Identification of metabolite compounds from ethanolic extract of the Red Gedi Leaves (Abelmoschus manihot L. Medik) by LC-ESI-MS

 

Juliet Tangka^1,2, Elisabeth N. Barung², Diana Lyrawati³*, Djoko W. Soeatmadji⁴, Nurdiana⁵

¹Doctoral Program of Medical Science, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia.

²Department of Pharmacy, Politeknik Kesehatan, Kementerian Kesehatan Manado, Manado, Indonesia.

³Department of Pharmacy, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia.

⁴Department of Internal Medicine, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia.

⁵Department of Pharmacology, Faculty of Medicine, Universitas Brawijaya, Malang, Indonesia.

 

ABSTRACT:

Abelmoschus manihot L. Medic, commonly called ‘‘red gedi’’, is an endemic species of Minahasa, Indonesia. The leaves of red gedi have been widely used in ethnomedicine and functional food as an antidiabetic.  In this study, the ethanolic extract of the red gedi leaves was characterized by using liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (LC–ESI-MS). Compounds identified were phenolic acid derivates, flavonoids, terpenoids, phytosterols, alkaloids, and lignans. The most abundant flavonoids in the extract sample were quercetin derivatives. In total, 38 metabolite compounds were identified in red gedi leaves and were reported for the first time, including alpha spinasterol which is newly identified in this particular Abelmoschus species.

 

1. INTRODUCTION:

The Abelmoschus manihot. L Medik) from Malvaceae grows endemically in North Sulawesi and known as “gedi”. The leaves of gedi empirically used by the locals to manage cardiovascular disease as an antidiabetic, antihypertension and anticholesterolemia ^{1, 2, 3}. Others reported some active flavonoids derived from the flowers and the whole plants of gedi used as traditional medicine^{4, 5}. However, little is known about the metabolite compounds of the gedi leaves and their pharmacologic activities, in particular the red gedi. The leaves of gedi has been reported to contain β-sitosterol⁵, eikodekana⁶, and heptadecanoic acid⁷. The flavonoid identified from ethyl acetate extract of gedi leaves were flavonoids of auron class i.e. 3’,4,6-trihydroxy, 4-alkoksi auron with functional groups aliphatic C-H, free -OH, alcohol C-O, aromatic C=C, aromatic C-He, ether C-O and dan C=O also substituted OH at C-4, C-6 and C-3’ as well as OR at C-4 as reported by Theodora⁸.

 

Ethanolic (96%) extract of gedi contained total flavonoid 41.56%⁹.  Specifically in red gedi, it was reported that the total phenolic, flavonoids, and tanin were 1003 mg/kg, 722 mg/kg and 1029 mg/kg, respevtively². In this study, we aimed to identify the metabolite compounds of the red gedi leaves following etanol extraction using liquid chromatography coupled with mass spectrometry.

 

2. MATERIAL AND METHODS:

2.1. Plant material:

The leaves of red gedi Abelmoschus manihot L. Medik were collected during August 2018 from North Tondano Plantation, North Sulawesi, Indonesia.  Determination of the plants were performed at the Center for Plant Conservation Botanic Gardens, Indonesian Institute of Sciences, Bogor, Indonesia (Letter B-3177/IPH.3./KS/IX/2018 on September 18, 2018).

 

2.2. Chemical and reagents:

Formic acid and methanol were obtained from Merck KGaA (Darmstadt, Germany), used for chromatographic analysis. All solutions were prepared with ultra-pure water (Millipore, MA, USA). The other solvents and reagents in this study were of analytical grade.

 

2.3. Extraction:

The extract was obtained by dynamic maceration extraction technique using 96% ethanol solvent. Fifty grams of the dried red leaves Abelmoschus manihot L. Medic, put into a 1-liter Erlenmeyer, then added 500 mL 96% ethanol (1:10) and placed on a digital hot plate magnetic stirrer. Extraction was performed at 30 to 40⁰C, stirred at 200 rpm for 6 hours. Macerate was collected by filtering the solution and transferred into a closed vessel. The macerate was then evaporated first at 50⁰C using a rotary evaporator, followed by oven evaporation at 80⁰C until a thick extract obtained without the smell of ethanol. The final extract was refrigerated and stored until used for further experiments.

