Health Beneficial Chemo Diversity and Antioxidant Potential of Philonotis revoluta Bosch & Sande Lac (A‐Moss)

ABSTRACT

The current study evaluated the phytochemical composition and antioxidant potential of Philonotis revoluta using methanolic, ethyl acetate, and n‐hexane extracts through analytical techniques. Preliminary qualitative analysis revealed secondary metabolites such as flavonoids, phenols, terpenoids, glycosides, alkaloids, and saponins. UV‐visible analysis supported the occurrence of compounds, and FTIR analysis confirmed the presence of diverse functional groups. GC‐MS identified various bioactive compounds, including ergost‐5‐en‐3‐ol, stigmasta‐5,22‐dien‐3‐ol, and other secondary metabolites. Quantitative analysis showed that the methanol extract had the highest total phenolic (21.97 ± 0.06 mg GAE/g) and total flavonoid content (38.97 ± 0.04 mg QE/g) compared to the ethyl acetate and n‐hexane extracts. Antioxidant assays (DPPH, ABTS, and FRAP) showed a solvent polarity‐dependent effect, with methanol showing stronger scavenging activity (IC₅₀ 229 ± 1.036 µg/mL ABTS; 568 ± 3.963 µg/mL DPPH), followed by the ethyl acetate and hexane extracts, while ethyl acetate (41.42 ± 0.000 µg FeSO₄ eq/mL) showed comparatively higher ferric‐reducing power than methanol and hexane extracts. All extracts showed significant antioxidant activity (p < 0.05). The study suggested P. revoluta contains numerous biologically active phytochemicals with promising antioxidant activity; therefore, there remains scope for this study to explore the therapeutic potential for the development of new drugs.

Methanolic, ethyl acetate, and n‐hexane extracts of Philonotis revoluta were evaluated for their phytochemical composition and antioxidant potential. Spectroscopic and GC–MS analyses revealed diverse bioactive metabolites. At the same time, antioxidant assays demonstrated notable free radical scavenging and reducing activities, highlighting the species as a promising natural source of antioxidant compounds.

1. Introduction

Bryophytes represent the second largest group of land plants and are considered pioneers in the evolution of terrestrial vegetation [1]. They comprise three major groups: liverworts (≈6000 species), hornworts (≈300 species), and mosses (≈13 000 species), which are distributed worldwide across tropical and temperate ecosystems [2]. Phylogenetically, they occupy an intermediate position between algae and pteridophytes [3]. Unlike higher vascular plants, bryophytes lack mechanical and structural protection but exhibit remarkable physiological and biochemical defense systems that help them to survive diverse abiotic and biotic stresses.

The key adaptive features of bryophytes are their ability to synthesize a variety of secondary metabolites, many of which have distinctive odors and potent biological activity. These biologically significant compounds include terpenoids, phenolics (such as flavonoids and bibenzyl derivatives), glycosides, fatty acids, alkaloids, saponins, steroids, and other aromatic compounds [4, 5]. Many significant biologically active compounds have been identified in several mosses collected from different localities in Rajasthan (India) using important analytical techniques such as UV‐Vis, TLC, FTIR, and GC‐MS [6, 7, 8, 9]. Among these, flavonoids are particularly important for their role in stress tolerance and defense against environmental challenges. Several studies have highlighted the antibacterial, antifungal, antioxidant, and pharmacologically relevant activities of bryophyte‐derived phytochemicals, suggesting their potential as a source of novel therapeutic agents [10, 11, 12]. More recently, comparative chemical profiling has extended to other moss genera: essential‐oil GC/FID/MS analysis combined with DPPH, FRAP, and total phenolic content (TPC) evaluation across eight Grimmia and Dicranum species confirmed their antioxidant and antimicrobial relevance [13], while GC–MS‐based solvent extraction of the Dicranaceae mosses Dicranoloma reflexum and Dicranella coarctata identified a comparably rich set of bioactive constituents across hexane, ethyl acetate, and methanol fractions [14], underscoring that chemodiversity assessment of mosses beyond well‐studied genera remains an active but still fragmentary area of investigation.

Oxidative stress, caused by an imbalance between the production of free radicals and the antioxidant defense system, is a main cause of chronic diseases. Reactive oxygen species (ROS), owing to their unpaired electrons, can damage proteins, DNA, and lipids, ultimately leading to tissue injury, inflammation, and degenerative conditions, such as hypertension, atherosclerosis, stroke, diabetes mellitus, Parkinson's, and Alzheimer's diseases [15, 16]. While synthetic antioxidants like BHA (butylated hydroxyanisole) and BHT (butylated hydroxytoluene) are heavily used in the food and pharmaceutical industries, their potential carcinogenic effects have prompted increasing interest in natural, plant‐derived alternatives [17]. Plants are rich in natural antioxidants, such as flavonoids, phenols, ascorbic acid, carotenoids, tocopherols, tocotrienols, and cinnamic acid derivatives [18].

Literature in hand suggested that bryophytes are rich sources of bioactive compounds of high medicinal value but remain underexplored regarding their phytochemical constituents and antioxidant activity; therefore, the current study was conducted to identify and explore the chemical compounds and their antioxidant potency of the moss Philonotis revoluta collected from different localities of Mount Abu (Rajasthan), India, using different standard analytical techniques.

2. Results and Discussion

2.1. Phytochemical Screening

2.1.1. Qualitative Tests of Phytochemicals

Qualitative analysis of the methanolic extract of Philonotis revoluta revealed the presence of secondary metabolites, as indicated by preliminary tests for phenols, flavonoids, terpenoids, glycosides, alkaloids, and saponins. However, primary metabolites such as proteins, amino acids, and carbohydrates, as well as secondary metabolites like gum, mucilage, and tannins, were absent (Table 1). The presence of phenols and flavonoids is important because they are strongly associated with bryophytes' antioxidant activity and stress tolerance [11, 19]. On the other hand, terpenoids and alkaloids are well known for their antimicrobial and medicinal properties. The absence of primary metabolites is consistent with earlier studies, as mosses generally accumulate secondary metabolites as their main defense mechanism [20, 21].

TABLE 1.

Preliminary phytochemical screening of Philonotis revoluta Bosch & Lac. extracts.

