INTRODUCTION:

The Asteraceae family represents one of the largest groups of flowering plants and is renowned for its phytochemical diversity and pharmacological relevance ¹. Species within this family produce a broad range of bioactive secondary metabolites, including essential oils, monoterpenes, sesquiterpenes, flavonoids, phenolic acids, diterpenes, triterpenes, and other biofunctional compounds. These metabolites contribute to the ecological roles of the plants and are extensively explored for medicinal applications ^2,3. The genus Inula, belonging to the Asteraceae tribe Inuleae, consists of over 100 species distributed across Asia, Africa, and Europe ⁴. These plants have gained attention due to their traditional use in ethnomedicine and the presence of biologically active constituents such as sesquiterpene lactones, diterpenes, and flavonoids. Inula britannica L. has been documented for its anti-inflammatory, hepatoprotective, antimicrobial, and cytotoxic activities, largely attributed to its secondary metabolites ⁵. Inula caspica F.K. Blum ex Ledeb, another species of interest, is found across Central Asia, including Kazakhstan, and remains underexplored in terms of its phytochemical composition ⁶. This study aims to perform a comprehensive phytochemical evaluation of Inula caspica and Inula britannica through advanced chromatographic techniques, including gas chromatography–mass spectrometry (GC-MS) and high-performance liquid chromatography coupled with UV and mass spectrometry detection (HPLC-UV-MS) ⁶. The work focuses on the identification and quantification of phenolic acids and flavonoids that are known for their relevance in biopharmaceutical applications and functional product development.

Plant Material:

Aerial parts of Inula caspica and Inula britannica were collected during flowering (June 2019) in southern and western Kazakhstan, respectively. Materials were air-dried, milled (3–5 mm), and stored under controlled temperature and humidity.

Chemicals and Standards:

HPLC-grade acetonitrile, formic acid, and Milli-Q water were used. Standard phenolic compounds included caffeic acid, gallic acid, chlorogenic acid, ferulic acid, rutin, quercetin, luteolin, kaempferol, and others.

Extract Preparation:

Three extracts were prepared with 70% ethanol: Extract 1 – I. caspica (ultrasound-assisted), Extract 2 – I. britannica (percolation), Extract 3 – I. caspica (percolation). Ultrasonic extraction involved 1 h treatments repeated 3 times, while percolation included swelling, soaking, and continuous extraction stages.

GC-MS Analysis:

Conducted on an Agilent 7890A with HP-5MS column and 5975C detector. Samples were prepared in ethanol (0.025 g/2 mL) and analyzed with helium carrier gas and standard NIST library identification.

HPLC-UV-MS Analysis:

Performed on Agilent 1260 Infinity with Zorbax Eclipse Plus C18 column. Mobile phase: 2.5% formic acid in water/acetonitrile with a gradient program. Detection: UV at 280 and 360 nm, and ESI-MS in negative mode using MRM. Extracts and standards were prepared in acetonitrile:water (1:1) and filtered through 0.45 µm membranes.

Identification and Quantification:

Flavonoids were preliminarily identified via bathochromic shifts with AlCl₃. Quantification used external standard calibration based on differential UV spectrophotometry. x = [s1 × m0 × 25 P ×100] ÷ [S0 × m1 × 25 × 100]

Where: S1 = peak area of sample; S0 = peak area of standard; m0 = weight of standard (g); m1 = weight of sample (g); P = content of standard (%).

RESULTS AND DISCUSSION:

GC MS analysis:

Extract 1:

A total of 15 compounds were identified in extract 1 using GC-MS analysis. The active constituents, along with their retention time (RT), arithmetic retention index (aRI), similarity percentage, and relative content, are presented in Table 1. The major identified compounds include 3-Hydroxy-6,2′,4′-trimethoxy-flavone, D-Prunasin, 2,4-Decadienal, E-14-Hexadecenal, Scoparone, n-Pentacosane, trans-Phytol, Oleic acid, E-11-Hexadecenal, Gazaniolide, Docosane, Tetradecanoic acid octyl ester, Tetracosane, Arglabin, and Ambrosin.

