Diterpenoid Constituents of Teucrium scordium L. Subsp. s cordioides (Shreb.) Maire Et Petitmengin

Abstract

A new scordidesin А (1) and 2 known furo-clerodane diterpenoids, teucrin A (2) and 6-ketoteuscordin (3), were isolated by the phytochemical investigation of the acetone extract from the aerial parts of Teucrium scordium L. subsp. scordioides (Lamiaceae). The structure and stereochemistry of the compounds were established by high-resolution electrospray ionization mass spectrometry, infrared spectroscopy, and various ¹H nuclear magnetic resonance (NMR) and 2-dimensional-NMR techniques. Additionally, we report ¹³C NMR spectral data of 3 that were not published before. Antimicrobial activity of the compounds was tested against Gram-positive and Gram-negative bacteria belonging to 5 different species and against 2 strains of yeast belonging to 2 different species of the genus Candida.

Keywords

terpenoids lamiaceae clerodane antimicrobial activity

The diterpenoid composition of Teucrium scordium subsp. scordium had been thoroughly studied in the period of 1981‐1985 by different collectives^1

-4. Ten known clerodane diterpenes in addition to 11 new derivatives were isolated. The decalin moiety in all representatives has 5:10 trans ring fusion. Among them, there is no compound with the 19-nor-clerodane skeleton. Only 2-keto-l9-hydroxyteuscordin⁵ contains 18,6-olide.

The diterpenoid fraction of T. scordium L. subsp. scordioides has not been studied. Cytotoxicity and antimicrobial activity of cyclohexane, dichloromethane (CH₂Cl₂), and methanol (CH₃OH) extracts of the plant had been tested.⁶ Cyclohexane and CH₂Cl₂ extracts showed high cytotoxicity against MDA-MB-361 cells (half-maximal inhibitory concentration [IC₅₀] = 130.33 ± 0.1 µg/mL and IC₅₀ = 189.89 ± 3.99 µg/mL). CH₂Cl₂ extract was more effective against MDA-MB-453 cell line with IC₅₀ = 130.33 ± 0.1 µg/mL. The CH₃OH extract possessed no cytotoxicity against breast cancer cell lines, MDA-MB-361 and MDA-MB-453. Herb extracts of the plant showed weak antibacterial activity on Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, and Bacillus subtilis and did not show any activity against Staphylococcus aureus, S. epidermidis, Micrococcus luteus, Enterococcus faecalis, and Candida albicans.

As a part of our search of biologically active diterpenoids from Teucrium species,⁷ we investigated T. scordium L. subsp. scordioides (Shreb.) Maire et Petitmengin. Herein, we report on the isolation and structural characterization of three furo-clerodane diterpenoids, the new scordidesin A (1), the known teucrin A^8
-10 (2), and 6-ketoteuscordin² (3) (Figure 1). Compounds 1 and 2, which were positional isomers, were isolated as a mixture. We also report the result of the antimicrobial activity test of the clerodans.

Figure 1

Structures of the 3 compounds (1-3) and teucrin H4 (4).

Results and Discussion

Two homogeneous substances (I and II) were obtained by thin-layer chromatography (TLC) of the acetone extract received from the aerial stems of T. scordium L. subsp. scordioides. Based on the observed pseudomolecular positive ion peak at m/z 367.1171 [M + Na]⁺ in the high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) (see Supplemental Material 1) for substance I, the molecular formula C₁₉H₂₀O₆ was established (calcd. 367.1158 for C₁₉H₂₀O₆Na) indicating 10 degrees of hydrogen deficiency. The molecular formula was also confirmed by the molecular ion peak at m/z 343.1173 [M − H]⁻ (calcd. for C₁₉H₁₉O₆: 343.1195) in HR-ESI-MS under negative conditions. A 19-nor-clerodane skeleton of the compound was suggested by the odd number of C-atoms.

The functional groups present in the compound were implied by the observed absorptions in the infrared (IR) spectrum at 1505, 1076, and 874 cm⁻¹ (furan), at 1746 br and 1180 cm⁻¹ (lactones), and at 3435 cm⁻¹ (hydroxyl group).