S. No.	Phytochemical	Phytochemical test	Results
			ME	EA	HE
1.	Terpenoids	Salkowski test	+	+	+
		Lieberman–Burchant	+	+	+
2.	Phenolic compounds	Ferric chloride test	˗	+	+
		Lead acetate test	+	+	−
3.	Glycosides	Glycoside test	+	−	−
		Keller–Killani test	+	−	−
4.	Protein and Amino acid	Ninhydrin test	−	−	−
		Xanthoproteic test	−	−	−
5.	Flavonoids	Lead acetate test	+	+	˗
		Alkaline reagent test	+	+	+
6.	Alkaloids	Dragendrof's test	+	+	+
		Wagner's test	+	+	+
7.	Gum and mucilage	Alcohol test	−	−	−
8.	Carbohydrates	Molisch's test	−	−	−
		Fehling's test	−	−	−
9.	Tannins	Gelatin test	−	−	˗
		Precipitate test	−	−	−
10.	Saponins	Foam test	—	−	−
		Olive oil test	+	−	−

2.1.2. Quantitative Analysis of TPC and TFC

The TPC of the extracts was measured using the FC assay, and the results were expressed as mg GAE/g (mg gallic acid equivalents per gram). The TPC was highest in the methanolic extract (21.97 ± 0.06^a mg GAE/g), followed by the ethyl acetate (10.29 ± 0.11^b mg GAE/g) and n‐hexane extract (9.22 ± 0.05^c mg GAE/g). Similarly, the total flavonoid content (TFC) of the plants was measured using the aluminum chloride method and expressed as mg QE/g dry weight. The order of TFC concentration was the same as that of TPC, with the methanolic extract showing the highest value, followed by ethyl acetate and n‐hexane: 38.97 ± 0.04^a > 23.41 ± 0.04^b > 22.62 ± 0.04^c mg QE/g. The results indicate that solvent polarity plays a crucial role in the extraction efficiency of phenolic and flavonoid compounds, as these metabolites are generally polar and are efficiently extracted by polar solvents such as methanol. Phenolics and flavonoids are well known for their antioxidant potential [22, 23] due to their ability to donate hydrogen atoms or electrons and neutralize free radicals. This suggests that the methanolic extract may exhibit stronger antioxidant activity than the other solvent fractions. The findings are presented in Figure 1.

FIGURE 1.

TPC and TFC of P. revoluta (Bosch & Lac.) extracts; values are expressed as mean ± SD (n = 3).

2.2. UV–Visible Spectroscopic Analysis

The UV‐Vis analysis of P. revoluta was conducted to examine the chromophoric phytochemicals present in different extracts (methanolic, ethyl acetate, and n‐hexane). Spectra were recorded over 200–800 nm to detect compounds exhibiting σ → σ*, π → π*, n → π*, and aromatic ring transitions. All three extracts showed clear baselines and distinct absorbance bands. Major peaks were observed at 200–290, 350–450, and 600–700 nm, with absorbance values ranging from 1.9547 to 0.0471 (Figure 2, Table 2). The 200–290 nm range, which is significant in both methanolic and hexane extracts, suggests the presence of phenolics, flavonoids, alkaloids, and other aromatic or conjugated metabolites, all of which absorb in the deep UV range. Although n‐hexane is a non‐polar solvent, the minor peaks found at 200–210 nm may be associated with non‐polar terpenoids or traces of lipid‐derived compounds, as saturated fatty acids and lipids absorb exclusively in the far‐UV region (< 210 nm). The 350–450 nm band in all extracts represents conjugated terpenoids, carotenoids, and extended phenolic compounds. Chlorophyll exhibited absorption between ∼430 nm and 660–665 nm; both extracts showed the presence of chlorophyll, but the n‐hexane extract had little chlorophyll due to its low solubility in non‐polar solvents. The ethyl acetate extract exhibited the highest peak intensity, particularly in the 200–250 nm range, indicating a significant concentration of phenolics, flavonoids, alkaloids, tannins, semi‐polar terpenoids, and small pigment components (Table 3).

FIGURE 2.

UV‐vis spectrum and peak values of Philonotis revoluta extracts.

TABLE 2.

UV‐vis spectroscopy peak values of P. revoluta (Bosch & Lac.) extracts.

S. No.	Wavelength (nm)	Abs.	Wavelength (nm)	Abs.	Wavelength (nm)	Abs.
	Methanol		Ethyl acetate		n‐Hexene
1.	200.5	1	232	0.6532	200.5	1.9547
2.	201.5	1.2553	244	0.727	203.5	1.9411
3.	206	1.7364	273.5	0.5596	206	1.9093
4.	211	1.5067	359	0.4127	225.5	1.058
5.	273	0.6112	411	0.7198	270	0.3878
6.	398.5	0.1656	536	0.0631	420.5	0.0742
7.	665.5	0.0471	604	0.051	—	—
8.	—	—	666	0.2355	—	—

TABLE 3.

Phytochemical classes potentially extracted from Philonotis revoluta using solvents of different polarities.

Solvents	Polarity	Phytochemical compounds (metabolites)	References
Methanol	High	Phenolics, alkaloids, flavonoids and their derivatives, glycosides, polar terpenoids, chlorophyll	[24, 25, 26, 27, 28, 29]
Ethyl acetate	Medium	Phenolics, alkaloids, flavonoid aglycones, semi‐polar terpenoids, tannins, carotenoids, chlorophyll
n‐Hexane	Low	Phenolics, alkaloids, flavonoids, lipids, fatty acids, non‐polar terpenoids

2.3. Fourier Transform Infrared (FTIR) Analysis

The FTIR spectrum was used to identify the functional groups of the active components present in the extract based on the peak values in the IR radiation region. When the extract was passed through FTIR, the functional groups of the components were separated based on their peak ratios [23]. The FTIR peak values and functional groups are presented in Table 4, and the FTIR spectrum is presented in Figure 3. The FTIR spectra of the extracts revealed the presence of functional groups, such as amines, alkanes, alkyl amines, alkyl aryl ethers, aldehydes, alcohols, phenols, carbonyls, aromatics, and nitro compounds. The methanolic extract showed distinctive N─H absorption at 3319 cm⁻¹ and C─N absorption at 1113–1021 cm⁻¹, indicating the presence of amine/alkaloid‐type polar compounds. The ethyl acetate extract exhibited a strong ester carbonyl band at 1736.77 cm⁻¹, along with C─O and C─N features, suggesting the presence of semi‐polar compounds such as esters, aldehydes, phenolics, and ether‐containing metabolites. The n‐hexane extract is dominated by aliphatic C─H stretching obtained at 2952‐2860 cm⁻¹ and shows carbonyl (1748 cm⁻¹) and free O─H at 3855–3615 cm⁻¹ signals, characteristic of hydrocarbons, lipid‐like esters, and trace free phenols [30, 31, 32, 33, 34]. Overall, the spectra confirmed that each solvent extract exhibited distinct classes of phytochemicals based on polarity.

TABLE 4.

FTIR peak values and functional groups present in Philonotis revoluta Bosch & Lac. extracts.

