Full-Spectrum Analysis of Bioactive Compounds in Rosemary (Rosmarinus officinalis L.) as Influenced by Different Extraction Methods.

Rosmarinus officinalis is a potent antioxidant herb rich in polyphenols. Ultra-high-performance liquid chromatography, coupled with electrospray ionization and quadrupole-time of flight mass spectrometry (UHPLC-ESI-QTOF-MS), enables an exhaustive, full-spectrum analysis of the molecular constituents of natural products. The study aimed to develop a rapid UHPLC method to contribute new insights into the phytochemical composition of rosemary and to assess the performance of nine different procedures for extraction. These include fresh tissue homogenization, fresh and dry leaf decoction, and their respective fermentation, Soxhlet extraction, and sonication using water and methanol. Different extraction methods were found to recover quite different groups of polyphenols within 11 min during 20 min of analysis. Soxhlet extraction, yielded very high concentrations of rosmarinic acid (33,491.33 ± 86.29 µg/g), luteolin-7-O-glucoside (209.95 ± 8.78 µg/g), carnosic acid (2915.40 ± 33.23 µg/g), carnosol (22,000.67 ± 77.39 µg/g), and ursolic acid (5144.27 ± 28.68 µg/g). UHPLC-ESI-QTOF-MS enabled the detection of more than 50 polyphenols, including phenolic acids, flavonoids, and terpenoids in the various extracts. Of these, sagerinic acid ([M - H]-m/z 719.16), salvianolic acid A ([M - H]-m/z 493.11) and B ([M - H]-m/z 717.15), and a pentacyclic triterpenoid corosolic acid ([M - H]-m/z 471.34) were detected for the first time in rosemary. Soxhlet extraction was found to be the most efficient method, followed by dry leaf decoction. The UHPLC-ESI-QTOF-MS methodology for the analysis proved to be very efficient in the identification and characterization of targeted and untargeted bioactive molecules in the rosemary.

1. Introduction

Rosemary (Rosmarinus officinalis L.) is an evergreen perennial culinary herb belonging to the family Lamiaceae and is popularly used as a spice and medicine. The herb is traditionally used to treat memory-related disorders, hypertension, headache, insomnia, and diseases related to the respiratory system [1,2]. Rosemary is considered as a powerful cardiac stimulant, a strong antiseptic, antispasmodic, carminative, emmenagogue, and nervine tonic, and is used to cure arthritis, dandruff, and skin diseases [3,4]. The essential oil from its leaves is used as a natural antimicrobial, pesticide, and insect repellent [5]. The therapeutic properties of rosemary have been attributed to its phytochemical constituents, such as phenolic acids, flavonoids, and terpenoids [6,7].

Ultra-high-performance liquid chromatography and electrospray ionization, coupled with quadrupole-time of flight mass spectrometry (UHPLC-ESI-QTOF-MS), is improved technology for separation and investigation of complex polyphenols in food samples [8]. UHPLC provides rapid, high-resolution, along with higher selectivity and sensitivity, while ESI-QTOF-MS identifies multiple targeted and untargeted constituents of the sample in real-time. Characterization of unknown compounds in UHPLC is based on their exact mass (m/z) and m/z fragmentation pattern with high m/z resolution; further, this technology could also be used to distinguish isobaric compounds by exact mass with different elemental positions [8,9]. Hence, the study was conducted to develop a rapid analytical methodology to provide new insights into the range of phytochemicals present in rosemary and the relative amounts of these compounds.

There are only a few studies reported on the phytochemical profiling of rosemary, and these mainly describe alcohol-based extraction. So far, minimal effort has been made to evaluate the quality of the herb and extracts hereof, based on traditional and industrial methods. There is some evidence in Ayurvedic classics that fermentation enhances the therapeutic and biochemical properties of herbal drugs [10,11,12]. At the same time, ultrasound extraction for a short period (1–2 h) at low frequencies (40 kHz) is reported to increase the yield of alkaloids in herbal extracts and to significantly reduce extraction time and solvent consumption, resulting in comparable or superior extracts to those obtained using decoction and maceration [13,14]. Hence, in the present investigation, an effort has been made to assess several extraction methods in terms of efficiency and final concentrations of critical bioactive constituents of rosemary. The extraction methods evaluated included aqueous extraction, decoction, Soxhlet’s extraction, Ayurvedic fermentation, and sonic extractions in rosemary.

2. Results and Discussion

2.1. Quantification of Bioactive Compounds by UHPLC-ESI-QTOF-MS

The concentrations of different bioactive compounds, caffeic acid, rosmarinic acid, luteolin-7-O glucoside, carnosic acid, ursolic acid, and carnosol (µg/g) analyzed through UHPLC-ESI-QTOF-MS as influenced by different extraction methods are presented in Table 1. Among all the extractions, Soxhlet extract (T7) recorded significantly higher rosmarinic acid (33,491.33 ± 86.29 µg/g), luteolin-7-O-glucoside (209.95 ± 8.78 µg/g), carnosic acid (2915.40 ± 33.23 µg/g), carnosol (22,000.67 ± 77.39 µg/g), and ursolic acid (5144.27 ± 28.68 µg/g). Soxhlet extraction combined with methanol solvent might enhance the solubility of polyphenols, flavonoids, and other bioactive compounds present in herbs, maximizing the extraction of phytochemical constituents [15]. All the extractions in the study yielded considerable amounts of rosmarinic acid ranging from 0.26 µg/g to 33.49 mg/g, contributing substantially to the high antioxidant potential of the extracts. The results are in good agreement with previous studies, in which rosmarinic acid concentrations were reported in the range of 5.6 µg–2.34 mg/g in rosemary leaf extracts from Serbia and Iraq [16,17]; rather, various extraction procedures of our study in rosemary (T4–T7, T9) recorded higher rosmarinic acid concentrations than the previous reports.

