Int J App Pharm, Vol 18, Issue 1, 2026, 421-431Original Article

GC-MS (GAS CHROMATOGRAPHY-MASS SPECTROMETRY) ANALYSIS SEED KEBIUL (CAESALPINIA BONDUC (L) ROXB)

DENSI SELPIA SOPIANTI1,6*, MUCHSIN DOEWES1,2, TATAR SUMANDJAR1,3, ETI PONCORINI PAMUNGKASARI1,4, PARAMASARI DIRGAHAYU1,5, RATIH PUSPITA FEBRINASARI1,2

1*Doctoral Program of Medical Sciences, Faculty of Medicine, Universitas Sebelas Maret, Surakarta, Indonesia. Jalan Ir. Sutami 36A Ir. Sutami 36A Surakarta Jawa Tengah Indonesia 57126,, Indonesia. 3Department of Internal Medicine, Faculty of Medicine, Universitas Sebelas Maret, Surakarta, Indonesia. 4Department of Public Health, Faculty of Medicine, Universitas Sebelas Maret, Surakarta, Indonesia. 5Department of Parasitology and Mycology, Faculty of Medicine, Universitas Sebelas Maret, Surakarta, Indonesia. 6Department of Pharmacy, Sekolah Tinggi Kesehatan Al-Fatah, Bengkulu, Indonesia
*Corresponding author: Densi Selpia Sopianti; *Email: dselpias@gmail.com

Received: 16 Jun 2025, Revised and Accepted: 15 Oct 2025

ABSTRACT

Objective: Kebiul (Caesalpinia bonduc (L) Roxb) is one of the medicinal plants that plays an important role in treating various diseases, but this plant is vulnerable to extinction. This study aims to explore the possibility of various biological compounds in the ethanol extract of the seed kernel and seed coat of kebiul using gas chromatography-mass spectrometry (GC-MS) analysis.

Methods: The seed coat was removed from the kernel of the kebiul seed. After being ground into a powder, the seed kernel and seed coat were dried in an oven set to 40 °C for 1 h, and grinding them into a fine powder. The powder was soaked in 70% ethanol at a 1:10 (w/v) ratio for 3 days at room temperature, with occasional stirring. The chemical composition of secondary metabolite in the ethanol extracts of both the seed kernel and seed coat of kebiul was analyzed using gas chromatography–mass spectrometry (GC-MS) instrumentation (ID ISQD1702517_1) at a temperature of 25.30 °C and humidity of 35%.

Results: In this GC-MS analysis, 87 bioactive phytochemical compounds were identified in the ethanol extract of seed kernel and 50 bioactive phytochemical compounds were identified in the ethanol extract of seed coat. Seven chemicals in the seed kernel ethanol extract obtained the highest relative peak area (>2%) from each chromatogram, with the highest compound at a retention time of 19.93 min, namely 6-Octadecenoic acid, and three compounds in the seed coat ethanol extract obtained the highest relative peak area (>2%) in this investigation, with the highest compound at a retention time of 19.65 min, namely 9,12-Octadecadienoic acid (Z,Z)-.

Conclusion: GC-MS analysis identified 87 phytochemical compounds were identified in the ethanol extract of kebiul seed kernel and 50 phytochemical compounds were identified in the ethanol extract of kebiul seed coat.

Keywords: Active compound, Caesalpinia bonduc (L) Roxb, GC-MS analysis

© 2026 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijap.2026v18i1.55764 Journal homepage: https://innovareacademics.in/journals/index.php/ijap

INTRODUCTION

In traditional medicine, various parts of the plant are consumed alone and formulated as polyherbs, along with other medicinal plants, which are further used to treat various diseases [1]. Kebiul seeds are bulging oblong pods measuring 5 to 7.5 cm, covered with spines, and containing one or two seeds per pod. The pods have short stems, and the seeds are 1-2 cm in size, round, hard, and bluish-gray in color with a smooth shiny surface [2]. All parts of the plant (roots, leaves, seeds, bark, and stem) have been used as herbal medicine for various diseases such as antipyretic, antiperiodic, anthelmintic, antibacterial, hydrocele, antioxidant, antitumor, and antidiabetic and also used for the treatment of skin diseases such as leprosy, as well as used for the treatment of paralysis and nervous complaints [2-4].

Medicinal plants are more prone to extinction [5]. However, the ability to regenerate entire plants from epicotyl explants using callus induction and shoot regeneration methods has been demonstrated. This provides an efficient and reproducible protocol for the regeneration of ethnomedicinal plants, which is important for in vitro propagation and large-scale species conservation [6]. Therefore, identifying the active ingredients in traditional medicinal plants used as therapeutic drugs is necessary for scientific validation purposes [7]. Gas Chromatography-Mass Spectrometry (GC-MS) has become a popular technique to identify volatile and non-volatile compounds in plant species. Many previous studies found phytochemical compounds in plant components using the GC-MS method [8]. There is currently relatively little scientific data on the characterization of chemicals found in seed kernels and seed coat kebiul. This study is an early attempt to use the GC-MS method to determine the secondary metabolite profile, which can reveal additional details about the distribution of bioactive chemicals in both seed sections. The main objective of this study is to identify the biological compounds present in the seed kernel and seed coat of kebiul, which are extracted using 70% ethanol and analyzed with the GC-MS method, allowing for their potential to be assessed through in vitro and in vivo tests aimed at discovering new compounds that may have therapeutic applications. Using different methods and solvents is expected to produce different compounds and concentrations.

MATERIALS AND METHODS

Sample preparation

Kebiul seeds from woods on Sumatra Island, Indonesia's Bengkulu province, served as the samples. At the General Hospital's Tawangmangu Traditional Health Service Functional Service Unit, tests for plant determination were conducted. Test report number TL.02.04/D. XI.6/22566.1061/2024, with synonym Caesalpinia bonduc (L) Roxb, was submitted by Dr. Sarjito of Karanganyar Regency, Central Java. After being separated into seed kernels and seed coats, the sample was ground into a powder. The seed coat was removed from the kernel of the kebiul seed. After being ground into a powder, the seed kernel and seed coat were dried in an oven set to 40 °C for 1 h, and grinding them into a fine powder. The powder was soaked in 70% ethanol at a 1:10 (w/v) ratio for 3 days at room temperature, with occasional stirring. After maceration, the mixture was filtered using whatman No. 1 filter paper. The residue was re-macerated twice under the same conditions. All filtrates were pooled and evaporated under reduced pressure at 40 °C using a rotary evaporator. The resulting extract was then stored in a sealed container at 4 °C for subsequent analysis.