Table 1: Component composition of the thick extract of Inula caspica F.K. Blum ex Ledeb (ultrasound, 70% ethanol) according to GC-MS analysis

Peak	R.T., min	^aIR	^АComponent	Similarity (%)	Content (%)
1	2	3	4		5
1	5.831				0.635
2	6.297				3.926
3	9.290	815	3-Hydroxy-6,2′,4′-trimethoxy-flavone	81	0.334
4	9.736				0.166
5	10.537				0.168
6	10.597				0.012
7	12.487				0.058
8	12.937				0.282
9	14.708				0.089
10	15.950				0.376
11	17.739				0.260
12	17.809	1132	D-Prunasin	78	0.138
13	17.932				0.855
14	20.904				0.193
15	22.261	1284	2,4-Decadienal	76	0.395
16	25.953				0.593
17	26.318				0.783
18	28.708				0.276
19	29.909				0.332
20	29.980				0.018
21	31.581				0.368
22	31.728	1710	E-14-Hexadecenal	82	0.720
23	32.823				7.434
24	32.871				5.663
25	33.746				0.407
26	33.812				0.059
27	34.898				1.063
28	34.924				0.624
29	35.340				0.126
30	35.791				1.072
31	35.838				0.169
32	36.003				0.416
33	36.505				15.958
34	36.722				1.204
35	37.570				5.197
36	37.645				22.566
37	37.906	1990	Scoparone	89	0.860
38	38.281				0.982
39	38.358				0.404
40	39.386				0.160
41	39.980				0.132
42	40.211	2105	n-Pentacosane	90	1.497
43	40.498	2114	trans-Phytol	75	0.256
44	40.758				1.087
45	40.870	2141	Oleic acid	90	4.992
46	41.272				0.026
47	41.362				0.261
48	41.476	2171	E-11-Hexadecenal	72	0.108
49	41.875	2190	Gazaniolide	82	2.225
50	41.990	2200	Docosane	82	1.536
51	43.099				0.354
52	43.702	2314	Tetradecanoic acid, octyl ester	81	1.463
53	43.804				0.246
54	43.955				0.208
55	44.425				0.599
56	45.353	2400	Tetracosane	77	3.045
57	46.076				0.163
58	46.254				0.169
59	47.231				0.602
60	48.160	2633	Arglabin	76	1.818
61	48.355				0.410
62	52.044	2739	Ambrosin	63	0.641
63	54.047				0.016
64	54.202				2.807

^AComponent: Listed in order of elution from HP-5MS column;

^aIR: Identification based on retention index and comparison of mass spectra.

Extract 2:

GC-MS analysis of extract 2 revealed 17 bioactive compounds, including 2-Cyclopenten-1-one, 2-hydroxy; 4H-Pyran-4-one; α-Monoacetin; Asarylaldehyde; n-Hexadecanoic acid; Hexadecanoic acid ethyl ester; trans-Phytol; 9,12-Octadecadienoic acid (Z,Z)-; cis-Oleic acid; linoleic acid ethyl ester; E-9-octadecadienoic acid ethyl ester; Butyl citrate; n-Butyl citrate; Arglabin; and Ambrosin. Their retention times, arithmetic retention indices, similarity indices, and relative contents are listed in Table 2.

Table 2: Component composition of the thick extract of Inula britannica L. (percolation, 70% ethanol) according to GC-MS analysis