Table 1

Polyphenol and terpenoid content (µg/g) in different extraction of Rosemary analyzed by ultra-high-performance liquid chromatography and electrospray ionization, coupled with quadrupole-time of flight mass spectrometry (UHPLC-ESI-QTOF-MS). (Figures S1–S6)

Treatment	Polyphenol and Terpenoid Content (µg/g) in Rosemary
	Caffeic Acid	Rosmarinic Acid	Luteolon-7-O-Glucoside	Carnosic Acid	Carnosol	Ursolic Acid
T1	6.02 ± 0.08 b	1.51 ± 0.07 a	1.59 ± 0.22 a	0.64 ± 0.01 a	112.06 ± 0.61 b	5.32 ± 0.17 a
T2	12.30 ± 0.33 c	1124.03 ± 13.62 b	7.14 ± 0.14 bc	1374.63 ± 7.72 b	171.52 ± 1.59 b	6.08 ± 0.17 a
T3	13.03 ± 0.70 c	2.51 ± 0.35 a	ND	0.25 ± 0.01 a	0.54 ± 0.01 a	0.36 ± 0.01 a
T4	38.56 ± 1.58 e	5428.47 ± 19.69 c	ND	0.97 ± 0.01 a	1.91 ± 0.04 a	2.36 ± 0.07 a
T5	322.02 ± 3.39 g	13,310.13 ± 26.12 d	130.53 ± 5.41 d	2671.83 ± 20.03 c	417.21 ± 1.99 c	89.20 ± 1.92 b
T6	106.83 ± 1.49 f	15,242.40 ± 43.62 e	4.67 ± 0.68 ab	5.39 ± 0.48 a	10.82 ± 0.59 a	6.09 ± 0.19 a
T7	40.55 ± 0.03 e	33,491.33 ± 86.29 g	209.95 ± 8.78 e	2915.40 ± 33.23 d	22,000.67 ± 77.39 d	5144.27 ± 28.68 d
T8	2.40 ± 0.06 a	0.26 ± 0.00 a	0.97 ± 0.01 a	ND	2.19 ± 0.19 a	10.37 ± 0.88 a
T9	23.77 ± 1.63 d	15,944.00 ± 36.39 f	9.11 ± 0.35 c	6.97 ± 0.34 a	34.98 ± 1.10 a	1042.88 ± 11.33 c
Mean	62.83	9393.85	52.99	872.01	2527.99	700.77
F Test	**	**	**	**	**	**
SEM ±	0.85	21.02	0.81	7.61	14.90	5.95
CD at 1 %	3.46	85.56	3.28	31.00	60.67	24.21

** Significant at 1% level, values followed by different letters indicate a significant difference between the treatments at p < 0.01; ND—Not detected. Treatment Details: T1: Fresh tissue homogenization; T2: Fresh leaf decoction; T3: Fresh homogenized tissue extract fermentation; T4: Fresh leaf decoction fermentation; T5: Dry leaf decoction; T6: Dry leaf decoction fermentation; T7: Soxhlet extraction; T8: Sonic extraction—aqueous; T9: Sonic extraction—methanol.

The decoction from dry leaf powder (T5) recorded significantly higher levels of caffeic acid (322.02 ± 3.39 µg/g) as compared to other treatments. Fresh leaf decoction also contained a considerable amount of rosmarinic acid, carnosic acid, and carnosol. Carrying out the decoction process using water helps to dissolve the maximum amounts of these water-soluble compounds [18]. Levels of polyphenols and terpenoid compounds were significantly higher in dry leaf decoction compared to fresh leaf decoction, primarily because the amount of biomass that could be extracted was immense. The conversion rate of fresh to dry rosemary was 33%. Among the traditional extraction methods, dry leaf decoction (T5) and its fermentation (T6) were found to yield higher levels of caffeic acid and rosmarinic acid. Fermentation significantly enhanced the rosmarinic acid levels in both T4 and T6. Fermentation also enhanced caffeic acid content in both fermented fresh homogenized tissue extract (T3) and fresh leaf decoction (T4). This may be due to the microbial transformation of chemical compounds and better extraction of herbal constituents due to the production of alcohol during fermentation. It may also be the case that extraction was facilitated by fermentation due to the release of bacterial enzymes that broke down cell walls of the rosemary plant, making compounds more accessible to extraction by a solvent [19,20]. In the present study, fermentation enhanced the phenolic acids; however, it reduced flavonoid content, luteolin-7-glucoside and diterpenoids, carnosic acid, and carnosol. It is likely that the oxidation of phenolic compounds during fermentation reduced the levels of certain polyphenols. Similar results were reported in Centella asiatica and Orthosiphon aristatus [21,22,23].

Ultrasound extraction using methanol (T9) resulted in significantly higher concentrations of rosmarinic acid and ursolic acid as compared to other fresh extraction and fermentation procedures. Ultrasound is known to disrupt plant cell walls, thereby facilitating the release of extractable compounds and enhancing mass transport of solvent from plant cells into the solvent phase. This effect boosts compound recovery, mostly when an optimal solvent, in this case, methanol, was used [13,24]. In contrast to sonication with methanol, sonication with water yielded the lowest levels of phenolic acids and flavonoids of all extraction methods employed. This is not surprising since the complex structures of phenolic compounds cause them to be rather insoluble in aqueous media [25]. Among aqueous and methanol extraction, methanol extracts showed significantly higher polyphenols and terpenoids, especially in Soxhlet and sonic extracts. This might be due to the higher solubility of complex bioactive compounds in organic solvents than the aqueous base [25,26]. The herb was found to contain a considerable quantity of rosmarinic acid and ursolic acid in most of the extractions, responsible for its healing properties, supporting traditional usage for treating gastrointestinal inflammation, colitis, colon cancer, and nervous system inflammation [27].

2.2. Identification and Characterization of Bioactive Constituents in R. officinalis

Rapid separation polyphenol molecules were achieved within the first 11 min of 20 min of UHPLC analysis duration. More than 50 polyphenolic compounds have been identified by UHPLC-ESI-QTOF-MS under negative electrospray ionization conditions [M − H]− based on their retention times, molecular weights, and mass (m/z) fragmentation patterns. The study was focused on negative ionization mode [M − H]− because it is reported to be more sensitive for analysis of phenolic acids and flavonoids, compared to positive ionization mode [28,29,30]. The phenolic compounds in rosemary extracts were mostly flavonoids, phenolic acids, and terpenoids. The terpenoids included diterpenoids largely, along with a few triterpenoids. The data are presented in three groups: polyphenols in homogenous aqueous extraction (T1) and its fermentation (T3) (Table 2); fresh and dry leaf decoctions (T2 and T5), and their respective fermentations (T4 and T6) (Table 3); industrial extractions Soxhlet (T7) and sonication with water and methanol (T8 and T9) (Table 4). Chromatograms depicting the intensity of polyphenols in different rosemary extracts (T1–T9) versus retention time (min) are presented in Figure 1a–i. The compounds without reference standards were identified tentatively by comparing the mass spectra data, ion fragmentation, and molecular weight (m/z) with data available in the literature [17,31] and the mass spectral library obtained from the National Institutes of Standards and Technology (NIST-2017), AOI (All-in-One) spectral library from Sciex, MoNA (MassBank of North America), and HILIC (Hydrophilic Interaction Liquid Chromatography) library database from University of California, Davis.

Table 2

Bioactive compounds in fresh tissue homogenization (T1) and its fermentation (T3) of Rosmarinus officinalis identified by UHPLC-ESI-QTOF-MS.

