Phytochemical profile of eleven peruvian Mentheae. Isolation of ursolic acid from Clinopodium revolutum (Ruiz &amp; Pavon) Govaerts and analysis of the aqueous infusions

doi:10.21203/rs.3.rs-2476132/v1

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Phytochemical profile of eleven peruvian Mentheae. Isolation of ursolic acid from Clinopodium revolutum (Ruiz & Pavon) Govaerts and analysis of the aqueous infusions

https://doi.org/10.21203/rs.3.rs-2476132/v1

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published 03 Jul, 2023

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The profile of secondary metabolites in eleven Mentheae (Nepetoideae, Lamiaceae) from Peru by liquid chromatography associated with Orbitrap mass spectrometry, UHPLC-OT-MS is presented. Precursors of and salvianolic acids have been found, particularly rosmarinic acid, as well as a diversity of free and glycosylated flavonoids as main substances, 127 tentatively identified structures. In addition, a method to obtain ursolic acid from Clinopodium revolutum (R. & P.) Govaerts and a quantitative analysis for rosmarinic acid and triterpenic acids in aqueous infusions of these plants are presented.

Biological sciences/Drug discovery

Biological sciences/Plant sciences

Mentheae- Chromatography UHPLC-OT-MS- ursolic acid- Peru

The tropical Andes are considered one of the most diverse areas on the planet in terms of vascular plants. The flora of Peru is extremely rich, its territory is home to some 25,000 species, almost 10% of all plants in the world. The large family Lamiaceae has twelve subfamilies. The Nepetoideae subfamily with 3400 species and 105 genera has three tribes [1]: Elsholtzieae, Ocimeae and Mentheae, the latter with 65 genera. The Mentheae tribe is chemically characterized by having volatile terpenoids and a phenolic acid called rosmarinic acid that makes these plants aromatic and with medicinal properties [2, 3]. The Mentheae can also be classified into 3 subtribes: Menthinae (43 genera), Salviinae (10 genera) and Nepetiinae (12 genera) [4, 5]. In Peru (Herbario Nacional Universidad de San Marcos-Perú, October 2017), the main genera of Mentheae are Clinopodium (29 species); Hedeoma (1 species); Lepechinia (11 species); Minthostachys (7 species) and Salvia (59 species). Clinopodium, Hedeoma, and Minthostachys belong to the Menthinae subtribe, while Lepechinia and Salvia belong to the Salviinae subtribe. In a previous work [6] the content of rosmarinic acid and triterpenic acids, oleanolic and ursolic was quantified in thirteen Peruvian Mentheae: The highest content of rosmarinic acid was observed in Lepechina meyenii (Walp.) Epling and the highest content of triterpenic acids in Clinopodium revolutum (Ruiz & Pavón) Govaerts. Subsequently [7], the metabolite profile was obtained in two Lepechinia species: L. meyenii and L. floribunda (Benth.) Epling, by UHPLC-Q-Exactive Orbitrap - MS, the presence of salvianolic acids and diterpenoids being notable, as well as a preparative method for rosmarinic acid by cold evaporative crystallization. In the present communication, the profile of secondary metabolites by UHPLC-Q-Exactive Orbitrap-MS is also described in eleven Mentheae: Clinopodium (4), Salvia (5), Hedeoma (1) and Minthostachys (1) and also a preparative method for ursolic acid from Clinopodium revolutum. In addition, the content of rosmarinic acid and triterpenic acids in the aqueous infusions of these plants was evaluated.

Phytochemical Profile

The metabolite profile of the ethanolic extracts of the eleven Mentheae was obtained in the negative mode and the detected compounds appear in Table 1. Assignments were made based on the literature [8–40]. Isomers of quinic acid (m/z 191), danshensu (m/z 197), protocatechuic aldehyde (m/z 137), and caffeic acid (m/z 179) occur in most plants. Equally abundant are the monocaffeoylquinic acids present in seven species. Minthostachys mollis contains four different monocaffeoylquinic acids. Furthermore, diversity of flavonoids (flavonols, flavones, flavanones, flavanonols) has been found in all the samples, both free and glycosylated. Minthostachys mollis, Clinopodium sericeum and Clinopodium pulchellum are the most diverse with respect to their flavonoids. In Salvia dombeyi, the presence of polymethoxylated flavones is notable. All samples contain rosmarinic acid (m/z 359). In Clinopodium revolutum, salvianic acid C (m/z 377) has been detected, which is the result of hydrating the double bond of rosmarinic acid, and in Salvia sagitatta, teucrol (m/z 315), which is a decarboxylated rosmarinic acid. Isorinic acid (m/z 343) which is a rosmarinic acid without the 3-OH is present in Clinopodium brevicalyx, Salvia sagitatta, Salvia cuspidata and Hedeoma mandoniana. Methyl (m/z 373) and ethyl (m/z 387) esters of rosmarinic acid are present in Salvia cuspidata and in Clinopodium brevicalyx. In Salvia cuspidata and Clinopodium revolutum, the dimer of rosmarinic acid, sagerinic acid (m/z 719), which is a molecule with a stabilized cyclobutane ring was found. In Clinopodium brevicalyx and Hedeoma mandoniana, the presence of salvianolic acid B (m/z 717) was observed, a particularly important substance due to its effect on neurodegenerative diseases [41]. Clinopodium pulchellum contains salvianolic acid A (m/z 493) and salvianolic acid F (m/z 313). But the plant with the greatest diversity of salvianolic acids is Clinopodium sericeum, "romero de jalca", since lithospermic acid (m/z 537), salvianolic acid B, two isomers of salvianolic acid A and two isomers of salvianolic acid F have been found in it. This type of substances is very important due to its effect on cell fibrosis (scar formation) in direct relation to cancer [42]. Among the other substances found, it should be noted that the Rosmarinus type diterpenoids, common in Lepechinia [7, 43] are scarce in this work, only Salvia sagitatta and Salvia cuspidata show the presence of carnosol (m/z 285) and the phenolic diterpenoid, rosmadial (m/z 343) in the last one. Salvia dombeyi turns out to be the most dissimilar plant in this work, since apart from a very low content of rosmarinic acid [6] and the presence of polymethoxylated flavones, it shows substances such as cimifugin (m/z 305), a substance with antiallergic and antipruritic activity [51, 52] and the lignan lariciresinol (m/z 359), common in oilseeds and in the Brassica and Allium genera [53]. Salvia haenkei contains the ent-(5R,9R)-15,16-epoxy-10S-hydroxycleroda-3,7,13(16),14-tetraene-17,12S; 18,19 diolide (m/z 355) [49], while Salvia cuspidata contains other lignan, isolariciresinol (m/z 359) present in Linum seeds [54], and also 5-epi-icetexone (m/z 341) described as anti Trypanosoma cruzii molecule [40]. Oleanolic and ursolic triterpenic acids, quantified in [6], do not appear in this analysis due to the elution program used, which does not reach 100% acetonitrile. Figure 1 shows the ESI(-) chromatogram of Salvia sagitatta and Fig. 2, the chromatogram of Clinopodium sericeum. Table 1 shows the 129 substances detected.