¹H and ¹¹C nuclear magnetic resonance (NMR) spectra of the substance I (see Figures 2s-5s in Supplemental Material 1) displayed the presence of duplicate distinct signals for all hydrogen and carbon atoms. As in many clerodanes isolated from Teucrium species, signals of a β-substituted furan ring (2 α-furan protons at δ _H 7.78/7.72 and 7.65/7.62, 1 β-furan proton at δ 6.59/6.55, and 4 carbons at δ _C 124.5/125.5, C-13; 108.4/108.5, C-14; 144.7/144.4, C-15; 141.0/140.4, C-16) were detected easily, leading to a 9/10 ratio of an inseparable mixture of 2 structurally related furo-clerodane derivatives (1/2).

The presence of 2 γ-lactone rings was supported by the specific signals for carbonyl groups at δ _C 180.6/175.6 (C-20) and 172.5/171.6 (C-18) and 2 oxygenated methine groups at δ _C 74.6/71.7//δ _H 5.85 m/5.62 t (CH-12) and δ _C 80.5/77.7//δ _H 4.93 ddd/4.91‐4.84 m (CH-6), forming a C-20, C-12; C-18, C-6 diolide. These assignments were based on the heteronuclear single quantum correlations (HSQCs) and were in agreement with the presence of heteronuclear multiple bond correlation (HMBC) from H-11A to C-12 and C-20, from H-12 to C-11, from the methine proton H-8β to C-6, C-7, C-9, C-10, C-17, and C-20, from methylene protons H₂-7 to C-6, C-8 and C-9, and from the methyl protons H₃-17 to C-6. Signals of just 1 methyl at δ _C 13.2/16.5 and δ _H 1.24 d/1.09 d (CH₃-17) pointed out the absence of C-19. The 2 unprotonated olefin carbons at δ _C 126.7/127.8 (C-4) and 158.3/166.1 (C-5) were part of the С-18 to С-6 α,β-unsaturated γ-lactone ring.

The β-position of the furan ring was established by the nuclear Overhauser effect spectroscopy (NOESY) interactions of the methyl protons H₃-17 with H-14 and H-16 and of H-12 with H-11A. The observed NOESY correlation of H-6β with H-8β and H-10β indicated the α bond between C-6 and the oxygen atom of the lactone ring.

The identity of the NMR data for the compound 2 in a larger quantity with those of the previously described diterpenoid teucrin A^8

-13 (compare data in Table 1 with Figures 14s and 15s, on page 11 in Supplemental Material 1) allows to exclude its signals and to consider the other signals separately for compound 1 only.

Table 1

Scordidesin A,^a Teucrin A,^a and 6-Ketoteuscordin^b (1-3) Nuclear Magnetic Resonance Data

