radial growth, ANOVA test and a multiple Tuke’s comparision test were performed. Meanwhile, biomass dry weight difference and sub-products generation was determined by Student T test. The former was done in the GrahPad Prism 5 software.

RESULTS

In the present work, we show the biotransformation of progesterone by the zygomicetus fungus Cunninghamella elegans as an effect of detoxification. First we evaluated the growth and morphology of C. elegans in the presence of progesterone. Figure 1 shows the growth of C. elegans in PDA medium (Figure 1A), ethanol (diluent control) (Figure 1B) and progesterone (Figure 1C). Colonies were white and cotton-like. In PDA medium, the colony cover the whole plate after 72 h of incubation, meanwhile a radial growth diminish was observed in ethanol as well as in progesterone.

Figure 2 shows the comparison of the radial growth of the fungi showed in Figure 1 as well as three repetitions, in PDA medium, ethanol and medium with progesterone. The maximum average growth, after 72 hours, was of 3.5 cm in control conditions.

The treatment with ethanol and progesterone induced an inhibition of the radial growth since the fungi decrease to an average size of 2.8 cm in ethanol and 2.4 cm in progesterone, which means a 20 % and 31.42% of inhibition, respectively.

The ANOVA test showed significative difference between the treatments (F=134.9, p=<0.001), meanwhile the Tukey’s multiple comparison test showed that there was no difference between ethanol and progesterone treatment at any time point.

Cellular morphology of C. elegans in the presence of progesterone or ethano is shown in Figure 3. The microscopic morphology in PDA medium (Figure 3A) shows erect and round sporangiophores with vesicles that contain spores. In the case of ethanol and progesterone (Figure 3B and C), after 72 hours of incubation, there is a decrease in the number of sporangiophores as well as in the thickness of the hypha, but in the case of ethanol, the decrease in the number of sporangiophores is less obvious than in the progesterone treatment.

Figure 1. Radial growth of Cunninghamella ellegans. Macroscopic morphology a) PDA medium, b) ethanol and c) progesterone. The orange line shows the points of growth measured at 24, 48 and 72 h.

Figure 2. Cunnighamella ellegans radial growth. One way ANOVA test show statistical difference (F(8,18)=134.9 p=<0.001) between PDA medium and ethanol and progesterone. The multiple comparision test statistical difference among treatments is shown with letters.



In order to confirm the observed growth inhibition, dry weight biomass was evaluated. Figure 4 shows the diminish of C. elegans biomass production in the presence of progesterone in liquid medium. There was a significant decrease in biomass production in the presence of progesterone, compared with PDA medium (p=0.0078) at all time points. As is observed, both cultures start with the same biomass that increase in the control condition, with a less obvious increase in the progesterone treatment, with an average percentage of inhibition of 50% of biomass production.

The biotransformation of progesterone at different ages of the fungus is shown in figure 5. The first two lanes show the progesterone and ethanol controls respectively. The red square shows the more polar by-products of the biotransformation (Figure 5A). Right after addition of progesterone, it penetrates to the mycelium as it can be seen in lane 3, but the absorption is not complete since some progesterone remains in the supernatant (lane 4). The biotransformation began at 8 hours since there is no progesterone neither in the mycelium nor at the supernatant, and the polar

by-product are seen (Red square lane 5). After 24 and 48, progesterone biotransformation is total and the more polar by-products increase at the mycelium (red square lanes 7 and 9), and the by-products are not excreted from the mycelium to the supernatant (lanes 8 and 10).

In order to confirm that the more polar by-products increase across the time, densitometric analysis of the image was carried out (Figure 5B). There is significant increase of the presence of the more polar by-product from 8 to 24 h as well as from 24 to 48 h (p=0.038 and p= 0.021 respectevily). It is known that progesterone remains in the mycelium until an activity of 14α oxygenase appears, according to the hydroxylation pathway of progesterone. The activity of oxygenase appears as a phenomenon of detoxification.14

|

Figure 3. Cellular morphology of Cunnighamella ellegans. A) PDA medium, B) ethanol and C) Progesterone.

Figure 5. Biotransformation of progesterone by C. elegans at different fungi ages. A) The by-product of biotransformation are marked in the red square. B) Densitometric analysis of by-production generation.

p=0.0078

Figure 4. Biomass inhibition. Comparison between the biomass production in PDA medium (blue line) and progesterone (red line).



DISCUSSION

We identified that progesterone has a toxic effect for C. elegans since it inhibits the growth of the fungus as we showed by the radial growth evaluation and provokes a change in the microscopic morphology. The fungus treated with this pollutant, exhibited a reduce number of sporangiophores which could be translated in a decrease in the spore production, thus a decrease in the reproduction of the organism. Our results are in agreement with previous works showed that progesterone can inhibit growth of the fungus Mucor rouxii.13,14,15

Even though, the fungus can recover from this condition since it continues growing, maybe because an adaptation of the fungus to the pollutant and the beginning of the biotransformation of the hormone as a detoxificant effect. The capability of Cuninghamella as a model for drug metabolism is well stablished, this metabolism is due to the expression of monooxigenase enzymes, that hydroxylate.8 Specifically, Cunninghamella fungi carry at least one gene of CYP enzimes,18 part of phase 1 metabolism reactions used in mammals metabolism, as a way to convert lipid-soluble compounds to more soluble ones.16

In fact, here we show by thin chromatography the presence of by-product originated from the biotransformation, these products are presumably less toxic since there are of greater polarity, thus more soluble.10 In order to prove that, is necessary to conduct toxicity assays with this by-product and to determine the nature of the by-product by mass spectrophotometry. As we already mention, is quite possible that the biotransformation is conducted by monooxygenase, a group of enzymes for detoxification which are induced as a co-metabolism in the presence of toxic compounds that in fact are not a carbon source to the fungi. 17,19

Is worth mention that after the penetration of progesterone to the mycelium, a toxic effects is visible in the first hours of exposition as we stated in the morphology evaluation, but the recovery is related to the time when the biotransformation begins. Also, the total of progesterone is transformed since no band is visible at 24 or 48 hours. From the progesterone hydroxylation pathway, the effect we show here is presumably due to 14α oxygenase.14 Other fungi genera, i. e Aspergillus,17 are capable of biotrasform progesterone, in the case of A. brasiliensis, after 7 days of exposition to progesterone, authors report the presence of 3 different hydroxylated metabolites, once of which corresponded to 14 αhydroxyporgesterone, and other two metabolites, that is worth mention, we wasn’t able to observed, this might be due to the time of exposition. It would be interesting to evaluated longer periods of exposition to find other by-products and also to evaluate a possible saturation on the enzyme activity.15

CONCLUSIONS

The progesterone is able to penetrate the mycelial cells of C. elegans and even it has toxic effect, the fungus is able to biotransform the pollutant. The more polar by-products, of more polarity, are excreted to the medium along with the residual progesterone. Evidence suggests that this biotransformation is due to monooxygenase enzymes as a detoxificant effect of the mycelial cells which are induced by a co-metabolism. The byproducts obtained are less toxic for living beings due to their greater polarity. Allowing them to be more soluble in water and increasing the ease of metabolizing them.

The results present here suggest that the genera Cuninghamella, specially C. elegans could be a good option for the treatment of wastewater from pharmaceutical industries.




Not funding received.

Special acknowledge to Julio Cesar Antonio Huerta and Sara Luz Vera Garcia fir their great technical support.

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