INTRODUCTION:

Selaginella is distributed throughout the continent except for the Antarctic continent, which is estimated to have 700-800 species^1–4. The growth forms of this genus are herbaceous, creeping, climbing, prostrate, upright, epiphytic, and rosette shapes⁵. The stem is branched dichotomous, with a rhinophores-positively gravitropic rooting structure⁶. Its distribution in tropical rainforests, deserts, alpines, and arctic habitats such as Selaginella doederleinii, Selaginella tamariscina, Selaginella pulvinata, Selaginella sinensis, and Selaginella bryopteris⁷.

In pharmacology, members of Selaginella have the potential to cure a variety of diseases. For example, Selaginella tamariscina (P. Beauv.) introduced the Chinese Pharmacopoeia for its effectiveness in improving blood circulation since its 1953rd edition⁸.

Selaginella doederleinii and Selaginella sinensis (Desv.) have anti-inflammatory, antibacterial, antiviral, immune-stimulating, antitumor, analgesic, antispasmodic biological properties, and antispasmodic^9,10. Selaginella trichoclada is a Traditional Chinese Medicine (TCM) for treating dysentery, jaundice, and coughing with lung heat^11,12.

The Selaginella family is a plant rich in bioflavonoids, aglycone flavonoids, alkaloids, lignins, polyphenol compounds selaginellin, diterpenoids, terpenoids, and steroid glycosides^1,11,13,14. To date, about 80 bioflavonoids have been found from the genus Selaginella including Brivaracetam (BRV) related to C-C; amentoflavone, robusta flavone, taiwaniaflavone, sumaflavone, 2′,8"-biapigenin, and C-O-C related Brivaracetam (BRV); ochnaflavone, delicaflavone, hinoki flavone, and isocryptomerin^15,16. Some can act as pharmacological antibacterial, anti-inflammatory, and potential anticancer molecules involving many factors, including apoptosis induction, angiogenic cascade retardation, and metastasis^3,9,16–19. Despite the many reports on the bioactivity of this plant, the complete profile of the phytoconstituents is still essential to decipher.

Recent reports mention that S. willdenowii (Desv.) Baker, a medicinal herb, has a high source of antioxidants^20,21. Looking at its toxicity value, S. Willdenowii (Desv.) Baker concentration of 50% cannot exert toxic effects on juvenile carp²². To complete the metabolite data, this study aims to analyze the metabolite profile of the roots, stems, and leaves of S. willdenowii (Desv.) Baker for the first time.

MATERIAL AND METHODS:

Sample:

All fresh plant parts of S. willdenowii (Desv.) Baker (leaves, roots, and stems) were obtained from the edge of the forest near Cibadak, Sukamakmur, Bogor, Indonesia (6°35'44.0"S 106°57'24.0"E) in mid-August 2022. Samples were taken directly and stored in the coolerbox to be taken to the laboratory for further analysis. Sample authentication was carried out at the Bogoriensi Herbarium Laboratory, BRIN (National Research and Innovation Agency), Indonesia, and the collection were stored with specimen voucher number BO-1560831.

Extract preparation:

Every part of S. willdenowii (Desv.) Baker was separated and washed using running equadest water to remove dirt. 50g of samples were oven-dried for 14 hours at 33^oC²³. The dry sample of each part was mashed with a blender machine until it became powder (40 mesh) following the previous study²⁴. Each part was macerated with ethanol solvent (99.8% p.a.) for five days. With the Rotary Evaporator (BUCHI), each extract (10ml) was put into Ependoft and dried at 60^oC. Finally, 200μL of the solid residue solution was used for GC-MS.

GC-MS Analysis:

Gas Chromatography (Agilent Technologies 7890) and 5975 Mass Selective Detector and Chemstation data system were implemented. following the procedures of the Spice and Medicinal Plants Research Institute (BALITRO). Briefly, the ethanol extract of each portion was filtered through a 5μL syringe filter in split mode (8:1). The helium gas was set at 1.2mL/min and the injector at 250°C. Then, the analyte is separated into a silica capillary column. The oven program and determination of the mass spectrum follow the previous method²⁴.