 

2.4. Identification of active compounds:

Characterization of compounds of ethanolic extract of red leaves Abelmoschus manihot L.Medik was conducted using an  UHPLC system connected to a Triple Quadrupole Mass Spectrometer LCMS-8040 (Shimadzu, Kyoto, Japan). The specific configuration included LabSolutions Ver. 5.00 Chromatography Workstation, using a column Shimadzu Shim-pack FC-ODS III (2.0 mm (I.D.) x 150 mm,3 μm), mobile phase A solution containing 0.1% formic acid in water, mobile phase B absolute methanol. Separation of compounds was carried out with gradient elution profile 0/0 at 0 min, 15:85 at 5 min, 20:80 at 20 min, 90:10 at 24 min. Mass spectra were simultaneously acquired using electrospray ionization (ESI) in the positive ionization modes, and full-scan mass spectra were acquired at a mass-to-charge ratio (m/z) of 50–1000. Fragmentation method used was at low energy CID with flow rate 0.5 mL/min; column temperature 35°C; and injection volume 1 μL. Other settings were used at values obtained by automatic adjustment.

 

Primary raw data of LC-ESI-MS assay was analyzed by data alignment, peak findings, peak integration, and retention time (Rt) correction using a LabSolutions Ver. 5.00 Chromatography Workstation, compared with commercial compounds based on the NIST/EPA/NIH mass spectral library.

 

3. RESULTS AND DISCUSSION:

3.1. Identification of metabolite compounds:

In this study, we identified in total 38 phenolic- and non-phenolic compounds of ethanolic extract red gedi leaves Abelmoschus manihot L.Medik, based on retention time and the LC-ESI-MS profiles (Figure 1 and Table 1).  Identification of major peaks was based on their molecular mass¹⁰. Chemical formulas were identified based on m/z precursor and fragment ions. Spectra data were compared to that of the NIST/EPA/NIH mass spectral library. The LC-ESI-MS and similar methods based on chromatographic profiles are commonly used for compounds identification^11-14.

 

Fig.1. LC-MS/MS peak chromatograms (positive ion mode) of the ethanolic extract of red gedi leaves Abelmoschus manihot L.Medik.

The peaks are labeled according to the compounds listed in Table 1.

 

Identified phenolic compounds from ethanolic extract of red gedi were of 6 phenolic acids and 14 flavonoids. The phenolic acids were two hydroxybenzoic acid derivates (vanilic acid, ficusol) dan four hydroxycinnamic acid derivates (p-coumaric acid, caffeic acid methyl ester, ferulic acid and syringic acid). The flavonoids were of mostly as flavonols (quercetin, myricetin, gossypetin, hibiscetin, glycoside –O-, hyperin, kaempferitrin and isoquercetin) and some of anthocyanin flavonoids (sambicyanin and cyanidin 3-O-rutinoside).

 

The non-phenolic compounds identified in the ethanolic extract of red gedi leaves were many. They were 1 of hydroxybutanedioic acid (malic acid), 9 terpenoids, 2 alkaloids (moupinamide, daphniphylline), two steroids (α-spinasterol, stigmasterol) and 4 lignans. The terpenoids were of sesquiterpenes (farnesol, hibiscone A, B, gmelofuran, hibisquinone A) and triterpenoids (myriceric acid A, B, C and hibicusin). The lignans were boehmenan, erythrocarolignan E, coumarinolignan (aquillochin), and phenylpropanoidlignan (syringaresinol).  All the compounds in red gedi leaves of the species Abelmoschus manihot L.Medik were for the first time identified and reported in this study.