Sl No	Compound	RT (min)	Mass[M − H]− (m/z)	Formula	Fragments	T1	T3
1.	Quinic acid	0.42	191.05681	C7H12O6	85.0297 (42) *, 127.0401 (24), 59.0165 (13)	+	+
2.	Caffeic acid	0.78	179.03520	C9H8O4	135.0438 (100), 134.0370 (21)	+	+
3.	p-Coumaric acid	1.44	163.04014	C9H8O3	119.0500 (100)	+	+
4.	Gallocatechin	1.65	305.07057	C15H14O7	225.1126 (69), 96.9597 (16), 98.9574 (8)	+	+
5.	Luteolin 7-O-rutinoside	2.18	593.15382	C27H30O15	297.0740 (8), 285.0410 (6)	+	−
6.	Salvianolic acid B	2.29	717.14274	C36H30O16	519.0900 (62), 339.0494 (33)	+	−
7.	Rosmarinic acid	2.58	359.07906	C18H16O8	161.0236 (100), 197.0449 (72), 179.0347 (68), 135.0448 (7)	+	+
8.	Isorhamnetin-3-glucoside	2.80	477.10646	C22H22O12	315.0695 (38)	+	−
9.	Apigenin-7-O-glucoside	2.88	431.109907	C21H20O10	269.0449 (100)	+	−
10.	Hesperidin	2.92	609.18546	C28H34O15	301.0695 (100)	+	+
11.	Hispidulin rutinoside	2.95	607.17094	C28H32O15	301.0699 (100), 299.0559 (24)	+	+
12.	Hispidulin-7-O-glucoside	3.03	461.11113	C22H22O11	283.0234 (13), 299.0561(8)	+	−
13.	6-Hydroxyluteolin-7-O-glucoside	3.10	463.08011	C21H20O12	301.0350 (100)	+	−
14.	Luteolin	3.11	285.03995	C15H10O6	133.0284 (12), 151.0029 (12), 175.0395 (9),199.0395 (8)	−	+
15.	Luteolin-7-O-glucuronide	3.11	461.07495	C21H18O12	285.0385 (100)	+	+
16.	Isorhamnetin	3.20	315.05001	C16H12O7	300.0255 (100), 301.0311 (39)	+	+
17.	Luteolin 3’-acetyl-O-glucuronide isomer I	3.29	503.08570	C23H20O13	285.0381 (100), 443.0587 (100), 381.0606 (35), 399.0720 (28)	+	+
18.	Luteolin 3’-acetyl-O-glucuronide isomer II	3.38	503.08550	C23H20O13	285.0366 (100)	+	+
19.	Apigenin	3.49	269.04559	C15H10O5	117.0356 (12), 149.0356 (8), 225.0560 (5)	−	+
20.	Hesperetin	3.58	301.07074	C16H14O6	242.0571 (87), 284.286.0468 (55), 164.0108(54), 151.0036 (35)	−	+
21.	Diosmetin	3.58	299.05595	C16H12O6	284.0310 (100)	−	+
22.	Luteolin 3’-acetyl-O-glucuronide	3.62	503.08594	C23H20O13	285.4652 (100), 443.0598 (76)	+	+
23.	Rosmanol isomer	4.07	345.16874	C20H26O5	301.1782 (100), 283.1673 (68), 284.1719 (29)	−	+
24.	Pectolinarigenin	4.15	313.07285	C17H14O6	298.0464 (100), 283.0235 (52), 255.0285 (17), 163.0034 (10), 227.0344 (6), 117.0350 (4)	+	+
25.	Rosmanol	4.30	345.17145	C20H26O5	301.1782 (100), 283.1673 (65), 284.1719 (32)	+	+
26.	Genkwanin	4.58	283.06224	C16H12O5	268.0381 (100), 240.0431 (6)	+	+
27.	Rosmanol isomer	4.60	345.17190	C20H26O5	284.1704 (100)	−	+
28.	Rosmadial isomer	4.98	343.15577	C20H24O5	299.1618 (55), 243.1010 (9)	+	+
29.	Rosmanol methyl ether	5.08	359.14801	C21H18O5	315.1577 (19)	−	+
30.	Rosmanol	5.15	345.16890	C20H26O5	283.1669 (69)	−	+
31.	Carnosol isomer	5.49	329.17480	C20H26O4	285.1825 (100)	−	+
32.	Rosmadial	5.61	343.15305	C20H24O5	299.1623 (100)	+	+
33.	Trihydroxy-methoxyflavone	5.70	299.16397	C16H12O6	284.0310 (100)	−	+
34.	Carnosol	5.75	329.17666	C20H26O4	285.1834 (100)	+	+
35.	Carnosic acid	5.76	331.18358	C20H28O4	287.1649 (100)	+	+
36.	Rosmaridiphenol	6.16	315.19780	C20H26O3	285.1843 (19)	+	+
37.	Rosmadial isomer	6.20	343.15249	C20H24O5	299.1598 (56)	+	+
38.	Rosmadial isomer	6.56	343.15233	C20H24O5	299.1602 (68)	+	+
39.	12-methoxy-carnosic acid	6.99	345.20823	C21H30O4	301.2157 (100), 286.1923 (65)	+	+
40.	Betulinic acid	8.05	455.34934	C30H48O3	−	+	−
41.	Ursolic acid	8.10	455.35307	C30H48O3	−	+	+

T1: Fresh tissue homogenization; T3: Fresh homogenized tissue extract fermentation; * Fragmentation values are followed by their intensity % in parenthesis.

Table 3

Analysis of bioactive compounds in fresh leaf (T2) and dry leaf decoction (T5) and their respective fermented extracts (T4 and T6) of R. officinalis by UHPLC-ESI-QTOF-MS.