Instrument GC-MS

This research is an exploratory study using ethanol extract from the seed kernel and seed coat of kebiul. The chemical composition of secondary metabolite in the ethanol extracts of both the seed kernel and seed coat of kebiul was analyzed using gas chromatography–mass spectrometry (GC-MS) instrumentation (ID ISQD1702517_1) at a temperature of 25.30 °C and humidity of 35%. The analysis was performed using a capillary column HP-5MS (30 m length × 0.25 mm internal diameter × 0.25 µm film thickness). Helium was used as the carrier gas at a constant flow rate of 1.0 ml/min. The injection volume was 1 µl** with a split ratio of 10:1. The GC oven temperature was programmed as follows: initial temperature 50 °C (held for 2 min), ramped to 280 °C at 10 °C/min, and held at 280 °C for 10 min. GC-MS analysis was conducted using electron ionization at 70 eV, scanning m/z values from 40 to 600. Detector and injector were maintained at 280 and 250 °C, respectively. Then, the compound identification was performed by comparing retention indices and mass spectra with entries in the National Institute of Standards and Technology (NIST) library database, as described [9].

RESULTS

By employing the maceration method to produce 70% ethanol extract of the seed kernel and seed coat of kebiul, 29.99 g of ethanol extract of the seed kernel with a yield of 2.14% and 26.81 g of ethanol extract of the seed coat of kebiul with a yield of 2.6% were obtained. The chromatogram of the kebiul seed kernel, along with its corresponding retention periods and peaks, was obtained by GC-MS analysis and is shown in fig. 2 and table 1.

a

b

c

Fig. 1: Preparation of seed kernel and seed coat of kebiul. a. seeds, b. seed coat, c. seed kernel of kebiul

Fig. 2: Chromatogram of ethanol extract of kebiul seed kernel by GC-MS

Fig. 2 shows the chromatogram identified in the ethanol extract of the seed kernel. Individual compounds were identified by comparing their mass spectra database with the main library alliance, ensuring the listed spectra had a Similarity Index (SI) of more than 80% (NIST), and by comparing them with values published in the literature. The GC-MS analysis indicated that 87 phytochemical compounds were identified in the ethanol extract of seed kernels (tables 1 and 2).

Table 1: Compound of ethanol extract of kebiul seed kernel by GC-MS

No.

