CHEMICAL ENGINEERING TRANSACTIONS VOL. 79, 2020 A publication of The Italian Association of Chemical Engineering Online at www.cetjournal.it Guest Editors: Enrico Bardone, Antonio Marzocchella, Marco Bravi Copyright © 2020, AIDIC Servizi S.r.l. ISBN 978-88-95608-77-8; ISSN 2283-9216 Toxicity in Artemia Salina by Hydroalcoholic Extracts of Monocotyledonous and Dicotyledonous Varieties of Medicinal Plants from the Peruvian Amazon Ana N. Sandoval*a, Jhonny W. Valverde Flores,b, Kriss M. Callaa, Rafael A. Albaa, Herry Llocllaa, Santos A. Soteroa Armando G. Ismiñoc, Marco L. Salazard a Universidad César Vallejo, 22201, Cacatachi, San Martín, Perú. b Universidad Nacional Agraria La Molina,15024, Lima, Perú. c Asociación de Productores Jardines de Palma, 22236, Pongo de Caynarachi, San Martin, Perú. d Universidad Nacional de Trujillo, 13006, Trujillo, Perú. asandoval@ucv.edu.pe Medicinal plants have been used since ancient times, acquiring broad interest in their healing properties due to their active compounds. The Peruvian Amazon rainforest has a great variety of flora, such as monocots (Dracontium loretense Krause and Commelina diffusa) and dicotyledons (Dysphania ambrosioides, Malva sylvestris, Origanum vulgare, Bixa orellana, Pinus edulis, Jatropha curcas L, and Brunfelsia). The study aimed to evaluate the toxicity in saline artemia by hydroalcoholic extracts of leaves of medicinal plants of the Peruvian Amazon. A phytochemical analysis of the leaves of the medicinal plants was performed to identify their active components. For the hydroalcoholic extraction, 300 g of leaves were used and dried extracts were obtained at concentrations of 10, 100 and 1000 μL/mL. The toxicity of the extract of each plant species in 10 larvae of Artemia was evaluated by triplicate tests. It was observed that chemical compounds such as steroids, triterpenes, quinones, phenolic compounds, flavonoids, lactones, alkaloids, reducing sugars, tannins and saponins, are the causes of toxicity in artemia salina, showing that the higher the concentration of the extract, the higher the index of mortality. 1. Introduction Medicinal plants have gained interest in their healing potential due to the presence of phytochemicals (Afsar et al., 2015; Bokhari and Khan, 2015). The use of plants as a therapeutic mechanism is based on popular culture. Today the consumption of synthetic medicines has grown with high costs and limited access for the population, opting for the use of medicinal plants to address diseases as part of primary health care (WHO, 2015; Teles and Costa, 2014). The use of botanical drugs and plant derivatives were valued at $ 23.2 and 24.4 billion between 2013 and 2014 respectively. This total market is expected to reach $ 25.6 billion in 2015 and almost $ 35.4 billion in 2020 (BCC, 2017) Although synthetic drugs are available in most countries and are efficient, there are still people who opt for traditional medicine. Therefore, research has shown that parts of the plant such as seeds, fruit, leaves and fruits have been used for disease control. (Moraia et al., 2019). Likewise, a phytochemical detection must be carried out to identify the bioactivity that each active compound possesses, since it will allow to identify its advantages and disadvantages. The ethnobotanical use of plants is important because it allows research and through it to discover new therapeutic alternatives, however, due to the daily consumption of plants for medicinal