Peak	R.T., min	^aIR	^АComponent	Similarity (%)	Content (%)
1	5.833				0.585
2	6.305				2.898
3	7.400				0.125
4	9.292				1.903
5	9.534				0.267
6	9.762				0.358
7	10.550	926	2-Cyclopenten-1-one, 2-hydroxy-	86	0.436
8	12.496	989	Pyranone	76	0.327
9	12.939				0.296
10	13.994				0.163
11	15.906				0.368
12	17.725	1123	3-Amino-2-oxazolidinone	70	0.287
13	17.862				0.126
14	17.927	1151	4H-Pyran-4-one	94	2.005
15	20.901	1241	α-Monoacetin	65	1.197
16	23.942	1315	Asarylaldehyde	90	0.151
17	25.124				0.240
18	26.000				1.736
19	26.203				0.187
20	26.360				0.157
21	26.417				0.039
22	29.913				0.131
23	30.122				3.431
24	30.333				0.643
25	30.435				0.242
26	30.713				0.359
27	31.029				2.397
28	31.324				0.556
29	31.518				0.525
30	33.153				0.389
31	33.370				0.346
32	33.572				0.362
33	33.745				0.612
34	33.802				0.305
35	34.281				0.116
36	34.306				0.084
37	35.213				0.322
38	35.353				0.252
39	35.792				0.218
40	35.861				0.361
41	36.049				0.225
42	37.649	1968	n-Hexadecanoic acid	90	8.238
43	37.715				1.535
44	38.282	1993	Hexadecanoic acid ethyl ester	78	2.220
45	40.500	2114	trans-Phytol	73	0.498
46	40.839	2133	9,12-Octadecadienoic acid (Z,Z)-	89	3.888
47	40.938	2141	cis-Oleic acid	87	5.703
48	41.181	2150	Butyl citrate	79	1.443
49	41.356	2162	Linoleic acid ethyl ester	82	2.788
50	41.472	2174	E-9-Octadecadienoic acid ethyl ester	75	1.303
51	41.932	2198	n-Butyl citrate	93	9.427
52	43.798				0.153
53	44.343				0.467
54	44.770				0.376
55	45.298				0.338
56	45.381				0.181
57	45.555				0.983
58	46.079				0.468
59	46.254				0.215
60	46.850				0.887
61	47.451				0.367
62	48.157	2633	Arglabin	77	2.118
63	48.359				1.095
64	48.831				0.897
65	52.032	2739	Ambrosin	81	2.593
66	54.174				26.090

^AComponent: Listed in order of elution from HP-5MS column;

^aIR: Identification based on retention index and comparison of mass spectra.

Extract 3: GC-MS analysis of extract 3 identified 17 compounds, including Chromelin, 4H-Pyran-4-one, α-Monoacetin, Asarylaldehyde, Fingolimod, α-Mannitol, δ-D-Gluconolactone, n-Hexadecanoic acid, trans-Phytol, 9,12-Octadecadienoic acid (Z,Z)-, cis-Oleic acid, Butyl citrate, Gazaniolide, Thebain, n-Butyl citrate, Arglabin, and Ambrosin. Their retention times, arithmetic retention indices, similarity percentages, and relative contents are summarized in Table 3.

Table 3: Component composition of the thick extract of Inula caspica (percolation, 70% ethanol) according to GC-MS analysis

Peak	R.T., min	^aIR	^АComponent	Similarity (%)	Content (%)
1	2	3	4		5
1	5.837				0.573
2	6.304				2.254
3	7.402				0.239
4	9.283	814	Chromelin	70	0.878
5	9.409				0.013
6	9.731				0.147
7	9.905				0.038
8	10.545				0.417
9	12.502				0.212
10	12.951				0.104
11	17.727				0.297
12	17.925	1151	4H-Pyran-4-one	95	1.594
13	20.145				0.191
14	20.291				0.158
15	20.507				0.240
16	20.883	1241	α-Monoacеtin	87	0.887
17	21.919				0.382
18	22.145				0.176
19	22.928				0.243
20	23.934	1315	Asarylaldehyde	93	0.256
21	25.123	1404	Fingolimod	84	0.259
22	26.003	1432	α-Mannitol	67	4.226
23	26.186				0.592
24	26.349				0.697
25	26.455				0.146
26	30.117				5.134
27	30.713				0.188
28	31.048	1637	δ-D-Gluconolactone	68	2.491
29	31.511				0.279
30	33.143				0.194
31	33.562				0.329
32	33.740				0.777
33	33.818				0.132
34	35.211				0.540
35	35.343				0.282
36	35.634				0.290
37	35.795				0.119
38	35.858				0.185
39	37.636	1968	n-Hexadecanoic acid	90	7.693
40	37.712				1.357
41	38.278				1.282
42	40.499	2114	trans-Phytol	71	0.471
43	40.834	2133	9,12-Octadecadienoic acid (Z,Z)-	90	5.307
44	40.932	2141	cis-Oleic acid	89	5.917
45	41.177	2150	Butyl citrate	72	0.834
46	41.343				1.592
47	41.462				0.409
48	41.865	2190	Gazaniolide	79	1.735
49	41.926	2198	n-Butyl citrate	94	9.078
50	43.790				0.212
51	44.760	2365	Thebain	89	0.509
52	45.005				0.138
53	45.297				0.290
54	45.386				0.161
55	45.557				0.362
56	46.027				0.154
57	46.075				0.229
58	46.262				0.343
59	48.156	2633	Arglabin	68	3.395
60	48.358				1.267
61	48.838				0.370
62	49.023				0.163
63	52.041	2739	Ambrosin	69	4.453
64	54.168				26.120

^AComponent: Listed in order of elution from HP-5MS column;

^aIR: Identification based on retention index and comparison of mass spectra.