Ret. time

min

 Hit# 1 Chemical formula Mol. weight SI Hit#1 Rel. area %

Area

counts*min
1 4,38 Sec-Butyl nitrite C4H9NO2 103 652 0,05 12356481,038
2 4,78 2-Nitro-1-buten-3-ol C4H7NO3 117 686 0,09 21175346,480
3 4,87 Isoxazolidine-3,5-dicarboxylic acid, dimethyl ester C7H11NO5 189 678 0,01 1435204,059
4 5,05 N-Isopentyl-N-nitroso-pentylamine C10H22N2O 186 664 0,13 30663445,304
5 5,43 Propanoic acid, 3-ethoxy-, ethyl ester C7H14O3 146 736 0,69 166938220,038
6 5,98 Alpha-l-rhamnopyranose C6H12O5 164 730 0,32 77240815,627
7 6,47 d-Glycero-d-ido-heptose C7H14O7 210 663 0,02 4290477,110
8 6,59 2-Deoxy-D-galactose C6H12O5 164 694 0,04 8648154,764
9 7,14 Isosorbide Dinitrate C6H8N2O8 236 706 0,33 80890210,831
10 7,45 Glycerin C3H8O3 92 739 0,49 118153069,053
11 8,30 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl- C6H8O4 144 842 1,05 254430558,682
12 8,71 a-D-Galactopyranose, 2-(acetylamino)-2-deoxy- C8H15NO6 221 694 0,03 7774744,820
13 9,42 Isosorbide Dinitrate C6H8N2O8 236 747 0,09 22094672,577
14 9,77 5-Hydroxymethylfurfural C6H6O3 126 853 1,29 314249191,294
15 10,15 6-Acetyl-ß-d-mannose C8H14O7 222 710 0,03 8442761,233
16 10,56 Ascaridole epoxide C10H16O3 184 733 0,09 21494313,997
17 10,92 l-Gala-l-ido-octonic lactone C8H14O8 238 712 0,07 16405387,642
18 11,10 Dithiocarbamate, S-methyl-,N-(2-methyl-3-oxobutyl)- C7H13NOS2 191 732 0,05 11323892,739
19 11,47 Melezitose C18H32O16 504 787 0,00 846197,660
20 11,77 Melezitose C18H32O16 504 787 0,15 36047631,307
21 12,43 1-Hexadecanol, 2-methyl- C17H36O 256 746 0,02 5128618,693
22 12,66 Melezitose C18H32O16 504 753 0,09 22299600,463
23 12,98 Melezitose C18H32O16 504 740 0,00 1121755,970
24 13,25 Desulphosinigrin C10H17NO6S 279 745 0,11 26612156,948
25 13,56 Dodecanoic acid, 3-hydroxy- C12H24O3 216 740 0,38 91361996,381
26 13,72 Melezitose C18H32O16 504 741 0,14 33739231,949
27 13,99 2-Myristynoyl pantetheine C25H44N2O5S 484 775 0,01 2514248,128
28 15,52 Ethyl a-d-glucopyranoside C8H16O6 208 850 3,79 919183671,051
29 15,94 Tetradecanoic acid C14H28O2 228 874 0,42 102582161,109
30 16,32 3-O-Methyl-d-glucose C7H14O6 194 761 0,10 23927031,993
31 16,42 3-O-Methyl-d-glucose C7H14O6 194 771 0,16 39138063,552
32 16,57 3-O-Methyl-d-glucose C7H14O6 194 766 0,41 100239213,835
33 16,76 3-O-Methyl-d-glucose C7H14O6 194 677 1,14 276170844,873
34 17,37 Hexadecanoic acid, methyl ester C17H34O2 270 900 0,17 41806131,566
35 17,72 9-Hexadecenoic acid C16H30O2 254 841 0,06 14857838,310
36 18,16 l-(+)-Ascorbic acid 2,6-dihexadecanoate C38H68O8 652 856 8,97 2177601507,849
37 18,75 Ethanol, 2-(9-octadecenyloxy)-, (Z)- C20H40O2 312 850 0,10 25223422,168
38 18,86 n-Hexadecanoic acid C16H32O2 256 817 0,05 13348651,915
39 19,02 9,12-Octadecadienoic acid (Z,Z)-, methyl ester C19H34O2 294 923 0,47 113340275,316
40 19,41 Androsta-4,16-dien-3-one C19H26O 270 714 0,99 240011900,417
41 19,64 9,12-Octadecadienoic acid, ethyl ester C20H36O2 308 886 5,86 1422586259,826
42 19,93 6-Octadecenoic acid C18H34O2 282 877 16,28 3951600262,929
43 20,50 1-Heptatriacotanol C37H76O 536 817 0,04 9685050,657
44 20,77 1-Heptatriacotanol C37H76O 536 825 0,09 22874119,034
45 21,02 Cyclopropanebutanoic acid, 2-[[2-[[2-[(2-pentylcyclopropyl)methyl]cyclopropyl]methyl]cyclopropyl]methyl]-, methyl ester C25H42O2 374 813 0,03 6862248,149
46 21,16 1-Heptatriacotanol C37H76O 536 811 0,05 11870633,118
47 21,29 1-Heptatriacotanol C37H76O 536 784 0,03 6601142,798
48 21,47 4,8,13-Cyclotetradecatriene-1,3-diol, 1,5,9-trimethyl-12-(1-methylethyl)- C20H34O2 306 780 0,37 89915392,029
49 21,60 1-Heptatriacotanol C37H76O 536 815 0,03 6871881,498
50 21,94 1-Heptatriacotanol C37H76O 536 811 0,03 7184095,749
51 22,09 2-Phenyl-4-tret-butyl-7-methylindene C20H22 262 746 0,36 88592175,622
52 22,30 1-Heptatriacotanol C37H76O 536 830 0,06 13499608,111
53 22,40 Curan, 16,17,19,20-tetradehydro- C19H22N2 278 664 0,09 21530222,532
54 22,68 2,4,6-Tri-t-butylbenzenethiol C18H30S 278 679 1,35 327352977,751
55 22,89 1-Heptatriacotanol C37H76O 536 756 0,14 33836126,080
56 23,00 1-Heptatriacotanol C37H76O 536 807 0,11 27317859,989
57 23,21 Pregna-5,17(20)-dien-3-ol, (3ß,17E)- C21H32O 300 728 2,52 611086882,463
58 23,39 Hexadecanoic acid, 1-(hydroxymethyl)-1,2-ethanediyl ester C35H68O5 568 765 0,10 23951481,918
59 23,64 1-Heptatriacotanol C37H76O 536 794 0,13 31028509,408
60 23,81 Gibbane-1,10-dicarboxylic acid, 4a,7-dihydroxy-1-methyl-8-methylene-, 1,4a-lactone, 10-methyl ester, (1a,4aa,4bß,10ß)- C20H26O5 346 678 0,58 139667652,116
61 23,95 1,4,9(11)-Pregnatriene-3,20-dione, 21-acetoxy-17-hydroxy- C23H28O5 384 668 1,51 367737480,175
62 24,07 1-Heptatriacotanol C37H76O 536 701 1,14 275818489,090
63 24,19 17Alpha-ethynyl-17beta-hydroxy-6beta-methoxy-3alpha,5-cyclo-5alpha-androstan-19-oic acid C22H30O4 358 701 1,03 251033153,944
64 24,90 Pregnenolone C21H32O2 316 720 11,77 2858353832,214
65 25,06 6ß-Hydroxymethandienone C20H28O3 316 707 0,07 18100793,977
66 25,42 Norethindrone Acetate C22H28O3 340 730 2,36 572240692,503
67 25,82 1H-Cyclopropa[3,4]benz[1,2-e]azulene-5,7b,9,9a-tetrol, 3-[(acetyloxy)methyl]-1a,1b,4,4a,5,7a,8,9-octahydro-1,1,6,8-tetramethyl-, 5,9,9a-triacetate, [1aR-(1aa,1bß,4aß,5ß,7aa,7ba,8a,9ß,9aa)]- C28H38O9 518 662 11,21 2720558837,592
68 26,11 1H-Cyclopropa[3,4]benz[1,2-e]azulene-5,7b,9,9a-tetrol, 3-[(acetyloxy)methyl]-1a,1b,4,4a,5,7a,8,9-octahydro-1,1,6,8-tetramethyl-, 5,9,9a-triacetate, [1aR-(1aa,1bß,4aß,5ß,7aa,7ba,8a,9ß,9aa)]- C28H38O9 518 722 9,58 2324559456,196
69 26,25 1H-Cyclopropa[3,4]benz[1,2-e]azulene-5,7b,9,9a-tetrol, 3-[(acetyloxy)methyl]-1a,1b,4,4a,5,7a,8,9-octahydro-1,1,6,8-tetramethyl-, 5,9,9a-triacetate, [1aR-(1aa,1bß,4aß,5ß,7aa,7ba,8a,9ß,9aa)]- C28H38O9 518 681 0,21 50305606,222
70 26,40 5,16,20-Pregnatriene-3beta,20-diol diacetate C25H34O4 398 756 0,16 37891353,402
71 26,63 1H-Cyclopropa[3,4]benz[1,2-e]azulene-5,7b,9,9a-tetrol, 1a,1b,4,4a,5,7a,8,9-octahydro-3-(hydroxymethyl)-1,1,6,8-tetramethyl-, 5,9,9a-triacetate, [1aR-(1aa,1bß,4aß,5ß,7aa,7ba,8a,9ß,9aa)]- C26H36O8 476 758 0,65 157287190,038
72 26,84 1H-Cyclopropa[3,4]benz[1,2-e]azulene-5,7b,9,9a-tetrol, 3-[(acetyloxy)methyl]-1a,1b,4,4a,5,7a,8,9-octahydro-1,1,6,8-tetramethyl-, 5,9,9a-triacetate, [1aR-(1aa,1bß,4aß,5ß,7aa,7ba,8a,9ß,9aa)]- C28H38O9 518 711 0,10 25002936,711
73 27,04 1H-Cyclopropa[3,4]benz[1,2-e]azulene-5,7b,9,9a-tetrol, 3-[(acetyloxy)methyl]-1a,1b,4,4a,5,7a,8,9-octahydro-1,1,6,8-tetramethyl-, 5,9,9a-triacetate, [1aR-(1aa,1bß,4aß,5ß,7aa,7ba,8a,9ß,9aa)]- C28H38O9 518 746 0,74 180109102,256
74 27,27 1H-Cyclopropa[3,4]benz[1,2-e]azulene-5,7b,9,9a-tetrol, 1a,1b,4,4a,5,7a,8,9-octahydro-3-(hydroxymethyl)-1,1,6,8-tetramethyl-, 5,9,9a-triacetate, [1aR-(1aa,1bß,4aß,5ß,7aa,7ba,8a,9ß,9aa)]- C26H36O8 476 784 2,32 563669675,482
75 27,44 4,13,20-Tri-O-methylphorbol 12-acetate C25H36O7 448 762 0,42 101842917,656
76 27,99 Phorbol 12,13,20-triacetate C26H34O9 490 747 1,15 278563075,943
77 28,28 Acetic acid, 17-(1-acetoxy-ethyl)-10,13-dimethyl-3-oxo-2,3,8,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-11-yl (ester) C25H34O5 414 721 0,18 42548366,987
78 28,57 2-Cyclopenten-1-one, 3,4-dihydroxy-5-(3-methyl-2-butenyl)-2-(3-methyl-1-oxobutyl)-4-(4-methyl-1-oxo-3-pentenyl)- C21H30O5 362 650 3,00 729450089,319
79 28,77 3-Pyridinecarboxylic acid, 2,7,10-tris(acetyloxy)-1,1a,2,3,4,6,7,10,11,11a-decahydro-1,1,3,6,9-pentamethyl-4-oxo-4a,7a-epoxy-5H-cyclopenta[a]cyclopropa[f]cycloundecen-11-yl ester, [1aR-(1aR*,2R*,3S*,4aR*,6S*,7S*,7aS*,8E,10R*,11R*,11aS*)]- C32H39NO10 597 698 0,61 149225718,612
80 29,02 1H-Cyclopropa[3,4]benz[1,2-e]azulene-5,7b,9,9a-tetrol, 1a,1b,4,4a,5,7a,8,9-octahydro-3-(hydroxymethyl)-1,1,6,8-tetramethyl-, 9,9a-diacetate, [1aR-(1aa,1bß,4aß,5ß,7aa,7ba,8a,9ß,9aa)]- C24H34O7 434 696 0,23 56766218,653
81 29,27 Acetic acid, 17-(1-acetoxy-ethyl)-10,13-dimethyl-3-oxo-2,3,8,9,10,11,12,13,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-11-yl (ester) C25H34O5 414 796 0,08 19652808,230
82 29,69 Ethyl iso-allocholate C26H44O5 436 770 0,11 26385262,922
83 29,84 Pregn-5-ene-3,11,12,14,20-pentol, 11-acetate 12-(3-methylbutanoate), (3ß,11a,12ß,14ß)- C28H44O7 492 717 0,38 91647832,814
84 30,11 Butanoic acid, 1a,2,5,5a,6,9,10,10a-octahydro-5,5a-dihydroxy-4-(hydroxymethyl)-1,1,7,9-tetramethyl-11-oxo-1H-2,8a-methanocyclopenta[a]cyclopropa[e]cyclodecen-6-yl ester, [1aR-(1aa,2a,5ß,5aß,6ß,8aa,9a,10aa)]- C24H34O6 418 782 0,02 6034424,424
85 30,67 Stigmasterol C29H48O 412 766 0,03 8062119,845
86 31,78 ?-Sitosterol C29H50O 414 802 0,10 23362737,090
87 31,96 9,12,15-Octadecatrienoic acid, 2-phenyl-1,3-dioxan-5-yl ester C28H40O4 440 717 0,01 1880362,172