Sl No	Compound	RT (min)	Mass[M − H]− (m/z)	Formula	Fragments	T2	T4	T5	T6
1.	Quinic acid	0.35	191.05670	C7H12O6	85.0301 (39), 93.0354 (18), 127.0406 (15)	+	+	+	+
2.	Syringic acid	0.42	197.04643	C9H10O5	135.0450 (100), 123.0450 (100), 72.9947 (84), 179.0349 (54)	+	+	+	+
3.	Chlorogenic acid	0.67	353.08621	C16H18O9	191.0560 (28)	+	−	+	−
4.	Caffeic acid	0.78	179.03600	C9H8O4	135.0444 (100), 134.0372 (19)	+	+	+	+
5.	4-O-Caffeoyl quinic acid	1.00	353.08940	C16H18O9	173.0439 (100), 179.0329 (37), 135.0434 (14)	+	−	+	−
6.	p-Coumaric acid	1.30	163.04015	C9H8O3	119.0509 (100)	+	+	+	+
7.	Gallocatechin	1.40	305.07127	C15H14O7	225.1123 (49), 96.9595 (24)	+	+	+	+
8.	6-Hydroxyluteolin-7-O-glucoside	1.89	463.08849	C21H20O12	286.0427 (100), 301.0350 (69), 285.7613 (44)	+	−	+	−
9.	Luteolin-7-O-glucoside	2.18	447.09508	C21H20O11	285.0413 (53)	+	−	+	+
10.	Luteolin 7-O-rutinoside	2.18	593.15454	C27H30O15	285.0431 (11)	+	+	+	+
11.	Scutellarin	2.21	461.07517	C21H18O12	285.0405 (100), 113.0252 (9), 175.0252 (6)	−	−	−	+
12.	Rosmarinic acid-3-O-glucoside	2.23	521.13273	C24H26O13	359.0792 (100), 324.0832 (78), 323.0785 (60)	+	+	+	+
13.	Salvianolic acid B	2.29	717.15054	C36H30O16	519.0891 (100), 339.0500 (15)	+	+	+	+
14.	Isorhamnetin-3-O-glucoside	2.33	477.10584	C22H22O12	315.0539(38)	+	+	+	+
15.	Sagerinic acid	2.52	719.16630	C36H32O16	359.0761 (100), 179.0336 (20), 161.0223 (16)	+	+	+	+
16.	Rosmarinic acid	2.56	359.07835	C18H16O8	161.0238 (100), 197.0447 (64), 179.0341 (57), 133.0290 (35), 72.9940 (6)	+	+	+	+
17.	Apigenin-7-O-glucoside	2.59	431.09779	C21H20O10	269.0420 (100), 149.0969 (3)	−	−	+	−
18.	Hesperidin	2.63	609.18493	C28H34O15	301.0714 (100)	+	+	+	+
19.	Salvianolic acid A	2.64	493.11382	C26H22O10	295.0615 (100), 185.0224 (43), 109.0289 (11)	+	+	+	+
20.	Diosmin	2.66	607.17017	C28H32O15	301.0704 (100), 299.0551 (59)	+	+	+	+
21.	Hispidulin-7-O-glucoside	2.68	461.11080	C22H22O11	283.0235 (12), 299.0552 (9)	+	−	+	−
22.	Luteolin-7-O-glucuronide	2.79	461.07517	C21H18O12	286.0430 (100), 285.0399 (38)	+	+	+	+
23.	Hesperetin	2.87	301.07245	C16H14O6	286.0459 (12), 164.0100 (4)	−	−	+	−
24.	Methyl rosmarinate	2.99	373.09453	C19H18O8	175.0403 (100), 357.0610 (61), 198.0477 (33), 179.0367 (22), 135.0465 (11)	+	+	+	+
25.	Luteolin 3’-acetyl-O-glucuronide isomer I	3.10	503.08463	C23H20O1	399.0721 (100), 285.7547 (6)	+	+	+	+
26.	Luteolin	3.11	285.04114	C15H10O6	133.0302 (18), 151.0051 (6), 175.0410 (5), 199.0414 (4)	+	+	+	+
27.	Isorhamnetin	3.17	315.05133	C16H12O7	300.0279 (100), 301.0332 (32)	+	+	+	+
28.	Luteolin 3’-acetyl-O-glucuronide isomer II	3.27	503.08550	C23H20O1	286.0415 (100), 285.7547 (62), 443.0607 (60), 399.0721 (7)	+	+	+	+
29.	Apigenin	3.47	269.04675	C15H10O5	117.0350 (8), 151.0043(7), 225.0576 (4)	+	+	+	+
30.	Luteolin 3’-acetyl-O-glucuronide	3.62	503.08530	C23H20O13	286.0415 (100), 443.0607 (47), 285.7547 (38)	+	+	+	+
31.	Diosmetin	3.64	299.05647	C16H12O6	284.0339 (100)	+	+	+	+
32.	Rosmanol isomer	4.07	345.17252	C20H26O5	301.1802 (100), 283.1698 (67)	+	+	+	+
33.	3,7 Dihydroxy-dimethoxyflavone	4.15	313.07320	C17H14O6	298.0473 (100), 283.0243 (70), 255.0306 (19), 269.0464 (12)	−	−	+	−
34.	Pectolinarigenin	4.15	313.07258	C17H14O6	298.0469 (100), 283.0240 (63)	+	+	−	+
35.	Rosmanol	4.30	345.17100	C20H26O5	283.8834 (18)	+	+	+	+
36.	Pectolinarigenin isomer	4.37	313.07211	C17H14O6	298.0471 (91), 283.0233 (63), 255.0290 (24)	+	−	−	−
37.	Rosmanol isomer	4.57	345.17200	C20H26O5	284.1750 (13), 283.1706 (11)	+	+	+	+
38.	Genkwanin	4.58	283.06218	C16H12O5	268.0398 (79), 240.0434 (5)	+	+	+	+
39.	Rosmadial isomer	4.80	343.15636	C20H24O5	299.1669 (32)	+	+	+	+
40.	Rosmanol isomer	5.17	345.17210	C20H26O5	283.8769 (39)	+	+	+	+
41.	Rosmanol methyl ether	5.07	359.18598	C21H28O5	283.1703 (100), 300.1747 (82)	+	+	&	&
42.	Asiatic acid	5.57	487.34312	C30H48O5	&	+	&	&	&
43.	Rosmadial	5.69	343.15590	C20H24O5	299.1645 (9)	+	+	+	+
44.	Trihydroxy- methoxyflavone	5.69	299.16340	C16H12O6	284.0333 (100)	&	+	&	&
45.	Carnosol	5.75	329.17690	C20H26O4	286.1870 (100), 285.1845 (92)	+	+	+	+
46.	Carnosic acid	5.76	331.19253	C20H28O4	287.2007 (100)	+	+	+	+
47.	Rosmadial isomer	6.04	343.17192	C20H24O5	299.1653 (15)	-	+	&	+
48.	Rosmaridiphenol	6.16	315.19689	C20H28O3	284.1860 (4)	+	+	+	+
49.	Carnosol isomer	6.17	329.17560	C20H26O4	286.1880 (100), 285.1852 (58)	+	&	+	&
50.	12-methoxy-carnosic acid	6.99	345.20853	C21H30O4	286.1943 (100), 301.2186 (81)	+	+	+	+
51.	Micromeric acid	7.64	453.33442	C30H46O3	&	+	&	+	&
52.	Betulinic acid	8.05	455.34921	C30H48O3	&	+	&	+	&
53.	Ursolic acid	8.10	455.35205	C30H48O3	&	+	+	+	+

T2: Fresh leaf decoction; T4: Fresh leaf decoction fermentation; T5: Dry leaf decoction; T6: Dry leaf decoction fermentation.

Table 4

Analysis of bioactive constituents by UHPLC-ESI-QTOF-MS in Soxhlet extract (T7) and sonicated extracts (water and methanol—T8 and T9) of R. officinalis.

