GC-MS metabolites profiling of anethole-rich oils by different extraction techniques: antioxidant, cytotoxicity and in-silico enzymes inhibitory insights

Figures & data

Figure 1. GC-MS analysis of volatile oils of (I) anise and (II) star anise extracted by different techniques, (a, d) solvent extraction, (b, e) microwave-assisted extraction, and (c, f): hydro-distillation, showing the predominance of trans-anethole in both oils.

Figure 2. A schematic stacked bar charts showing relative percentages of total identified peak area (A) different classes of compounds except for phenyl propanoids and (B) phenyl propanoids class identified by GC-MS in anise and star anise using different extraction techniques.

Table 1. GC-MS analyses of volatiles of anise and star anise oils prepared by different methods (solvent extraction, microwave and hydrodistillation), whereas n = 3.

Peak no.	RT (min)	KI (obs.)	KI (lit.)	Compound name	% Composition (Mean of 3 independent runs ± SD)
					Anise			Star anise
					SEA	MA	HDA	SESA	MSA	HDSA	Identification
1	7.34	916	917	α-Pinene	–	–	–	–	–	0.396 ± 0.03	MS, KI
2	9.63	999	1004	3-Carene	–	–	–	–	–	0.09 ± 0.01	MS, KI
3	10.05	1018	1018	D-Limonene	-	-	-	0.95 ± 0.05	1.09 ± 0.09	3.21 ± 0.09	MS, KI
4	10.27	1020	1023	Eucalyptol	–	–	–	–	0.45 ± 0.04	0.20 ± 0.03	MS, KI
5	12.47	1090	1082	Linalool	–	–	–	0.43 ± 0.04	0.63 ± 0.10	0.78 ± 0.16	MS, KI
6	14.87	1168	1177	terpinene 4-ol	–	–	–	0.04 ± 0.01	0.15 ± 0.03	0.23 ± 0.02	MS, KI
7	15.27	1181	1185	α-terpineol	–	–	–	0.15 ± 0.03	0.10 ± 0.00	0.07 ± 0.02	MS, KI
8	15.51	1189	1195	Estragole	0.16 ± 0.08	-	-	1.57 ± 0.13	3.44 ± 0.12	1.63 ± 0.35	MS, KI
9	17.29	1250	1251	P-anisaldehyde	–	–	–	1.69 ± 0.19	–	–	MS, KI
10	17.18	1246	1245	Cis-anethole	–	–	–	0.13 ± 0.02	–	–	MS, KI
11	18.25	1283	1283	t-Anethole	82.91 ± 0.59	93.21 ± 2.61	87.35 ± 0.22	82.48 ± 1.01	93.78 ± 0.42	93.39 ± 1.34	MS, KI
12	19.52	1327	1337	δ-Elemene	0.05 ± 0.01	0.06 ± 0.01	0.31 ± 0.01	–	–	–	MS, KI
13	20.62	1366	1376	α-Copaene	–	–	–	0.15 ± 0.00	–	–	MS, KI
14	20.95	1378	1381	Anisketone	–	–	–	0.57 ± 0.01	–	–	MS, KI
15	21.49	1379	1383	Cis-caryophyllene	–	–	–	0.23 ± 0.01	–	–	MS, KI
16	21.67	1403	1405	Trans-α-bergamotene	–	–	–	0.96 ± 0.02	–	–	MS, KI
17	22.82	1448	1446	β-Sesquiphellandrene	–	–	–	0.07 ± 0.00	–	–	MS, KI
18	22.62	1440	1447	α-Himachalene	0.42 ± 0.00	0.30 ± 0.06	0.46 ± 0.02	–	–	–	MS, KI
19	23.4	1470	1470	γ-Himachalene	4.80 ± 0.01	3.54 ± 1.01	5.99 ± 0.11	-	-	-	MS, KI
20	23.52	1475	1467	trans-caryophllene	0.24 ± 0.01	0.15 ± 0.03	–	0.30 ± 0.01	–	–	MS, KI
21	23.76	1485	1495	Zingiberene	0.41 ± 0.01	0.48 ± 0.09	1.25 ± 0.12	–	–	–	MS, KI
22	23.97	1493	1499	β-Himachalene	0.28 ± 0.03	0.17 ± 0.05	0.40 ± 0.03	–	–	–	MS, KI
23	24.12	1499	1500	β-Bisabolene	0.27 ± 0.01	0.36 ± 0.00	0.39 ± 0.01	0.35 ± 0.01	–	–	MS, KI
24	24.22	1503	1668	unidentified	–	–	–	0.13 ± 0.01	–	–	MS, KI
25	25.47	1551	1554	Nerolidol	–	–	–	0.10 ± 0.08	–	–	MS, KI
26	27.80	1647	1580	α-cadinol	–	–	–	0.07 ± 0.01	–	–	MS, KI
27	31.89	1828	2028	unidentified	–	–	–	0.05 ± 0.01	–	–	MS, KI
28	31.92	1830	1822	Pseudoisoeugenol 2- methyl butanoate	4.81 ± 0.08	1.42 ± 0.15	3.10 ± 0.08	-	-	-	MS, KI
29	33.13	1887	1882	Thellungianin- G	2.87 ± 0.08	0.19 ± 0.03	0.75 ± 0.06	–	–	–	MS, KI
30	38.67	1670	1677	Foeniculin	-	-	-	7.41 ± 0.98	-	-	MS, KI
31	40.75	2254	2467	Verimol H	0.37 ± 0.03	–	–	0.13 ± 0.01	–	–	MS, KI
32	42.85	2403	2394	Unidentified	–	–	–	0.22 ± 0.01	–	–	MS, KI
33	43.53	2414	2286	Verimol C	–	–	–	0.26 ± 0.01	–	–	MS, KI
34	44.13	2486	2358	Bemotrizinol	–	–	–	0.27 ± 0.01	–	–	MS, KI
35	44.84	2531	2704	Unidentified	–	–	–	0.26 ± 0.03	–	–	MS, KI
36	46.99	2670	2700	Heptacosane	0.65 ± 0.08	–	–	–	–	–	MS, KI
37	49.89	2850	2900	Nonacosane	0.99 ± 0.06	–	–	–	–	–	MS, KI
38	52.58	3029	3000	Triacontane	0.66 ± 0.03	–	–	–	–	–	MS, KI
39	52.97	3055	3187	β-sitosterol	–	0.12 ± 0.01	–	0.08 ± 0.03	–	–	MS, KI
40	56.43	3276	3290	γ-sitosterol	–	–	–	0.36 ± 0.06	–	–	MS, KI
Monoterpene hydrocarbons					0	0	0	0.95	1.09	3.69
Oxygenated monoterpenes					0	0	0	0.62	1.68	1.28
Sesquiterpene hydrocarbons					6.47	5.06	8.80	2.06	0	0
Oxygenated sesquiterpenes					2.87	0.19	0.75	0.17	0	0
Phenyl propanoids					88.25	94.63	90.45	94.24	97.22	95.02
Hydrocarbons					2.30	0	0	0	0	0
Miscellaneous					0	0.12	0	0.71	0	0
% Identified of total peaks area					99.89	100.00	100.00	98.75	99.99	99.99