Table 2 summarizes the seven compounds in the seed kernel ethanol extract with the highest relative peak areas in this investigation, whereas fig. 4-10 summarize the apex pecan compound and real-time.

Ret. time 19,635
Hit# 1
Component: 9,12-Octadecadienoic acid, ethyl ester
SI: 886
RSI: 928
Library: mainlib
Hit# 2
Component: 9,12-Octadecadienoic acid (Z,Z)-, methyl ester
SI: 863
RSI: 886
Library: mainlib
Hit# 3
Component: Butyl 9,12-octadecadienoate
SI: 862
RSI: 882

Fig. 4: Apex peak 9,12-octadecadienoic acid, ethyl ester

Table 2: Compounds with various retention times, peak area percentages, molecular formulas, and molecular weights appeared after GC-MS analysis of the ethanol extract of kebiul seed kernel

No Name

RT*

(Min)

 Peak area (%) Molekuler formula Molecular weight

Structure

(2D PubChem)

SI

(NIST)
1 l-(+)-Ascorbic acid 2,6-dihexadecanoate 18,16 8,97 C38H68O8 652 856
2 9,12-Octadecadienoic acid, ethyl ester 19,64 5,86 C20H36O2 308 886
3 6-Octadecenoic acid 19,93 16,28 C18H34O2 282 877
4 Pregna-5,17(20)-dien-3-ol, (3ß,17E)- 23,21 2,52 C21H32O 300 728
5 Pregnenolone 24,90 11,77 C21H32O2 316 720
6

1H-Cyclopropa[3,4]benz[1,2-e]azulene-5,7b,9,9a-tetrol, 3-[(acetyloxy)methyl]-

1a,1b,4,4a,5,7a,8,9-octahydro-1,1,6,8-tetramethyl-, 5,9,9a-triacetate, [1aR-(1aa,1bß,4aß,5ß,7aa,7ba,8a,9ß,9aa)]