Table 1

Detected compounds in eleven peruvian Mentheae
N° peak	assignment	Rt	[M-H]-	experimental mass (error)	fragments	Detected in	Reference
1	Quinic acid	1.33	C₇H₁₁O₆	191.0559(1.57)	127.0394	Cb, So, Mm, Sc, Cr, Cs, Sd, Cp.	[8]
2	Malic acid	1.36	C₄H₅O₅	133.0139 (1.5)		Ss, Sc, Sd	[8]
3	Quinic acid isomer	1.44	C₇H₁₁O₆	191.056(2.09)	127.8695	Cb, Mm, Cr, Cs, Cp	[8]
4	Citric acid	1.77	C₆H₇O₇	191.0196(2.09)	111.0081	So	[8]
5	Pyroglutamic acid	1.87	C₅H₆O₃N	128.0348 (0)		Sh	[8]
6	Succinic acid	1.98	C₄H₅O₄	117.0187(0.85)		So, Mm, Ss, Sc, Cr, Cs, Sh, Sd, Hm	[8]
7	monoacetylglycerol	2.09	C₅H₉O₄	133.0502 (0.75)		Ss
8	Mesaconic acid	2.96	C₅H₅O₄	129.0190(1.55)		Cp
9	3,4-dihydroxyphenyl lactic acid “danshensu”	4.05	C₉H₉O₅	197.0454(2.02)	135.0446[M-H-H₂0-CO₂]⁻, 179.0346 [M-H-H₂O]⁻, 123.0445	So, Cb, So, Mm, Sc, Cr,Cs, Hm, Sh	[9], [10]
10	Protocatechuic acid	4.64	C₇H₅O₄	153.019(1.31)	135.0448[M-H-H₂O]⁻, 109.0289[M-H-CO₂]⁻	So, Sc, Hm, Cp	[9]
11	Caffeoyl quinic acid	6.39	C₁₆H₁₇O₉	353.0883(2.83)	135.0447, 179.0347[caffeic acid – H]⁻, 191.0559[quinic acid – H]⁻	Mm	[11], [12], [13], [50], [14], [15]
12	protocatechuic aldehyde	7.73	C₇H₅O₃	137.0239(0)	109.0289 [M-2H-CO]⁻	Cb,So, Mm,Ss, Cr, Cs, Sh, Hm, Cp	[9]
13	Hydroxyheptandioic acid	8.78	C₇H₁₁O₅	175.0611(2.28)		So, Ss
14	Coumaroyl quinic acid	8.83	C₁₆H₁₇O₈	337.0934(2.96)	119.0496, 163.0398, 173.0453, 191.0559	Mm
15	Caffeoyl quinic acid	8.99	C₁₆H₁₇O₉	353.0883(2.83)	173.0453, 179.0559[caffeic acid – H]⁻, 191.056[quinic acid – H]⁻	Mm	[11], [12], [13], [50], [14], [15]
16	Caffeic acid –O-hexoside	9.02	C₁₅H₁₇O₉	341.0883(2.93)	179.0347[caffeic acid –H]⁻, 251.0564, 233.0458, 281.0670	So, Ss, Sc	[16], [17]
17	Caffeoyl quinic acid	9.04	C₁₆H₁₇O₉	353.0883(2.83)	191.056[quinic acid-H]⁻	Mm, Cr	[11], [12], [13], [50], [14], [15]
18	Coumaric acid	9.28	C₉H₇O₃	163.0399 (2.45)	119.0497 [M-carboxyl]⁻	Ss	[11], [12]
19	Caffeoyl quinic acid	9.41	C₁₆H₁₇O₉	353.0883(2.83)	179.0347[caffeic acid-H]⁻, 173.0452	Cb, So, Mm, Cr, Hm, Cp	[11], [12], [13], [50], [14], [15]
20	Eucomic acid	9.54	C₁₁H₁₁O₆	239.0561(2.09)	195.0660, 178.0586	Cb
21	Caffeoylquinic acid	9.56	C₁₆H₁₇O₉	353.0882(2.55)	135.0446 [caffeic acid-H-CO₂]⁻, 179.0346 [caffeic acid-H]⁻, 191.055 [quinic acid-H]⁻	Hm	[11], [12], [13], [50], [14], [15]
22	Caffeic acid	9.64	C₉H₇O₄	179.0348(1.67)	135.0446 [M-H-CO₂]⁻, 161.0446 [6,7-dihydroxy-1H-inden-1-one – H]⁻	So,Mm, Ss, Sc, Cr, Cs, Sh, Sd	[11], [12], [17]
23	Caffeic acid –O-hexoside I	9.77	C₁₅H₁₇O₉	341.088 (2.05)	179.0345[caffeic acid –H]⁻, 281.0667, 251.0561,235.0453	Sc	[16], [17]
24	Tuberonic acid glucoside	9.86	C₁₈H₂₇O₉	387.1665(2.58)	206.9725, 163.0033, 101.5668	So, Ss, Cr, Sh, Sd	[8]
25	coumaroylquinic acid	10.1	C₁₆H₁₇O₈	337.0934(2.97)	173.0454, 163.0397[coumaric acid – H]⁻	So, Mm	[11], [12], [15], [18]
26	salvianic acid C	10.21	C₁₈H₁₇O₉	377.0882(2.39)	359.0776 [M-H₂O]⁻, 161.0240[6,7-dihydroxy-1H-inden-1-one – H]⁻, 179.0347 [caffeic acid –H]⁻	Cr	[17]
27	p-coumaroyl hexoside	10.38	C₁₅H₁₇O₈	325.093(1.85)	119.0496 [coumaric acid-H-CO₂]⁻, 163.0396 [coumaric acid-H]⁻	Sc	[16]
28	Feruloylquinic acid	10.38	C₁₇H₁₉O₉	367.104(3.0)	149.0240 [ ferulic acid-H-CO₂]⁻, 191.0560 [quinic acid-H]⁻, 193.0504 [ferulic acid – H]⁻, 173.0453	Mm
29	Quercetin-3,7-di-O-glucoside	10.43	C₂₇H₂₉O₁₇	625.1407(0.32)	303.1084, 463.0882	Cs, Cp	[19]
30	Caffeoylquinic acid	10.45	C₁₆H₁₇O₉	353.088 (1.12)	135.0445 caffeic acid-H-CO₂]⁻, 179.0345 [caffeic acid –H]-, 191.0557[quinic acid-H]⁻	Sc	[11], [12], [13], [50], [14], [15]
31	p-coumaroyl hexoside I	10.59	C₁₅H₁₇O₈	325.093(1.85)	119.0496, 163.0396	Sc
