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Erythroid Induction Activity of Aquilegia Fragrans and Aquilegia Pubiflora and Identification of Compounds Using Liquid Chromato

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Erythroid Induction Activity of Aquilegia Fragrans and Aquilegia Pubiflora and Identification of Compounds Using Liquid Chromato Journal of King Saud University – Science 33 (2021) 101227 Contents lists available at ScienceDirect Journal of King Saud University – Science journal homepage: www.sciencedirect.com Original article Erythroid induction activity of Aquilegia fragrans and Aquilegia pubiflora and identification of compounds using liquid chromatography-tandem mass spectrometry Nudrat Aziz a, Muhammad Noman Khan a, Faraz Ul Haq a, Fayaz Ahmed b, Arslan Ali b, ⇑ ⇑ Hesham R. El-Seedi c,d, , Syed Ghulam Musharraf a,b,e, a H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan b Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan c Pharmacognosy Group, Department of Pharmaceutical Biosciences, BMC, Uppsala University, SE-751 23 Uppsala, Sweden d International Research Center for Food Nutrition and Safety, Jiangsu University, Zhenjiang 212013, China e T.C.M. Hospital of Southwest Medical University, Luzhou, Sichuan, China article info abstract Article history: Aquilegia fragrans (AF) and Aquilegia pubiflora (AP) are the two medicinally important species of genus Received 11 May 2020 Aquilegia used for the treatment of various diseases and infections. This paper describes the potential Revised 20 October 2020 of fetal hemoglobin induction activity of the methanolic extracts of AF and AP in K562 cell line. AF and Accepted 28 October 2020 AP have shown 27.147 ± 1.376 and 32.786 ± 1.048 percent erythroid induction, respectively at 15.625 Available online 4 November 2020 (mg/mL) concentration which suggested that both plants can be the source of potential fetal hemoglobin inducers and may be used for the treatment of b-thalassemia. Phytochemical analyses of both species Keywords: were also evaluated by using high-resolution LC-ESI-QTOF-MS/MS techniques. A Total of thirty com- Aquilegia pubiflora pounds were identified using positive and negative ionization modes. The identification was based on Aquilegia fragrans b-thalassemia the matching of high-resolution masses, isotopic pattern, and MS/MS fragmentation. Several statistical Fetal hemoglobin analyses were performed to evaluate the distribution of compounds in both species. Identified com- pounds belong to various classes including flavonoids, steroids, lignans, terpenoids, benzofuran and cou- marins. The established chemical fingerprints will be helpful in standardization and quality control of plant extracts. Ó 2020 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1. Introduction excessive a-globin chains, get accumulated in cells and form inclu- sion bodies that affect the membrane integrity and cause the pre- b-thalassemia, an inborn genetic disorder, is characterized by mature death of erythroid cells (Sankaran and Orkin, 2013). This total absence or insufficient synthesis of b-globin chain (Thein, leads to ineffective erythropoiesis, which results in chronic anemia 2018). Due to reduced synthesis or total absence of b-globin chains, associated with b-thalassemia (Nienhuis and Nathan, 2012). Current treatment options for b-thalassemia are mostly symp- tomatic, including blood transfusion and iron chelation therapy. ⇑ Corresponding authors at: H.E.J. Research Institute of Chemistry, International Bone marrow transplantation and gene therapy could be promis- Center for Chemical and Biological Sciences, University of Karachi, Karachi-75270, Pakistan (Syed Ghulam Musharraf). Pharmacognosy Group, Department of Phar- ing, but these methods have limitations due to the high cost and maceutical Biosciences, BMC, Uppsala University, SE-751 23 Uppsala, Sweden availability to common man, especially in developing countries (Hesham R. El-Seedi). (Cavazzana-Calvo et al., 2010). Therefore, current research focuses E-mail addresses: [email protected] (H.R. El-Seedi), musharra- on a cost-effective and safe therapeutic approach to treat b- [email protected] (S.G. Musharraf). thalassemia, which is fetal hemoglobin (HbF) induction. Fetal Peer review under responsibility of King Saud University. hemoglobin, a predominant form of hemoglobin during fetal stage of development, and about one year after birth, it is gradually replaced with adult hemoglobin as predominant form. This devel- opmental suppression of fetal hemoglobin can be reversed by Production and hosting by Elsevier using inducers to ameliorate the clinical symptoms of thalassemia https://doi.org/10.1016/j.jksus.2020.10.024 1018-3647/Ó 2020 The Author(s). Published by Elsevier B.V. on behalf of King Saud University. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). N. Aziz, M.N. Khan, Ul Haq et al. Journal of King Saud University – Science 33 (2021) 101227 (Rees et al., 1999). Several potential HbF inducers, like hydrox- bought from ‘‘DaeJung Chemical and Metals”, (Korea) and type I yurea, butyrates, erythropoietin, and 5-azacytidine have been dis- water was obtained from BarnsteadTM GenPure (USA) water purifi- covered, but their therapeutic usage is limited due to their cation assembly. Reagents for erythroid induction activity are Fetal cytotoxicity and carcinogenic properties (Osti et al., 1997). There- Bovine Serum (Gibco), RPMI-1640 (Gibco), Pen/Strip (Gibco), fore, there is a strong need to search for potential HbF inducers DMSO (Sigma Aldrich, France), Benzidine hydrochloride (Perkin which are affordable and associated with minimal side effects. Chemicals, China), Hydrogen Peroxide-30% (Sigma Aldrich, Ger- Plants have been the source of lead molecules that are being many), Glacial acetic acid (Lab Scan), Trypan Blue 4% (Sigma used for the treatment of various diseases. Aquilegia (common Aldrich, UK), Hydroxyurea (Sigma Aldrich USA). name columbine) is one of the phytochemically important, pheno- typically diverse and experimentally a tractable genus that belongs 2.3. Sample preparation to family Ranunculaceae. Aquilegia fragrans is a perennial herb that mostly grows in northern regions of Pakistan, north-west Himalaya, Shade dried whole plants were ground using liquid nitrogen eastern Afghanistan, north-western India and highly distributed at into homogenized powder. Exactly 1 g of each sample was higher regions of Kashmir valley (Hussain et al., 2011). A. fragrans is weighed and extracted with 10 mL methanol in a 15 mL falcon commonly called as sweet-scented columbine and commonly tube using a ultra-sonicator for 30 min which were then cen- called as Jangli kuth and Kalumb. The roots of A. fragrans are used trifugated for 15 min at room temperature at 12,000 RPM in to treat wounds and various inflammatory diseases like, gout, cys- order to remove solid particles. 1 mL of upper layer from each titis, eczema, psoriasis, blood sugar, kidney stones, and veterinary sample was filtered through a PTFE syringe-driven filter diseases such as bovine mastitis (Ganie et al., 2019). Dried flower (0.22 mm) and transferred to 1.5 mL centrifuge tubes for LC-MS paste is used for the relief of headache while seeds and the whole analysis. Filtered sample extracts were further diluted by taking plant are used to cure jaundice and pneumonia, respectively 50 mL of sample and diluted with 950 mL (20 times) of methanol (Khan et al., 2018). Aquilegia pubiflora is another medicinally impor- in HPLC vial. tant herb widespread in the Himalaya in India, Afghanistan, and northern Pakistan. It is commonly known as Hairy-flowered colum- 2.4. Erythroid induction activity bine while locally called as Domba and Thandi buti. This species is reported to have many medicinal values including anti-asthmatic, K562 cells (CCL243, ATCC,), were cultured in RPMI-1460 com- astringent, stimulant, cardio-tonic, febrifuge, jaundice, and dyspep- plete medium containing 10% FBS, and 1% penicillin/streptomycin sia. Dried roots are used to treat toothache, eye diseases, snakebite solution, in a 5% CO2 at 37 °C under humid conditions. 10 mg/mL and in homeopathy, especially for the nervous system (Dhar and stock solutions of methanolic extracts of each plant were prepared Samant, 1993; Hazrat et al., 2011). in sterile deionized water as a solvent to prepare a stock solution. Previous phytochemical studies on Aquilegia species, including To determine the erythroid induction, the cells were seeded at den- A. vulgaris L., A. panicii, A. oxysepala, A. canadensis, A. glandulosa, A. sity of 4x104 cells/mL in culture and treated in concentrated excimia were reported based on various extraction, isolation and depend manner range from 250 mg to 15.625 mg of each extract. analytical methods. These studies have shown identification of fla- The cells were then placed in 5% CO2 incubator at 37 °C for five vonoids (Refaat et al., 2015), alkaloids, cyanogenic constituents, days. cycloartane-type glycosides (Yoshimitsu et al., 1999); butenolides For erythroid induction benzidine-hydrogen peroxide was used. (Guerriero and Pietra, 1984); terpenoids, tannins and unusual Briefly, 0.2% benzidine solution was prepared in 0.5 M acetic acid, unsaturated fatty acids (Bylka and Matlawska, 1999). Until now, and 20 mL of hydrogen peroxide was added per 1 mL of ice-chilled reported protocols for phytochemical analysis of Aquilegia species 0.2% benzidine solution. On day five, the cells were washed with by mass spectrometric techniques were either low-resolution
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