Position	1			2			3
Position	δ ¹³C, nH	δ ¹H	m, J (in Hz)	δ ¹³C, nH	δ ¹H	m, J (in Hz)	δ ¹³C, nH	δ ¹H	m, J (in Hz)
1α	24.7, CH₂	1.44	m	21.7, CH₂	1.61	m	23.0, CH₂	1.60 (α)	ov m^d
1β	24.7, CH₂	2.42	m	21.7, CH₂	1.99	ov m^d	23.0, CH₂	2.06 (β)	ov m^d
2α	58.1, CH	4.42 (eq)	br s	19.5, CH₂	1.73	m	29.7, CH₂	1.22 (α)	ov m^d
2β	58.1, CH	-	-	19.5, CH₂	2.03	ov m^d	29.7, CH₂	1.57 (β)	ov m^d
3α	19.3, CH₂	2.17	m	31.2, CH₂	1.66	m	23.8, CH₂	1.47 (α)	ov m^d
3β	19.3, CH₂	2.20	ov m^d	31.2, CH₂	1.95	ov m^d	23.8, CH₂	1.96 (β)	ov m^d
4	126.7, C	-	-	127.8, C	-	-	49.2, CH	2.00	dd, 13.0, 3.3
5	166.1, C	-	-	158.3, C	-	-	55.4, C	-	-
6β	77.7, CH	4.89	ddd, 9.1, 3.1, 2.2	80.5, CH	4.93	dddd, 6.0, 3.9, 2.6, 1.3	208.1, C	-	-
7α	35.09, CH₂	2.24	ov m^d	72.1, CH	-	-	41.6, CH₂	3.41 (α)	t, 14.1
7β	35.09, CH₂	2.18	ov m^d	72.1, CH	4.09 (eq)	ddd, 11.2, 4.6, 2.6	41.6, CH₂	2.39 (β)	dd, 14.5, 4.5
8β	35.08, CH	2.12	m	38.3, CH	2.31	qd, 7.1, 4.5	41.4, CH	2.07 (β)	ov m^d
9	56.7, C	-	-	53.6, C	-	-	50.8, C	-	-
10β	42.0, CH	3.13	td, 6.8, 3.1	41.7, CH	2.79	br dd, 10.2, 4.0	40.5, CH	2.86 (β)	dd, 10.9, 6.4
11A	40.2, CH₂	2.78 A	dd, 14.3, 9.3	42.1, CH₂	2.85 A	dd, 14.2, 8.0	41.0, CH₂	2.46	br t, 9.3
11B	40.2, CH₂	2.65 B	dd, 14.1, 9.0	42.1, CH₂	2.87 B	dd, 14.2, 8.0	41.0, CH₂	2.46	br t, 9.3
12α	71.7, H	5.62	t, 8.6	74.6, CH	5.85	m	72.1, CH	5.44 (α)	t, 8.4
13	125.5, C	-	-	124.5, C	-	-	124.5, C	-	-
14	108.5, CH	6.55	dd, 1.8, 0.8	108.4, CH	6.59	dd, 1.8, 0.8	107.8, CH	6.40	br s
15	144.4, CH	7.62	t, 1.7	144.7, CH	7.65	t, 1.7	144.5, CH	7.47	br s
16	140.4, CH	7.72	dt, 1.5, 0.7	141.0, CH	7.78	m	139.7, CH	7.48	br s
Me-17	16.5, CH₃	1.09	d, 6.6	13.2, CH₃	1.24	d, 7.1	17.2, CH₃	1.14	d, 6.6
18	171.6, C	-	-	172.5, C	-	-	176.8, C	-	-
19A^c	-	-	-	-	-	-	69.3, CH₂	4.80 A^c	d, 11.3
19B	-	-	-	-	-	-	69.3, CH₂	4.49 B	d, 11.2
20	175.6, C	-	-	180.6, C	-	-	23.0, CH₂	-	-
OH		4.04	d, 11.0	OH	4.62	d, 11.2

Abbreviations: Abbreviations: br, broad; ov, overlapped.

^aDeuterated acetone, ¹H 600.13 MHz, δ _ref 2.05 ppm; ¹³C 150.9 MHz, δ _ref 206.26 (CO), 30.60 (CH₃) ppm.

^bDeuterated chloroform, ¹H 600.13 MHz, δref 7.26; ¹³C 150.9 MHz, δref 77.0 ppm.

^cEndo hydrogen with respect to ring B.

^dData from heteronuclear single quantum correlation.

The same molecular formula for the other constituent¹ of the mixture I was assumed based on both the positive and negative HR-ESI-MS. The ¹³C NMR spectrum displayed the presence of 19 carbon atoms, and a distortionless enhancement by polarization transfer (DEPT) experiment identiﬁed 1 methyl (δ _C16.5, CH₃-17), 4 methylenes, 8 methines (3 aromatic at δ _C 140.4 [C-16], 144.4 [oxygenated C-15], 108.5 [oxygenated C-14]; 3 more oxygenated at δ _C58.2 [C-2], 77.7 [C-6], 71.7 [C-12]; at δ _C 35.08 [C-8], 42.0 [C-10]) and 6 unprotonated (including 2 carbonyl, at δ _C 175.6 for γ-lactone [C-20] and 171.6 for α, β-unsaturated γ-lactone [C-18]; 2 olefin at δ _C 126.7 [C-4] and 166.1 [C-5]; 1 aromatic at δ _C 125.5 [C-13], and 1 quaternary at δ _C 53.6 [C-9]) carbon atoms.

The assignments of the hydrogen atoms to the corresponding carbon atoms were in agreement with the results from the HSQC experiment. The presence of a furan ring in the molecule was confirmed by the correlations between the signals at δ _C 108.5/δ _H 6.55 dd (CH-14), 144.4/7.62 t (CH-15), and 140.4/7.72 dt (CH-16). The correctness of the assignments was proved by the observed HMBC from H-14 to C-13, C-15 and C-16, from H-15 to C-13, and from H-16 to C-14 and C-15. Other correlations from the signal of proton H-12 (δ _H 5.62 t) to carbons C-13, C-14, C-16 and from the signals of the methylene protons H₂-11 (δ _H 2.78 and 2.65) to C-13 were present. In the correlation spectroscopy (COSY) experiment interactions, H-14/H-15, H-14/H-16, and H-11A/H-12 were observed.