Extract	Peak values (cm− 1)	Vibration/bond	Functional group	Probable compound class
Methanolic extract	3319.25	N─H stretching	Secondary amine	Amine/alkaloid‐type compounds
	2940.48, 2830.29	C─H/O─H stretching	Aliphatic, minor O─H	Alkanes, alcohols/phenolics (low)
	1449.48	C─H bending	Alkane	Aliphatic chains
	1113.62, 1020.63	C─N stretching	Alkyl amines	Nitrogenous metabolites
Ethyl acetate extract	2984.68	C─H stretching	Alkane	Aliphatic components
	1736.77	C═O stretching	Ester/aldehyde	Esters, aldehyde‐type metabolites
	1450.07, 1372.03	C─H bending	Alkane	Aliphatic chains
	1231.96, 1100.21, 1043.39	C─O/C─N stretching	Alkyl aryl ether, phenol, amine	Semi‐polar phenolics, ether‐containing compounds
	930.27	C═C bending	Alkene	Unsaturated/aromatic compounds
n‐Hexane extract	3855.15, 3741.64, 3615.17	Free O─H stretching	Alcohol/phenol	Trace free phenols/alcohols
	2952.14, 2920.85, 2860.55	C─H stretching	Alkane	Hydrocarbons, lipid components
	1748.59, 1695.76, 1648.65, 1514.93	C═O stretch; C═C stretch; aromatic region	Carbonyl, alkene, aromatic	Fatty esters/ketones, unsaturated/aromatic compounds
	1459.99, 1378.03	C─H bending	Alkane	Hydrocarbon chains
	1203.62, 1066.92	O─H bending, C─N stretch	Phenol, amine	Minor polar constituents

FIGURE 3.

FTIR spectrum of methanol, ethyl acetate and n‐hexane extracts of Philonotis revoluta.

2.4. Gas Chromatography–Mass Spectrometry (GC–MS) Analysis

GC–MS analysis of the methanolic, ethyl acetate, and n‐hexane extracts of Philonotis revoluta identified 53, 55, and 56 compounds, respectively. The GC‐MS chromatograms show the peaks of identified compounds (Figure 4), and Tables 5, 6, and 7 present the compounds, along with their retention times, peak areas, molecular formulas, and molecular weights. Prominent compounds in methanol include 2,2‐dimethylpropanoic acid, 3‐chloroprop‐2‐enyl ester (34.24), hexadecanoic acid, methyl ester (8.10), n‐hexadecanoic acid (6.56), ergost‐5‐en‐3‐ol, (3β,24R)‐ (5.65), stigmasta‐5,22‐dien‐3‐ol (5.74), hexadecanoic acid, 2‐hydroxy‐1‐(hydroxymethyl)ethyl ester (4.52). In ethyl acetate, ergost‐5‐en‐3‐ol, (3β,24R)‐ (13.57), stigmasta‐5,22‐dien‐3‐ol (12.70), stigmast‐5‐en‐3‐ol, (3β)‐ (6.02), 4‐campestene‐3‐one (7.45), stigmasterone (4.54), cyclopropa[5,6]stigmast‐22‐en‐3‐ol, 3',6‐dihydro‐, (3β,5β,6α,22E)‐ (6.43), phytol (4.69), and octacosanol (12.21) were present. In n‐hexane, nonacosanal (8.70), ergost‐5‐en‐3‐ol (3β) (6.43), stigmasta‐5,22‐dien‐3‐ol (7.01), Heneicosane (6.16), and tris (2,4‐di‐tert‐butylphenyl) phosphate (6.12) were present. Some sterol compounds, such as ergost‐5‐en‐3‐ol, stigmasta‐5,22‐dien‐3‐ol (stigmasterol), and related ergosterol derivatives, were commonly detected in the extracts. These sterols play a crucial role in membrane stabilization and are associated with antioxidant, anti‐inflammatory, antimicrobial, and anticancer properties in higher plants and model systems [35, 36, 37, 38]. Although direct studies on bryophytes are limited, the presence of these compounds suggests that they play comparable functional and protective roles in moss lipid metabolism and stress responses. Notably, several of the constituents identified here overlap with those reported in other moss genera: hexadecanoic acid (palmitic acid), a major fatty acid detected across the present extracts, was similarly among the principal bioactive compounds identified in both the n‐hexane (3.70%–4.17%) and ethyl acetate (7.21%) extracts of Dicranella coarctata, while the isoprenoid ketone 2‐pentadecanone, 6,10,14‐trimethyl‐, and the diterpene neophytadiene, both detected in the present study, were likewise reported in the ethyl acetate extract of Dicranoloma reflexum and the methanol extract of Dicranella coarctata, respectively [14], and the essential oils of eight Grimmia and Dicranum species identified 80 compounds dominated by aldehydes, monoterpene hydrocarbons, and terpene‐related constituents, with the methanol extract of Dicranum scoparium [13].

FIGURE 4.

GC‐MS Chromatogram of methanolic, n‐hexane, and ethyl acetate extract of Philonotis revoluta.

TABLE 5.

Phytoconstituents identified in the methanolic extract of Philonotis revoluta Bosch & Lac.