 25,82 11,21 C28H38O9 518 662
7

1H-Cyclopropa[3,4]benz[1,2-e]azulene-5,7b,9,9a-tetrol, 3-

[(acetyloxy)methyl]-1a,1b,4,4a,5,7a,8,9-octahydro-1,1,6,8-tetramethyl-, 5,9,9a-triacetate, [1aR-(1aa,1bß,4aß,5ß,7aa,7ba,8a,9ß,9aa)]-

 26,11 9,58 C28H38O9 518 722
Ret. Time: 19,928
Hit# 1
Component: 6-Octadecenoic acid
SI: 877
RSI: 894
Library: mainlib
Hit# 2
Component: cis-13-Octadecenoic acid
SI: 876
RSI: 880
Library: mainlib
Hit# 3
Component: cis-Vaccenic acid
SI: 876
RSI: 879

Fig. 5: Apex peak 6-octadecenoic acid

Ret. Time: 18,159
Hit# 1
Component: l-(+)-Ascorbic acid 2,6-dihexadecanoate
SI: 856
RSI: 856
Library: mainlib
Hit# 2
Component: n-Hexadecanoic acid
SI: 830
RSI: 835
Library: mainlib
Hit# 3
Component: Palmitic anhydride
SI: 818
RSI: 819

Fig. 6: Apex peak-(+)-ascorbic acid 2,6-dihexadecanoate

Ret. Time: 23,213
Hit# 1
Component: Pregna-5,17(20)-dien-3-ol, (3ß,17E)-
SI: 728
RSI: 758
Library: mainlib
Hit# 2
Component: Retinoic acid
SI: 715
RSI: 735
Library: mainlib
Hit# 3
Component: Androst-4-en-11-ol-3,17-dione, 9-thiocyanato-
SI: 709
RSI: 714
Library: mainlib

Fig. 7: Apex peak pregna-5,17(20)-dien-3-ol, (3ß,17E)-

Ret. Time: 26,108
Hit# 1
Component: 1H-Cyclopropa[3,4]benz[1,2-e]azulene-5,7b,9,9a-tetrol, 3-[(acetyloxy)methyl]-1a,1b,4,4a, 5,7a,8,9-octahydro-1,1,6,8-tetramethyl-, 5,9,9a-triacetate, [1aR-(1aa,1bß,4aß,5ß, 7aa,7ba,8a,9ß,9aa)]-
SI: 722
RSI: 722
Library: mainlib
Hit# 2
Component: 5,16,20-Pregnatriene-3beta,20-diol diacetate
SI: 702
RSI: 725
Library: mainlib
Hit# 3
Component: 17Alpha-ethynyl-17beta-hydroxy-6beta-methoxy-3alpha,5-cyclo-5alpha-androstan-19-oic acid
SI: 689
RSI: 739

Fig. 8: Apek peak 1H-cyclopropa[3,4]benz[1,2-e]azulene-5,7b,9,9a-tetrol,3-[(acetyloxy)methyl]-1a,1b,4,4a,5, 7a,8,9-octahydro-1,1,6,8-tetramethyl-, 5,9,9a-triacetate, [1aR-(1aa,1bß,4aß,5ß,7aa,7ba,8a, 9ß,9aa)]-

Ret. Time: 24,897
Hit# 1
Component: Pregnenolone
SI: 720
RSI: 750
Library: mainlib
Hit# 2
Component: 6ß-Hydroxymethandienone
SI: 709
RSI: 747
Library: mainlib
Hit# 3
Component: Bolasterone
SI: 708
RSI: 738

Fig. 9: Apek Peak pregnenolone

Ret. Time: 25,822
Hit# 1
Component: 1H-Cyclopropa[3,4]benz[1,2-e]azulene-5,7b,9,9a-tetrol, 3-[(acetyloxy)methyl]-1a,1b,4,4a,5, 7a,8,9-octahydro-1,1,6,8-tetramethyl-, 5,9, 9a-triacetate, [1aR-(1aa,1bß, 4aß,5ß,7aa,7ba,8a,9ß,9aa)]-
SI: 662
RSI: 663
Library: mainlib
Hit# 2
Component: 17Alpha-ethynyl-17beta-hydroxy-6beta-methoxy-3alpha,5-cyclo-5alpha-androstan-19-oic acid
SI: 645
RSI: 704
Library: mainlib
Hit# 3
Component: 1H-Cyclopropa[3,4]benz[1,2-e]azulene-5,7b,9,9a-tetrol, 3-[(acetyloxy)methyl]-1a,1b,4,4a,5,7a,8,9-octahydro-1,1,6,8-tetramethyl-, 9,9a-diacetate, [1aR-(1aa,1bß, 4aa,5ß,7aa,7ba,8a,9ß,9aa)]-
SI: 642
RSI: 642
Library: mainlib

Fig. 10: Apek Peak1H-Cyclopropa[3,4]benz[1,2-e]azulene-5,7b,9,9a-tetrol,3-[(acetyloxy)methyl] 1a,1b,4,4a,5,7a,8,9-octahydro-1,1,6,8-tetramethyl-,5,9,9a-triacetate, [1aR(1aa,1bß,4aß,5ß,7aa,7ba,8a,9ß,9aa)]-

Table 3: Chromatogram of ethanol extract of kebiul seed coat by GC-MS.

No.