The ¹H and ¹³C NMR spectral data of 1 were close to the data оf teucrin A (2). A significant difference was observed in the ¹H NMR spectrum for the H-6 signal (δ _H 4.93 dtd, J = 3.9, 2.6, 1.3) in 2 which was shifted upfield in 1 at 4.89 and was modified into a ddd with 2 larger coupling constant (J = 9.1, 6.7, 2.0). The ddd at δ _H 4.09 (H-7β) in 2 was not observed among the signals of compound 1. A new downfield shifted br s at δ _H 4.42 appeared instead. The hydroxyl group in 1 was located at C-2 (δ _C 58.2). This assumption was in agreement with the observed HMBC—from H-3β (δ _H 2.20) to C-2 and from OH to C-2, from the COSY correlation between H-2α/H-3α, H-2α/H-3β, and H-2α/OH, and from the NOESY interactions between H-1α/H-2α, H-2α/H-3α, and H-2α/H-3β. The NOESY interactions of H-2α with the adjacent axial protons H-1α and H-3α emphasized the equatorial position of the proton H-2α.

The ¹H and ¹³C NMR spectral data of 1 were very close to those reported previously for teucrin H₄ (4).¹⁴ A noticeable difference was observed for the signal of H-6 which appeared in 4 at δ _H 5.98 and was shifted in 1 with 1.10 ppm in the high field at δ _H 4.88. The β-position of H-6 in 1 was supported by the observed NOESY correlations of H-6 with H-8β and H-10β. Another significant difference was found for the methine group CH-2 and the adjacent CH₂-1 and CH₂-3 methylene groups. In compound 4, these signals were reported at δ _C 68.6 (CH-2)/δ _H 4.44 dddd (J _{1α(ax), 2β(ax)} = 10.4, J _{1β(eq), 2β(ax)} = 3.7, J _{2β(ax), 3α(ax)} = 9.5, J _{2β(ax), 3β(eq)} = 6.5) for the axial proton H-2β. The signals for the adjacent methylene groups were quoted at δ _C 34.3 (C-1)/δ _H 1.96 dddd (J _{1α , 1β} = 11.8, J _{1α, 10β} = 10.1, H-1α), δ _H 2.76 dt (J _{1β, 10β} = 3.7, H-1β), δ _C 30.4 (C-3)/δ _H 2.39 dddd (J _{3α , 3β} = 17.9, H-3α), and δ_H 3.00 br dd (H-3β). In compound 1, the signal of proton H-2 at δ _H 4.42 was reduced into broad singlet compared with the dddd at δ _H 4.44 in teucrin H₄ (4). The small value of the coupling constants for H-2 in 1 required the proton to be in the equatorial position, in this case, α-oriented. This conclusion was confirmed by the NOESY interaction of H-2α with H-1α and H-3α, indicating that these protons are α-oriented. Such correlations were impossible if the H-2 had been β-oriented in the axial position. Also, no interaction was observed in the NOESY spectrum between the H-2 and H-10β, which would be expected if the proton H-2 had been in beta position, in this instancе, axial. Based on all the above data, scordidesin A was assigned the 2β-hydroxyteucvin structure 1 (Figure 1)

The recorded ¹H and ¹³C NMR spectra disclosed substance II as a single compound.³ Based on the pseudomolecular positive ion peak at m/z 381.1308 [M + Na]⁺ in the HR-ESI-MS (see Supplemental Material 1), the molecular formulae C₂₀H₂₂O₆ was established for compound 3 (calcd. for C₂₀H₂₂O₆Na: 381.1314) indicating 10 degrees of hydrogen deficiency.

Absorptions consistent with the presence of a furan ring (1504 and 875 cm⁻¹), lactones (1786, 1760, 1182 cm⁻¹), and hydroxyl group (3436 cm⁻¹) were observed in the IR spectrum.

The ¹³C NMR spectrum displayed the presence of 20 carbons, and a DEPT experiment identiﬁed 1 methyl, 6 methylene (one of them oxygenated, CH₂-19), 7 methines (including 3 aromatic and 3 oxidized), and 6 unprotonated (including 2 quaternary, 1 aromatic, and 3 for carbonyl groups—1 fоr a ketone group and 2 fоr γ-lactones).