32	Quercetin-O-vicianoside	10.64	C₂₆H₂₇O₁₆	595.1301 (0.34)	301.0356 [M-vicianose-H]-, 433.2081	Sd
33	Salvianic acid C isomer	10.67	C₁₈H₁₇O₉	377.0881(2.12)	359.0775 [M-H₂O]⁻, 347.1708, 197.0453 [ HOOC-CHOH-CH₂-C₆H₃(OH)₂ – H]⁻	Cr	[17]
34	Tuberonic acid	10.67	C₁₂H₁₇O₄	225.1132(2.22)	134.8648, 146.9382, 168.8359, 187.9417, 213.0961	Mm, Sh, Sd	[8]
35	Quercetin-O-rutinoside	10.78	C₂₇H₂₉O₁₆	609.1458 (0.33)	301.0363 [M-rutinosa-H]⁻, 447.1302	Mm, Sd, Cp	[16]
36	Eriodyctiol rutinoside (eriocitrin)	10.89	C₂₇H₃₁O₁₅	595.1661 (0.34)	287.0562, 151.0397	Cs, Sh, Hm	[20]
37	Luteolin rutinoside	11.00	C₂₇H₂₉O₁₅	593.1504 (0.51)	285.0403 [M-rutinose-H]⁻, 447.0928	Cb, Sc, Cr, Cp	[21]
38	Apigenin rutinoside	11.01	C₂₇H₂₉O₁₄	577.1556 (0.35)	269.103 [M-rutinose-H]⁻	Sc, Cr	[15], [21]
39	Luteolin-O-hexoside	11.02	C₂₁H₁₉O₁₁	447.0936(1.78)	285.0406 [M-hexose-H]⁻	Cb, Ss, Cs	[22]
40	Quercetin-O-hexoside	11.02	C₂₁H₁₉O₁₂	463.0886(1.94)	301.0358 [M-hexose-H]⁻	So, Mm, Ss, Sc, Cp	[16], [23]
41	Quercetin-O-glucuronide	11.09	C₂₁H₁₇O₁₃	477.0679(2.1)	301.0356 [M-GlcA-H]⁻	So, Ss, Sd	[23]
42	Feruoyl hexoside	11.12	C₁₆H₁₉O₉	355.1036 (1.97)	193.0502 [ferulic acid – H]⁻, 149.0240 43[ferulic acid-H- CO₂]⁻	Sc	[16]
43	Pentahydroxymethoxyflavone glycoside	11.12	C₂₂H₂₁O₁₃	493.0989(1.42)	331.0827, 315.1089, 163.0397, 162.8387	Cs
44	Isorhamnetin-O-glucoside	11.23	C₂₂H₂₁O₁₂	477.1046 (2.72)	315.0824 [isorhamnetin-H]⁻, 357.0352, 462.0768	Ss	[22]
45	Naringenin-7-O-Glc-Rha (narirutin)	11.33	C₂₇H₃₁O₁₄	579.1714 (0)	271.0612, 235.0611, 151.0030	Mm, Cb, Cs, Hm, Cp
46	Eriodictiol-O-neohesperidoside (neoeriocitrin)	11.40	C₂₇H₃₁O₁₅	595.1665(0.34)	287.0564, 151.0033	Cs
47	Luteolin-O-glucuronide	11.56	C₂₁H₁₇O₁₂	461.0729(1.95)	285.0407 [M-GlcA-H]⁻, 151.0395, 133.0290	So, Ss, Cr, Sh	[23]
48	Kaempferol-O-hexoside	11.57	C₂₁H₁₉O₁₁	447.0937 (2.01)	285.0407 [Kf-hexose]⁻	Mm, Cr, Sd	[16]
49	dihydrobaicalin	11.57	C₂₁H₁₉O₁₁	447.0936 (1.79)	403.1613 [M-H-CO2]⁻, 271.0250[M-glucuronic acid]⁻	Sc
50	Sagerinic acid	11.58	C₃₆H₃₁O₁₆	719.16(1.67)	539.1186 [M – HOOC-CH-CH₂-C₆H₃(OH)₂]⁻, 359.0715 [ M- rosmarinic - H]⁻, 179.0348, 161.0239 [6,7-dihydroxy-1H-inden-1-one – H]⁻	Sc, Cr	[11], [12], [24], [25], [26]
51	Caffeoyldihydroxyhexyl ester	11.63	C₁₅H₁₉O₆	295.1193(3.73)	161.0242 [C₉H₅O₃]-	Ss
52	Hesperetin-7-O-rutinoside	11.64	C₂₈H₃₃O₁₅	609.1819(0.16)	301.0718	Mm, Cb, Hm, Cp
53	Apigenin-O-rutinoside	11.66	C₂₇H₂₉O₁₄	577.1557(0.17)	269.0453	Cr
54	Dimethylrosmarinic acid	11.67	C₂₀H₁₉O₈	387.1091(2.84)	179.0347 [caffeic acid − 1]-, 135.0447 [caffeic acid-H-CO₂]⁻,, 161.0452 [6,7-dihydroxy-1H-inden-1-one – H]⁻	So, Sh
55	Isorhamnetin-3-O-glucuronide	11.7	C₂₂H₁₉O₁₃	491.0834(1.62)	299.0565, 301.0358, 302.0388	So
56	Salvianolic acid A isomer	11.75	C₂₆H₂₁O₁₀	493.1142(1.42)	179.0344 [caffeic acid-H]⁻, 197.0450 [danshensu-H]-, 269.0821, 295.1192 [salvianolic acid F – OH]⁻, 313.0723 [salvianolic acid F-H]⁻, 359.0778 [rosmarinic acid –H]⁻	Cs	[9], [10], [17], [33]
57	Tetrahydroxymethoxyflavone –O-hexoside	11.78	C₂₂H₂₁O₁₂	477.1041(1.68)	315.1451, 163.8391, 162.8398	Cr
58	Trihydroxymethoxyflavone hexoside	11.89	C₂₂H₂₁O₁₁	461.1093 (1.95)	299.0559[M-H-glucose]-	Sh, Sd
59	Caffeoyldihydroxylhexyl ester	11.93	C₁₅H₁₉O₆	295.1193(3.73)	161.0245 [C₉H₅O₃]-	Ss
60	Acetylglycitin	11.96	C₂₄H₂₃O₁₁	487.1250(1.85)	267.0720, 241.1080, 444.0807	Hm
61	Salvianolic acid B isomer	11.99	C₃₆H₂₉O₁₆	717.1443 (1.81)	321.0616 [ M – 2 danshensu- 3H]⁻, 519.0945 [M-danshensu-2H]⁻	Cs	[10], [15], [17], [26], [27]
62	Apigenin-6-C-hexósido	12.01	C₂₁H₁₉O₁₀	431.0984(1.16)	331.1396, 341.1960, 283.1192	Cr
