Bioactive Compounds Profile Of Alkaloid On Elaeocarpus Sphaericus Schum .

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Advances in Social Science, Education and Humanities Research, volume 6302nd International Conference on Education and Technology (ICETECH 2021)Bioactive Compounds Profile of Alkaloid onElaeocarpus sphaericus Schum Seeds by LiquidChromatography-Mass SpectrometryCicilia Novi Primiani1*, Pujiati1, Mohammad Arfi Setiawan21Department of Biology Education, Universitas PGRI Madiun, East Java IndonesiaDepartment of Chemical Engineering, Universitas PGRI Madiun, East Java Indonesia*Corresponding author. Email: primiani@unipma.ac.id2ABSTRACTElaeocarpus sphaericus Schum with the local name genitri has medicinal plant properties. Therefore, this study aimsto identify the diversity of alkaloid compounds in genitri seeds using the Liquid Chromatography-Mass Spectrometry(LC-MS) method. The Extract Purification through Solid Phase Extraction (SPE) was conducted using C18 Sep-Pak.Based on the LCMS results, several alkaloids were obtained in the genital seeds with different compositions namely,Elaeokanine C 0.90%, ( )-elaeocarpine 1.59%, Elaeocarpenine 1.88%, Isoelaeocarpine 1.36%, Grandisine B 0.86%,Isoelaeocarpiline 1.14%, Grandisine D 1.51%, Elaeocarpidine 1.03%, Isoelaeocarpicine 1.89%, Grandisine F 1.39%,Grandisine A 0.89%, Grandisine C 0.98%, Grandisine E 1.14%, Habbemine A 1.24%, Habbemine B 1.46%,Grandisine G 0.85%. Furthermore, the bioavailability of these compounds was determined through PhytochemicalAnalysis.Keywords: alkaloids, compounds profile, Elaeocarpus sphaericus, LCMS1. INTRODUCTIONBiodiversity of plants can thrive in Indonesia due togeographical conditions, especially with a tropicalclimate, and they have medicinal properties. Previousstudies showed that plants have various activecompounds used as medicine. Elaeocarpus sphaericusSchum medicinal plant is one of the 360 species of theElaeocarpaceae family that can grow in tropical andsub-tropical areas. This species is a local endemic plantthat has not been fully utilized, and it is used as shade,firewood, seeds are used for jewelry. Several studiesshowed that genitri can be used as a medicinal plant [13] due to the complex compounds [4-5]. Furthermore,the chemical compounds have pharmacologicalactivities that can be used in the treatment of diseases[3],[6]. The ethanolic extract of E. ganitrus seed of 250mg/kg and 500 mg/kg (p.o) affected the normal kidneytissue structure of rabbits fed with cholesterol [7].Meanwhile, E. tectorius seed extract 40.06 mg hasantioxidant and antimicrobial properties [8]. E.tonkinesis leaf extract has anti-influenza ability [5]. Oneof the chemical compounds in plants are alkaloids, andthey contain heterocyclic nitrogen atoms [9-10] that aresynthesized in various plants. Alkaloids in purecompounds have the characteristics of colorless, nonvolatile, crystalline, tend to taste bitter [11], and theyhave various physiological effects on the human body[12-13]. Qualitative and quantitative methods areconducted to identify the phytochemical properties ofnatural materials [14]. Liquid Chromatograph MassSpectrometry (LC-MS) is one of these analyticalmethods that combines the separation capabilities ofliquid chromatography (LC) against a target compound.Furthermore, this compound is obtained from themixture of mass spectroscopic capabilities (massspectrometry/MS) which can read the spectrum of theseparated target compound [15]. This study aims toanalyze the compounds of the alkaloid group ofElaeocarpus sphaericus Schum seeds using the LC-MSmethod.2. METHOD2.1. MaterialsDried genetri seeds were obtained from a forest areain Magetan, East Java, Indonesia.Copyright 2022 The Authors. Published by Atlantis Press SARL.This is an open access article distributed under the CC BY-NC 4.0 license 0