Sl No	Compound	RT (min)	Mass[M − H]− (m/z)	Formula	Fragments	T7	T8	T9
1.	Quinic acid	0.35	191.05628	C7H12O6	85.0299 (34), 93.0353 (17), 127.0403 (12)	+	+	+
2.	Syringic acid	0.42	197.04569	C9H10O5	135.0450 (100), 123.0450 (75), 72.9947 (60), 179.0349 (54)	+	-	+
3.	Chlorogenic acid	0.56	353.08506	C16H18O9	191.0545 (26)	&	&	+
4.	Caffeic acid	0.82	179.03589	C9H8O4	135.0441 (100), 134.0370 (23)	+	&	+
5.	4-O-Caffeoyl quinic acid	0.85	353.08949	C16H18O9	173.0437 (100), 191.0544 (27), 179.0334 (6)	&	&	+
6.	p-Coumaric acid	1.28	163.04014	C9H8O3	119.0509 (100)	+	+	+
7.	Gallocatechin	1.81	305.06932	C15H14O7	96.9588 (65), 225.1109 (59)	+	+	+
8.	6-Hydroxyluteolin-7-O-glucoside	1.97	463.08518	C21H20O12	301.0350 (69), 285.7613 (44)	&	&	+
9.	3-p-coumaroylquinic acid	2.1	337.10122	C16H18O8	163.0397 (100), 119.0506 (32)	+	&	&
10.	Luteolin-7-O-glucoside	2.23	447.09286	C21H20O11	285.0377 (29)	+	+	+
11.	Luteolin 7-O-rutinoside	2.24	593.15126	C27H30O15	285.0380 (2)	+	&	+
12.	Rosmarinic acid-3-O-glucoside	2.25	521.12957	C24H26O13	359.0740 (100), 323.0737 (89), 179.0337 (15)	+	&	+
13.	Apigenin-7-O-glucunoride	2.51	445.07602	C21H18O11	269.0437 (100), 113.0255 (10), 175.0252 (9)	&	+	&
14.	Sagerinic acid	2.52	719.15575	C36H32O16	359.0761 (100), 179 (50), 161.0223 (17)	+	&	+
15.	Rosmarinic acid	2.56	359.07651	C18H16O8	161.0229 (100), 197.0434 (78), 179.0330 (60), 133.0285 (15), 72.9934 (8)	+	+	+
16.	Isorhamnetin-3-O-glucoside	2.56	477.10320	C22H22O12	315.0466 (32), 300.0246 (5)	+	&	+
17.	Apigenin-7-O-glucoside	2.59	431.09762	C21H20O10	269.0435 (100)	+	+	+
18.	Isoferulic acid	2.62	193.05102	C10H10O4	134.0386 (89), 133.0295 (75), 178.0271 (12)	+	&	&
19.	Isorhamnetin-3-O-rutinoside	2.63	623.15851	C28H32O16	315.0471 (9)	+	&	+
20.	Hispidulin-7-O-glucuronide	2.64	475.08642	C22H20O12	299.0543 (100), 285.0367 (50), 283.0313 (3)	&	+	&
21.	Hesperidin	2.64	609.18172	C28H34O15	301.0674 (100)	+	&	+
22.	Salvianolic acid A	2.64	493.11132	C26H22O10	295.0615 (100), 185.0224 (43), 109.0289 (11)	+	&	&
23.	Diosmin	2.66	607.16666	C28H32O15	299.0527 (57), 284.0309 (4)	+	+	+
24.	Hispidulin-7-O-glucoside	2.69	461.11138	C22H22O11	298.0477 (18), 283.0234 (7)	+	&	+
25.	Kaempferol-7-O-hexoside	2.72	447.09094	C21H20O11	285.0382 (100)	+	&	+
26.	Luteolin-7-O-glucuronide	2.83	461.06888	C21H18O12	285.7541 (100)	+	+	+
27.	Phlorizin	2.95	435.13210	C21H24O10	273.0767 (100), 167.0349 (28), 125.0247 (5)	+	&	&
28.	Luteolin	3.12	285.04024	C15H10O6	133.0300 (14), 151.0042 (10), 175.0409 (8), 199.0406 (6)	+	+	+
29.	Isorhamnetin	3.21	315.04996	C16H12O7	300.0248 (100), 301.0286 (41)	+	&	+
30.	Luteolin 3’-acetyl-O-glucuronide isomer I	3.29	503.08148	C23H20O13	399.0707(100), 285.4652 (9), 443.0598 (5)	&	+	&
31.	Methyl rosmarinate	3.30	373.08946	C19H18O8	135.0441 (100), 175.0397 (100), 179.0346 (85), 197.0449 (79)	+	&	+
32.	Luteolin 3’-acetyl-O-glucuronide II	3.38	503.08230	C23H20O13	286.0407 (100), 285.4649 (38)	+	+	+
33.	Apigenin	3.50	269.04386	C15H10O5	117.0347 (11), 151.0036 (11), 225.0557 (5)	+	+	+
34.	Salvianolic acid B	2.29	717.14025	C36H30O16	519.0912 (57), 339.0471 (37)	+	&	&
35.	Hesperetin	3.58	301.07242	C16H14O6	164.0114 (11), 286.0827 (5)	&	+	&
36.	Luteolin 3’-acetyl-O-glucuronide	3.62	503.08259	C23H20O13	443.0607 (100), 285.7547 (68)	+	+	+
37.	Diosmetin	3.64	299.05429	C16H12O6	284.0298 (100)	+	+	+
38.	Rosmanol isomer	4.07	345.16839	C20H26O5	301.1773 (100), 283.1668 (65)	+	&	+
39.	Pectolinarigenin	4.15	313.06990	C17H14O6	298.0474 (100), 283.0237 (59), 255.0295 (24), 163.0037 (15), 117.0345 (7)	+	+	+
40.	Rosmanol	4.30	345.16852	C20H26O5	301.1773 (100), 283.1668 (57)	+	&	+
41.	Pectolinarigenin isomer	4.37	313.06892	C17H14O6	298.0471 (91), 283.0233 (63), 255.0290 (24)	+	&	&
42.	Triptolidenol	4.45	375.15421	C20H24O7	331.1526 (13), 244.1082 (9), 313.1430 (7)	&	&	+
43.	Rosmadial isomer	4.54	343.15249	C20H24O5	299.1610 (9)	+	&	+
44.	Genkwanin	4.58	283.06017	C16H12O5	268.0379 (89), 117.0353 (5), 151.0039 (4)	+	+	+
45.	Rosmanol isomer	4.59	345.16789	C20H26O5	284.1687 (40), 283.8801 (23)	+	&	+
46.	Rosmanol isomer	4.80	345.16793	C20H26O5	283.1668 (19)	+	&	+
47.	Asiatic acid	5.47	487.34312	C30H48O5	&	+	&	&
48.	Rosmadial	5.71	343.15314	C20H24O5	299.1616 (10)	+	+	+
49.	Rosmanol isomer	5.71	345.16811	C20H26O5	283.1670 (12)	&	+	&
50.	Carnosol	5.75	329.17532	C20H26O4	285.1833 (100)	+	+	+
51.	Epirosmanol methyl ether	5.85	359.18381	C21H28O5	283.1665 (97), 329.1719 (16), 300.1713 (15)	+	&	+
52.	Rosmadial isomer	6.05	343.15233	C20H24O5	299.1621 (11)	+	&	+
53.	Carnosic acid	6.58	331.19123	C20H28O4	287.1982 (100)	+	&	+
54.	Corosolic acid	6.61	471.34212	C30H48O4	&	&	&	+
55.	12-methoxy-carnosic acid	6.99	345.20630	C21H30O4	301.2170 (100), 287.1938 (64)	+	&	+
56.	Micromeric acid	7.84	453.34261	C30H46O3	&	+	&	+
57.	Betulinic acid	8.05	455.34934	C30H48O3	&	+	&	+
58.	Ursolic acid	8.10	455.35011	C30H48O3	&	+	+	+