63	cimifugin	12.03	C₁₆H₁₇O₆	305.1035 (3.28)	274.1170[M-hydroxymethyl-H]⁻, 215.0098[M-CH3COH-CH3-H]⁻	Sd	[28]
64	Rosmarinic acid	12.04	C₁₈H₁₅O₈	359.0775(2.23)	161.0240 [6,7-dihydroxy-1H-inden-1-one – H]^−, 179.0345 [caffeic acid-H]⁻, 197.0452 [danshensu-H]⁻	Cb, So, Mm, Ss, Sc, Cr, Cs, Sh, Sd, Hm, Cp	[10–12], [24–25], [27]
65	isorhamnetin	12.50	C₁₆H₁₁O₇	315.0513 (2.54)	301.0306, 300.0272 [M-Me-H]⁻, 271.1550[(M-CH3-H)- (H + CO)]⁻,179.0346, 151.0394[A]⁻	So, Sh	[29–30]
66	Salvianolic acid B isomer	12.53	C₃₆H₂₉O₁₆	717.1443(1.81)	295.0611, 321.0408 [ M – 2 danshensu- 3H]⁻, 339.0512, 493.1137, 519.0930[M-danshensu-2H]⁻, 537.1024 [M-caffeic acid- 2H]⁻	Cb, Hm	[10], [15], [17], [26], [27]
67	isorhamnetin acetyldihydroxybutyl ester	12.61	C₂₂H₂₁O₁₀	445.1144 (2.02)	387.1090[M-acetyl]⁻, 315.0514[M-acetyl,hydroxybutyl]⁻, 151.0396 [A]⁻	Sd
68	Luteolin-O-acetylglucoside	12.71	C₂₃H₂₁O₁₂	489.1039(1.23)	285.0404, 447.0935	Cr
69	artemetin	12.80	C₂₀H₁₉O₈	387.1089 (2.32)	372.1184[M-H-Me]⁻, 357.0992[M-H-2Me]⁻, 342.1067[M-H-3Me]⁻⁷⁰327.1241[M-H-4Me]⁻	Sh, Sd
70	heptamethoxyflavone	12.81	C₂₂H₂₃O₉	431.1352 (2.32)	417.1189, 402.1563, 387.1089, 372.1174	Sd
71	caffeoil-4’-hydroxyphenyllactic acid (isorinic acid)	12.95	C₁₈H₁₅O₇	343.0827(2.62)	161.0241 [6,7-dihydroxy-1H-inden-1-one – H]⁻, 327.2181[ M-OH]⁻	Cb, Ss, Sc Hm	[17], [31]
72	Lithospermic acid	13.03	C₂₇H₂₁O₁₂	537.1038(0.93)	493.1147 [M-CO₂-H]⁻, 295.0610 [M-CO₂-H – danshensu]⁻	Cs	[9], [17]
73	Isosakuranetin-O-rutinoside (Neoponcirin)	13.15	C₂₈H₃₃O₁₄	593.1874(0.51)	594.1905, 593.1873, 285.077	Mm, Cp
74	Caffeoyloxohydroxyhexyl ester	13.23	C₁₅H₁₇O₆	293.1035 (3.41)	161.0242 [C₉H₅O₃]-	Ss
75	Methyl rosmarinate	13.28	C₁₉H₁₇O₈	373.0935 (2.95)	359.0778 [rosmarinic acid –H]-, 194.0540, 179.0347 [danshensu –H]⁻	Sc	[11], [12], [21]
76	pentamethoxyflavone	13.36	C₂₀H₁₉O₇	371.114 (2.43)	357.1349, 327.1241, 311.0934	Sd
77	Quercetin-3-O-(3’-O-p-coumaroyl)-hexoside (Helichrisroside)	13.51	C₃₀H₂₅O₁₄	609.1242 (0.49)	301.0719, 447.0940, 462.0747, 594.1343	Cr
78	Eriodictiol	13.60	C₁₅H₁₁O₆	287.0563(2.44)	135.0446, 151.0030	Cb, Cp
79	Luteolin	13.62	C₁₅H₉O₆	285.0408(3.16)	133.0289, 151.0032	Cb, Hm
80	dihydrophilonotisflavone	13.63	C₃₀H₁₉O₁₂	571.0883 (1.05)	285.0410, 286.0441[M-luteolin-H]⁻	So, Ss
81	Ferulic acid	13.68	C₁₀H₉O₄	193.0504 (1.55)	134.0367, 149.0239 [M-H-CO₂]⁻, 178.022 [M-H-CH₃]⁻	Sc	[10], [21], [32]
82	pentamethoxyflavone	13.79	C₂₀H₁₉O₇	371.114 (2.43)	357.1339, 327.1240	Sd
83	Salvianolic acid A isomer	13.87	C₂₆H₂₁O₁₀	493.1141(1.22)	159.8595, 179.0345 [caffeic acid – H]⁻, 197.0451[danshensu-H]⁻, 269.0821, 295.0612, 313.0719, 359.0774 [rosmarinic acid –H]⁻	Cs, Cp	[9], [10], [17], [33]
84	Protocatechuic acid − 7-O-(4-hydroxybenzoyl)glucoside	13.94	C₂₀H₁₉O₁₁	435.0935(1.61)	297.1346, 153.0191, 315.1452, 137.0239	Cr
85	Chrysoeriol (trihydroxymethoxyflavone)	14.00	C₁₆H₁₁O₆	299.0565 (3.01)	285.0413 [M-H-methyl]⁻, 151.0397 [A]⁻, 255.0698 [M-H-CO₂]⁻	So, Mm, Ss, Sh
86	Hesperetin-O-hexoside	14.27	C₂₂H₂₃O₁₁	463.1250(2.06)	285.0773, 301.0720	Cp
87	pentamethoxyflavone	14.38	C₂₀H₁₉O₇	371.114 (2.43)	357.1348, 327.1241	Sd
88	caffeic acid ethyl ester	14.66	C₁₁H₁₁O₄	207.0661(1.44)	179.0347	Cb, Sc
89	kaempferol	14.72	C₁₅H₉O₆	285.0406 (2.46)	161.0240, 151.0396 [A]⁻, 135.0444	Sc, Cr	[29]
90	lariciresinol	14.84	C₂₀H₂₃O₆	359.1505 (2.78)	419.1715	Sd	[19]
91	quercetin	14.97	C₁₅H₉O₇	301.0356 (2.33)	179.0344, 121.0288,273.1719 92	Sc	[29]
92	Caffeic acid dimethyl derivative	15.01	C₁₁H₁₁O₄	207.0661(1.45)	16931.0239, 151.940396, 147.069552	Cs