Data Analysis:

Data analysis and constituent identification were performed by comparing the mass fragments and standard mass spectra in Agilent MassHunter Qualitative Analysis Software. International library databases such as PubChem, FOODB, Chemistry WebBook, and SpectraBase are used to study the potential of compounds²⁵.

RESULT:

GC–MS is still a powerful analytical tool for analyzing phytochemicals, natural products, foods, and metabolomics. Identification of metabolites based on GC-MS can be carried out perfectly because it has sensitive detection, fast work, and efficiency in separating the complexity of phytoconstituents^26,27. Analysis of many plant compounds has been well done with GC-MS, for example,Cinnamomum malabatrum²⁸, Diospyros virginiana²⁹, Tephrosia villosa³⁰, Achnatheruminebrians³¹, Azima tetracantha³², Terminalia catappa³³, Citrus medica³⁴, and many more.The phytoconstituents of the leaves, stems, and roots of S. willdenowii (Desv.) Baker was well confirmed by Gas Chromatogram (Fig. 1). Compounds present with varying retention times, molecular weights, and peak areas. Interestingly, there are new compounds whose activities are unknown based on chemical library data. There were 22 compounds detected in the extract on the leaves that had a percentage of more than 1%, for the most compounds were Phytol (peak area: 14.98%), Glycerin (peak area: 14.95%), 2,6,10-Trimetyl, 14-Ethylene-14-Pentadecne (peak area: 8.84%), 9,12,15-Octadecatrienoic Acid, Cyclopropane Carboxamide, 2-Cyclopropylethyl-2-Methyl-N-(1-Cyclopropylethyl)- (peak area: 5.09%), Ethyl Ester (peak area: 8.12%), and Hexadecanoic Acid, Methyl Ester (peak area: 4,87%). A complete list of compounds can be seen in Table 1.

Phytol belongs to the class of prenol lipids, with the subclass of diterpenoids with the highest % of the area. The most common group of sugar alcohols found in leaves is glycerin with the subclass carbohydrates and carbohydrate conjugates. The compounds 9,12,15-Octadecatrienoic Acid, 2-Cyclopropylethyl-2-Methyl-N-(1-Cyclopropylethyl)-, and Ethyl Ester and Cyclopropane Carboxamide have a reasonably high percentage and these two compounds are not found in other parts.

24 different compounds were present in the stem extract. The main phytochemical compounds include Stigmast-5-En-3-Ol (peak area: 9.96%), Stigmasterol (peak area: 9.53%), 2,6,10-Trimethyl, 14-Ethylene-14-Pentadecne (peak area: 8.35%), Hexadecanoic Acid, Ethyl Ester (peak area: 7.67%), and Linoleic Acid Ethyl Ester (peak area: 7.22%%). Of the five most common compounds, Linoleic Acid Ethyl Ester is not found in other parts. Some compounds are only present in the stem, such as Formamide, N-Methoxy- (peak area: 6.3%), 4,4-Dimethylcholest-7-En-3-One (peak acre: 2.79%), 13-Docosenamide, (Z)-, (peak area: 2.55%), and N-Ethyl-N-.Beta., . Beta., .Beta.-D3-Ethylacetamide (peak area: 2.44%). On the stem found, quite a lot of compounds have not been reported

Figure 1: Chromatogram analysis of GC-MS secondary metabolites obtained from Selaginella willdenowii (Desv.) Baker

Table 1: Identified similar phytocompounds from Selaginella willdenowii(Desv.) Baker