Advances in Social Science, Education and Humanities Research, volume 6302.2. Sample PreparationThe seeds were washed and dried using a microwaveat 60 oC, then mashed using a blender, and 100-250 gwere taken after being smooth. Furthermore, the seedpowder was dissolved in 95% methanol in a ratio of 1:5(sample: methanol), and the solution was stirred untilhomogeneous. It was left to stand for 24 hours at a coldtemperature in a closed bottle, and the filtration processwas further conducted to obtain the filtrate and dregsusing an Erlenmeyer vacuum filter. The process wasrepeated three times by dissolving with methanol untilthe dregs were obtained. The combined filtrate wasevaporated from the methanol to obtain a semi-viscousextract using half of the previous solution.2.3. Extract DilutionA vortex was used to dissolve the extract withmethanol to a concentration below 100 ppm until ahomogeneous solution was obtained. Furthermore, thesolids were separated by centrifuging at a speed of 8000rpm for 10 minutes, and the obtained supernatant wasused in the next stage.2.4. Protein PrecipitationIn the centrifuge tube, 2 ml of supernatant extractwas added to 3 ml of acetonitrile acidified with 0.2%formic acid. The centrifugation was conducted at aspeed of 8000 rpm for 30 seconds to obtain anothersupernatant.2.4. Purification with SPE (Solid PhaseExtraction)The supernatant was put into a Sep-Pak C18Cartridge (1 ml, 100 mg) which had been conditionedwith 1 ml 80:20 (acetonitrile: water), and about 0.5 mlof the solution was accommodated with 1 ml of thesample into the Sep – Pak column. Furthermore, 0.5 mlof 80:20 acetonitrile/water solution was added into theSep – Pak column, and 0.5 ml of the solution that comesout was accommodated. Then, 0.25 ml of 200 mMammonium formate was added in a 50:50 solution ofacetonitrile/methanol to the Sep-Pak column. Theobtained 0.5 ml was added with 0.2 ml of 25:75acetonitrile: buffer (25 mM ammonium formate pH 4.5),and the solution was used for injection in LCMS.Furthermore, the filtration was conducted using amembrane filter of 0.45 m cellulose acetate, and Table 1showed the LCMS specifications.Table 1. Specifications of LC-MSLC-MS apparatusmodelColumnInjection volumeCapillary voltageColumntemperatureMobile phasemodeFlow rateSampling coneSolventMS focused ionmodeCollison energyDesolvation nizationScanningSourcetemperatureRun timeLCMS methodShimadzu LC-MS – 8040LC/MSShimadzu Shim Pack FC-ODS(2 mm x 150 mm, 3 µm)1 µl3,0 kV35 CIsocratic0,5 ml/min23,0 VEthanol 95%Io type [M] 5,0 V60 ml/hr350 CLow energy CIDESI0,6 sec/scan (mz: 10-1000)100 C80 minutesLCMS method3. RESULTSTable 2 showed the analysis of genitri seed alkaloidsusing the LC-MS method, with specifications. Inaddition, it showed the composition of each compoundbased on the analysis of the alkaloid group using theLC-MS method of genitri seeds.Table 2. Results of LC-MS analysis of compounds from the seed alkaloid group Elaeocarpus sphaericus SchumPeaknumberRT(min)Similarityindex (%)Curve areaComposition(%)Compound Result156,40992605,718860,90496elaeokanine C168,034921068,82281,59685( )-elaeocarpineAnalysisStructure121

Advances in Social Science, Education and Humanities Research, volume 630PeaknumberRT(min)Similarityindex (%)Curve nd ine tureisoelaeocarpiline218,322grandisine ine F122

Advances in Social Science, Education and Humanities Research, volume 630PeaknumberRT(min)Similarityindex 4561,13645grandisine E299,9792831,555371,24237habbemine A309,97392976,886861,45949habbemine B3410,5292568,088280,84874grandisine G92Curve area596,84565Composition(%)0,891704. DISCUSSIONThe analysis results of the alkaloid group using theLC-MS method of genitri seeds showed differentcompounds, namely Elaeokanine C, ( )-elaeocarpine,Elaeocarpenine, Isoelaeocarpine, Grandisine B,Isoelaeocarpiline, Grandisine D, Elaeocarpidine,Compound ResultAnalysisStructuregrandisine Agrandisine CIsoelaeocarpicine, Grandisine F, Grandisine A,Grandisine C, Grandisine E, Habbemine A, HabbemineB, Grandisine G. Alkaloids are one of the activecompounds in genitri seeds, and the results of theLCMS analysis showed that the highest content wasIsoelaeocarpicine compound, which is about 1.89%.Figure 1 showed the structural analysis ofIsoelaeocarpicine, with a chemical formula ofC16H21NO3 and a molecular weight of 275.3480.123