93	salvianolic acid F isomer	15.46	C₁₇H₁₃O₆	313.0721(2.88)	269.082196 [M-CO₂-H]⁻, 15979.0656	Sc, Cs	[17]
94	dimethylquercetin	15.49	C₁₇H₁₃O₇	329.0673 (3.34)	314.0756[M-H98-methyl]⁻, 9996299.0200[M-H-2 100methyl]-	Cb
95	Trihydroxydimethoxyflavone	15.53	C₁₇H₁₃O₇	329.0672(3.04)	151.0398, 201.8020, 257.8197, 283.0612, 313.0722, 314.0754, 299.0201	Mm
96	Trihydroxylinoleic acid	16.03	C₁₈H₃₁O₅	327.2183(2.75)	269.0457	Cb, Mm, Ss, Sc, Hm
97	Ethyl caffeate	16.14	C₁₁H₁₁O₄	207.0660(0.97)	179.0346 [caffeic acid-H]⁻	Ss
98	apigenin	16.15	C₁₅H₉O₅	269.0459 (3.35)	151.0396 A⁻, 117.0187 B⁻	Ss, Sc, Cr	[15], [29]
99	naringenin	16.39	C₁₅H₁₁O₅	271.0616 (3.32)	151.0397 A⁻, 177.0190 [M-H-ring B]⁻	Ss, Cp	[34]
100	Salvianolic acid F isomer	16.87	C₁₇H₁₃O₆	313.0719(2.23)	269.0822 [M-CO₂-H]⁻, 159.0658	Sc	[17]
101	Ethyl rosmarinate	17.23	C₂₀H₁₉O₈	387.1088(2.07)	359.0777 [M-ethyl]⁻, 206.9724 [M – caffeic acid]⁻, 179.0344 [caffeic acid-H]⁻	Cb	[11], [12], [21]
102	3,7-quercetindimethylether	17.59	C₁₇H₁₃O₇	329.0673 (3.34)	314.0756[M-H-methyl]⁻, 299.0200[M-H-2 methyl]-	Ss, Sd, Cp	[32]
103	Hesperetin	17.66	C₁₆H₁₃O₆	301.0722(3.32)	286.0495, 257.0822, 241.7579	Cp
104	Salvianolic acid F isomer	17.87	C₁₇H₁₃O₆	313.0721(2.87)	269.0821 [M-CO₂-H]⁻, 159.0448	Cs	[17]
105	15,16-epoxi-10S-hidroxicleroda-3,7, 13(16), 14 tetraeno-17, 12S; 18,19 diolido	17.94	C₂₀H₁₉O₆	355.119 (2.25)	311.1291[M-H-CO2]⁻	Sh	[49]
106	Sanleng acid (6,9,10-trihydroxy-7-octodecenoic acid)	18.13	C₁₈H₃₃O₅	329.2336(2.43)	171.0195, 224.7632, 250.1448	Mm, Cb Cs
107	Hydroxypentamethoxyflavone	18.47	C₂₀H₁₉O₈	387.1087(1,81)	285.0403	Sc
108	8-Hydroxyhexadecandioic acid	18.63	C₁₆H₂₉O₅	301.2025(3.32)		Cs
109	Trihydroxy-trimethoxyflavone	18.73	C₁₈H₁₅O₈	359.0766(0.28)	344.0546, 329.0299, 314.2232, 301.6655	Mm, Cb, Cp
110	10-(1-hydroxyhexoxy)-10-oxodecanoic acid	18.80	C₁₆H₂₉O₅	301.2023(2.66)	201.8017	Cr
111	Trihydroxymethoxyflavanone (hesperetin isomer)	19.15	C₁₆H₁₃O₆	301.0721(2.87)	161.0240, 139.0032	Cp
112	trihydroxymethoxyflavone	19.23	C₁₆H₁₁O₆	299.0565(3.01)	151.0397 [A]⁻, 179.0346, 284.0327 [M-CH3-H]⁻	So, Mm, Cr, Sh, Cp	[35]
113	cirsimaritin	19.34	C₁₇H₁₃O₆	313.0724 (3.19)	298.0488[M-H-methyl]-, 283.0249[M-H-2methyl]-	Ss, Cr	[36]
114	isolariciresinol	19.61	C₂₀H₂₃O₆	359.1502 (1.95)	345.1346[M-Me]⁻, 344.1582[M-H-Me]⁻, 313.0714	Sc	[16], [37]
115	Salvianolic acid F isomer	19.77	C₇H₁₃O₆	313.0722(3.19)	269.0459 [M-CO₂-H]⁻, 159.8597	Cp	[17]
116	rosmadial	20.03	C₂₀H₂₃O₅	343.1552 (1.75)	299.1652 [M-H-CO₂]⁻, 315.1598 [M-formyl]⁻	Sc
117	eupatorin	20.06	C₁₈H₁₅O₇	343.0829 (3.21)	328.0595[M-H-Me]⁻, 313.0359[M-H-2Me]⁻, 298.0125[M-H-3Me]⁻	Cb, Mm, Ss, Cr, Cp	[27], [38]
118	teucrol	20.3	C₁₇H₁₅O₆	315.088 (3.5)	179.0349[Caffeic acid-H]⁻, 135.0447[caffeic acid -H-CO2]⁻, 161.0244 [6,7-dihydroxy-1H-inden-1-one – H]⁻	Ss	[39]
119	Dihydroxymethoxyflavone	20.32	C₁₆H₁₁O₅	283.0617(3.53)	268.0386, 151.0034, 107.0327	Mm,Cp
120	Dihysroxydimethoxyflavanone	20.36	C₁₆H₁₃O₅	285.0773(3.51)	153.0190, 161.0453, 179.0349, 151.0397, 243.0668, 270.0535, 164.0012	Mm
121	genkwanin	20.47	C₁₆H₁₁O₅	283.0616 (3.18)	268.0386[M-H-Me]⁻, 151.0398 A⁻	Ss, Cr, Sh	[22]
122	Sakuranetin	20.57	C₁₆H₁₃O₅	285.0771(2.81)	119.0497, 165.0188, 151.0396	Cr
123	Octadecendioic acid	20.68	C₁₈H₃₁O₄	311.2232(2.89)	310.2107	So, Sh, Sd	[8]
124	Octadihydroxyoctadecadienoic acid	21.15	C₁₈H₃₁O₄	311.2229 (1.93)	197.8076	Sc	[8]
125	Carnosol	22.2	C₂₀H₂₅O₄	329.1761(2.7)	285.1861[M-H-CO₂]⁻	Ss, Sc	[11–12]
126	5-epi-icetexone	22.45	C₂₀H₂₁O₅	341.1396 (0.88)	297.1500 [M-H-CO2]⁻, 299.1652 [M-H-CO]⁻	Sc	[40]
127	9,10-dihydroxystearic acid	23.47	C₁₈H₃₅O₄	315.2547 (3.47)		Ss	[8]