T7: Soxhlet extraction; T8: Sonic extraction—aqueous; T9: Sonic extraction—methanol.

Figure 1

Chromatogram representing relative abundance of polyphenols and terpenoids in different extracts of rosemary leaves (T1–T9) analyzed through ultra-high-performance liquid chromatography with electrospray ionization and quadrupole-time of flight mass spectrometry (UHPLC-ESI-QTOF-MS) (intensity versus elution time); (a) Fresh tissue homogenization (T1); (b) Fresh homogenized tissue extract fermentation (T3); (c) Fresh leaf decoction (T2); (d) Fresh leaf decoction fermentation (T4); (e) Dry leaf decoction (T5); (f) Dry leaf decoction fermentation (T6); (g) Soxhlet extraction (T7); (h) Sonication with water (T8); (i) Sonication with methanol (T9). Peak numbers refer to: 1—quinic acid; 2—caffeic acid; 3—coumaric acid; 4—gallocatechin; 5—rosmarinic acid-3-O-glucoside; 6—luteolin-7-O-glucoside; 7—salvianolic acid B; 8—rosmarinic acid; 9—hesperidin; 10—Salvianolic acid A; 11—Sagerinic acid; 12—Luteolin 3’-acetyl-O-glucuronide; 13—Apigenin; 14—Diosmetin; 15—Rosmanol; 16—Pectolinarigenin; 17—Rosmadial; 18—Carnosol; 19—Carnosic acid; 20—Corosolic acid; 21—12-methoxy-carnosic acid; 22—Micromeric acid; 23—Betulinic acid; 24—Ursolic acid.

As shown in Table 2, there were about 41 polyphenols detected in fresh homogenized tissue extraction (T1) and its fermentation (T3); among them, T1 contained 30 and T3 contained 33 polyphenols. The chromatogram in Figure 1a,b represents the relative intensity of phenolic compounds in T1 and T3, respectively. The fermented sample (T3) was found to have a higher intensity of rosmanol and rosmadial compared to T1, whereas the relative intensities of luteolin 3-acetyl-O-glucuronide and carnosol were high in T1. Rosemary leaf decoctions (fresh and dry, T2, and T5, respectively) were found to be a more efficient extraction method for polyphenol content. In total, 54 phenolic compounds were identified in T2 and T5 and their fermented extracts (T4 and T6) (Table 3). The intensities of phenolic compounds and terpenoids were lower in the fermented decoctions compared to the fresh and dry leaf decoctions (Figure 1c–f). This is consistent with earlier reports with other herbs, indicating that prolonged fermentation can break down phenolic compounds resulting in decreased antioxidant potential [22].

A large group of phenolic compounds was observed in Soxhlet and sonicated methanol extracts (T7 and T9, respectively). Of the 59 polyphenols, 11 were tentatively identified as phenolic acids and seventeen as terpenoids (Table 4). In methanolic samples, the intensity of terpenoid compounds, rosmanol, rosmadial, carnosol, carnosic acid, and ursolic acid, was found to be very high, as is depicted in their chromatograms in Figure 1g,i. Sonication of rosemary in water (T8) resulted in a much lower number of polyphenols, compared to methanolic extraction (Figure 1h). This is likely due to the lower solubility of complex terpenoids and phenolic molecules in water compared to methanol [24]. Out of 11 identified phenolic compounds in methanol extracts, quinic acid, syringic acid, chlorogenic acid, caffeic acid, 4-O-caffeoylquinic acid, p-coumaric acid, and rosmarinic acid have been reported before [16,30]. However, isoferulic acid ([M − H]− m/z 193.05), sagerinic acid ([M − H]− m/z 719.16), and salvianolic acid A ([M − H]− m/z 493.11) and B ([M − H]− m/z 717.15) were reported herein for the first time in rosemary extracts, based on comparison of the m/z ion fragmentation pattern of the observed compounds compared to those in the NIST MS library. Sagerinic acid was found in very high intensities (T2, T4, T5, T6, T7, and T9—Figure 1), and it shared some (m/z) MS/MS ion fragments (359.08) with rosmarinic acid ([M − H]− m/z 359.08). Lu and Foo reported sagerinic acid as possible derivatives of rosmarinic acid, since they are structurally related [32]. Similarly, syringic acid ([M − H]− at m/z 197.05), 4-O-caffeoylquinic acid ([M − H]− at m/z 353.09), rosmarinic acid ([M − H]− at m/z 359.08), and methyl rosmarinate ([M − H]− at m/z 373.09) all shared many of the same MS/MS (m/z) ion fragments (179.03), since they were found to be dimers of caffeic acid.

A large group of flavonoids has been reported in this study, and most of them were derivatives of luteolin ([M − H]− at m/z 285.04), hesperidin ([M − H]− at m/z 609.18), and apigenin ([M − H]− at m/z 269.04). Similar results were obtained from LC/MS analysis of rosemary herb from the USA and Iraq [17,31]. Very high intensities of gallocatechin ([M − H]− at m/z 305.07) were observed in all rosemary extracts (Figure 1a–i), and this was reported in previous studies [6,17]. Gallocatechin is a flavan-3-ol found predominantly in fruit peels, and gallocatechin was reported to be responsible for the high antioxidant potential of the herb [33,34,35]. In the present study, some flavonoid compounds have been detected for the first time in rosemary, viz., phlorizin ([M − H]− at m/z 435.13) in Soxhlet extract (Table 4) and pectolinarigenin ([M − H]− at m/z 313.07) in all the extracts (T1–T9). Phlorizin was earlier found in tree barks of the Rosaceae family, and the studies indicated high antidiabetic property of the drug [36]. Pectolinarigenin was also reported before in rosemary as dimethoxyflavone with similar fragment ions, and it was found to have potent anti-inflammatory and anticancer properties [31,37,38]. Further, these newly detected flavonoids and phenolic acids can be confirmed by procuring respective standards or by using advanced techniques like nuclear magnetic resonance (NMR) spectroscopy for identification and confirmation of unknown molecules. The presence of three peaks for luteolin 3’-acetyl-O-glucuronide ([M − H]− at m/z 503.08) eluted at 3.29, 3.38, and 3.62 min with similar m/z fragments (443.06, 245.47) could be observed in chromatograms of all extracts (T1–T9). Previously, multiple peaks for luteolin 3’-acetyl-O-glucuronide in rosemary extract were reported by Borras-Linares [17]. These are probably due to the existence of multiple positional isomers of this compound in rosemary.