 

The identified compounds were not all uniquely found in in red gedi leaves. For instance, quercetin (compound 13) identified based on peak m/z 302.0427 and characteristic peak at m/z 303.0460, 304.0469 and 304.0494 were also found in other species. Similar peaks were reported in extract of other plants including Capparis spinosa^{14, 15}. The quercetin derivatives (compound 22, 23 and 24), i.e., quercetin-3-O-rhamnoside, hyperin and isoquercetin respectively, were also reported by others earlier^{14, 16-18}. The quercetin-3-O-rhamnoside (compound 22), showed precursor ion at peak m/z 448,1006, however, was identified first time in this genus.

 

Several identified compounds of gedi leaves may explain the antidiabetic properties of the extract. Further studies using in silico and in vitro, followed by in vivo studies, as commonly performed ^19-23, will serve as pharmacological evidence of such properties.

 

Table 1. Peak assignment of metabolites in ethanolic extract of red gedi leaves Abelmoschus manihot L.Medik using LC-ESI-MS in positive ion mode

Peak No	RT (min)	Molecular Formula	Calculated mass (M)	Experimental mass [M+H]+  m/z	Tentative Identified Compounds	PubChem CID
1	1.473	C6H6O5	134.0215	135.0249	Malic acid	525
2	1.839	C9H8O3	164.0473	165.0507	p-Coumaric acid	637542
3	2.799	C8H8O4	168.0423	169.0456	Vanillic acid	8468
4	5.043	C10H10O4	194.0579	195.0613	Ferulic acid	445858
5	5,044	C10H10O4	194.0579	195.0613	Caffeic acid methyl ester	689075
6	5.177	C9H10O5	198.0528	199.0562	Syringic acid	10742
7	5.967	C15H26O	222.1984	223.2017	Farnesol	445070
8	7.338	C11H14O5	226.0841	227.0875	Ficusol	100955863
9	7.616	C15H20O2	232.1463	233.1497	Hibiscone A	102239770
10	8.004	C15H18O3	246.1256	247,1289	Gmelofuran	156117
11	8.011	C15H20O3	248.1412	249.1446	Hibiscone B	102090463
12	8.246	C15H14O4	258.0892	259.0926	Hibiscoquinone A	442745
13	11.427	C15H10O7	302.0427	303.0460	Quercetin	5280343
14	11.502	C18H19NO4	313.1314	314.1348	Moupinamide	5280537
15	11.514	C15H10O8	318.0376	319.0409	Myricetin	5281672
16	11.518	C15H10O8	318.0376	319.0409	Gossypetin	5280647
17	12.038	C15H10O9	334.0325	335.0358	Hibiscetin	15559735
18	15.635	C29H48O	412.3705	413.3739	α-Spinasterol	5281331
19	15.638	C29H48O	412.3705	413.3739	Stigmasterol	5280794
20	17.423	C21H20O9	416.1107	417.1141	Aquillochin
21	17.456	C22H26O8	418.1628	419.1661	Syringaresinol	443023
22	22.616	C21H20O11	448.1006	449.1039	Quercetin -3 –O- rhamnoside	5353915
23	24.027	C21H19O12	463.0882	464.0916	Hyperin	5281643
24	24. 032	C21H20O12	464.0955	465.0988	Isoquercetin	5280804
25	24.762	C30H48O4	472.3553	473.3586	Myricerol
26	25.891	C21H20O13	480.0904	481.0937	Gossypetin -3-glucoside	44259979
27	25.913	C21H20O13	480.0904	481.0937	Myricetin-3-glucoside	44259426
28	25.961	C21H20O14	496.0853	497.0887	Hibiscetin-3-glucoside	44259992
29	29.67	C32H49NO5	527.3611	528.3644	Daphniphyllin	21627122
30	33.483	C27H30O14	578.1636	580, 1678	Kaempferitrin	5486199
31	33.569	C27H31O15+	581.1501	582.1535	Sambicyanin	44256719
32	34.063	C27H31O15+	595.1657	596.1691	Cyanidin-3-O-rutinoside	441674
33	36.983	C39H52O7	632.3713	633.3747	Myriceric acid A
34	37.042	C39H54O7	634.3870	635.3903	Myriceric acid B	15767724
35	46.229	C40H40O12	712.2520	713.2553	Boehmenan	5274624
36	46.253	C40H42O13	730.2625	731.2659	Erythro Carolignan E	5274622
37	49.702	C48H60O9	780.4237	781. 4271	Hibicusin	5274618
38	49.883	C48H60O10	796.4186	797,4220	Myriceric acid C	15767725