Ursolic acid purification from Clinopodium revolutum

In a previous work [6] the content of triterpenic acids was analyzed in thirteen plants of the Mentheae tribe, finding that Clinopodium revolutum “Flor de Arena” is the one with the highest ursolic acid content, suggesting that this substance could be isolated from it. Therefore, from dry, pulverized and defatted material, by alcoholic extraction and subsequent filtration of the extract on SiGel with increasing polarity gradient, ursolic acid crystals of approximately 90% purity were obtained. The purification of this material, by recrystallization from ethanol, was done taking into account the solubilities described in [44] as shown in Table 2.

Table 2

Ursolic acid (UA) solubility in ethanol [44].
temperature °C	solubility (mg UA/g ethanol)	solubility (mg UA/mL ethanol)	g of ethanol to disolve 100 mg of UA	mL of ethanol to disolve 100 mg of UA
20.45	5.10	4.08	19.61	24.51
26.95	7.61	6.09	13.14	16.43
32.05	7.30	5.84	13.70	17.12
36.85	7.56	6.05	13.23	16.53
46.95	10.39	8.31	9.62	12.03
50.05	10.41	8.33	9.61	12.00
56.55	16.34	13.07	6.12	7.65

Experiments with 50 mg of 90% ursolic acid at concentrations of 0.8 to 1.4% solute:solvent ratio show that at a percentage of 0.99 approximately 50% of the acid used is recovered with a purity greater than 98% as seen in table 3 and Fig. 3.

Tabla3. Recovery and purity of recrystallized ursolic acid according to the material/solvent volume ratio. (crystallizand with 91.15% ursolic acid and 4.65% oleanolic acid).

% (mass/volume) of crystallizand in ethanol	Recovery %	UA Purity % in the recrystallized
0.84	39.53	95.51
0.88	40.46	95.57
0.99	51.09	98.52
1.39	38.07	99.47

It is then seen that 1 g of ursolic acid of approximately 90% purity by recrystallization in 100 mL of 96% ethanol, will give us 0.5 g of ursolic acid with a purity greater than 98%.

Quantification of rosmarinic acid and triterpenic acids in the aqueous infusions of eleven Mentheae

As the form of use of the majority of medicinal plants is the aqueous infusion, it is interesting to know the amount of phenolics, rosmarinic acid and triterpenic acids in said infusions. Thus, the content of total phenols (expressed as µg of gallic acid/10 mL of infusion), the total antioxidant capacity (as µg of ascorbic acid/10 mL of infusion) and the mg of rosmarinic acid/10 mL of infusion were determined. [6]. The results appear in Table 4.