There were about 17 terpenoid compounds that have been tentatively identified in methanolic extracts of rosemary, out of which 12 were diterpenoids (Table 4). Rosmanol ([M − H]− at m/z 345.17), rosmadial ([M − H]− at m/z 343.15), carnosol ([M − H]− at m/z 329.18), carnosic acid ([M − H]− at m/z 331.19), and 12-methoxy carnosic acids ([M − H]− at m/z 345.21) were the major diterpenoids present in higher intensities in T2, T3, T4, T5, T6, T7, and T9 (Figure 1). The presence of more than one peak corresponding to the same molecular mass but different elution times was due to the presence of isomers, especially in rosmanol and rosmadial. Rosmanol ([M − H]− m/z 345.17) eluted at four different retention times, with the same ion fragmentation (MS2 m/z fragments 301.1779, 183.1668). Rosmadial ([M − H]− m/z 343.15) and its isomers also resulted in three to four peaks with similar fragmentation patterns (MS2 m/z 299.16). Similar peaks were obtained in rosmadial of sage and rosemary extracts during the chromatographic determination of polyphenols [31,39]. A diterpenoid, triptolidenol ([M − H]− at m/z 375.15), was detected only in T9. Five pentacyclic triterpenoid compounds viz., asiatic acid ([M − H]− at m/z 487.33), corosolic acid ([M − H]− at m/z 471.34), micromeric acid ([M − H]− at m/z 453.34), betulinic acid, and ursolic acid ([M − H]− at m/z 455.35) were tentatively detected in methanolic samples (Table 4). Previously, betulinic acid, ursolic acid, and micromeric acids were determined in rosemary leaves [17,31]. Betulinic acid in the herbs was found to have potent antiviral activity against severe acute respiratory syndrome coronavirus [40]. A triterpenoid corosolic acid was tentatively identified for the first time in the rosemary extract of T7. Asiatic acid was present in T2 and T7; micromeric acid and betulinic acid were detected in T2, T5, T7, and T9; ursolic acid was found in all extracts. Pentacyclic triterpenoids reported having several medicinal properties, especially anti-inflammatory, anticancer, and antidiabetic potential [41,42]. Even though ursolic acid and betulinic acid have the same pseudomolecular weight ([M − H]− at m/z 455.35), the former was identified through the reference standard, and the later molecule was confirmed by comparison to the NIST mass spectral library. Besides, several other compounds were detected in significant amounts in certain extracts that were not represented in the mass spectral databases available.

Even though comparison of high resolution, accurate mass, LC-MS/MS chromatograms and m/z fragmentation patterns of observed compounds with high-resolution mass spectral libraries is a very effective approach for the identification and characterization of known and previously unknown compounds, this approach is limited to those compounds represented in MS/MS libraries. Our analyses generated mass spectral data for a large number of yet-to-be-identified phenolic compounds present in the rosemary extracts, which we analyzed. As mass spectral libraries expand, the data that we have already gathered can be further analyzed to structurally identify additional polyphenols based on m/z fragmentation patterns. In addition, if further inspection of our data identifies unnamed compounds that are of particular interest, possibly due to a high abundance of other features of interest, then additional work can be done to isolate and identify those compounds using nuclear magnetic resonance (NMR) spectroscopy and other approaches.

3. Materials and Methods

3.1. Herb Collection

Rosemary (Rosmarinus officinalis L.) leaves after eight months of planting were harvested from the Regenerative Organic Farm, Maharishi University of Management, Fairfield, Iowa. Freshly harvested leaves were used for fresh extractions, whereas air-dried leaf powder was used for dry extractions. The sample was submitted to Ada Hayden Herbarium (ISC/IA), Iowa State University, Iowa, USA, and obtained the accession no. ISC-454695.

3.2. Chemicals

LCMS grade acetonitrile and methanol were purchased from Honeywell, Burdick, and Jackson, USA. LCMS grade formic acid and glacial acetic acid were procured from Merck, Germany. Caffeic acid, rosmarinic acid, carnosic acid, ursolic acid, and luteolin 7-glucoside were purchased from Toronto Research Chemicals, Canada. Carnosol and 13C- caffeic acid were purchased from Cayman Chemical, USA. Ultrapure water from the Milli-Q, A10 water purification system (Millipore Sigma, Madison, WI, USA) was used throughout the experiment.

3.3. Preparation of R. Officinalis Extracts

There were nine different sample extraction methods used as treatments for liquid chromatographic analysis.

T1: Fresh aqueous extraction by tissue homogenization—10 g of fresh leaf samples was macerated in 100 mL Milli-Q water at room temperature and fresh leaf juice was extracted by filtering through cellulose filter paper.

T2: Fresh leaf decoction—10 g of fresh leaves was chopped into 1–2 cm pieces and boiled in 200 mL Milli-Q water at 100–110 °C temperature until the volume was reduced to 100 mL. The extract was cooled, filtered, and used for the analysis.

T3: Fresh tissue homogenized extract fermentation—homogenized fresh leaf tissue extract (T1) was fermented by adding 24% sugar and 10 mg of the activated wine yeast Saccharomyces cerevisiae for 60 days; the resultant clear fermented extract was filtered and used for analysis.

Preparation of yeast (Saccharomyces cerevisiae) inoculum: 10 mg of commercial wine yeast culture (Lalvin EC-1118 strain—produced in Canada from grape skin) was dissolved in 2 mL of warm water (43 °C) for 10 min; as the yeast activates at warm water, it starts producing small bubbles. A total of 2 mL of such activated Saccharomyces cerevisiae culture was added into the rosemary extracts for fermentation.

T4: Fresh leaf decoction fermentation—T2 samples were fermented by adding 24% sugar and the activated wine yeast Saccharomyces cerevisiae culture for 60 days.

T5: Dry leaf decoction—10 g of leaf powder was boiled in 200 mL Milli-Q water at 100–110 °C temperature until the volume was reduced to 100 mL, and the extract was cooled, filtered, and used for further analysis.

T6: Dry leaf decoction fermentation—T5 samples were fermented by adding 24% sugar and the activated wine yeast Saccharomyces cerevisiae culture for 60 days.

