Abstract

Mutisia L. f. (Mutisiinae, Mutisieae, Mutisioideae, Asteraceae) is a South American genus that contains several medicinal species. Mutisia acuminata Ruiz & Pav. and M. orbignyana Wedd. are known sources of the 12-dihydroxy-5-methylcoumestan and 5-methylcoumarin-4-glucoside respectively. The 12-dihydroxy-5-methylcoumestan exhibited notable antihepatotoxic activity in a previous study. In the present work, 5-methylcoumarin-4-glucoside and 12-dihydroxy-5-methylcoumestan were isolated from a Peruvian collection of M. orbignyana. Additionally, the relative concentrations of those two isolated compounds in six Peruvian species of Mutisia (M. acuminata, M. cochabambensis Hieron., M. lanata Ruiz & Pav., M. orbignyana, M. venusta S.F. Blake and M. wurdackii Cabrera) were determined by an HPLC method. The highest concentrations of both constituents were observed in the sample of M. orbignyana.

Keywords:
Mutisia; Asteraceae; 5-methylcoumarin; coumestan

GRAPHICAL ABSTRACT

HIGHLIGHTS

• A 5-methylcoumarin and a coumestan were isolated from M. orbignyana.

• An HPLC method was established for the quantification of both compounds.

• The concentrations of both compounds were assessed in six samples of Mutisia.

• Mutisia orbignyana displayed the highest contents of both isolated compounds.

INTRODUCTION

Among the main antihepatotoxic agents of natural origin silymarin stands out for its great commercial impact and wedelolactone due to its high dissemination because it comes from Eclipta alba (Asteraceae), a common weed that occurs in the lowlands of India subcontinent [¹1 Antunes C, Dutra M, Konrath E. Hepatoprotective native plants documented in brazilian traditional medicine literature: current knowledge and prospects. Chem Biodivers. 2022 May;19(6):e202100933.]. In 1988, Daily and coauthors [²2 Daily A, Seligmann O, Nonnenmacher G, Fessler B, Wong S, Wagner H. New chromone, coumarin, and coumestan derivatives from Mutisia acuminata var. Hirsuta. Planta Med.1988 Feb;54(1):50-2.] reported the presence of 11,12-dihydroxy-5-methylcoumestane among other substances in the methanolic extract of Mutisia acuminata (Mutisieae, Mutisioideae, Asteraceae). Furthermore, in 2009, Flores and coauthors [³3 Flores Y, Rodrigo G, Mollinedo P, Akesson B, Sterner O, Almanza GR. A 5-methylcoumarin glucoside and a coumestan derivative from Mutisia orbignyana. Rev. Boliv. Quím. 2009 Aug;26(1):21-6.], established the presence of the 5-methylcoumarin-4-glucoside (1) and 11,12-dihydroxy-5-methylcoumestane (2) in M. orbignyana (Figure 1). The chemical structure of compound 2 is related to wedelolactone and a remarkable antihepatotoxic effect has also been demonstrated for it [²2 Daily A, Seligmann O, Nonnenmacher G, Fessler B, Wong S, Wagner H. New chromone, coumarin, and coumestan derivatives from Mutisia acuminata var. Hirsuta. Planta Med.1988 Feb;54(1):50-2.]. Regarding compound 1, a high concentration of it has recently been reported in Vernonia glaberrima (Vernoniaeae, Cichoroideae, Asteraceae) [⁴4 Alhassan AM, Ahmed QU, Latip J, Shah SAA, Khan AYF, Sarian MN, Wahab RA, Taher M, Abdullahi MI, Khatib A. Phytoconstituents from Vernonia glaberrima Welw. Ex O. Hoffm. Leaves and their cytotoxic activities on a panel of human cancer cell lines. S. Afr. J. Bot. 2018 May;116:16-24.] showing cytotoxic activity. The presence of 5-methylcoumarins in member of Mutisioideae subfamily has also been extensively reviewed by Vestena and coauthors 2022 [⁵5 Vestena A, Meirelles G, Zuanazzi J, von Poser G. Taxonomic significance of coumarins in species from the subfamily Mutisioideae, Asteraceae. Phytochem. Rev. 2022 Jul;22:85-112.], and members of Mutisia genus stands out as potential sources of 5-methylcoumarins with potential antihepatotoxic activity. Therefore, in the present work, both 5-methylcoumarin-4-glucoside (1) and 11,12-dihydroxy-5-methylcoumestane (2) were purified from a Peruvian assession of M. orbignyana. Additionally, the relative concentrations of both substances in six Peruvian species of Mutisia genus (M. acuminata; M. cochabambensis; M. lanata; M. orbignyana, M. venusta and M. wurdackii) were determined by an HPLC method as part of our program for the search of new source of 5-methylcoumarins.