 

 

4. CONCLUSION:

In this study, the ethanolic extract of the red gedi leaves was characterized by using liquid chromatography coupled to electrospray ionization-tandem mass spectrometry (LC–ESI-MS). Compounds identified were phenolic acid derivates, flavonoids, terpenoids, phytosterols, alkaloids, and lignans. In total, 38 metabolite compounds were identified in red gedi leaves and were reported for the first time, including alpha spinasterol which is newly identified in this particular Abelmoschus species. The most abundant flavonoids in red gedi extract were quercetin derivatives. This study may serve as the basis for further research elucidating the pharmacological activities underlying the use of the plant as traditional medicine for cardiovascular disease.

 

5. ACKNOWLEDGMENTS:

The authors would like to thank Dr. Didik Widyatmoko, M.Sc. from Center for Plant Conservation Botanic Gardens – Indonesia Institute of Sciences, Bogor, Indonesia for determination of the red gedi leaves, and Head of Laboratories at Faculty of Mathematics and Natural Sciences of Universitas Brawijaya and Faculty of Pharmacy Universitas Muhammadiyah, Malang, Indonesia for allowing the LC-ESI-MS works done in their facilities. This study was funded partly by DIPA for Development of Health Human Resources, Ministry of Health, Republic of Indonesia.

 

6. REFERENCES:

1.     Sarwar M, Attitalla IH, Abdollahi M. A review on the recent advances in pharmacological studies on medicinal plants; animal studies are done but clinical studies needs completing. Asian Journal of Animal and Veterinary Advances. 2011;6:867-883 doi.org/10.3923/ajava.2011.867.883

2.     Suoth E, Kaempe H, Tampi A. Evaluasi kandungan total polifenol dan isolasi senyawa flavonoid pada daun gedi merah (Abelmoschus manihot l.),. Chemistry Progress. 2013;6:86-91 doi.org/10.35799/cp.6.2.2013.3500

3.     Nurrani L, Kinho J. Utilization of natural plant by the North Sulawesi community as a lowering of diabetic. International Conference on Forest and Biodiversity. 2013:443-452

4.     Luan F, Wu Q, Yang Y, Lv H, Liu D, Gan Z, et al. Traditional uses, chemical constituents, biological properties, clinical settings, and toxicities of Abelmoschus manihot l.: A comprehensive review. Frontiers in pharmacology. 2020;11:1068 doi.org/10.3389/fphar.2020.01068

5.     Mamahit L. Satu senyawa steroid dari daun gedi (Abelmoschus manihot l. Medik) asal sulawesi utara. Chemistry Progress. 2009;2:33-38

6.     Mamahit L. Eikodekana dari daun tumbuhan gedi (Abelmoschus manihot l. Medik) asal sulawesi utara. Chemistry Progress. 2009;2:122-125 doi.org/10.35799/cp.2.2.2009.4972

7.     Mamahit LP, Soekamto NH. Satu senyawa asam organik yang diisolasi dari daun gedi (Abelmoschus manihot l. Medik) asal sulawesi utara. Chemistry Progress. 2010;3:42-45 doi.org/10.35799/cp.3.1.2010.73

8.     Theodora CT, Gunawan IWG, Swantara IMD. Isolasi dan identifikasi golongan flavonoid pada ekstrak etil asetat daun gedi (Abelmoschus manihot l.). Jurnal Kimia (Journal of Chemistry). 2019:131-138 doi.org/10.24843/JCHEM.2019.v13.i02.p02

9.     Pine ATD, Alam G, Attamimi F. Standardisasi mutu ekstrak daun gedi ( Abelmoschus manihot (l.) medik) dan uji efek antioksidan dengan metode dpph JurnalFarmasi. 2015;3:111-128 doi.org/10.24252/jfuinam.v3i3.2214