Peaks	R. time	Area%	MW	MF	Name
1.	8.677	126	C6H6O3	1.50	5‐Hydroxymethylfurfural
2.	9.110	158	C9H18O2	0.18	Nonanoic acid
3.	10.985	184	C13H28	0.17	Tridecane
4.	12.210	176	C8H13ClO2	34.24	2,2‐Dimethylpropanoic acid, 3‐chloroprop‐2‐enyl ester
5.	12.784	139	C5H9N5	0.50	Pyridine‐2,3,5,6‐tetraamine
6.	13.015	200	C12H24O2	0.35	Dodecanoic acid
7.	13.498	254	C18H38	0.35	Octadecane
8.	14.105	226	C13H22O3	1.45	Methyl (3‐oxo‐2‐pentylcyclopentyl) acetate
9.	14.343	162	C6H10O5	3.42	3‐Deoxy‐d‐mannoic lactone
10.	14.556	180	C6H12O6	1.02	3‐Deoxy‐d‐mannonic acid
11.	15.304	228	C14H28O2	0.80	Tetradecanoic acid
12.	15.541	196	C11H16O3	0.42	6‐Hydroxy‐4,4,7a‐trimethyl‐5,6,7,7a‐tetrahydrobenzofuran‐2(4H)‐one
13.	15.681	266	C19H38	0.17	1‐Nonadecene
14.	15.753	240	C17H36	0.22	Heptadecane
15.	15.985	270	C17H34O2	0.61	Isopropyl tetradecanoate
16.	16.141	278	C20H38	0.62	Neophytadiene
17.	16.196	268	C18H36O	0.42	2‐Pentadecanone, 6,10,14‐trimethyl‐
18.	16.600	226	C15H30O	0.29	Pentadecanal‐
19.	16.942	250	C17H30O	0.31	Butanol, 1‐[2,2,3,3‐tetramethyl‐1‐(3‐methyl‐1‐penynyl) cyclopropyl]
20.	17.043	270	C17H34O2	8.10	Hexadecanoic acid, methyl ester
21.	17.121	292	C18H28O3	1.08	Benzenepropanoic acid, 3,5‐bis(1,1‐dimethylethyl)‐4‐hydroxy‐, methyl ester
22.	17.422	256	C16H32O2	6.56	n‐Hexadecanoic acid
23.	17.732	366	C20H37F3O2	0.20	Octadecyl trifluoroacetate
24.	18.683	294	C19H34O2	1.99	9,12‐Octadecadienoic acid (Z,Z)‐, methyl ester
25.	18.742	292	C19H32O2	4.16	9,12,15‐Octadecatrienoic acid, methyl ester, (Z,Z,Z)‐
26.	18.842	296	C20H40O	1.44	2‐hexadecen‐1‐ol, 3,7,11,15‐tetramethyl‐, (R‐(R*,R*))‐
27.	18.980	298	C19H38O2	1.05	Methyl stearate
28.	19.112	306	C20H34O2	0.35	8,11,14‐Eicosatrienoic acid, (Z,Z,Z)‐
29.	19.231	280	C20H40	0.13	1‐Eicosene
30.	20.172	318	C21H34O2	0.62	5,8,11,14‐Eicosatetraenoic acid, methyl ester, (all‐Z)‐
31.	20.382	353	C22H43NO2	0.29	2‐(Dimethylamino)ethyl vaccenoate
32.	20.497	338	C21H38O3	0.28	9‐Octadecenoic acid (Z)‐, oxiranylmethyl ester
33.	20.759	326	C21H42O2	0.54	Eicosanoic acid, methyl ester
34.	21.866	213	C12H23NO2	0.30	3‐Cyclopentylpropionic acid, 2‐dimethylaminoethyl ester
35.	21.958	132	C3H12Si3	0.20	1,3,5‐Trisilacyclohexane
36.	22.111	412	C26H52O3	0.67	Ethyl 3‐hydroxytetracosanoate
37.	22.281	330	C19H38O4	4.52	Hexadecanoic acid, 2‐hydroxy‐1‐(hydroxymethyl)ethyl ester
38.	23.651	308	C20H36O2	0.74	Linoleic acid ethyl ester
39.	23.712	318	C21H34O2	0.70	Methyl (Z)‐5,11,14,17‐eicosatetraenoate
40.	23.858	358	C21H42O4	0.66	Octadecanoic acid, 2,3‐dihydroxypropyl ester
41.	24.534	410	C30H50	0.50	Squalene
42.	24.822	332	C22H36O2	0.16	5,8,11,14‐Eicosatetraenoic acid, ethyl ester, (all‐Z)‐
43.	24.905	490	C35H70	0.31	17‐Pentatriacontene
44.	25.134	410	C28H58O	0.67	Octacosanol
45.	25.600	630	C45H74O	1.11	Solanesol
46.	26.743	298	C20H42O	0.21	1‐Eicosanol
47.	27.091	386	C27H46O	0.18	Cholesterol
48.	28.205	400	C28H48O	5.65	Ergost‐5‐en‐3‐ol, (3β,24r)‐
49.	28.495	412	C29H48O	5.74	Stigmasta‐5,22‐dien‐3‐ol
50.	29.241	414	C29H50O	2.43	Stigmast‐5‐en‐3‐ol, (3β)‐
51.	29.800	225	C15H31N	1.10	N‐Tetradecyl methyl imine
52.	30.354	442	C30H50O2	0.36	Betulin

TABLE 6.

Phytoconstituents identified in the ethyl acetate extract of Philonotis revoluta Bosch & Lac.