No.	Compund	leaves		Stem		Root
No.	Compund	RT	% of Area	RT	% of Area	RT	% of Area
1	2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-Hexamethyl-(All-E)-	-	-	36,447	3,42	36,04	19,83
2	2,6,10-Trimethyl, 14-Ethylene-14-Pentadecne	29,406	8,84	29,40	8,35	29,40	6,73
2	2,6,10-Trimethyl, 14-Ethylene-14-Pentadecne	29,765	4,38	29,758	3,97	29,765	3,11
3	2-Methyl-Z,Z-3,13-Octadecadienol	31,896	1,20	-	-	36,04	1,26
3	2-Methyl-Z,Z-3,13-Octadecadienol	32,551	2,22	-	-	-	-
4	2-Propenoic Acid, 3-(4-Methoxyphenyl)-, 2-Ethylhexyl Ester	-	-	32,544	2,46	32,544	1,29
5	3,7,11,15-Tetramethyl-2-Hexadecen-1-Ol	29,62	1,67	29,613	1,29	-	-
6	Ergost-5-En-3-Ol	-	-	44,535	3,85	44,5	1,18
7	Hexadecanoic Acid, Methyl Ester	30,096	1,78	30,082	1,36	30,089	2,22
7	Hexadecanoic Acid, Methyl Ester	30,544	4,87	-	-	-	-
8	Hexadecanoic Acid, Ethyl Ester	-	-	30,537	7,67	30,537	5,14
9	Octadecanoic Acid, Ethyl Ester	-	-	31,682	2,42	31,689	2,72
10	Oleic Acid	32,454	1,63	-	-	32,82	22,2
10	Oleic Acid	33,792	3,23	-	-	-	-
12	Phytol	31,31	14,98	31,296	6,91	-	-
12	Stigmast-5-En-3-Ol	-	-	47,224	9,96	40,218	1,58
12	Stigmast-5-En-3-Ol	-	-	-	-	47,176	5,35
13	Stigmastan-3, 5-Diene	41,37	1,00	-	-	41,342	1,25
14	Stigmasterol	45,445	1,27	45,438	9,53	45,404	3,85
15	Trans-13-Octadecenoic Acid, Methyl Ester	31,227	2,76	-	-	31,22	5,49
16	Vitamen E	-	-	41,88	2,41	41,88	4,8