Table 4

Total phenols, antioxidant capacity and rosmarinic acid content of the eleven *Mentheae* infusions (three repetitions).
Plant	Total phenols (mg gallic acid/10mL)	Antioxidant capacity (ascorbic acid mg/10 mL)	Rosmarinic acid mg/10 mL
Clinopodium brevicalyx	4.08 ± 0.05	2.72 ± 0.02	0.90 ± 0.01
Salvia oppositiflora	2.19 ± 0.01	1.82 ± 0.26	0.21 ± 0.01
Minthostachys mollis	2.07 ± 0.00	1.53 ± 0.24	0.26 ± 0.00
Salvia sagitatta	3.25 ± 0.05	2.19 ± 0.04	0.79 ± 0.01
Salvia cuspidata	3.96 ± 0.09	2.84 ± 0.16	0.41 ± 0.00
Clinopodium revolutum	3.72 ± 0.03	2.46 ± 0.17	0.32 ± 0.01
Clinopodium sericeum	4.20 ± 0.12	2.96 ± 0.06	0.39 ± 0.01
Salvia haenkei	2.08 ± 0.01	1.63 ± 0.18	0.22 ± 0.00
Salvia dombeyi	1.65 ± 0.02	0.88 ± 0.13	undetected
Hedeoma mandoniana	2.00 ± 0.03	1.65 ± 0.22	0.02 ± 0.00
Clinopodium pulchellum	1.49 ± 0.01	1.14 ± 0.15	0.05 ± 0.00

Also, in all cases the content of triterpenic acids was analyzed but it was not found in any of the eleven aqueous infusions.

It is the first time that the phytochemical profile has been obtained in these Peruvian Mentheae (Lamiaceae). The genera studied were Salvia (Salviinae), Clinopodium, Hedeoma and Minthostachys (Menthinae). While Salvia and Clinopodium are genera of worldwide distribution, Hedeoma and Minthostachys are American and South American genera, respectively. All Salvia species in this work belong to the Salvia subgenus: Calosphace Benth. (Epling) [46]. The phytochemical profile of these Mentheae are quite similar to their European and Asian relatives. All the species analyzed show the presence of rosmarinic acid and, quinic acid, 3,4-dihydroxyphenyl-lactic acid “danshensu”, protocatechuic aldehyde and caffeic acid are present in most of the samples. Monocaffeoylquinic acids, also called chlorogenic acids, are also frequent but better expressed in Minthostachys. Di or tricaffeoylquinic acids have not been detected. All samples contain flavonoids with more diversity in Minthostachys and Clinopodium. Flavonoid-free aglycones predominate in several plants: In Salvia sagitatta, cirsimaritin is abundant [58] while eupatorin predominates in Clinopodium revolutum [38], chrysoeriol in Salvia haenkei [59], 3,7-quercetindimethylether [60] in Salvia dombeyi and hesperetin in Clinopodium pulchellum [61]. In several plants, rosmarinic acid is the main peak: Clinopodium brevicalyx, Salvia oppositiflora, Clinopodium sericeum and Hedeoma mandoniana. Some type of salvianolic acid is present in all the samples, although in some cases they are very small modifications of the rosmarinic acid molecule. Dimers and trimers of rosmarinic acid are present in Clinopodium brevicalyx, Salvia oppositiflora, Salvia cuspidata, Clinopodium sericeum, Hedeoma mandoniana and Clinopodium pulchellum. In Clinopodium sericeum, not only is the diversity of salvianolic acids important, but also their abundance in salvianolic acid A, which would allow the preparation of said substance from it [62].

In all cases, triterpenic acids, oleanolic and ursolic are present, which do not appear with the present methodology used, but did appear in a previous work [6], where it is reported that Clinopodium revolutum contains 2.55 and 4.81 percent of these acids. As it is now known that ursolic acid has great pharmacological importance [47], it was decided to study the purification of this substance [48], now more focused on its recrystallization in ethanol. These procedures serve to have a ursolic acid chromatographic standard that could be applicable on a larger scale, to treat cheaper raw materials such as apple peels (Malus), eucalyptus (Eucalyptus) or olive (Olea) leaves [55].

The aqueous infusion of these plants shows the presence of phenolic substances with antioxidant capacity and specifically the presence of rosmarinic acid, with Clinopodium brevicalyx and Clinopodium sericeum being the ones with the highest rosmarinic acid content and also the highest total antioxidant capacity. In none of the aqueous infusions could the presence of triterpenic acids be detected. It means that the effect of the infusion of these plants is due to the action of salvianolic acids and other water-soluble compounds and not due to the presence of triterpenic acids.

Plant material

The plants used in this study are: Clinopodium brevicalyx Epling (Harley & Granda) (Menthinae), Salvia oppositiflora (R. and P.) (Salviinae), Minthostachys mollis Griseb. (Menthinae), Salvia sagittata R. and P. (Salviinae), Salvia cuspidata subsp. cuspidata (R. and P.) (Salviinae), Clinopodium revolutum (R. and P.) (Menthinae), Clinopodium sericeum (Briq. et Benth) Govaerts (Menthinae), Salvia haenkei Benth. (Salviinae), Salvia dombeyi Epl. (Salviinae), Hedeoma mandoniana Wedd. (Menthinae), Clinopodium pulchellum Kunth (Govaerts) (Salviinae). All of them were collected in Peru (2014-2018) by the author (C.S.) according to the Universidad San Antonio Abad procedures and following the guidelines of the Herbarium Truxillense of the Universidad Nacional de Trujillo-Perú https://facbio.unitru.edu.pe. Specimens were identified and deposited by the botanist Eric Frank Rodríguez.

Sample preparation for metabolite profiling

50 mg of pulverized aerial parts are subjected to an ultrasonic bath for five minutes with 1 mL of ethanol three times. The filtrates are evaporated in vacuo and stored at 4°C until use.