purposes, it is necessary to study their toxicology to avoid side effects (Ullah et al, 2014). The Peruvian Amazon rainforest has a great diversity of flora, including monocotyledons (Dracontium loretense Krause and Commelina diffusa) and dicotyledons (Dysphania ambrosioides, Malva sylvestris, Origanum vulgare, Bixa orellana, Pinus edulis, Jatropha curcas L, Brunfraia L, Brunfraia L, Brunfraia L, Brunfraia L, Brunfraia L.) (MINAM, 2019); species that are used as an option to traditional medicine necessary DOI: 10.3303/CET2079062 Paper Received: 17 July 2019; Revised: 15 December 2019; Accepted: 22 February 2020 Please cite this article as: Sandoval A., Valverde Flores J., Calla K., Alba R., Lloclla H., Sotero S., Ismino A., Salazar M., 2020, Toxicity in Artemia Salina by Hydroalcoholic Extracts of Monocotyledonous and Dicotyledonous Varieties of Medicinal Plants from the Peruvian Amazon, Chemical Engineering Transactions, 79, 367-372 DOI:10.3303/CET2079062 367 for its active compounds, such as steroids, phenolic compounds, flavonoids, terpenes, reducing sugars, lactones among others (Pereira et al, 2016). Research has shown that leaf extracts of some species are toxic for human consumption due to the combination of their active compounds (Leite et al, 2015; Paredes et al, 2018). Artemia salina is a light brown-bodied shrimp of the Crustaceae family, it has a size of 1 to 7 mm. This species is cosmopolitan and they live in salt water at a temperature of 6ºC to 35ºC. It feeds on algae and bacteria. Its physiology was studied as a preliminary test for toxicological investigations having its advantage, reliable results, low cost, easy handling, minimum requirements for laboratory manipulation, and highly sensitive to a wide variety of toxic agents; This trial being a practical, sustainable and sustainable method for evaluating the pharmacological potential of synthetic and natural compounds measured through their toxicity in plants (Ávalos et al, 2014 and Andrade et al, 2014). The toxicity of plants in Artemia salina implies only life or death, and the absence of toxicity is a starting point that biological systems can tolerate the plant (Silva et al, 2015). Therefore, the objective of the study was to evaluate the toxicity in saline artemia by hydroalcoholic extracts of medicinal plants of the Peruvian Amazon. 2. Methods 2.1 Vegetal material 500 grams of plant samples of each species (Dysphania ambrosioides, Commelina diffusa, Malva sylvestris, Dracontium loretense Krause, Origanum vulgare, Bixa orellana, Pinus edulis, Jatropha curcas L, Brunfelsia grandiflora) were used, which were collected by researchers in the Cacatachi district San Martin region, 295 m. above sea level and 12 km northern of Tarapoto (6 ° 29'40 "south latitude and 76 ° 27'57" west longitude), Peru. The samples were placed in vacuum bags that were labeled with their names at a temperature of 37 °C, subsequently taken to the Truxillense Herbarium of the National University of Trujillo for identification and deposit with a registration code for each species: Dysphania ambrosioides (COD. 59598), Commelina diffusa (COD. 59600), Malva sylvestris (COD. 59601), Dracontium loretense Krause (COD. 59602), Origanum vulgare (COD. 59604), Bixa orellana (COD. 59605), Pinus edulis (COD. 59606), Jatropha curcas L (COD. 59607), Brunfelsia grandiflora (COD. 59610). 