Peaks	R. time	Area%	MW	MF	Name
1.	7.603	150	C9H10O2	0.16	Acetic acid, phenylmethyl ester
2.	8.743	134	C5H10O4	0.51	1,2,3‐Propanetriol, 1‐acetate
3.	8.901	140	C7H12N2O	0.65	3‐Amino‐5‐t‐butylisoxazole
4.	9.907	140	C10H20	0.28	Cyclohexane, 1,1,4,4‐tetramethyl‐
5.	10.879	182	C13H26	0.51	1‐Tridecene
6.	11.963	172	C11H24O	0.52	1‐Undecanol
7.	12.390	206	C14H22O	0.59	Phenol, 3,5‐bis(1,1‐dimethylethyl)‐
8.	12.790	180	C11H16O2	0.24	2(4H)‐Benzofuranone, 5,6,7,7a‐tetrahydro‐4,4,7a‐trimethyl‐, (R)‐
9.	14.110	226	C13H22O3	1.03	Methyl (3‐oxo‐2‐pentylcyclopentyl) acetate
10.	14.344	234	C15H22O2	0.43	1‐(4‐Isopropylphenyl)‐2‐methylpropyl acetate
11.	14.563	338	C22H42O2	1.30	Erucic acid
12.	15.540	196	C11H16O3	0.98	6‐Hydroxy‐4,4,7a‐trimethyl‐5,6,7,7a‐tetrahydrobenzofuran‐2(4H)‐one
13.	15.681	238	C17H34	1.48	1‐Heptadecene
14.	15.951	226	C15H30O	1.15	Pentadecanal‐
15.	16.142	278	C20H38	1.48	Neophytadiene
16.	16.196	268	C18H36O	1.26	2‐Pentadecanone, 6,10,14‐trimethyl‐
17.	16.606	242	C16H34O	1.28	1‐Hexadecanol
18.	17.043	270	C17H34O2	3.17	Hexadecanoic acid, methyl ester
19.	17.718	284	C18H36O2	3.06	Hexadecanoic acid, ethyl ester
20.	18.611	270	C18H38O	0.48	1‐Octadecanol
21.	18.682	322	C21H38O2	0.41	cis‐11,14‐Eicosadienoic acid, methyl ester
22.	18.744	348	C23H40O2	1.01	8,11,14‐Docosatrienoic acid, methyl ester
23.	18.844	296	C20H40O	4.69	Phytol
24.	18.982	298	C19H38O2	0.63	Methyl stearate
25.	19.606	322	C23H46	0.48	9‐Tricosene, (Z)‐
26.	19.765	338	C22H42O2	1.15	2‐Hexadecen‐1‐ol, 3,7,11,15‐tetramethyl‐, acetate, [R‐[R*,R*‐(E)]]‐
27.	20.443	312	C21H44O	0.20	1‐Heneicosanol
28.	20.974	324	C21H40O2	0.62	4,8,12,16‐Tetramethylheptadecan‐4‐olide
29.	22.104	380	C19H40O3S2	1.04	D‐Ribose, 2‐deoxy‐bis(thioheptyl)‐
30.	22.286	330	C19H38O4	1.27	Hexadecanoic acid, 2‐hydroxy‐1‐(hydroxymethyl)ethyl ester
31.	23.393	361	C24H27NO2	1.33	Octocrylene
32.	23.682	430	C22H39F5O2	0.15	Nonadecyl pentafluoropropionate
33.	23.863	358	C21H42O4	0.25	Octadecanoic acid, 2,3‐dihydroxypropyl ester
34.	24.530	410	C30H50	0.42	Squalene
35.	24.765	462	C29H50O4	0.53	α‐Tocospiro A
36.	24.903	462	C29H50O4	0.71	α‐Tocospiro B
37.	25.136	410	C28H58O	1.82	Octacosanol
38.	25.603	630	C45H74O	1.36	Solanesol
39.	25.925	536	C37H76O	0.69	1‐Heptatriacotanol
40.	26.123	452	C31H48O2	0.58	3β‐Acetoxystigmasta‐4,6,22‐triene
41.	26.334	416	C28H48O2	0.26	γ‐Tocopherol
42.	27.095	386	C27H46O	1.14	Cholesterol
43.	28.201	400	C28H48O	13.57	Ergost‐5‐en‐3‐ol, (3β,24r)‐
44.	28.490	412	C29H48O	12.70	Stigmasta‐5,22‐dien‐3‐ol
45.	28.787	412	C29H48O	0.76	Stigmasta‐7,24(28)‐dien‐3‐ol, (3β,5α)‐
46.	29.237	414	C29H50O	6.02	Stigmast‐5‐en‐3‐ol, (3β)‐
47.	29.796	398	C28H46O	7.45	4‐Campestene‐3‐one
48.	30.063	430	C32H46	0.42	1,1'‐Biphenyl, 4‐(4‐pentylcyclohexyl)‐4'‐(4‐propylcyclohexyl)‐
49.	30.148	410	C29H46O	4.54	Stigmasterone
50.	30.354	426	C30H50O	1.25	9,19‐Cyclolanost‐24‐en‐3‐ol, (3β)‐
51.	31.086	412	C29H48O	2.65	γ‐Sitostenone
52.	31.232	440	C31H52O	1.44	9,19‐Cyclolanostan‐3‐ol, 24‐methylene‐, (3β)‐
53.	32.219	306	C20H34O2	1.48	4,8,13‐Cyclotetradecatriene‐1,3‐diol, 1,5,9‐trimethyl‐12‐(1‐methylethyl)‐
54.	32.646	426	C30H50O	6.43	Cyclopropa[5,6]stigmast‐22‐en‐3‐ol, 3',6‐dihydro‐, (3β,5β,6alpha.,22E)‐

TABLE 7.

Phytoconstituents identified in the n‐hexane extract of Philonotis revoluta Bosch & Lac.

Peaks	R. time	Area%	MW	MF	Name
1.	5.894	142	C10H22	2.76	3‐Ethyl‐3‐methylheptane
2.	6.238	224	C16H32	0.24	3‐Hexadecene, (Z)‐
3.	6.497	184	C13H28	0.76	Nonane, 5‐(2‐methylpropyl)‐
4.	6.603	170	C12H26	2.02	Decane, 3,7‐dimethyl‐
5.	9.248	226	C16H34	2.43	Hexadecane
6.	9.371	198	C14H30	0.57	Tetradecane
7.	9.456	240	C17H36	0.53	Heptadecane
8.	9.618	200	C13H28O	0.28	1‐Tridecanol
9.	9.829	296	C12H25I	0.31	Dodecane, 1‐iodo‐
10.	9.908	198	C14H30	4.81	Dodecane, 4,6‐dimethyl‐
11.	10.298	226	C16H34	0.37	2,6,10‐Trimethyltridecane
12.	10.985	212	C15H32	0.21	Pentadecane
13.	12.482	242	C16H34O	0.90	1‐Decanol, 2‐hexyl‐
14.	12.696	296	C21H44	12.79	Heneicosane
15.	14.109	226	C13H22O3	0.26	Methyl (3‐oxo‐2‐pentylcyclopentyl) acetate
16.	15.141	282	C20H42	9.57	Eicosane
17.	15.751	254	C18H38	0.26	Octadecane
18.	15.930	184	C13H28	0.13	Nonane, 5‐methyl‐5‐propyl‐
19.	15.986	270	C17H34O2	0.22	Isopropyl myristate
20.	16.144	278	C20H38	0.31	Neophytadiene
21.	16.607	242	C16H34O	0.55	1‐Hexadecanol
22.	16.908	492	C26H53I	1.78	Hexacosane, 1‐iodo‐
23.	17.215	380	C27H56	0.31	2‐Methylhexacosane
24.	19.329	450	C32H66	4.03	Dotriacontane
25.	19.480	282	C20H42	0.91	Hexadecane, 2,6,10,14‐tetramethyl‐
26.	19.545	548	C30H61I	0.26	Triacontane, 1‐iodo‐
27.	19.729	338	C24H50	0.26	11‐Methyltricosane
28.	22.282	330	C19H38O4	0.44	Hexadecanoic acid, 2‐hydroxy‐1‐(hydroxymethyl)ethyl ester
29.	22.813	758	C54H110	0.98	Tetrapentacontane
30.	23.394	361	C24H27NO2	0.34	Octocrylene
31.	23.693	492	C35H72	1.54	Pentatriacontane
32.	23.863	358	C21H42O4	0.16	Octadecanoic acid, 2,3‐dihydroxypropyl ester
33.	24.408	506	C36H74	0.93	Hexatriacontane
34.	25.138	410	C28H58O	12.21	Octacosanol
35.	26.090	268	C19H40	0.23	5,5‐Diethylpentadecane
36.	26.214	422	C29H58O	8.70	Nonacosanal
37.	27.097	386	C27H46O	0.50	Cholesterol
38.	28.203	400	C28H48O	6.43	Ergost‐5‐en‐3‐ol, (3β)‐
39.	28.501	412	C29H48O	7.01	Stigmasta‐5,22‐dien‐3‐ol
40.	28.784	562	C40H82	1.01	Tetracontane
41.	28.907	438	C30H62O	0.59	1‐Triacontanol
42.	29.241	414	C29H50O	2.16	Stigmast‐5‐en‐3‐ol, (3β)‐
43.	29.642	480	C32H64O2	0.44	Hexadecanoic acid, hexadecyl ester
44.	29.795	398	C28H46O	0.90	4‐Campestene‐3‐one
45.	30.149	410	C29H46O	0.87	Spinasterone
46.	32.688	562	C38H74O2	0.60	Phytyl stearate
47.	33.128	662	C42H63O4P	6.12	Tris(2,4‐di‐tert‐butylphenyl) phosphate

2.5. Antioxidant Analysis

The antioxidant analysis of the plant extracts, as IC₅₀ values, was determined to have high potential in methanol, followed by ethyl acetate and n‐hexane against ABTS and DPPH, respectively. In FRAP, the ethyl acetate extract exhibited the highest reducing capacity, followed by the methanol and n‐hexane extracts (Table 8).