T7: Soxhlet extraction—10 g of leaf powder was extracted using 250 mL LCMS grade methanol in the Soxhlet apparatus at 70 °C for 6 h, and the volume was further reduced to 100 mL by a vacuum evaporator and filtered through a 0.2 µ Nalgene filter unit from Thermo Fisher Scientific Inc. (Waltham, MA, USA).

T8 and T9: Sonic/ultrasound extraction in water and methanol, respectively—10 g of leaf powder was extracted in 100 mL Milli-Q water and methanol (50 °C) for 2 h with a frequency of 40 kHz in a Bransonic-52 ultrasonic bath unit from Branson, USA.

All extractions were made in triplicates and stored protected from light at −20 °C until chromatographic analysis.

3.4. UHPLC-ESI-QTOF-MS Method Development

R. officinalis samples were analyzed by ultra-high-performance liquid chromatography, electrospray ionization coupled with quadrupole-time of flight mass spectrometry (UHPLC-ESI-QTOF-MS). The analysis was carried out by reverse-phase UHPLC (Shimadzu Nexera, Kyoto, Japan) directly connected to a quadrupole Time-of-Flight (QTOF) Triple TOF 5600 mass spectrometer (AB SCIEX, Concord, ON, Canada). The autosampler (Shimadzu SIL30AC, Kyoto, Japan) was operated in direct injection mode, filling a 50 µL loop with 10 µL analyte for optimal sample delivery reproducibility. Samples were passed through the C18 column (Kinetex XB, 1mm I.D. × 5 cm, 2.6 µm, particle size, 100 Å) and eluted at a flow rate of 250 µL/min. Pumps (Shimadzu LC30AD, Kyoto, Japan) were operated in the following multi-step linear gradient with different proportion of mobile phase B: 0 min, 10% B; 10 min, 90% B; 12.5 min, 90% B; 15 min, 10% B; 20 min, 10% B, with a total runtime of 20 min including mobile phase equilibration. Mobile phases A and B used were 0.1% of acetic acid made in Milli-Q water and acetonitrile, respectively. The column oven (Shimadzu CTO30A, Kyoto, Japan) was set to 40 °C.

3.5. Identification and Quantification of Polyphenols

Mass spectra and tandem mass spectra data were recorded in electrospray ionization (ESI), “negative-ion” mode with a resolution of ~35,000 full-width half-maximum on the QTOF 5600. The ion spray needle voltage was at −4500 V with drying gas temperature 600 °C, and ion source Gas 1 (nebulizer) and Gas 2 (heater) values were 50 psi each. The collision-energy values for QTOF MS were at 5 eV and for MS/MS experiments at 25 eV with a spread of 15 eV. For collision-induced dissociation tandem mass spectrometry, the mass window for precursor ion selection of the quadrupole mass analyzer was set to ±1 m/z. The precursor ions were fragmented in a collision cell using nitrogen as the collision gas. Data independent acquisitions (DIA) with SWATH-MS2 cover the mass range of m/z 50–1000 in 16 segments (15 × 48.5 ms), yielding a cycle time of 0.8268 s, which includes one 50 msec MS1 scan. During the execution of the liquid chromatography method, the mass spectrometer was externally calibrated using a known mixture of masses from Sciex (P/N 4460134, AB SCIEX, Concord, ON, Canada).

Quantitative analysis was performed by diluting the extracted samples with 0.1% formic acid (1/10 to 1/10,000) in order to quantify the samples within the linearity range of the standard calibration curve, avoiding MS signal saturation. The method was validated for sensitivity and precision. The standard calibration curves were constructed for quantification of caffeic acid, rosmarinic acid, luteolin-7-O-glucoside, carnosol, carnosic acid, and ursolic acid. Table 5 represents calibration parameters, including limits of quantification (LOQ), calibration range, equations, and slope. All samples were extracted and analyzed in triplicate. Unknown polyphenolic compounds and flavonoids were identified based on their accurate mass (m/z) and molecular (m/z) ion fragmentation patterns using Peak view Software (ver.2.2, AB SCIEX, Concord, ON, Canada), Master view, Library view (AB SCIEX, Concord, Canada), National Institute of Standards and Technology (NIST), and the AOI database.

Table 5

Results of analysis of calibration curve and limits of quantification.

	Standard	Purity (%)	Formula	Molecular Weight	LOQ (ng/mL)	Calibration Range (ng/mL)	Calibration Equations	Slope (R2)
1	Caffeic acid	98.0	C9H8O4	180.00	6.0	6–250	y = 0.00523x + 0.00157	0.9993
2	Rosmarinic acid	98.0	C18H16O8	360.31	6.0	6–250	y = 0.00374x − 0.00269	0.9998
3	Luteolin-7-O-glucoside	98.0	C21H20O11	448.38	6.0	6–250	y = 0.00347x − 0.00156	0.9994
4	Carnosol	100.0	C20H26O4	330.40	6.0	6–250	y = 0.00673x + 0.03215	0.9982
5	Carnosic acid	96.0	C20H28O4	332.43	24.0	24–1000	y = 9.06876 × 10−5x + 0.00303	0.9984
6	Ursolic acid	97.0	C30H48O3	456.70	24.0	24–1000	y = 0.00147x − 0.03017	0.9938

Limits of quantification (LOQ).

3.6. Statistical Analysis

The results of polyphenol quantification were expressed as mean ± SD. The data were analyzed statistically by using single-factor ANOVA in MS Excel software. The critical difference at 1% level of significance or Tukey’s HSD (Honestly Significant Difference) test (at p < 0.01) was used to compare the significant difference between the treatments [43].

4. Conclusions

Rapid separation of most of the polyphenols was achieved within the first 11 min during 20 min of UHPLC analysis. Among all the extraction methods, Soxhlet extraction yielded significantly higher levels of polyphenols, both in terms of numbers of compounds and levels of these compounds. Dry leaf decoction was found to be the next best extraction method for rosemary, yielding significantly higher caffeic acid, rosmarinic acid, carnosol, carnosic acid, and flavonoids. This might be the best method for large-scale commercial extraction. Sonic extraction with methanol was found to be the second-best for the extraction of rosmarinic acid and ursolic acid. Most of the extractions in the study yielded a high concentration of rosmarinic acid up to 33.49 mg/g, contributing substantially to the high antioxidant potential of the extracts. As compared to previous studies, the rosemary extract of our study recorded a higher concentration of bioactive constituents, indicating the quality of the herb grown in Fairfield, Iowa, USA. The present study also helps to choose an efficient extraction method for obtaining maximum polyphenolic and terpenoid content, not only in rosemary but also in similar herb species. UHPLC-ESI-QTOF-MS methodology for the analysis proved to be very efficient in the identification and characterization of targeted and untargeted phenolic compounds present in the rosemary. However, there is substantial scope to investigate structurally and functionally the many potentially interesting but yet-unidentified phenolic compounds present in rosemary.