Table 2. Biological activities of Sellaginellawildenowii (Desv.) Baker

Plant part	Metabolite compounds	Biological activities
Leaf	Glycerin	Increase body fluids, osmotic laxatives, lubricants or³⁷
Leaf, stem, root	2,6,10-Trimetyl, 14-Ethylene-14-Pentadecne	Not Found
Leaf, stem	3,7,11,15-Tetramethyl-2-Hexadecen-1-Ol	Anti-inflammatory, anticancer, antieczemic, Anti-inflammatory, Hypocholesterolemic, Hepatoprotective, Nematicide Insectifuge, ^38,39
Leaf, stem, root	Hexadecanoic Acid, Methyl Ester	anti-inflammatory and anticancer, treating type 2 diabetes, ulcerative colitis, psoriasis, and rheumatoid arthritis ^36,40
Leaf	Pyrrolo [1,2-A] Pyrazine, 1,4-Dimethyl-	Antibacterial, antimicrobial and anticancer ^41,42
Leaf, root	Trans-13-Octadecenoic Acid, Methyl Ester	Anti-inflammatory and cancer prevention ³⁶
Leaf, stem	Phytol	Anticancer, antioxidant, anti-inflammatory, antitumor, antimicrobial, diuretic, and chemopreventive and used in vaccine formulations ^36,43
Leaf	9,12,15-Octadecatrienoic Acid, Ethyl Ester	Cell survival and antiplasmodical^39,44,45
Leaf	Heptadecanoic Acid, 15-Methyl-,Ethyl Ester	Antibacterial, antimycobacterial, and antioxidant activity ^45,46
Leaf	12-methyl-E,E-2,13-Octadecadien-1-Ol	Not Found
Leaf , root	2-Methyl-Z,Z-3,13-Octadecadienol	Not Found
Leaf	1,3-Cyclohexadecanedione,6-Nitro	Not Found
Leaf, root	Oleic Acid	Antitumor, antidiabetic and anticancer ^47–50
Leaf	Cyclopropane Carboxamide, 2-Cyclopropylethyl-2-Methyl-N-(1-Cyclopropylethyl)-	Not Found
Leaf	17-(1,5-Dimethyl-Hexyl)-10,13-Dimethyl-4-Vinyl-Hexadecahydro-Cyclopenta [A] Phenanthren-3-Ol	Not Found
Leaf, root	Stigmastan-3, 5-Diene	Not Found
Leaf, stem, root	Stigmasterol	Anti-inflammatory ^51,52
Leaf	.Beta. – Sitosterol	Anticancer potential ^38,44
Stem	Methanecarbothiolic Acid	Not Found
Stem	Formamide, N-Methoxy-	Not Found
Stem	Azetidine, 2-Methyl-	Anti-inflammatory ^17,53
Stem	N-Ethyl-N-.Beta., .Beta., .Beta.-D3-Ethylacetamide	Not Found
Stem, root	Hexadecanoic Acid, Ethyl Ester	Antibacterial, antimycobacterial, and low antioxidant activity ^45,46
Stem	Oxirane, 2-Decyl-3-(5-Methylhexyl)-, Cis-	Not Found
Stem	Linoleic Acid Ethyl Ester	Anti-inflammatory ^54,55
Stem, root	Octadecanoic Acid, Ethyl Ester	Antibacterial, antimycobacterial, and low antioxidant activity ^45,46
Stem	1-Nonadecene	Antimicrobial and antioxidant ¹⁵
Stem, root	2-Propenoic Acid, 3-(4-Methoxyphenyl)-, 2-Ethylhexyl Ester	Not Found
Stem	Cyclopropaneoctanal, 2-Octyl-	Not Found
Stem	1-Docosene	Not Found
Stem	13-Docosenamide, (Z)-	Antifungal and antibacterial ⁵⁶
Stem, root	2,6,10,14,18,22-Tetracosahexaene, 2,6,10,15,19,23-Hexamethyl-(All-E)-	Antibacterial, antioxidant, antitumor, anticancer, immunostimulant and lipoxygenase inhibitor (Zayed et al., 2019)
Stem, root	Vitamin E	Antioxidant, anti-inflammatory and anti-fibroblastic ^57,58
Stem, root	Ergost-5-En-3-Ol	Anti-inflammatory, anti-diabetic and antioxidant ^59,60
Stem, root	Stigmast-5-En-3-Ol	Anticancer, antitumor, and anti-diabetic ^61,62
Stem	4,4-Dimethylcholest-7-En-3-One	Not Found
Root	6,6-Dimethyl-4-Cycloocten-1-One 6,6-Dimethyl-Cyclooct-4-Enone	Not Found
Root	Trans-13-Octadecenoic Acid, Methyl Ester	Anti-inflammatory and anti-cancer ³⁶
Root	(2E)-2,7,11,15-Tetramethyl-2-Hexadecen-1-Ol	Not Found
Root	(9E)-9-Octadecanoic Acid	Antibacterial, antimycobacterial, and low antioxidant activity ^45,46
Root	1-Eicosene	Anticancer, antifungal and antioxidant ^63,64
Root	2-[4 (E)-Formylcyclohex-(E)-YL]-3,5,6-Trimethyl)-1,4-Benzoquinone	Not Found
Root	3,7,11,Trimethyl-Dodeca-2,4,6,10-Tetraenal	Not Found
Root	Octacosane	Anti-diabetic and antibacterial ^14,65