Advances in Social Science, Education and Humanities Research, volume 630Figure 1. Isoelaeocarpicine, one of the alkaloid compounds in genitri seeds, indicated by the adsorption value andchemical structureLiquid Chromatography-Mass Spectrometry (LCMS) is an analytical technique that combines raphy with the detection specificity of massspectrometry [16]. It separates the components of thesample and the charged ions are detected by a massspectrometer, and the result can be used to provideinformation about the molecular weight, structure,identity, and quantity of certain sample components[17]. Furthermore, the compounds are separated basedon their relative interaction with the chemical layer ofthe particles (stationary phase) and the elution of thesolvent through the column (mobile phase). Thismethod can analyze a wider range of components, suchas thermolabile compounds, high polarity or highmolecular mass, and proteins. The font in the rowheader should be bold and you can use the styleavailable from the style palette.Elaeocarpidine1.03%, Isoelaeocarpicine 1.89%,Grandisine F 1.39%, Grandisine A 0.89%, GrandisineC 0.98%, Grandisine E 1.14%, Habbemine A 1.24%,Habbemine B 1.46%, Grandisine G 0.85%.Alkaloids are one of the secondary metabolites ingenitri seeds, and they have medicinal plants ynchophylla have been identified as an anti-denguevirus [20]. These compounds can inhibit the growth ofStaphylococcus aureus and Pseudomonas aeruginosabacteria in Callistemon citrinus and Vernonia adoensisleaves [19]. Previous studies showed that alkaloidcompounds have immunomodulatory properties [22].[2] S. Jain, K. Jatwa, V. Jain, A. Sharma, S.C.Mahajan, A review on elaeocarpus sphaericus(Rudraksha). PharmaTutor, vol. 2(7), 2014, pp. 8391.5. CONCLUSIONA phytochemical analysis is an important factor usedto determine the components of chemical compounds inplants. Furthermore, Liquid Chromatography-MassSpectrometry (LC-MS) was used to analyze the alkaloidcompounds of genitri seeds, and different compoundswere obtained, namely Elaeokanine C 0.90%, ( )elaeocarpine1.59%, Elaeocarpenine1.88%,Isoelaeocarpine 1.36%, Grandisine B0.86%,Isoelaeocarpiline 1.14%, Grandisine D1.51%,ACKNOWLEDGMENTSThank you as well to Ministry of Research,Technology and Higher Education of Indonesia forsupporting in research funding.REFERENCES[1] M. Pant, A. Lal, P. Bisht, A. Rani, Elaeocarpussphaericus: A tree with curative powers: anoverview. Research Journal of Medicinal 0.3923/rjmp.2013.23.31[3] S. Hardainiyan, B.C Nandy, K. Kumar,Elaeocarpus ganitrus (Rudraksha): A reservoirplant with their pharmacological effects. Int JPharm Sci Rev Res, vol. 34(1), 2015, pp. 55-64.[4] D.H. Geetha, M. Rajeswari, I. Jayashree, Chemicalprofiling of Elaeocarpus serratus L. by GCMS. AsianPacificjournaloftropicalbiomedicine, vol. 3(12), 2013, pp. 985-987. 5] N.T Dao, Y. Jang, M. Kim, H.H. Nguyen, et.al.,Chemical Constituents and Anti-influenza ViralActivity of the Leaves of Vietnamese PlantElaeocarpus tonkinensis. Records of NaturalProducts, vol. 13(1), 2019, pp. 71-80. DOI:https://doi.org/10.25135/rnp.76.18.04.266124