HPLC-UV-MS analysis of phenolic compounds

The HPLC-UV-MS analysis revealed the presence of phenolic compounds with considerable amounts in all the 3 extracts (Fig. 4, Fig. 5 and Fig. 6).

In the thick extract of Inula caspica F.K. Blum ex Ledeb, prepared via percolation with 70% ethanol, HPLC-UV-MS analysis identified and quantified 10 phenolic compounds. Among these, four were phenolic acids—caffeic acid, gallic acid, chlorogenic acid, and ferulic acid—while six were flavonoids, including rutin (quercetin-3-O-rutinoside), cinaroside (luteolin-7-O-glucoside), dihydroquercetin, quercetin, apigenin, and luteolin. Moreover, four additional peaks with retention times of 12.116, 14.005, 15.287, and 15.579 minutes were detected but remained unidentified, as they did not match the reference standards employed (Table 6).

DISCUSSION:

Inula species are a prolific source of chemical diversity, encompassing over 400 secondary metabolites including more than 100 newly discovered natural products with demonstrated pharmacological potential ⁷. Notably, these plants show promising neuroprotective effects due to their relatively low toxicity ^8-13. Inula britannica L. is particularly known for its wide spectrum of secondary metabolites and has been traditionally used to treat various diseases ¹⁴. The present GC-MS analysis identified 34 compounds across three extracts, each associated with diverse biological activities such as anti-inflammatory, antimicrobial, and cytotoxic effects. The detailed compound list and their respective activities are summarized in Table 7. High-performance liquid chromatography (HPLC) coupled with UV-MS detection proved effective for profiling phenolic constituents in Inula species. Previous studies have identified phenolic acids and flavonoids such as chlorogenic acid, caffeic acid, rutin, myricetin, quercetin, luteolin, and kaempferol in methanolic extracts of various Inula plants ^15,16. In this study, ethanolic extracts of Inula caspica and Inula britannica revealed the presence of 11 major phenolics and flavonoids, including caffeic acid, gallic acid, chlorogenic acid, ferulic acid, rutin, cinaroside, dihydroquercetin, quercetin, apigenin, luteolin, and kaempferol. Caffeic acid, known for its antioxidant and anti-inflammatory properties, plays a role in scavenging free radicals and mitigating oxidative stress. Its presence in Inula helenium has previously been confirmed via HPLC ¹⁷. Gallic acid demonstrates strong antioxidant, antimicrobial, and anticancer activity, validating traditional uses of Inula species in oxidative stress management ¹⁸. Chlorogenic acid, identified in Inula cappa, Inula viscosa, and Inula helenium, contributes to metabolic health through its antioxidant, anti-inflammatory, and antimicrobial actions ^18-20. Ferulic acid protects against UV-induced oxidative damage and is also observed in Inula helenium roots ²¹. Flavonoids such as rutin detected in flowers of Inula viscosa, Inula montbretiana, and Inula helenium offer potent vascular and antioxidant benefits ¹⁶. Cinaroside, a luteolin glycoside, exhibits bioactivity against conditions like paralysis and flu, and has been reported in Vicia subvillosa and Scutellaria ocellata ^22,23. Additional flavonoids like quercetin, apigenin, luteolin, and kaempferol are renowned for their multifaceted therapeutic properties, including anti-inflammatory and cytotoxic effects^24-28. Secondary metabolites and plant-derived antioxidants play a crucial role in disease prevention and treatment, and their significance in pharmaceuticals and human health is increasingly recognized. In summary, the phenolic acids and flavonoids identified in Inula caspica and Inula britannica support their medicinal significance. These phytochemicals validate traditional uses and suggest potential for development into therapeutic agents. The widespread distribution and ethnomedicinal relevance of Inula caspica and Inula britannica reinforce their value as pharmacologically potent plants. This study confirms that the rich profile of polyphenols and flavonoids in these species underpins their therapeutic potential and supports further exploration in drug discovery and development.