UHPLC- Q- Exactive – Orbitrap – MS

Chromatographic separation was performed on a Thermo Scientific Dionex Ultimate 3000 UHPLC system with an Acclaim RPC18 150 x 4.6 mm x 1.8 µm chromatographic column at 25°C and a gradient of a) 0.1% H2CO2 in water and b) acetonitrile: (time, % b)): (0.5); (5,5); (10.30); (15.30); (20,70); (25.70); (35.5) and 12 min of equilibration before each injection. The flow was 1 mLmin-1 and the injection volume was 10 μL. The extracts are dissolved in 1.5 mL of methanol and filtered through 0.22 µm PTFE. For mass spectrometry, a Q-Exactive MS (Thermo Fisher Germany) equipped with electrospray ionization (ESI) in negative mode was used. The MS collection parameters were: spray voltage 2500 V. Capillary temperature 400°C. Sheath gas flowed at a rate of 75 units. Auxiliary gas flowed at 20 units. Scanning range of 100/1500 m/z. Resolution 35,000. The mass tolerance threshold was 5 ppm. Data acquisition and processing was done with XCalibur 2.3 (Thermo Fisher Scientific).

Isolation of ursolic acid from Clinopodium revolutum

Dry and ground material with a content of 4.48% ursolic acid and 1.27% oleanolic acid was used. 53.9 g are Soxhlet defatted with petroleum ether and then extracted with 96% ethanol until exhausted. The dry ethanolic extract (15 g) is impregnated in 40-63 μm Si Gel and placed on a 1 cm high bed of the same sorbent in a 9 cm diameter Büchner funnel and eluted with 1.- ether of petroleum, 2.- ethyl acetate and 3.- 96% ethanol. The 7.5 g of alcoholic eluate by dissolution in 80 mL of boiling ethanol in 24 hours produces 1200 mg of ursolic acid needles of 90.91% purity. Recrystallization of this product from 100 mL of ethanol produced 900 mg of 95.34% pure crystals. The purity of ursolic acid was verified by HPLC [45] and its identity by ¹HNMR and ¹³CNMR spectroscopy.

¹H-NMR (in CD3OD, 400 MHz): δ 5.22, 1H, m, H-12; δ 3.15, 1H, m, H-3; δ 2.20, 1H, J = 11.2 Hz, H-18; δ 2.02-1.15, m, H-22; δ 1.12, 3H, s, H-23; δ 0.972, 3H, s, H-27; δ 0.966, 3H, s, H-26; δ 0.956, 3H, s, H-24; δ 0.88, 3H, d, H-29; δ 0.84, 3H, d, H-30; δ 0.77, 3H, s, H-25. ¹³C-NMR (in CD3-OD, 101 MHz): δ 38.12 (C-1), 27.91 (C-2), 79.71 (C-3), 40.43 (C-4), 58.33 (C-5), 19.49 (C-6), 34.35 (C-7), 40.80 (C-8), 49.04 (C-9), 38.15 (C-10), 17.82 (C-11), 126.90 (C-12), 139.64(C-13), 43.26 (C-14), 29.23(C-15), 25.34(C-16), 48.37(C-17), 54.38(C-18), 40.01(C-19), 39.85 (C-20), 31.78 (C-21), 32.70 (C-22), 28.78 (C-23), 16.03 (C-24), 16.39 (C-25), 17.65 (C-26), 24.10 (C-27), 181.64 (C-28), 16.39 (C-29), 21.58 (C-30). [56, 57].

This last recrystallization was found to be crucial. Subsequent experiments showed that 50 mg samples of 90% purity ursolic acid recrystallized in 6.3-3.6 mL (0.8-1.4%) of ethanol in sealed vials at 60°C and then cooled to 15.8°C over a period of 24 hours produce 25 mg of ursolic acid with a purity greater than 98%.

Preparation and Characterization of aqueous infusions in eleven Mentheae

A 50 mg plant sample was infused with 10 mL of boiling water for 10 minutes. It was filtered (0.22 μm) and in the filtrate total phenols (Folin Ciocalteau) [6], antioxidant capacity (Blue Mo(V)) [6] and the content of rosmarinic acid and triterpenic acids were quantified by UHPLC [6, 45 ].

For rosmarinic acid quantification, DAD-UHPLC (Dionex Thermoscientific Ultimate 3000 UHPLC with Chromeleon 7.2 software) was used with a 100 x 2.1 mm x 1.8 µm RPC₁₈ Zorbax Eclipse Rapid Resolution column and a 1.5 x 2.1 mm x 1.8 µm UHPLC Zorbax Eclipse column guard. The working conditions were: Separation temperature: 40°C, Flow: 0.4 mL/minute. Gradient: a) 0.1% H2CO2; b) acetonitrile; (time, %b)): (0,0); (1,0); (6.40); (9,100); (13,100); (14,0); (17.0). The quantification was done with the readings obtained at 330 nm. In parallel, a calibration curve was made with standard rosmarinic acid (Aldrich) of 2, 1.5, 1, 0.5 and 0.2 mg/mL with a linearity of 0.999. The analysis was run in triplicate.

Quantification of triterpenic acids by HPLC [45] (Dionex Thermoscientific Ultimate 3000 UHPLC, with Chromeleon 7.2 software) was performed using a Phenomenex Lichrospher RPC₁₈ 25 x 0.46 cm x 5 µm column and Zorbax Eclipse 1.5 x 4.6 mm x 5 µm HPLC column guard. . The analysis conditions were as follows: Analysis time: 20 minutes. Temperature: 30°C. Elution mode: isocratic (acetonitrile:water, 8:2). Detection at 209 nm. A calibration curve was made with a mixed standard of oleanolic acid and ursolic acid (Chromadex) of 2, 1, 0.5 and 0.3 mg/mL of AO and AU. The analysis was run in triplicate.

Data Availability Statement

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Competing Interest Statement

The authors declare no conflict of interest.

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Phytochemical profile of eleven peruvian Mentheae. Isolation of ursolic acid from Clinopodium revolutum (Ruiz & Pavon) Govaerts and analysis of the aqueous infusions

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