Ret. time

min

 Hit# 1 Chemical formula Mol. weight SI Hit#1

Rel. area

%

Area

counts*min
1 4,40 Butanoic acid, 4-butoxy- C8H16O3 160 690 0,65 17149251,603
2 4,90 1-(3,3,3-Trifluoro-2-hydroxypropyl)piperidine C8H14F3NO 197 678 1,98 52600361,831
3 5,46 Propanoic acid, 3-ethoxy-, ethyl ester C7H14O3 146 809 2,92 77439842,012
4 6,15 e-N-Formyl-L-lysine C7H14N2O3 174 719 0,24 6348137,883
5 7,05 DL-Arabinose C5H10O5 150 665 2,72 72235304,066
6 7,52 Glycerin C3H8O3 92 834 5,19 137676830,908
7 7,97 Isosorbide Dinitrate C6H8N2O8 236 695 0,28 7424173,969
8 8,38 Acetamide, N-(2-acetyl-3-oxo-4-isoxazolidinyl)- C7H10N2O4 186 687 1,75 46451880,017
9 9,37 2-Myristynoyl pantetheine C25H44N2O5S 484 718 0,26 6865816,496
10 9,89 6-Acetyl-ß-d-mannose C8H14O7 222 702 0,37 9807341,141
11 10,57 Ascaridole epoxide C10H16O3 184 720 0,20 5377155,199
12 11,10 Z-(13,14-Epoxy)tetradec-11-en-1-ol acetate C16H28O3 268 689 0,26 6789871,482
13 11,41 d-Mannose C6H12O6 180 765 0,31 8239081,820
14 12,56 1,2,3-Benzenetriol C6H6O3 126 795 3,86 102304036,550
15 12,99 Melezitose C18H32O16 504 665 0,01 266193,201
16 13,40 Melezitose C18H32O16 504 747 1,07 28348426,477
17 13,58 d-Gala-l-ido-octonic amide C8H17NO8 255 719 0,04 933308,843
18 13,75 Melezitose C18H32O16 504 724 0,10 2628173,803
19 14,81 Desulphosinigrin C10H17NO6S 279 718 0,46 12147900,801
20 15,02 Melezitose C18H32O16 504 751 1,94 51576313,489
21 15,28 [1,1'-Bicyclopropyl]-2-octanoic acid, 2'-hexyl-, methyl ester C21H38O2 322 695 2,44 64609697,162
22 15,89 Desulphosinigrin C10H17NO6S 279 733 2,64 70056482,665
23 16,35 3-O-Methyl-d-glucose C7H14O6 194 725 8,01 212444222,592
24 16,41 3-O-Methyl-d-glucose C7H14O6 194 731 0,81 21420479,779
25 16,56 3-O-Methyl-d-glucose C7H14O6 194 714 4,69 124442347,240
26 16,75 7-Methyl-Z-tetradecen-1-ol acetate C17H32O2 268 707 1,02 27033533,752
27 16,90 Dodecanoic acid, 2,3-bis(acetyloxy)propyl ester C19H34O6 358 737 0,08 2119033,345
28 17,05 Dodecanoic acid, 2,3-bis(acetyloxy)propyl ester C19H34O6 358 726 0,38 10019404,547
29 17,37 Hexadecanoic acid, methyl ester C17H34O2 270 824 0,51 13473523,905
30 17,96 n-Hexadecanoic acid C16H32O2 256 875 6,45 171053124,069
31 18,82 Heptadecanoic acid C17H34O2 270 822 0,36 9504109,889
32 19,00 9,12-Octadecadienoic acid (Z,Z)-, methyl ester C19H34O2 294 909 0,99 26351755,956
33 19,65 9,12-Octadecadienoic acid (Z,Z)- C18H32O2 280 886 33,68 893553961,822
34 19,81 Octadecanoic acid C18H36O2 284 895 3,51 93133812,412
35 21,13 9,12,15-Octadecatrienoic acid, 2,3-dihydroxypropyl ester, (Z,Z,Z)- C21H36O4 352 800 0,31 8280821,105
36 21,32 7-Methyl-Z-tetradecen-1-ol acetate C17H32O2 268 792 0,27 7034298,457
37 21,43 Ethyl iso-allocholate C26H44O5 436 778 0,53 14171794,774
38 22,09 1,4-Naphthoquinone, 2-acetyl-5,8-dihydroxy-3-methoxy- C13H10O6 262 719 0,37 9832281,596
39 22,62 Hexadecanoic acid, 1-(hydroxymethyl)-1,2-ethanediyl ester C35H68O5 568 791 0,64 17030916,494
40 23,15 Ethyl iso-allocholate C26H44O5 436 760 0,50 13224711,648
41 23,99 9,12-Octadecadienoic acid (Z,Z)-, 2,3-dihydroxypropyl ester C21H38O4 354 826 0,89 23739730,221
42 24,68 6ß-Hydroxymethandienone C20H28O3 316 737 1,64 43534942,669
43 25,32 7aH-Cyclopenta[a]cyclopropa[f]cycloundecene-2,4,7,7a,10,11-hexol, 1,1a,2,3,4,4a,5,6,7,10,11,11a-dodecahydro-1,1,3,6,9-pentamethyl-, 2,4,7,10,11-pentaacetate C30H44O11 580 745 0,28 7490606,831
44 25,56 1H-Cyclopropa[3,4]benz[1,2-e]azulene-3-carboxaldehyde, 9a-(acetyloxy)-1a,1b,4,4a,5,7a,7b,8,9,9a-decahydro-4a,7b,9-trihydroxy-1,1,6,8-tetramethyl-5-oxo-, [1aR-(1aa,1bß,4aß,7aa,7ba,8a,9ß,9aa)]- C22H28O7 404 613 1,64 43411487,584
45 27,82 Prednisolone Acetate C23H30O6 402 713 0,28 7344743,487
46 28,32 Ethyl iso-allocholate C26H44O5 436 711 0,73 19360722,136
47 28,51 (+)--Tocopherol, O-methyl- C29H50O2 430 778 0,44 11561126,095
48 29,74 9,12,15-Octadecatrienoic acid, 2-phenyl-1,3-dioxan-5-yl ester C28H40O4 440 702 0,05 1402279,047
49 30,07 Ethyl iso-allocholate C26H44O5 436 800 0,36 9572451,194
50 31,75 -Sitosterol C29H50O 414 865 0,91 24167148,635

Table 4 summarizes the three compounds in the ethanol extract of the seed coat that have the highest relative peak areas (%) in this investigation, whereas fig. 12-14 summarizes the apex peak compound and real-time.

Fig. 11: Chromatogram of ethanol extract of kebiul seed coat by GC-MS

The chromatogram of the kebiul seed coat, as illustrated in fig. 11 and detailed in table 3, showcases the retention times and peaks identified through GC-MS analysis.

Fig. 11 presents the chromatogram obtained from the ethanol extract of the kebiul seed coat. Compounds were identified by comparing their mass spectra with a primary database, ensuring a Similarity Index (SI) exceeding 80% (NIST), and by cross-referencing with published literature values. GC-MS analysis identified 50 phytochemical compounds in the seed coat extract (table 3).