2.2 Preparation of hydroalcoholic extract The leaves were washed with distilled water and disinfected with 96% ethanol. They were fractionated to an approximate size of 3 mm. For the extraction of the hydroalcoholic extract, the method was used by maceration with 300 g of leaves and 500 mL of 96% ethanol for 15 days under stirring with a vertical rotary evaporator (Scilogex RE-100) at 70 revolutions per minute to obtain dry extracts. With the sample obtained, dilutions were prepared at concentrations of 10, 100 and 1000 μg/mL. 2.3 Phytochemical analysis The hydroalcoholic leaf extract was evaluated with the purpose of identifying its active ingredients such as: steroids, triterpenes, quinones, phenolic compounds, flavonoids, lactones, alkaloids, reducing sugars, tannins and saponins, using the protocol described by Lock (2016). 1 mL of pure extract was added to 10 test tubes to see the metabolites in each hydroalcoholic extract, and tests were performed to identify secondary metabolites through color change, being classified as light, moderate or strong. The tests used to determine the presence of each type of secondary metabolite are listed in Table 1. 2.4 Obtaining and breeding Artemia salina. The eggs of Artemia salina 20 days old were provided by the Animal Physiology Department, National University of Trujillo, washed with filtered seawater to remove impurities and were transported to a glass incubation chamber with abundant oxygenation was used one gram of eggs (equivalent to 700-800 eggs), allowing to incubate in 5 liters of filtered seawater under artificial fluorescent light at 110 Watts, temperature of 25 °C and adjusted to a pH of 7-8 for 24 hours. Artemia salina eggs were fed commercial yeast extract to hatch and continue their biological cycle for approximately seven days. Then 10 larvae were used for each concentration of 7 old days (10, 100 and 1000 μg / mL) in stage III as toxicity markers due to their high sensitivity (Silva et al, 2015; Ravi et al, 2014; Jaramillo et al, 2016). 368 Table 1. Phytochemical analysis of the hydroalcoholic extract of the medicinal plants leaves from the Peruvian Amazon Test Secondary Metabolites Monocotyledons Dicotyledons Commelina diffusa Dracontium loretense L Brunfelsia grandiflora Malva sylvestris Dysphania ambrosioides Pinus edulis Bixa Orellana Jatropha curcas L Origa num vulga re Lieberm an- Bouchar d Steroids and triterpens ++ ++ + + ++ + ++ ++ ++ Borntrag er Quinones - - - - - - - - - ferric Chloride phenolic Compounds +++ ++ + ++ ++ + + ++ ++ Shinoda Flavonoids ++ ++ - ++ ++ - ++ ++ +++ Baljet lactones - - + - + - + - - Dragend orff Alkaloids - - - - - - - - - Mayer Alkaloids - - - - - - - - - Fehling reducing Sugars ++ ++ + + - - ++ + + Gelatine tannins - - - - - - + - - Foam Saponins - - + - - - - - - Note: Color changes of secondary metabolites in (+) = Light, (++) = moderate and (+++) = strong 2.5 Toxicity tests The concentrations of 10, 100 and 1000 µg/mL of filtered seawater were prepared according to the protocol described by Seremet et al (2018). Then 5 µg of extract was diluted in 5 mL of filtered seawater, equivalent to 10 μg/mL, 50 µg of extract in 5 mL of filtered seawater, equivalent to 100 µg/mL, and 500 µg of extract in 5 mL of filtered seawater, equivalent to 1000 µg/mL. The larvae were placed in a test tube containing 10 mL of filtered seawater and 0.5 mL of the hydroalcoholic extract. 