TABLE 8.

Antioxidant activity of Philonotis revoluta extracts as determined by ABTS, DPPH, and FRAP.

Solvents	ABTS	DPPH	FRAP
IC50 Me	229 ± 1.036c µg/mL	568 ± 3.963c µg/mL	33.10 ± 0.002b
IC50 EA	338 ± 4.298b µg/mL	671 ± 6.573b µg/mL	41.42 ± 0.001a
IC50 n‐H	527 ± 5.472a µg/mL	814 ± 9.872a µg/mL	32.12 ± 0.055c
Ascorbic acid	3.38 ± 0.05d µM	3.08 ± 0.01d µM	—

Note: IC₅₀ values were obtained by interpolation from linear regression analysis for the ABTS and DPPH assays. Values are expressed as mean ± SD (n = 3). FRAP values are expressed as µg FeSO₄ equivalents/mL extract.

Different superscript letters (a–d) indicate different statistical groups and significant differences among means (DMRT, p < 0.05).

2.5.1. ABTS Radical Scavenging Assay

The ABTS radical scavenging test is widely used to determine the antioxidant activities of both hydrophilic and lipophilic compounds. The ABTS test is a valuable tool for evaluating the antioxidant activity of chain‐breaking and hydrogen‐donating antioxidants [39]. At 50‐250 µg/mL, the scavenging abilities of methanolic, ethyl acetate, and n‐hexane extracts on ABTS radicals increased from 10.92% to 52.66%, 1.16% to 33.72%, and 0.58% to 22.51%, respectively (Figure 5). The methanolic extract showed better scavenging activity as compared to ethyl acetate and n‐hexane extracts at 250 µg/mL. The ABTS radical scavenging activity of P. revoluta exhibited a solvent‐dependent trend, with the methanolic extract exhibiting the highest activity (IC₅₀ = 229 ± 1.04 µg/mL), followed by ethyl acetate and n‐hexane extracts. The standard, ascorbic acid, exhibited a concentration‐dependent increase in ABTS radical scavenging activity, with inhibition values ranging from 23.57% to 86.76% over the tested concentration range of 2–12 µM. The IC₅₀ value of ascorbic acid was 3.38 ± 0.05 µM, indicating its strong antioxidant capacity. The observed activity was comparable to that of several bryophyte species. This reflects a moderate antioxidant potential, which correlates well with the higher total phenolic and flavonoid contents of the methanolic extract. Therefore, the ABTS radical‐scavenging activity of various extracts indicates their ability to scavenge free radicals, thereby preventing lipid oxidation via a chain‐breaking reaction.

FIGURE 5.

(A)ABTS radical‐scavenging activity of Philonotis revoluta extracts; values are expressed as mean ± SD (n = 3). (B) ABTS radical inhibition (%) of ascorbic acid standard at different concentrations; values are expressed as mean ± SD (n = 3).

2.5.2. DPPH Free Radical Scavenging Assay

The DPPH free radical is a stable free radical that has been widely accepted as a tool for estimating the free radical scavenging activities of antioxidants [40]. The DPPH scavenging activities of the extracts are shown in Figure 6. The different extracts of P. revoluta showed an increase in DPPH radical‐scavenging activity with increasing concentration. The methanol, ethyl acetate, and n‐hexane extracts of the moss were seen to scavenge the stable DPPH radicals directly to different extents over a concentration range of 17.98%–23.59%, 16.95%–20.34%, and 15.01%–17.54%, respectively. At 250 ug/mL, the DPPH scavenging activities of the methanol extract were more effective, followed by ethyl acetate and n‐hexane extracts. The standard, ascorbic acid, showed a concentration‐dependent increase in DPPH radical scavenging activity, with inhibition values ranging from 14.24 % to 96.28% over the tested concentration range of 10–120 µM (6 A). The lowest IC₅₀ value (3.08 ± 0.01 µM) was recorded for ascorbic acid, confirming its superior free radical scavenging activity compared with the tested moss extracts. The methanolic extract exhibited the lowest IC₅₀ value (568 ± 3.96 µg/mL), indicating the highest DPPH radical scavenging activity among the tested solvents, followed by the ethyl acetate extract, while the n‐hexane extract showed the weakest activity. As expected, the plant extracts showed higher IC₅₀ values than the reference antioxidant, ascorbic acid, in both the ABTS and DPPH assays. This difference is attributable to the fact that ascorbic acid is a purified antioxidant, whereas the extracts are a complex mixture of bioactive and non‐bioactive constituents. The methanolic extract of Philonotis revoluta exhibited weaker DPPH radical‐scavenging activity than ABTS scavenging. Such differences are commonly reported in bryophytes due to the preferential reactivity of DPPH with lipophilic antioxidants, whereas ABTS is more responsive to hydrophilic phenolic compounds.

FIGURE 6.

(A)DPPH radical‐scavenging activity of P. revoluta extracts; values are expressed as mean ± SD (n = 3). (B) DPPH radical inhibition (%) of ascorbic acid standard at different concentrations, values are expressed as mean ± SD (n = 3).