Table 4: Compounds with various retention times, percentage of peak area, molecular formula, and molecular weight appeared after GC-MS analysis of ethanol extract of kebiul seed coat

No Name

RT*

(Min)

 Peak area (%) Molekuler formula Molecular weight Structure SI
1 n-Hexadecanoic acid 17,96 6,45 C16H32O2 256 875
2 9,12-Octadecadienoic acid (Z,Z)- 19,65 33,68 C18H32O2 280 886
3 Octadecanoic acid 19,81 3,51 C18H36O2 284 895
Ret. Time: 17,955
Hit# 1
Component: n-Hexadecanoic acid
SI: 875
RSI: 880
Library: mainlib
Hit# 2
Component: l-(+)-Ascorbic acid 2,6-dihexadecanoate
SI: 859
RSI: 859
Library: mainlib
Hit# 3
Component: Palmitic anhydride
SI: 810
RSI: 810

Fig. 12: Apex peak GC-MS hexadecanoic acid

Ret. Time: 19,652
Hit# 1
Component: 9,12-Octadecadienoic acid (Z,Z)-
SI: 886
RSI: 886
Library: mainlib
Hit# 2
Component: (Z)-18-Octadec-9-enolide
SI: 883
RSI: 896
Library: mainlib
Hit# 3
Component: Linoelaidic acid
SI: 852
RSI: 922

Fig. 13: Apek peak 9,12-octadecadienoic acid (Z,Z)-

Ret. time: 19,812
Hit# 1
Component: Octadecanoic acid
SI: 895
RSI: 899
Library: mainlib
Hit# 2
Component: Octadecanoic acid, 2-(2-hydroxyethoxy)ethyl ester
SI: 817
RSI: 830
Library: Mainlib
Hit# 3
Component: L-Ascorbic acid, 6-octadecanoate
SI: 769
RSI: 769

Fig. 14: Apex peak octadecanoic acid

In contrast to the main library, fig. 12-14 shows the compound's apex peak at a certain moment as well as its resemblance to other compounds.

DISCUSSION

The kingdom of plants contains an endless supply of active chemicals for medications. In the quest for resources and raw materials for the pharmaceutical sector, phytochemical approaches are crucial. Finding new drugs is a drawn-out process that includes several steps that must be taken one after the other. Fresh and dried plant materials can be extracted using various of methods, either with or without the use of solvents (expression, sublimation, distillation, etc). These methods may include the use of water, ether, acetone, methanol, ethanol, chloroform, etc. A key step in the analysis of medicinal plants is isolation, which involves several basic procedures, including prewashing, drying or freeze-drying plant materials, grinding to produce a homogenous sample, and frequently enhancing the kinetics of analytic extraction and increasing sample surface contact with the solvent system. Qualitative assays for screening phytochemical compounds are the main way to start phytochemical characterization. The last stages in the photochemical study of plants are the characterization and identification of the isolated and separated constituents [10].

The selection of plant material, phytochemical analysis, characterization, and pharmacological investigation are all steps in the drug development process. A thorough preclinical inquiry is then conducted before clinical trials begin. Until 1996, naturally occurring chemicals either inspired or directly derived almost 80% of pharmaceuticals. Between 1981 and 2019, 1881 new medications were approved; of these, about 23.5% were natural products or semisynthetic derivatives of natural products, and about 25% were either pharmacophores or natural product mimics. The assessment of natural products in novel drug discovery has been revolutionized by the emergence of new skills [11].

By utilizing both mass spectra and retention periods, GC-MS is an effective method for compound identification. The GC-MS chromatogram examination of the ethanol extract revealed that the ethanol extract of seeds contained 87 bioactive phytochemical compounds (fig. 2), whereas the ethanol extract of the seed coat contained 50 bioactive phytochemical compounds (fig. 11). Tables 1-4 highlight the three compounds in the ethanol extract of the seed coat and the seven compounds in the ethanol extract of the seed kernel with the largest relative peak areas, as well as the apex peak compound and real time. The amount of time it takes for a chemical to move from the GC column to the detector is known as the retention time. It is an essential criterion for compound identification. When compared to the mainlib collection, the mass spectrum's apex peak similarity and retention duration are matched to different chemicals (fig. 4-13).

The bioactive chemicals from the coral Junceella delicata were separated and then processed for structure elucidation using GC-MS and FTIR. These substances include ethyl aminomethyl formimidate, Gly-Gly, and 2-(2-Pyridyl)-4-methylthiazole-5-carboxylic acid, 7-Methoxy-2-methylquinolin-4-ol, fraxidin, 2-methyl-3-trans-propenylpyrazine, 3-tert-butylpyridine, acetaldehyde benzyl ethyl acetal, α-methylcinnamicacid, 4-ethoxycoumarin, 3-hydroxycoumarin, 2,4,7,9-tetramethyl-5-decyne-4,7-diol, 2,2-bis(3-allyl-4-hydroxyphenyl) propane, phenyltriethylammonium cation, and dodecanedioic acid [12]. Using spectroscopic data from the literature, five lignans (1–5), two phloroglucinols (6–7), five flavonoids (8–12), and four phenolics (13–16) were discovered from the leaves of Caesalpinia bonduc. The genus Caesalpinia yielded compounds 1–4, 6–7, 11, and 13–14, which are useful chemotaxonomic markers for Caesalpinia bonduc [13]. Determine secondary metabolites and analyze the methanol extract of Caesalpinia bonduc seeds using Gas Chromatography-Mass Spectrometry (GC-MS) and Fourier Transform Infrared (FTIR) as well as antibiotic activity. Alkaloids, flavonoids, saponins, tannins, and phenolics are detected by phytochemical screening of the methanol extract from Caesalpinia bonduc seeds. Three major chemicals were identified by the GC-MS analysis: 4-Methyl-1,3-Dioxolan-2-One, 2-Trimethylsilyl-1,3-dithiane, and Cholesta-5,7,9(11)-trien-3-ol acetate. The FTIR study revealed that the extract contains the functional groups O-H, N-H, C-N, C-H, C-O, C=C, C=O, C-S, and C-Si. Escherichia coli and Staphylococcus aureus growth was inhibited by the antibacterial activity methanol extract from Caesalpinia bonduc seeds at different doses [14].