10.  Hemchand S, Babu RRC, Annapurna MM. Separation, characterization and quantification of impurities and identification of stress degradants of cabazitaxel by RP-HPLC and LC-ESI-MS techniques. Research J. Pharm. and Tech. 2018;11:4683-4698 doi.org/10.5958/0974-360X.2018.00857.0

11.  Anandan A, Eswaran R, A.Doss, Sangeetha G, Anand SP. Investigation for bioactive compounds of Aerva lanata (l.) juss. Ex schultes. Asian J. Research Chem. 2012;5:469-471

12.  Hemchand S, Babu RRC, Annapurna MM. Separation, characterization and quantification of impurities and identification of stress degradants of cabazitaxel by RP-HPLC and LC-ESI-MS techniques. Research J. Pharm. and Tech. 2018;11:4683-4698 doi.org/10.5958/0974-360X.2018.00857.0

13.  Mohanasrinivasan V, D S, V S, R K, A. N. Studies on bioactive compounds and therapeutic potential of Terminalia chebula seed extract. Research J. Pharm. and Tech. 2018;11:1889-1893 doi.org/10.5958/0974-360X.2018.00351.7

14.  Prakash O, Mahapatra DK, Singh R, Singh N, Ved. NVA. Development of a new isolation technique and validated RP-HPLC method for quercetin and kaempferol from Azadirachta indica leaves. Asian J. Pharm. Ana. 2018;8:164-168 doi.org/10.5958/2231-5675.2018.00030.3

15.  Bakr RO, Bishbishy MHE. Profile of bioactive compounds of Capparis spinosa var. Aegyptiaca growing in Egypt. Revista Brasileira de Farmacognosia. 2016;26:514-520 doi.org/10.1016/j.bjp.2016.04.001

16.  Crupi P, Genghi R, Antonacci D. In-time and in-space tandem mass spectrometry to determine the metabolic profiling of flavonoids in a typical sweet cherry (Prunus avium l.) cultivar from southern Italy. Journal of Mass Spectrometry. 2014;49:1025-1034 doi.org/10.1002/jms.3423

17.  Felipe DF, Brambilla LZ, Porto C, Pilau EJ, Cortez DA. Phytochemical analysis of Pfaffia glomerata inflorescences by LC-ESI-MS/MS. Molecules (Basel, Switzerland). 2014;19:15720-15734 doi.org/10.3390/molecules191015720

18.  Ding S, Dudley E, Plummer S, Tang J, Newton RP, Brenton AG. Quantitative determination of major active components in Ginkgo biloba dietary supplements by liquid chromatography/mass spectrometry. Rapid communications in mass spectrometry : RCM. 2006;20:2753-2760 doi.org/10.1002/rcm.2646

19.  Das K, Krishna P, Sarkar A, Ilangovan SS, Sen S. A review on pharmacological properties of Solanum tuberosum. Research J. Pharm. and Tech. 2017;10:1517-1522 doi.org/10.5958/0974-360X.2017.00267.0

20.  Pattan SR, Dighe NS, Musmade DS, Gaware VM, Jadhav S, Khade MA, et al. Synthesis and antidiabetic activity of some new benzopyrone derivatives. Asian J. Research Chem. 2010;3:126-128

21.  Shah BR, Sen DJ, Bahekar R. Design, synthesis and pharmacological evaluation of potent and selective dipeptidyl-derived inhibitors as new class of antidiabetic drugs. Asian J. Research Chem. 2011;4:50-54

22.  Suganya J, Radha M, Manoharan S, V V, Francis A. Virtual screening and analysis of bioactive compounds of Momordica charantia against diabetes using computational approaches. Research J. Pharm. and Tech. 2017;10:3353-3360 doi.org/10.5958/0974-360X.2017.00596.0

23.  Usman, M MA, Erika F, Nurdin M, Kuncoro H. Antidiabetic activity of leaf extract from three types of mangrove originating from sambera coastal region Indonesia. Research J. Pharm. and Tech. 2019;12:1707-1712 doi.org/10.5958/0974-360X.2019.00284.1

 

 

Accepted on 22.02.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2022; 15(11):5164-5167.