Vol. 1 No. 1 Natural Products and Biotechnology pp. 38-48 (2021) Determination of Toxic and Anthelmintic Activities of Ornithogalum nutans L., Sternbergia lutea (L.) Ker-Gawl. ex Spreng. and Allium stylosum O. Schwarz Mehmet Ozgur Atay1 , Buse Ardil1* , Mehlika Alper1 , Olcay Ceylan2 1 Department of Moleculer Biology and Genetics, Faculty of Science, Muğla Sıtkı Koçman University, Muğla, Turkey 2 Department of Biology Faculty of Science, Muğla Sıtkı Koçman University, Muğla, Turkey Article History Abstract Received : May 22, 2021 In this study, toxic and anthelmintic activities of methanol extracts of aerial and Revised : June 03, 2021 underground parts of Ornithogalum nutans L., Sternbergia lutea (L.) Ker-Gawl. ex Accepted : June 15, 2021 Spreng. and Allium stylosum O.Schwarz were investigated. In order to determine the anthelmintic activity, the time elapsed for the duration of paralysis and death was Keywords determined after the extracts of different concentrations (10, 20 and 30 mg/mL) were added to the Tubifex tubifex in petri dishes. Each concentration of A. stylosum aerial Ornithogalum nutans, and underground parts extracts showed high anthelmintic activity. In addition, aerial Sternbergia lutea, extract of O. nutans at a concentration of 30 mg/mL showed high anthelmintic Allium stylosum, activity. A. stylosum extracts showed a higher activity than the standard anthelmintic Artemia salina, drug. The toxic activity was determined against Artemia salina with brine shrimp Anthelmintic activity lethality test. Among all extracts, the underground extract of S. lutea showed the highest activity with 0.002 mg/mL, LC50, the aerial extract of O. nutans showed the lowest activity with 0.03 mg/mL, LC50. Aerial and underground methanol extracts of A. stylosum showed high anthelmintic and lethal activity, but more specific studies are needed. Corresponding Author: Buse Ardil, Department of Moleculer Biology and Genetics, Faculty of Science, Muğla Sıtkı Koçman University, Muğla, Turkey, [email protected] Cite this article as: Atay, M. O., Ardil, B., Alper, M., & Ceylan, O. (2021). Determination of Toxic and Anthelmintic Activities of Ornithogalum nutans L., Sternbergia lutea (L.) Ker-Gawl. ex Spreng. and Allium stylosum O. Schwarz. Natural Products and Biotechnology, 1(1), 38-48. 1. INTRODUCTION The use of plant or plant-based products as medicine dates back to prehistoric times (Osama et al., 2020). Medicinal plants are natural resources that provide valuable phytochemical products that are frequently used in the treatment of various diseases (Sahgal et al., 2010). Therefore, using natural products and herbal medicines is an effective, beneficial, and economical way to treat different diseases (Azad et al., 2019). Part of traditional medicine often involves the use of crude plant extracts, which can contain a wide variety of molecules with uncertain biological effects (Sahgal et al., 2010). The health benefits of medicinal plants are related to their antioxidant activities and biological effects on cellular processes (Ukwade et al., 2020). Plants offer medicinal value due to the presence of substances such as alkaloids, essential oils, tannins, resins. These substances have a physiological effect on the human body. The use of local herbs as a source of medicine may have some negative effects mainly as a result of the lack of sufficient information about safe dosage and the presence of harmful by- products in some plants (Naher et al., 2019). Despite the worldwide recognition of herbal medicines and their use in different diseases, the safety of these treatments is still a major concern (Yang, 2020). More research is needed to determine the toxic effects of herbs used to treat various ailments (Ukwade et al., 2020). In the last decade, toxicity screening of a wider 38 ISSN: 2791-674X Research Article Atay, Ardil, Alper & Ceylan variety of food and plant products has been carried out using BSLT (Brine Shrimp Lethality Test), a method chosen by many researchers (Ajibola et al., 2020). The brine shrimp (Artemia salina) experiment has been proposed as a valid method to evaluate the toxic activity of plant extracts (Aydın et al., 2017; Suryawanshi et al., 2020). Combined with a reference standard, the brine shrimp test provides a fast, simple, and more importantly, inexpensive and reproducible bioassay (Tawaha, 2006). Helminth infections are global problems with serious social and economic repercussions (Dey & Ghosh, 2010). Helminth and parasitic worm infections affect over two million people worldwide. Anthelmintic drugs facilitate excretion from the body by killing (vermicide) or stunning (vermifuge) antistatic helminths (Verma et al., 2013). The resistance developed by helminth parasites to existing drugs becomes a problem to treat these diseases (Pal & Dey, 2011). The antihelmintic drugs used today mainly have little effect on microfilaricidal and adult worms (Murugamani et al., 2012; Hossain et al., 2015). In addition, various side effects such as epigastric pain, diarrhea, nausea, vomiting, headache, dizziness