Figure 1
Structures of 5-methylcoumarin-4-glucoside (1) and 11,12-dihydroxy-5-methylcoumestan (2)

MATERIAL AND METHODS

Plant material

Aerial parts (leaves) of M. acuminata Wedd., M. cochabambensis Hieron., M. lanata Ruiz & Pav. and M. venusta S.F. Blake were collected in Cusco region, Perú. Mutisia orbignyana was collected in Moquegua region, Perú whereas M. wurdackii Cabrera was collected in Amazonas region, Perú. The plants were translated to the phytochemistry laboratory (Chemistry Department, UNSAAC), and dried at room temperature.

Isolation of 5-methylcoumarin-4-glucoside (1) and 11,12-dihydroxy-5-methylcoumestan (2)

50 g of dried M. orbignyana were exhausted with 96% ethanol. The dried ethanolic extract was suspended in hot water (85 ºC) and vacuum filtered to remove waxes and chlorophylls. The aqueous extract was partitioned with ethyl acetate. The ethyl acetate extract was evaporated and vacuum liquid chromatographed with a gradient of hexane and ethyl acetate, the fraction of hexane:ethyl acetate (7:3) yielded by ethanol recrystallization 15.4 mg of 2 (0.034%) and the ethyl acetate fraction yielded by ethanol recrystallization 27 mg of 1 (0.054%). The structures of the isolated compounds were established based on NMR spectroscopy, high-resolution MS and comparison with literature data as 5-methylcoumarin-4-β-glucoside (1) and 11,12-dihydroxy-5-methylcoumestan (2) [²2 Daily A, Seligmann O, Nonnenmacher G, Fessler B, Wong S, Wagner H. New chromone, coumarin, and coumestan derivatives from Mutisia acuminata var. Hirsuta. Planta Med.1988 Feb;54(1):50-2.,³3 Flores Y, Rodrigo G, Mollinedo P, Akesson B, Sterner O, Almanza GR. A 5-methylcoumarin glucoside and a coumestan derivative from Mutisia orbignyana. Rev. Boliv. Quím. 2009 Aug;26(1):21-6.].

5-Methylcoumarin-4-β-glucoside (1): ¹HNMR (DMSO-d₆, 400MHz) δ 5.98 (s, 1H, H-3), δ 7.16 (d, 1H, H-6), δ 7.51 (t, 1H, H-7), δ 7.25 (d, 1H, H-8), δ 2.71 (s, 3H, C₅-CH₃), δ 5.20 (d, 1H, H-1’), δ 3.41 (t, 1H, H-2’), δ 3.39 (t, 1H, H-3’), δ 3.20 (t, 1H, H-4’), δ 3.49 (m, 1H, H-5’), δ 3.72 (t, 1H, H-6’), δ 4.63 (m, 1H, -OH-6’), δ 5.11, 5.20, 5.49 (3d, 3H, -OH- 2’,3’,4’). ¹³CNMR (DMSO-d6, 100 MHz) δ 161.7 (C-2), δ 93.4 (C-3), δ 167.0 (C-4), δ 114.2 (C-5a), δ 137.5 (C-5), δ 128.2 (C-6), δ 132.4 (C-7), δ 115.3 (C-8), δ 154.8 (C-8a), δ 23.6 (CH₃), δ 100.3 (C-1’), δ 73.5 (C-2’), δ 77.0 (C-3’), δ 70.0 (C-4’), δ 77.8 (C-5’), δ 61.0 (C-6’). LC-HRESIMS negative mode m/z 383.0984 [M - H]^- (calcd for C₁₆H₁₈O₈+CH₂O₂, 383.0978).

11,12-Dihydroxy-5-methylcoumestan (2): ¹HNMR (DMSO-d₆, 400MHz) δ 9.60 (br, 2H, 11,12-OH), δ 7.53 (t, 1H, H-7), δ 7.40 (d, 1H, H-6) δ 7.30 (s, 1H, H-10), δ 7.30 (d, 1H, H-8), δ 7.23 (s, 1H, H-13), δ 2.85 (s, 3H, C₅-CH₃). ¹³CNMR (DMSO-d6, 100 MHz) δ 158.8 (C-2), δ 104.7 (C-3), δ 157.5 (C-4), δ 113.4 (C-5), δ 111.8 (C-5a), δ 134.1 (C-6), δ 130.5 (C-7), δ 126.7 (C-8), δ 153.0 (C-8a), δ 105.5 (C-9), δ 114.7 (C-10), δ 149.4 (C-11), δ 146.4 (C-12), δ 98.8 (C-13), δ 144.7 (C-14), δ 20.8 (-CH₃). LC-HRESIMS negative mode m/z 281.0445 [M - H]^- (calcd for C₁₆H₁₀O₅, 383. 281.0450).