This work used gas chromatography and mass spectroscopy to identify the bioactive chemicals found in the ethanol extracts of kebiul seed kernel and kebiul seed coat. Retention time (RT), molecular formula, molecular weight (BM), and concentration (peak area%) are all active principles. An intriguing technique for determining the concentration of certain active ingredients in herbs used in the food, pharmaceutical, cosmetics, environmental, and forensic industries is the GC-MS method, which is employed to analyze the extracted materials [15]. This method analyzes a combination of chemical components by combining two analytical techniques into one. The components of the mixture are separated by gas chromatography, and each component is examined independently by mass spectroscopy.

Utilize HPLC and GC-MS methodologies to examine the bioactive constituents of Matricaria chamomilla (chamomile) flowers, encompassing flavonoids, terpenoids, and polyphenols [16]. 20 chemicals were identified as a result of the n-hexane extract's gas chromatography-mass spectrometry (GC-MS) investigation. These comprised siloxanes, fatty acid esters, sterols (e. g., stigmasterol and ergost-5-en-3-ol), terpenoids (e. g., phytol and caryophyllene oxide), alkanes, and other hydrocarbons. Numerous substances, such as erucamide, phytol, and squalene, have been investigated for their known antibacterial and anticancer properties.

Caesalpinia bonduc is utilized in ointments including castor oil and seed powder, which can be applied topically to address hydrocele and orchitis [17]. Seed-derived oil can be applied topically to address convulsions and paralysis. The consumption of equal quantities of pepper and Caesalpinia bonduc seed powder has demonstrated antimalarial properties [18]. They utilized such grains as necklaces [19]. Seed oil moisturizes the skin and alleviates acne. Caesalpinia bonduc seeds, as a polyherbal formulation, are utilized in the treatment of diphtheria (7 g of mixed seeds) and pneumonia (5 g of mixed seeds), as indicated by an ethnobotanical study [20].

The seeds are utilized for several medicinal conditions, including hemostatic, laxative, anthelmintic, inflammation, colic, malaria, skin disorders, and leprosy, according to Singh and Raghav (2012). The seeds' extracts are also utilized as anthelmintic and anti-blennorrhagic medicines, and they are also used as tonics [22]. Traditionally, the seed extract is used with castor oil or honey to make this anthelmintic medication [23]. Traditional folk pre-treat intermittent fever with Caesalpinia bonduc juice for two weeks. To treat hydrocele and orchitis topically, seed powder has also been combined with castor oil to create an ointment [24]. According to a recent assessment by Sasidharan et al. (2021), the best method of treating health issues is to take roasted and powdered seeds orally. Similarly, a review examined the potential of Caesalpinia bonduc in treating polycystic ovarian syndrome (PCOS) [2]. To energize the body and ease pain, the seeds are roasted and ground into a powder. The seeds, which are made as a poultice with asafoetida, ghee, and salt to taste, are also used to treat postpartum stomach pain.

Colitis, diarrhea, and dysentery have all been treated using the seed coat. Amra, Haridra, and Palasa can be taken with roasted seed powder or leaf juice to treat worm infestation-induced irritation in the anal area. In India, the roasted seeds are marketed as Latakaranja after being mixed 1:1 with pippali. An approved herbal treatment for malaria, it can be administered at intervals of 0.5 g per day for 3–4 d [26]. The extract of this plant is an excellent treatment for an enlarged spleen caused by malaria [27]. Additionally, they reported that the plant's seeds are used to treat menstrual pain in the abdomen, boost menstrual flow during oligomenorrhea, and enhance uterine function. Because the seed coat is so effective at treating fluoride, it is utilized to absorb the fluoride present in drinking water. Therefore, the first step in identifying the active chemicals in this plant is to analyze the ethanol extract of the seed kernel and seed coat of kebiul using GC-MS.

CONCLUSION

GC-MS analysis revealed the ethanol extract of the kebiul seed coat included 50 phytochemical compounds, with the highest compound at a retention time of 19.65 min, namely 9,12-Octadecadienoic acid (Z,Z)-. Subsequently, n-Hexadecanoic acid exhibits a retention time of 17.96 min, while Octadecanoic acid has a retention time of 19.81 min. While the ethanol extract of the kebiul seed kernel contained 87 phytochemical compounds. Since these results are preliminary and only based on chemical identification, more investigation is required, with the highest compound at a retention time of 19.93 min, namely 6-Octadecenoic acid. Subsequently Pregnenoloneexhibits a retention time of 24.90 min, 1H-Cyclopropa[3,4]benz[1,2-e]azulene-5,7b,9,9a-tetrol,3-[(acetyloxy)methyl]-1a,1b,4,4a,5,7a,8,9-octahydro-1,1,6,8-tetramethyl-, 5,9,9a-triacetate, [1aR-(1aa,1bß,4aß,5ß,7aa,7ba,8a,9ß,9aa)]-exhibits a retention time of 25.82 min,1H-Cyclopropa[3,4]benz[1,2-e]azulene-5,7b,9,9a-tetrol, 3-[(acetyloxy)methyl]-1a,1b,4,4a,5,7a,8,9-octahydro-1,1,6,8-tetramethyl-, 5,9,9a-triacetate, [1aR-(1aa,1bß,4aß,5ß,7aa,7ba,8a,9ß,9aa)]-has a retention time of 23.21 min, l-(+)-Ascorbic acid 2,6-dihexadecanoate has a retention time of 18.16 min, and 9,12-Octadecadienoic acid, ethyl ester has a retention time of 19.64 min, and Pregna-5,17(20)-dien-3-ol, (3ß,17E)-has a retention time of 23.21 min.

ACKNOWLEDGMENT

The authors would like to express their gratitude to the Center for Higher Education Funding and Assessment, Ministry of Higher Education, Science, and Technology of the Republic of Indonesia, which has provided financial support for this study. The authors also extend their gratitude to thank Al Fathah Foundation Bengkulu and all those who helped with this research process.

AUTHORS CONTRIBUTIONS

Densi Selpia Sopianti: Conceptualization, Writing–Original Draft; Muchsin Doewes: Writing the Original Draft and conducting the Review and Editing; Tatar Sumandjar: Writing–Original Draft, Writing–Review and Editing; Eti Poncorini Pamungkasari: Methodology, Writing–Original Draft; Paramasari Dirgahayu: Methodology, Writing–Original Draft; Ratih Puspita Febrinasari: Methodology, Writing–Original Draft.

CONFLICT OF INTERESTS

The authors declare no conflict of interest

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