Furan ring in the structure was confirmed by the signals at δ _C 124.5 (C-13), δ _C 107.8/δ _H 6.40 (br s, CH-14), δ _C 144.5/δ _H 7.47 (br s, CH-15), and δ _C 139.7/δ _H 7.48 (br s, CH-16) in the ¹³C and ¹H NMR spectra. Assignments of the aromatic protons to the corresponding carbon atoms were in agreement with the data from the HSQC spectrum and observed HMBCs (see Supplemental Material 1) from H-14 to C-15 and C-16, from H-15 to C-13, and from H-16 to C-14 and C-15. Additional correlations from the signal at δ _H 5.44 t (H-12) to carbons C-13, C-14, C-16 and from the methylene proton signals at δ _H 2.54 and 2.46 (CH₂-11) to C-13 were observed. Correlations between H-14/H-15, H-12/H-11A, H-12/H-11B were represented in the COSY spectra. The presence of 2 γ-lactone rings C-20 to C-12 as well as C-18 to C-19 was supported by the specific signals in the ¹H and ¹³C NMR spectrums: for the carbonyl functions, the signals were at δ _C 177.4 (C-20) and 176.8 (C-18), for oxygenated methine group at δ _C 72.1/δ _H 5.44 t (CH-12), and for oxygenated methylene group at δ _C 69.3/δ _H 4.80 d and 4.49 d (CH₂-19). These assignments were in agreement with the presented HMBCs: for the С-20 to С-12 γ-lactone ring, from H₂-11 (δ _H 2.46 br t) to C-12 and C-13, from H-12 to C-11 (δ _C41.0), C-13, C-14, and C-16, from H-10β (δ _H 2.86 dd) to C-20, as well as for the С-18 to С-19 γ-lactone ring, from H-7β (δ _H 2.39 dd) to C-5 (δ _C 55.4), from H-19A (δ _H 4.80, d) to C-18, and from H-19B (δ _H 4.49, d) to C-4 (δ _C49.2). In the COSY spectrum, cross-peaks between H₂-11/H-12, H₂-11/H-8, H-2β/H-4, and H-3α/H-4 were observed.

The ketone group, which resonated at δ _C 208.1, was located at C-6 on the ground on the downfield shifting of the methylene protons H₂-7 signals at δ _H 3.41 t and δ _H 2.39 dd, which were with the less complicated multiplet structure than the usual dt and ddd in diterpenoids with a hydroxyl group at C-6, due to the lack of adjacent protons at C-6. This conclusion was supported by the observed HMBC corrections to C-6 from the protons H-7α, H-7β, H-19A, and H-19B.

The relative configuration of 3 was determined by the NOESY experiment. The correlations between H-12/1α, H-12/1β, Me-17/H-14, and Me-17/H-16, indicating that the furan ring was in the β-configuration. Observed interaction of H-4 with H-10 and of H-8 with H-10 showed their co-facial relationship and were assigned in β-position. The NOESY correlations of H-1α with H-19A, H-7α with H₃-17, and H-19B conﬁrmed the α-orientation of all these protons.

Finally, the ¹H spectroscopic data of compound 3 were found to be identical to those reported previously by Papanov and Malakov² for 6-ketoteuscordin. The ¹³C NMR data of 6-ketoteuscordin had not been previously reported so they were included in Table 1 in order to facilitate the identification of this compound by subsequent isolation.

Antimicrobial activity of the isolated diterpenoids was tested against 7 strains, S. aureus ATCC 1805, Streptococcus pyogenes ATCC 12344, Listeria monocytogenes ATCC 8632, E. coli ATCC 3397, Salmonella abony ATCC 6017, C. albicans ATCC 10231, and Candida glabrata ATCC 90030. As seen (Table 2), both tested samples 1 and 2 demonstrated equal antimicrobial activity. Nevertheless, the carbon skeleton of the studied compounds is different and the functional groups are the same, which probably is the reason for equal antimicrobial activities. Antimicrobial activity of the tested samples against both Gram-positive and Gram-negative bacteria was almost equal, but antifungal activity against Candida species was weaker in comparison with antibacterial activity. This is probably due to the different composition and structure of bacterial and yeast cell walls.

Table 2

Antimicrobial Activity of Diterpenoid Constituents of Teucrium scordium L. Subsp. scordioides (Shreb.)