Quantification of 5-methylcoumarin-4-glucoside (1) and 11,12-dihydroxy-5-methylcoumestan (2)

For quantification purposes, 500 mg of each dried plant material was macerated for 24 hours with 20 mL of 70% ethanol, this operation was repeated 3 times and the filtrates were made up to 60mL. Five mL of each extract were evaporated at 40°C, weighed and redissolved in one mL of methanol and filtrated by 0.22μm PTFE membrane for conditioning in chromatographic vials. The chromatographic method was developed in a Thermo Scientific Ultimate 3000 UHPLC chromatograph with automatic injection and DAD detector. A Zorbax Eclipse Plus C₁₈ Column (1.8μm particle size, 4.6 × 100 mm) was used. Column temperature was set at 35°C. Mixtures of 0,1% formic acid in water (A) and acetonitrile (B) were used as mobile phase with the following gradient program (minutes, %B): 0,2; 5,30; 10,80; 12,80; 15,100; 17,100; 19,2; 20,2. DAD: 200-400 nm: 280, 345, 254, 330 nm. The total run time was 20 minutes and a flow rate of 0.4 mL/min. The relative concentrations of both compounds were quantified using calibration curves prepared with standard solutions of 1 and 2 dissolved in methanol with six data points. The calibrations curves were obtained by potting the peak area signals as a function of the concentration. The equation curves were obtained (Y=64.55X-2.71 for 1, Y=115.84X-1.43 for 2) and linearity was evaluated by least-squares regression analysis (r ² = 0.9996 for 1, and r ² = 0,9995 for 2). For quantification of 1 the chromatograms at 280 nm were used, whereas 2 was quantified at 345nm.

RESULTS AND DISCUSSION

In the Figure 2, typical UHPLC chromatograms of M. orbignyana are displayed. Compound 1 eluted at a retention time of 8.20 min whereas compounds 2 eluted at 12.09 min. In addition to the isolated constituent additional chromatographic peaks were observed, highlighting the occurrence of two peaks with retention times of 6.99 (peak a) and 8.89 (peak b) which displayed the characteristic UV spectra of the phenylpropanoids. Although phenylpropanoid are ubiquitous constituent in members of Asteraceae family, there is no previous report on the occurrence of phenylpropanoids in M. orbignyana. The phenylpropanoids 3-O-caffeoylquinic acid and 5-O-caffeoylquinic acid were previously describe in M. friesiana [⁶6 Viturro C, Molina M, Schmeda-Hirschmann G. Free radical scavengers from Mutisia friesiana (Asteraceae) and Sanicula graveolens (Apiaceae). Phytother. Res. 1999 Aug;13(5):422-4.].

Figure 2
Typical chromatograms of standard compounds and Mutisia orbignyana. (A) Chromatogram of a standard solution of 5-methylcoumarin-4-β-glucoside (1) monitored at 280nm. (B) Chromatogram of the ethanolic extract of M. orbignyana monitored at 280nm. (C) Chromatogram of a standard solution of 11,12-dihydroxy-5-methylcoumestan (2) monitored at 345 nm. (D) Chromatogram of the ethanolic extract of M. orbignyana monitored at 345nm. In the bottom, four selected UV spectra of unidentified phenylpropanoid derivatives (a and b) and the standard compounds 1 and 2.

The identity of those phenylpropanoids in M. orbignyana as well as their occurrence in other member of Mutisia genus need to be elucidated in further investigations. The Table 1 displays the content of 1 and 2 in six Peruvian species from Mutisia. Mutisia orbignyana is the plant with the highest content of both substances while in M. cochabambensis neither of those compounds were present. According to Cabrera [⁷7 Cabrera AL. Revisión del genero Mutisia (Compositae). Opera Lilloana. 1965 Oct;13:1-227.], who divided the genus Mutisia into six sections based on morphological aspects, M. orbignyana belongs to the Isantha section, the most basal. Mutisia orbignyana has a particularly high concentration of 1 comparable to the African plant Vernonia glaberrima (Cichoroideae). On the other hand, to our knowledge there is no other genus apart from Mutisia that displays the presence of 2.

CONCLUSION

Mutisia acuminata and especially M. orbignyana are candidate species to extract both 5-methylcoumarin-4-glucoside (1) and 11,12-dihydroxy-5-methylcoumestan (2). The other Peruvian Mutisia have none or small amounts of these compounds. However, further analysis of samples from different geographical origin and collection times are required.

Acknowledgments

We acknowledge Dr. Eric Frank Rodriguez, Herbarium Truxillense, National University of Trujillo, Trujillo, Peru, for plant identification. Dr. Helena Maruenda Castillo, Chemistry Department, Pontificia Universidad Católica del Perú, Lima, Peru, is acknowledge for NMR analyses. This research was carried out with the collection permit of SERFOR, Peru (RD No D000154-2022-MIDAGRI-SERFOR-DGGSPFFS-DGSPF)

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