Advances in Social Science, Education and Humanities Research, volume 630[6] S. Jawla, D.V Rai, Pharmacognostic Studies onRudraksh (Elaeocarpus angustifolius Blume)Fruit. Advances in Biological Research, vol.10(6),2016, pp. 382-387.[7] P.K. Jain, P. Sharma, S.C Joshi, Effect ofelaeocarpus ganitrus roxb. On kidney function ofcholesterol fed rabbits. Advances in Pharmacologyand Toxicology, vol. 18(2), 2017, pp. 1-9.[8] A.L. Manoharan, S. Thamburaj, K. Muniyandi,et.al., Antioxidant and antimicrobial investigationsof Elaeocarpus tectorius (Lour.) Poir. fruits againsturinary tract infection pathogens. Biocatalysis andAgricultural Biotechnology, vol. 20, 2019, 101260.DOI: https://doi.org/10.1016/j.bcab.2019.101260[9] N. Bribi, Pharmacological activity of alkaloids: areview. Asian Journal of Botany, vol. 1(1), 2018.[10] J. Himanshu, Jadon, G., Bhadauria, R. S., &Kishor, A. A review article on: plant alkaloids,International Journal of Pharmaceutical Erudition,vol. 9(4), 2020, pp. 1-5.[11] P.B. Saxena, Chemistry of alkaloids. DiscoveryPublishing House, 2007.[12] C.P. Kumar, J. Sachin, Pharmacological action ofplant alkaloids in female reproductive system oftest animals and/or human beings: A review. Int. J.Pharm. Sci. Rev. Res, vol. 23(2), 2013, pp. 98-107.[19] D. Mabhiza, T. Chitemerere, S. Mukanganyama,Antibacterial Properties of Alkaloid Extracts fromCallistemon citrinus and Vernonia adoensis a. International Journal of MedicinalChemistry, 2016.DOI:https://doi.org/10.1155/2016/6304163[20] T. Hishiki, F. Kato, S. Tajima, et.al., Hirsutine, anindole alkaloid of uncaria rhynchophylla, inhibitslate step in dengue virus lifecycle. Frontiers inmicrobiology, vol. 8, 2017, 1674. DOI:https://doi.org/10.3389/fmicb.2017.01674[21] W. Wang, X. Yang, Y. Chen, et.al., Seneciphylline,a main pyrrolizidine alkaloid in Gynura japonica,induces hepatotoxicity in mice and primaryhepatocytes via activating mitochondria‐mediatedapoptosis. Journal of Applied Toxicology, 0.1002/jat.4004[22] D.S. Senchina, J.E. Hallam, M.L. Kohut, et.al.,Alkaloids and athlete immune function: caffeine,theophylline, gingerol, ephedrine, and theircongeners. Exercise immunology review, vol. 20,2014, pp. 68-93[13] K.L. Kohnen-Johannsen, O. Kayser, Tropanealkaloids: chemistry, pharmacology, biosynthesisand production. Molecules, vol. 24(4), 2019, 96[14] K.P. Ingle, A.G. Deshmukh, D.A. Padole, et.al.,Phytochemicals: Extraction methods, identificationand detection of bioactive compounds from plantextracts. JournalofPharmacognosyandPhytochemistry, vol. 6(1), 2017, pp 32-36.[15] W.M.A. Niessen, Liquid chromatography-massspectrometry. Crc Press, 2006.[16] P.R. Kumar, S.R. Dinesh, R. Rini, LCMS-a reviewand a recent update. J Pharm Pharm Sci, vol. 5(5),2016, pp. 377-391.[17] N. Zhang, S.T. Fountain, H. Bi, D.T Rossi,Quantification and rapid metabolite identificationin drug discovery using API time-of-flightLC/MS. Analytical chemistry, vol. 72(4), 2000, pp.800-806. DOI: https://doi.org/10.1021/ac9911701[18] D.E. Okwu, F.U. Nnamdi, CannabinoidDronabinol alkaloid with antimicrobial activityfrom Cassia alata Linn. Der Chemica Sinica, vol.2(2), 2011, pp. 247-254.125

Liquid Chromatography-Mass Spectrometry (LC-MS) is an analytical technique that combines the physical separation capabilities of liquid chromatography with the detection specificity of mass spectrometry [16]. It separates the components of the sample and the charged ions are detected by a mass

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