10 larvae were used for each plant species and concentration; each test was performed in triplicate. A control group of 10 larvae in 10 mL of filtered seawater without extract was used to make the respective comparisons. The analyses were performed in the biological chemistry laboratory of the National University of Trujillo, the larvae were exposed to the treatments for 24 hours, after this time the number of larvae was considered dead only if there was no movement of their appendages for 10 seconds (Socea et al, 2015). For this, a stereoscope was used (Eurolab NSD-405). The toxicity criteria for Artemia salina samples were classified as follows: ˃ 1000 µg/mL (non-toxic), 500 DL 50 ≤ 1000 (low toxicity), 100 ˂ DL 50 ≤ 500 (moderate toxicity), DL 50 ˂ 100 (high toxicity) (Arana et al, 2016; Alonso et al, 2017 and Monteiro et al, 2018). The organisms toxicity percentage exposed to the effect of the extracts was estimated as follows: Toxicity (percentage) = (TNA - NAA) / TNA*100 (1) Where: TNA = Total number of Artemia NAA = Number of Artemia alive (Jan and Khan, 2016) 2.6 Ethical statement Artemia salina does not represent a danger to the environment, it is not an endangered species, because it does not appear on the red list of the International Union for the Conservation of Nature (IUCN), the species is used for scientific purposes (IUCN, 2019). 3. Results In Figure 1, it is observed that of 9 species of medicinal plants only 7 have high toxicity (HT), being at least 56.7% death in Artemia salina caused by Bixa Orellana and the maximum percentage 86.7% caused by Commelina diffusa. 369 Figure 1. Percentage of toxicity of 10 µg/mL of hydroalcoholic extract of medicinal plants. In Figure 2, it is observed that of 9 species of medicinal plants only 6 have moderate toxicity (MT), being at least 53.3% of death in Artemia salina is caused by Dracontium loretense Krause and the maximum percentage 66.7% is by Commelina diffusa. Figure 2. Percentage of toxicity of 100 µg/mL of hydroalcoholic extract of medicinal plants. In Figure 3, it is observed that the 9 species of medicinal plants have low toxicity (LT), finding that the minimum percentage 17% of death in Artemia salina is caused by Pinus edulis and the maximum percentage 43.3% is by Origanum vulgare. 370 Figure 3. Percentage of toxicity of 1000 µg / mL of hydroalcoholic extract of medicinal plants. 4. Conclusions If the concentration is greater than 10 µg/mL, toxicity is high in hydroalcoholic extracts of 7 plant species in Artemia salina. Also, if the concentration is less than 1000 µg/mL, toxicity is low in all hydroalcoholic extracts. The consumption of medicinal plants has been increasing due to its probable effectiveness. However, its indiscriminate use is a latent risk due to the toxicity of some compounds within the plant that can cause collateral damage. For this reason, it may be useful to study extracts of medicinal plants in order to show therapeutic or toxic activity of their active compounds using Artemia salina as an evaluation procedure (Simões and Almeida, 2015). References Afsar, T., Khan, M., Razak, S., Ullah, S., Mirza, B., 2015, Antipyretic, anti-inflammatory and analgesic activity of Acacia hydaspica R. Parker and its phytochemical análisis, 15: 136. Doi: 10.1186 / s12906-015-0658-8. bmccomplementalternmed.biomedcentral.com/track/pdf/10.1186/s12906-015-0658-8 Alonso, A., Domínguez, F., Ruíz, A., Campos, N., Zapata, J., Carranza, C., Maldonado, J. 2017, Medicinal Plants from North and Central America and the Caribbean Considered Toxic for Humans: The Other Side of the Coin. 2017: 9439868. doi: 10.1155 / 2017/9439868. www.ncbi.nlm.nih.gov/pmc/articles/PMC5688365/#B1 Andrade, F., Ribeiro, AR., Medeiros, M., Fonseca, S., Athayde, AC., Ferreira, A., Rodrigues, O., Silva, W., 2014, Anthelmintic action of the hydroalcoholic extract of the root of Tarenaya spinosa (Jacq.) Raf. for Haemonchus contortus control in sheep. Pesquisa Veterinária Brasileira. 