The leaves of S. willdenowii (Desv.) Baker may promote some pharmacological effects due to the interaction between plant molecules and organic systems. The effects that S. willdenowii (Desv.) Baker exhibits include the main phytol compounds that have anticancer, antioxidant, diuretic, antitumor, antimicrobial, and anti-inflammatory properties^36,43. Diterpenoid derivatives such as Phytol⁶⁶, which acts as a precursor of vitamin E in plants⁶⁷. Phytol can cause oxidative cell death of opportunistic pathogenic bacteria such as Pseudomonas aeruginosa. Thus S. willdenowii (Desv.) Baker leaves can be used as an important anti-bacterial agent that causes nosocomial infections⁶⁸. Glycerin is the second most common compound that can increase body fluids, osmotic laxatives, and lubricants⁶⁸. Literature studies reveal Hexadecanoic Acid, Methyl Ester acts as an anti-inflammatory and cancer prevention and treats type 2 diabetes, ulcerative colitis, rheumatoid arthritis, and psoriasis⁴⁰. The presence of phytocomponents in the leaves can be used as anti-inflammatory and antioxidants, as explained in previous reports^51,52, antibacterial^41,42, antitumor^47,49,50, and anticancer^36,40. Uniquely, some compounds still have not been reported, which can be further studied to determine their potential.

The potential for important biological activity in S. willdenowii(Desv.) Baker stems is dominated by Stigmast-5-En-3-ol which can inhibit total cholesterol, Low-Density Lipoprotein (LDL), and triglycerides, and Stigmasterol can increase High-Density Lipoprotein (HDL)⁶¹, providing significant antihyperlipidemic and antitumor activity⁵¹. Stigmasterol belongs to the group of sterols⁶⁹ with the primary function of maintaining the shape of cell membranes⁷⁰ and can be used as oleogelators leading to the formation of lipid structures in plant organelles⁷¹. For the human body, Stigmasterol acts as an anti-inflammatory⁷², antidiabetic⁷³, lowering cholesterol⁷⁴, antitumor⁷⁵.

The main compound Squalene on the root S. willdenowii (Desv.) Baker is pharmacological potential in protecting the liver, fighting fatigue, antioxidants, anticancer, lowering cardiovascular diseases, and boosting the immune system⁷⁶, and antibacterial⁷⁷. This phytocomponent is a natural triterpene hydrocarbon with great potential as an adjuvant to induce an immune response⁷⁸. Squalene-based adjuvant MF59 compounds have been used in human influenza vaccines⁷⁹. The compound (9E)-9-Octadecanoic acid acts as an antibacterial. There is proven inhibition in three strains of Salmonella sp., Staphylococcus aureus, and Escherichia coli in vitro⁸⁰. In closing, we believe S. willdenowii (Desv.) Baker is one of the sources of natural products that have important constituents in pharmacology.

CONCLUSION:

S. willdenowii (Desv.) Baker (leaves, stems and roots) is an important source of phytoconstituents in pharmacology. GC-MS analysis revealed that various main compounds in leaves, such as phytol (14.98%) have a lot of potential to be developed. Stigmast-5-en-3β-ol and Stigmasterol which are dominant in stems can be used as a source of diabetes drugs. The triterpenoids group in roots has pharmacological potential in protecting the liver, fighting fatigue, antioxidants, anticancer, and boosting the immune system. The results of this study also show that 2,6,10-Trimethyl, 14-Ethylene-14-Pentadecne is a compound that is always present in all parts of the plant where its bioactivity is unknown.To the best of our knowledge, these GC-MS results provide the most complete metabolite distribution data from S. willdenowii (Desv.) Baker. However, our present results are the first stage in the identification of the biochemical components of the natural product S. willdenowii (Desv.) Baker. Future studies need to be expanded for the development of the pharmaceutical and bioceutical industries.

CONFLICT OF INTERESTS:

The authors declare that no competing interests.

ACKNOWLEDGMENTS:

The author would like to thank the ELSA Botanical Identification Services and Herbarium Bogoriense, National Research and Innovation Agency (BRIN), Indonesia.

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Received on 26.01.2023 Modified on 29.05.2023

Research J. Pharm. and Tech 2023; 16(12):5978-5985.

DOI: 10.52711/0974-360X.2023.00970