The identified compounds are listed as per their retention time on column RTX-5 GC. RT: retention time (min), KI (obs.): Kovat index observed experimentally on RTX-5 column compared to n-alkanes C₈–C₂₈. KI. (Lit.): Kovat index reported from previous data. All identified volatiles referenced to identification based on the comparison of their mass spectral data and KI to those published retention indices in the NIST (National Institute of Standards and Technology) library, adams and other literature data. SEA: solvent extraction anise; SESA: solvent extraction star anise; MA: microwave anise; MSA: microwave star anise; HDA: hydro-distillation anise; HDSA: hydro-distillation star anise. Bold values are the major constituents in each oil sample. All GC-MS runs for anise and star anise oils extracted by three extraction methods were assessed in independent triplets (Each GC-MS run for one replicate of each extraction method).

Figure 3. The score plot of principal component analysis (PCA) (A) and hierarchical clustering analysis (HCA) dendrogram (B) originated from the GC data set of anise and star anise samples obtained by different extraction methods.

Figure 4. Heatmap represents the relative abundance of volatile metabolites extracted from anise and star anise fruits. Hierarchical clustering was done using Pearson’s correlation and average linkage. Heatmap colours represent log₁₀ and Pareto-scaled values of relative metabolite abundance as indicated in the colour key. SEA: solvent extraction anise; MA: microwave extraction anise; HDA: hydro-distillation anise; SESA: solvent extraction star anise; MSA: microwave extraction star anise; HDSA: hydro-distillation star anise.

Figure 5. (I) 2 D-Binding diagram of trans-anethole (A), estragole (B) and D-limonene (C) with the active sites of tyrosinase enzyme. (II) 2 D-Binding diagram of trans-anethole (A), estragole (B) and D-limonene (C) with the active sites of NAD(P)H oxidase enzyme.

Figure 6. Correlation coefficients of different volatiles correlated to trans-anethole.

Figure 7. The effect of trans-anethole enriched Star anise oil on the phases of the cell cycle of HeLa cells.

Table 2. The effect of trans-anethole enriched Star anise oil on the phases of the cell cycle of HeLa cells.

	% G0–G1	%S	% G2/M	%Sub-G1
MSA oil	37.19	23.09	29.96	9.76
Paclitaxel	58.71	15.79	19.47	5.26

Figure 8. Triplicate Annexin V-FITC apoptosis assay for trans-anethole enriched Star anise oil (MSA) (B) using flow cytometry as compared to Doxorubicin control (A).

Table 3. The results of the Annexin V-FITC apoptosis assay for trans-anethole enriched Star anise oil (MSA).

	Early apoptosis (Lower Right %)	Late Apoptosis (Upper Right %)	Total (L.R % + U.R %)
MSA oil	0.97	0.71	1.68
Doxorubicin	0.31	0.64	0.95

Figure 9. 2 D-Binding diagram of trans-anethole (A), estragole (B) and D-limonene (C) with the active sites of CDK2 (I), CDK4 (II) or EGFR (III).