CONCLUSION:

This study analyzed Inula caspica and Inula britannica using GC-MS and HPLC-UV-MS, identifying 34 bioactive compounds, including chlorogenic acid, rutin, quercetin, and cinaroside. Ultrasound-assisted and percolation extraction methods proved efficient in recovering diverse phytochemicals. The results highlight the therapeutic and nutraceutical potential of these species, warranting further in vivo validation and standardization studies.

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Received on 28.08.2025 Revised on 02.10.2025

Accepted on 03.11.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):233-240.

DOI: 10.52711/0974-360X.2026.00033

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Peak	t_R (min)	M−H⁻ (m/z)	Identified components	Content
Peak	t_R (min)	M−H⁻ (m/z)	Identified components	Weight of the extract (%)	Weight of the extract (mg g^-1)
1	3.827	179	Caffeic acid	0,028±0,01	0,28±0,1
2	4.773	169	Gallic acid	0,245±0,05	2,45±0,5
3	12.896	353	Chlorogenic acid	8,123±0,32	81,23±3,2
4	14.292	609	Rutin (Quercetin-3-O-rutinoside)	0,388±0,06	3,88±0,6
5	14.673	447	Cinaroside (Luteolin-7-O-glucoside)	1,513±0,07	15,13±0,7
6	16.194	303	Dihydroquercetin	4,622±0,11	46,22±1,1
7	16.744	193	Ferulic acid	0,047±0,03	0,47±0,3
8	22.663	301	Quercetin	0,165±0,04	1,65±0,4
9	27.291	269	Apigenin	0,016±0,01	0,16±0,1
10	28.053	285	Luteolin	0,005±0,001	0,05±0,01
11	28.508	285	Kaempferol	0,018±0,01	0,18±0,1

Peak	t_R (min)	M−H⁻ (m/z)	Identified components	Content
Peak	t_R (min)	M−H⁻ (m/z)	Identified components	Weight of the extract (%)	Weight of the extract (mg g^-1)
No. peak	t_R, (min)	M−H⁻ (m/z)	Identified components	Content
No. peak	t_R, (min)	M−H⁻ (m/z)	Identified components	in%, by weight of the extract	in mg per 1 g of extract (mg/g)
1	3.827	179	Caffeic acid	0,175±0,03	1,75±0,3
2	4.773	169	Gallic acid	0,367±0,06	3,67±0,6
3	12.896	353	Chlorogenic acid	8,749±0,34	87,49±3,4
4	14.292	609	Rutin (Quercetin-3-O-rutinoside)	0,295±0,03	2,95±0,3
5	14.673	447	Cinaroside (Luteolin-7-O-glucoside)	3,623±0,08	36,23±0,8
6	16.194	303	Dihydroquercetin	2,544±0,10	25,44±1,0
7	16.744	193	Ferulic acid	0,018±0,01	0,18±0,1
8	22.663	301	Quercetin	0,127±0,03	1,27±0,3
9	27.291	269	Apigenin	0,007±0,001	0,07±0,01
10	28.053	285	Luteolin	0,002±0,001	0,02±0,01

Peak	t_R (min)	M−H⁻ (m/z)	Identified components	Content
Peak	t_R (min)	M−H⁻ (m/z)	Identified components	Weight of the extract (%)	Weight of the extract (mg g^-1)
No. peak	t_R, (min)	M−H⁻ (m/z)	Identified components	Content
No. peak	t_R, (min)	M−H⁻ (m/z)	Identified components	in%, by weight of the extract	in mg per 1 g of extract (mg/g)
1	3.827	179	Caffeic acid	0,271±0,02	2,71±0,2
2	4.773	169	Gallic acid	0,338±0,05	3,38±0,5
3	12.896	353	Chlorogenic acid	8,800±0,28	88,00±2,8
4	14.292	609	Rutin (Quercetin-3-O-rutinoside)	0,301±0,02	3,01±0,2
5	14.673	447	Cinaroside (Luteolin-7-O-glucoside)	3,041±0,07	30,41±0,7
6	16.194	303	Dihydroquercetin	2,529±0,12	25,29±1,2
7	16.744	193	Ferulic acid	0,019±0,01	0,19±0,1
8	22.663	301	Quercetin	0,128±0,04	1,28±0,4
9	27.291	269	Apigenin	0,008±0,001	0,08±0,01
10	28.053	285	Luteolin	0,002±0,001	0,02±0,01