34 (10): 942–946. dx.doi.org/10.1590/S0100-736X2014001000003. Arana, J., Villacrés, J., García, D., Sotero V., Vasquez D., Monteiro U., Gonzalez, G., Mego, R., Vigo, W., 2016, Toxicidad, actividad antioxidante in vitro e hipoglicemiante in vitro e in vivo del extracto acuoso de Juglans neotrópica Diels (nogal peruano). Revista Peruana de Medicina Integrativa. 1, 4, 16-24. DOI: 10.26722/rpmi.2016.14.37 www.researchgate.net/publication/319660542_Toxicidad_actividad_antioxidante_in_vitro_e_hipoglicemiante_i n_vitro_e_in_vivo_del_extracto_acuoso_de_Juglans_neotropica_Diels_nogal_peruano Ávalos, J., Treviño, J., Verde, MJ., Rivas, C., Oranday, A., Moran, J., Serrano, L., Morales, ME., 2014, Cytotoxic evaluation of Azadirachta indica (A. Juss) ethanolic extracts against differents cells lines. Revista mexicana de ciencias farmacéuticas. 45(3): 39–44. www.scielo.org.mx/scielo.php?script=sci_arttext&pid=S1870-01952014000300004 BCC., 2017, Botanical and Plant-Derived Drugs: Global Markets. BCC BIO022G. www.bccresearch.com/market-research/biotechnology/botanical-plant-derived-drugs-report-bio022g.html Bokhari, J and Khan, M., 2015, Evaluation of anti-asthmatic and antioxidant potential of Boerhavia procumbens in toluene diisocyanate (TDI) treated rats. 85, 172: 377. doi: 10.1016/j.jep.2015 .06.049. www.sciencedirect.com/science/article/pii/S0378874115300143 371 Goncalves Reis De Melo A.C., Vasconcelos Dos Santos M.D., De Carvalho Neto M.F., Takahashi J.A., Ferraz V.P., Chagas E.A., Chagas Cardoso P., De Melo Filho A.A., 2019, Phytochemical Trial and Bioactivity of the Essential Oil from Coriander Leaves (coriandrum Sativum) on Pathogenic Microorganisms, Chemical Engineering Transactions, 75, 403-408 DOI:10.3303/CET1975068. www.cetjournal.it/index.php/cet/article/view/CET1975068 IUCN, (International Union for Conservation of Nature’s Red List). 2019, www.iucnredlist.org/ Jan, S., Khan, MR., 2016, Protective effects of Monotheca buxifolia fruit on renal toxicity induced by CCl4 in rats. 17, 16, 1, 289. doi: 10.1186/s12906-016-1256-0. www.ncbi.nlm.nih.gov/pubmed/27530158 Jaramillo, C., Jaramillo, A., D’Armas, H., Troccoli, L., Rojas, L., 2016, Concentrations of alkaloids, cyanogenic glycosides, polyphenols and saponins in selected medicinal plants from Ecuador and their relationship with acute toxicity against Artemia salina. Revista de Biología Tropical. 64,3, 1171-1184. doi.org/10.15517/rbt. v64i3.19537. revistas.ucr.ac.cr/index.php/rbt/article/view/19537 Leite, S., Dantas, AF., Oliveira GL., Gomes AL, de Lima SG., Citó, AM., de Freitas, RM., Melo-Cavalcante AA., Dantas, JA., 2015, Evaluation of toxic, cytotoxic, mutagenic, and antimutagenic activities of natural and technical cashew nut shell liquids using the Allium cepa and Artemia salina bioassays. Biomed Res Int. 2015: 626835. doi: 10.1155 / 2015/626835. www.ncbi.nlm.nih.gov/pubmed/25861638 Lock, O., 2016, Investigación Fitoquímica: Métodos en el estudio de productos naturales. 