2.5.3. Ferric Reducing Antioxidant Power Assay

The antioxidant potential of Philonotis revoluta extracts showed a clear concentration‐ and solvent‐dependent pattern across FRAP, DPPH, and ABTS assays. In the FRAP test, all extracts showed an increase in ferric reducing power with concentration (50–250 µg/mL), and the ethyl acetate extract showed the highest reducing capacity, at 41.42 ± 0.00 µg FeSO₄ eq/mL at a concentration of 250 µg/mL, followed by the methanol and n‐hexane extracts (Figure 7). In contrast, radical scavenging assays revealed stronger activity of the methanolic extract, which showed lower IC₅₀ values in both ABTS (229 ± 1.04 µg/mL) and DPPH (568 ± 3.96 µg/mL) assays. Thus, the antioxidant potential of P. revoluta, particularly in methanol and ethyl acetate extracts, can be considered comparable to or better than many bryophytes reported in the literature. For example, the Antarctic moss Sanionia uncinata presented DPPH and ABTS IC₅₀ values of approximately 356 and 181 µg/mL [41], respectively, having moderate antioxidant activity. Also, moss species like Meteoriopsis undulatum, Mitthyridium reclinata, Leucobryum scalare, and Hypnum plumiforme [42] have DPPH IC₅₀ values greater than 1000 µg/mL, indicating much lower antioxidant efficacy in comparison to Philonotis revoluta. When compared with other moss genera, however, the antioxidant potential of P. revoluta reported here is comparatively modest: the methanol extract of Dicranum scoparium was recently reported to achieve a DPPH SC50 of 0.145 mg/mL (145 µg/mL), a potency exceeding that of the synthetic antioxidant standard Trolox [13], whereas the most active P. revoluta extract in the present study (methanol) showed a DPPH IC₅₀ of 568 µg/mL, nearly four‐fold higher (i.e., less potent). This disparity may reflect differences in extraction approach (essential‐oil plus methanol dual extraction vs. solvent‐only extraction), species‐specific phenolic/flavonoid content, or genuine interspecific variation in antioxidant capacity among mosses, and highlights D. scoparium as a notably more potent benchmark for future comparative bryophyte antioxidant studies.

FIGURE 7.

FRAP assay of Philonotis revoluta extracts; values are expressed as mean ± SD (n = 3) in µg FeSO₄ eq/mL.

2.6. Statistical Analysis

Statistical analysis indicated significant differences (p < 0.05) among the solvent extracts for total phenolic and total flavonoid content, and antioxidant potential. The correlation matrix revealed a strong relation between the variables (Figure 8). TPC and TFC showed a strong positive correlation with r = 0.999, indicating a close association between them. A strong negative correlation was identified between TPC and TFC, and between ABTS and DPPH, suggesting that higher phytochemical content leads to stronger antioxidant activity (lower IC₅₀ values). FTIR also showed a clear positive correlation with ABTS and DPPH (r = 0.999, 0.995), suggesting the occurrence of functional groups is linked to antioxidant potential. In addition, there was a very good positive correlation between ABTS and DPPH (r = 0.998), indicating that the tests are compatible. The FRAP assay showed poor correlations with the other parameters, suggesting a different antioxidant mechanism.

FIGURE 8.

Correlation heat map of phytochemical and antioxidant parameters.

3. Conclusions

This study provides a detailed overview of the phytochemical composition and antioxidant capacity of P. revoluta. Qualitative phytochemical screening and FTIR analysis confirm the presence of various secondary metabolites, and GC–MS profiling reveals multiple bioactive compounds, including sterol derivatives and related metabolites with significant bioactivity. The methanolic extract exhibited high levels of phenolics and flavonoids, which are closely associated with its strong radical scavenging ability, as demonstrated by the ABTS and DPPH assays. This highlights the important role of phenolic compounds in antioxidant activity. Conversely, the FRAP activity was more potent in the ethyl acetate extract, suggesting that moderately polar, redox‐active compounds likely contribute to this effect. The antioxidant activity of P. revoluta is comparable to or exceeds that of several moss species previously documented. Overall, our findings position P. revoluta as a promising yet underexplored bryophyte, providing a solid foundation for future research focused on isolating and characterizing its bioactive components identified by GC–MS for potential pharmaceutical applications.

4. Materials and Methods

4.1. Collection of Plant Materials

Fresh specimens of the moss Philonotis revoluta were collected from Guru Shikhar Road (24.6259°N, 72.7408°E; ∼1650 m), Nakki Lake (24.5965°N, 72.7067°E; ∼1160 m), and Sunset Point (24.5894°N, 72.7033°E; ∼1195 m), Mount Abu, Sirohi District, Rajasthan, India, in August and September 2024–2025. After collection, the plant material was thoroughly cleaned to remove soil particles and debris. Specimens were identified and authenticated based on detailed morpho‐taxo‐anatomical characteristics, examined with a dissecting and a compound microscope (ZEISS Primo Star 3) at 10× and 40× magnifications. Microscopic observations were conducted on both fresh and preserved materials. Species identification was confirmed using standard bryological floras, including Mosses of Eastern India and Adjacent Regions [43] and Moss Flora of Rajasthan, India [44]. Bryophyte experts further confirmed the taxonomic identification, and the specimen was assigned voucher number LTPD86 under the accepted name Philonotis revoluta Bosch & Sande Lac. (synonym: Philonotis thwaitesii Mitt.). The authenticated voucher specimen has been deposited in the Herbarium of the Department of Botany, Mohanlal Sukhadia University, Udaipur, Rajasthan, India.

4.2. Plant Extract Preparation of Philonotis revoluta

Plant samples were then air‐dried at room temperature in the shade until all of the moisture had evaporated. The fully dried material was ground into a fine powder with the help of an electric blender. The powdered material (5 g) was extracted with the Soxhlet apparatus with solvents (100 mL each), including methanol, ethyl acetate, and n‐hexane. After that, the extracts were concentrated, dried, and kept at 4°C. The plant extracts were analyzed using standard analytical techniques suited for phytochemical profile and antioxidant assays.

4.3. Phytochemical Analysis

4.3.1. Qualitative Tests for Phytochemicals

Phytochemical screening of the prepared extract in methanol, ethyl acetate, and n‐hexane was carried out through the standard protocol provided by Harborne [45]. The presence or absence of compounds such as alkaloids, flavonoids, phenols, steroids, terpenoids, glycosides, tannins, saponins, amino acids, and proteins.

4.3.2. Quantitative Analysis

4.3.2.1. TPC Analysis

The TPC was evaluated using the Folin‐Ciocalteu method with slight modifications, as described in Singleton [21]. For the assay, 0.2 mL of each extract (concentration mg/mL) was combined with 0.1 mL of 50% Folin‐Ciocalteu reagent, and the mixture was allowed to incubate for 5‐10 min at RT (room temperature). Subsequently, 0.4 mL of a 20% solution of sodium carbonate was added, and the total volume was made up to 5 mL with double‐distilled water (DDW). The mixtures were incubated for 1 h in the dark, and the absorbance was taken at 760 nm against a blank. The results of TPC are quantified as gallic acid equivalents (mg/g GAE) and are reported as the mean ± SD.