and allergic events have been reported in hosts after treatment with albendazole or mebendazole (Pal & Dey, 2011; Verma et al., 2013). Therefore, new drugs are urgently needed (Murugamani et al., 2012). Plant-derived drugs act as a prototype for developing more effective and less toxic drugs. Several medicinal herbs have been used to treat parasitic infections in humans and animals (Pal & Dey, 2011; Hossain et al., 2015). Tubifex tubifex shows anatomical and physiological similarities with the intestinal parasites (roundworm, annelid) of human. Due to their easy availability, T. tubifex is widely used for the initial evaluation of anthelmintic compounds in vitro (Chowdhury et al., 2015). Sternbergia lutea (L.) Ker Gawl. ex Spreng, a member of the Amaryllidaceae family, is a geophyte plant that generally spreads around the Mediterranean basin and blooms bright yellow in autumn (Gage & Wilkin, 2008; Güner et al., 2012). It has different pharmacological effects due to the alkaloids it contains (Berkov et al., 2009). Antioxidant, antimicrobial, antidiabetic, anti-inflammatory activities and total phenolic amount have been reported in the studies (Ünver et al., 2005; Aydın et al., 2015; Ağca et al., 2021). Ornithogalum L., which has more than 140 species in the world, is belonging the Hyacinthaceae family. Of the 45 Ornithogalum L. species found in the Flora of Turkey, 17 are endemic. In a study conducted on Ornithogalum nutans L., it was reported that seventeen cardenolides were isolated from its leaves and bulbs (Plancic et al., 2015). There are 146 Allium L. species in the Amaryllidaceae family and 65 of them are endemic. Allium stylosum O. Schwarz is a species that blooms between May and June and grows in forested openings and fields (Aydın, 2012). In this study, it was aimed to determine toxic (against Artemia salina) and anthelmintic (against Tubifex tubifex) activities of methanol extract obtained from the aerial and underground parts of Sternbergia lutea, Ornithogalum nutans and Allium stylosum. 2. MATERIAL and METHODS 2.1. Plant Materials and Extract Preparation Sternbergia lutea was gathered in the Gülağzı neighborhood of Menteşe, Muğla providence, Turkey in 2020. Dr. Olcay Ceylan identified the plant sample were deposited in Muğla Sıtkı Koçman University Herbarium (MSKUH) under herbarium number of OC.2119. Allium stylosum was collected from Denizli in August-September in 2011. Identified the plant sample in Laboratory of Botanic of Pamukkale University. Ornithogalum nutans was collected from around the Denizli Babadağ district center. The aerial (flowers, leaves) and underground (tuber) parts of the collected plants were dried separately. After the dried plants were cut into small pieces, 100 mL methanol was added to the sample weighed 10 g. Each sample were kept in a shaking incubator at 55 ºC for 6 hours. The extraction mixture was separated from the residue by filtration through Whatman filter paper. The plant residue was left under the same conditions for second and third time with methanol added again and filtered. The solvent was removed from the obtained filtrate with the help of a rotary evaporator (Heidolph-Laborota 39 Natural Products and Biotechnology 4011) (at 50 ºC). Then, the water in the extract was removed by lyophilization (Thermo Savant). The extracts obtained were stored at -20ºC (Turan & Mammadov, 2021). 2.2. Toxic Activity Assay against Artemia salina (Brine Shrimp Lethality Test) Determination of the toxic activity assay of plant extracts against A. salina (Brine Shrimp Lethality Test (BSLT)) was made by modifying Krishnaraju et al. (2005) method. Methanol extract obtained from the aerial and underground parts of the plants was used. Two days before the assay, one pack of Artemia commercially available was added to the jar of 500 mL distilled water. An air motor was used to keep the air in the jar continuously. The aquarium was kept at 28 °C and in a bright environment. Artemia eggs were expected to hatch for 2 days. After 2 days 0.5 M sea saline solution was prepared. With different concentration (0.01, 0.05, 0.1, 0.5, and 1.0 mg/mL) was added to the test tubes containing 4.5 mL of saline solution. Later, 12 Artemia added to the tubes. After the tubes placed in the tube stand were placed in the aquarium on 24 hours for the emergence of Artemia. At the end of this time, living and non-living Artemia poured into Petri dishes were counted and noted. 2.3. Anthelmintic Activity against Tubifex tubifex Anthelmitic activity assay was carried out using T. tubifex, which belongs to the same annelid group as the roundworm parasite. T. tubifex is used for the in vitro evaluation of anthelmintic compounds because of its easy availability and low cost. Six of the 1-2cm tall T. tubifex were placed in a Petri dish containing 20 mL of methanol extract. Test samples of the extracts were prepared in distilled water at concentrations of 10, 20, 30 mg/mL. Albendazole (2.5, 5, 10 mg/mL) was used as the reference standard. Distilled water was used as a negative control.