3era edición, Editorial del Departamento de Ciencias. Universidad Pontificia Católica del Perú, p 6-8. MINAM, 2019, Sexto informe nacional sobre Diversidad biológica: La biodiversidad en cifras. Lima – Perú. www.gob.pe/institucion/minam/informes-publicaciones/281709-sexto-informe-nacional-sobre-diversidad- biologica Monteiro, J; Ferreira, J; Oliveira IR, Batista FLA, Pinto CCC, Silva AAS, Morais SM, Silva MGV., 2018, Bioactivity and Toxicity of Senna cana and Senna pendula Extracts. Biochem Res Int. 2018: 8074306. doi: 10.1155 / 2018/8074306. www.ncbi.nlm.nih.gov/pubmed/29808121 Morais Santana K., Morais Santana B., Oliveira Vilarinho Braga L., Chagas Cardoso P., Goncalves Reis De Melo A.C., Takahashi J.A., De Melo Filho A.A., 2019, Bioactive Extracts of Capsicum Chinense in the Northern Amazon, Chemical Engineering. Transactions, 75, 433-438 DOI:10.3303/CET1975073. www.cetjournal.it/index.php/cet/article/view/CET1975073 Paredes, B., Zegarra, J., Zanabria, J., Cuadros, F., Chavez, J., Huanca, P., 2018, Consumo de plantas medicinales de uso habitual y posible efecto adverso en la salud de la población, como resultado de la contaminación de las plantas. Arequipa, Perú. Revista avances en Ciencias e ingeniería. 9(3), 13-22. dialnet.unirioja.es/servlet/articulo?codigo=6597221 Pereira, J., Barros, K., Lima, L., MArtins, JM., Araujo, YC., da Silva Oliveira, GL., de Souza Aquino, J., Ferreira PMP., 2016, Phytochemistry Profile, Nutritional Properties and Pharmacological Activities of Mauritia flexuosa. J Food Sci. 81 (11): R2611 – R2622. doi: 10.1111/1750-3841.13529. www.ncbi.nlm.nih.gov/pubmed/30240016/# Seremet, O., Olaru OT, Gutu CM, Nitulescu GM, Ilie M, Negres S, Zbarcea CE, Purdel CN, Spandidos DA, Tsatsakis AM, Coleman MD, Margina DM., 2018, Toxicity of plant extracts containing pyrrolizidine alkaloids using alternative invertebrate models. Mol Med Rep. 17, 6, 7757–7763. doi: 10.3892 / mmr.2018.8795. www.ncbi.nlm.nih.gov/pubmed/29620235 Silva, E., De Castro, R., Barreto, F., de Moraes, MO., Souza, SA., Feitosa, A., Cerqueira, M., 2015, Estudo in vitro do potencial citotóxico da Annona muricata L. Journal of Applied Basic Pharmaceutical Sciences. 36 (2): 277-283. seer.fcfar.unesp.br/rcfba/index.php/rcfba/article/view/242 Simões, R., Almeida, S., 2015, Estudio fitoquímico Bauhinia forficata (Fabaceae). Biota amazónica. 5, 1, 27- 31. doi.org/10.18561/2179-5746/biotaamazonia. v5n1p27-31 www.redib.org/recursos/Record/oai_articulo1087489-estudo-fitoqu%C3%ADmico-bauhinia-forficata-fabaceae Socea, L; Socea, B; Saramet, G., Barbuceanu, SF., Draghici, C., Constantin, VD., Tudorel, O., 2015, Synthesis and cytotoxicity evaluation of new 5h-dibenzo<7>annulen-5-yl acetylhydrazones. Rev Chim. 66, 1122–1127. www.revistadechimie.ro/article_eng.asp?ID=4591 Teles, D., Costa, M., 2014, Estudo da ação antimicrobiana conjunta de extratos aquosos de Tansagem (Plantago major L., Plantaginaceae) y Romã (Punica granatum L., Punicaceae) e interferencia de dos meses en una dosis de amoxicilina in vitro. Revista Brasileira de Plantas Medicinais. 16 (2): 323–328. doi: 10.1590 / 1983-084X / 11_123. www.scielo.br/scielo.php?script=sci_arttext&pid=S1516-5722014000500003&lng=pt&tlng=pt Ullah, S., Khan, M., Shah, N., Shah S.,Majid, M., Farooq, M., 2014, Ethnomedicinal plant use value in the Lakki Marwat District of Pakistan. J Ethnopharmacol. 158: 412–22. doi: 10.1016 / j.jep.2014.09.048. https://www.sciencedirect.com/science/article/pii/S037887411400703X?via%3Dihub WHO. 2015, Traditional Medicine Strategy 2014-2023. Geneva, Switzerland. www.who.int/global_health_histories/seminars/Dr_Zhangs_Presentation_GHHSeminar_86.pdf 372