4.3.2.2. Total Flavonoid Content Analysis

Flavonoid content was evaluated by using the aluminum chloride colorimetric method, as defined by Chang [20], with some modifications. For this assay, 0.2 mL of extract was taken, to which 0.8 mL of methanol and 1 mL of deionized distilled water (DDW) were added. Subsequently, 0.2 mL of 10 percent aluminum chloride and 0.2 mL of 1 M potassium acetate were added, and DDW was added to bring the final volume of the mixture to 5 mL. The reaction mixture was allowed to incubate for 30‐45 min at room temperature, and absorbance was taken at 415 nm using methanol as a blank. Results are expressed as quercetin equivalents (mg/g QE) and reported as the mean ± SD.

4.4. UV‐Vis Spectroscopic Analysis

UV–visible spectrophotometric analysis of the extracts was conducted using a double‐beam spectrophotometer (Motras Scientific, India). Extracts were scanned under the wavelength range of 200–800 nm under both UV and visible light to detect characteristic absorption peaks coupled with phytochemicals, including flavonoids, phenolics, alkaloids, and other conjugated compounds. Prior to analysis, all extracts were centrifuged and filtered through Whatman filter paper. The filtrates were then diluted 1:10 with the respective solvents before being measured.

4.5. Fourier‐Transform Infrared Spectrophotometry (FTIR) Analysis

FTIR analysis works on the principle that molecular vibrations (stretching, bending, or twisting of bonds) absorb infrared radiation at characteristic frequencies, thereby producing spectra that are used to recognize the functional groups of phytoconstituents present in the plant extracts. The moss extracts were examined using a Fourier Transform Infrared spectrometer of Thermo Scientific, which was configured to operate within a wavelength range of 600–4000 cm^{− 1} with a resolution of 4 cm^{− 1}. This wavelength range was employed to determine the functional groups of phytoconstituents.

4.6. Gas Chromatography–Mass Spectrometry (GC–MS) Analysis

Phytochemical profiling of P. revoluta extracts was performed using a Shimadzu GC‐MS‐QP2010 Ultra system and an AOC‐20i+s autosampler. Extracts (1 µL) were injected in split mode at 260°C using helium as the carrier gas with a flow rate of 1.21 mL/min. The oven temperature was programmed to increase from 70°C to 300°C at 10°C/min, with a final hold of 17 min. The mass spectrometer was operated in electron impact mode at 70 eV, with an ion source temperature of 220°C and an interface temperature of 280°C. Spectra were recorded in the m/z range of 40–600, with a solvent delay of 2.5 min. The chromatograms of the samples were determined by examining the mass spectra with the NIST library and the GC retention time with an established standard. Mass spectra and chromatograms were used to determine the structures, molecular weights, and identities of the unknown bioactive substances.

4.7. Antioxidant Analysis

4.7.1. ABTS Radical Scavenging Assay

The ABTS (2,2'‐azinobis (3‐ethylbenzothiazoline‐6‐sulfonic acid)) radical‐scavenging activity of the extracts was determined according to Re [46], with slight modifications. For this assay, a 7 mM ABTS solution was combined with an equal quantity of 2.45 mM potassium persulfate and incubated at room temperature in the dark for 12–14 h to generate ABTS+• radicals. Before use, the solution was adjusted with methanol to achieve a pH of 7.4 and an absorbance of 0.700 ± 0.025 at 734 nm. Plant extracts were prepared in methanol at concentrations of 50, 100, 150, 200, and 250 µg/mL and then added to 1 mL of ABTS+• solution. The mixture was vortexed and incubated at room temperature for 5–10 min. The decrease in absorbance was measured at 734 nm using UV‐Vis spectrophotometry. Ascorbic acid was used as the standard (positive control) under the same experimental conditions. The % inhibition of ABTS+• radicals was calculated using the formula as follows:

$$\begin{array}{ccc}& & \mathrm{Inhibition}\left(\%\right)\hfill \\ & =& \left[\left(\mathrm{Abs}.\mathrm{Control}-\mathrm{Abs}.\mathrm{Sample}/\mathrm{Abs}.\mathrm{Control}\right)\times 100\right]\hfill \end{array}$$

4.7.2. DPPH Free‐Radical Scavenging Assay

The free radical‐scavenging potential of the plant extracts with DPPH radicals was investigated using the method described by Moraes‐de‐Souza [47] with slight modifications. Plant extract solutions of different concentrations (50–250 µg/mL) were prepared in methanol to a final volume of 1 mL, to which one mL of methanolic DPPH solution (0.1 mM) was added. The solution was thoroughly vortexed, and after incubation in the dark for 30–40 min, absorbance was measured at 517 nm using a UV–visible spectrophotometer; for the standard, ascorbic acid was used. The percentage inhibition was evaluated using the following equation:

$$\begin{array}{ccc}& & \mathrm{Inhibition}\left(\%\right)\hfill \\ & =& \left[\left(\mathrm{Abs}.\mathrm{Control}-\mathrm{Abs}.\mathrm{Sample}/\mathrm{Abs}.\mathrm{Control}\right)\times 100\right]\hfill \end{array}$$

4.7.3. Ferric Reducing Antioxidant Power Assay

The ferric‐ion‐reducing potential of the extracts was measured using the procedure described by Sudha [48], with some modifications. The FRAP reagent was made by dissolving 300 mM acetate buffer (pH 3.6), 10 mM TPTZ in 40 mM HCl, and 20 mM FeCl3 in a 10:1:1 (v/v/v) ratio. Afterward, 1 mL of the FRAP reagent was added to 1 mL of the plant extracts at various concentrations (50–250 µg/mL), and the solution was thoroughly mixed. The absorbance was taken at 593 nm after 30 min of incubation at 37°C. The increase in absorbance indicated the presence of reducing power in the extracts.

4.8. Statistical Analysis

4.8.1. ANOVA

The experiments were performed in three replicates, and the results are shown as mean ± SD (standard deviation). The collected data were evaluated using IBM SPSS Statistics. One‐way ANOVA and Duncan's multiple range test were used to compare the results, with a level of significance of p < 0.05.

4.8.2. Correlation Matrix

Pearson's correlation analysis was used to examine the correlations between TPC, total flavonoid content (TFC), FTIR, and antioxidant capacity (ABTS, DPPH, and FRAP). The analysis was conducted using MS (Microsoft) Excel, and correlation coefficients (r) were calculated from the mean values (triplicate). The correlation coefficients ranged from ‐1 to +1, indicating negative and positive relationships.

Author Contributions

Sheela Parihar: investigation, methodology, software analysis, data curation, writing – original draft. G. S. Deora: conceptualization, supervision, validation, writing – review and editing. Jagrati Agrawal: validation, formal analysis, writing – review and editing. Sarswati and Monika K. Shekhawat: visualization, writing – review and editing. Sandhya Deora: data curation, investigation, formal analysis, and writing – review and editing.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data supporting the findings of this study are available within the manuscript.