Test microorganism	MIC (µg/mL)
Test microorganism	1	2
Staphylococcus aureus ATCC 1805	500	500
Streptococcus pyogenes ATCC 12344	250	250
Listeria monocytogenes ATCC 8632	500	500
Escherichia coli ATCC 3397	500	500
Salmonella abony ATCC 6017	500	500
Candida albicans ATCC 10231	1000	1000
Candida glabrata ATCC 90030	1000	1000

Abbreviation: Abbreviation: MIC, minimum inhibitory concentration.

Conclusion

From the acetone extract of T. scordium L. subsp. scordioides (Shreb.) Maire et Petitmengin, a new neo-clerodane diterpenoid scordidesin A, besides the previously known teucrin A and 6-ketoteuscordin were isolated. Their structures were established by intensive spectral methods. ¹³C NMR data of 6-ketoteuscordin were published for the first time. Although scordidesin A and teucrin A were isolated as a mixture, their structures were unambiguously assigned, thanks to their varying amounts and by comparing with the published spectral data of the known teucrin A and with the relation teucrin H₄.

The substances showed moderate antimicrobial activity against the strains tested.

Experimental

Structural Data

¹H NMR spectra were recorded on Bruker Avance II + spectrometers, operating at 600.130 MHz. ¹³C NMR spectra were recorded at 150.903 MHz spectrometer. Trimethylsilane was used as an internal standard and deuterated acetone and deuterated chloroform as solvents. Chemical shifts (δ) are expressed in ppm and coupling constants (J) in Hertz. The IR spectra were registered in potassium bromide (KBr) pellet on a Vertex 70 spectrometer from 4000 cm^–1 to 400 cm^–1 at a resolution of 4 cm^–1 with 25 scans. The mass spectra were measured on Hewlett Packard 6890 GC System Plus/5973 MSD.

Plant Material

The aerial parts of T. scordium L. subsp. scordioides (Shreb.) Maire et Petitmengin were collected in June 2019 around the village Staro Orehovo near Varna, Bulgaria, and voucher specimens (n. 7212) were deposited in the Herbarium of the Higher Institute of Agriculture at Plovdiv, Bulgaria.

Extraction and Isolation

Dried and finely powdered material (2.3 kg) were extracted with Me₂CO (3 × 8 L) at room temperature for a week. After filtration, the solvent was evaporated to dryness under reduced pressure and low temperature (<40 °C) yielding a gum (28.0 g), which was dissolved in aqueous Me₂CO (40% H₂O, v/v, 300 mL). This solution was cooled to 4 °C for 24 hours and filtered. The filtrate was extracted with chloroform (3 × 300 mL), and the organic layer was dried (sodium sulfate) and evaporated in a vacuum to afford a residue (7.3 g, bitter fraction). This residue was subjected to column chromatography (100 g silica gel Merck n. 7734, deactivated with 10% H₂O, w/w). Pure petroleum ether (15 L), followed by a gradient of CH₂Cl₂-CH₃OH mixtures (10:0-9.7:0.3) were used as eluting solvents. Eluting with CH₂Cl₂-CH₃OH mixtures 9.8:0.2 resulted in the isolation of 15 mg of homogenous substance I. Eluting with CH₂Cl₂-CH₃OH mixtures 9.7:0.3 yielded 20 mg of substance II. Recrystallization from acetone gave 12 mg of a mixture of the positional isomers scordidesin A (1)/teucrin A (3), and 16 mg of 6-ketoteuscordin (3).

Scordidesin A (1)

Colorless prisms. TLC: R_f 0.40 (ethylacetate [EtOAc]). IR (1 + 2) ν_max (KBr): 3435, 2931, 2878, 1746, 1505, 1453, 1355, 1251, 1180, 1159, 1026, 875, 804, 602 cm⁻¹. ¹H and ¹³C NMR: see Tables 1 and Table 1s in the Supplemental Material 1. Positive ESI-MS (1 + 2) (70 eV, direct inlet) m/z (rel. int. in %): 367 [M + Na]⁺ (93.4), 257 (5.8), 217 (1.2). HR-ESI-MS m/z 367.1171 [M + Na]⁺ (calcd. for C₁₉H₂₀O₆Na: 367.1158). Negative ESI-MS (1 + 2) (70 eV, direct inlet) m/z (rel. int. in %): 343 [M − H]⁻ (97.6), 313 (18.3), 249 (10.2). HR-ESI-MS m/z 343.1174 [M − H]⁻ (calcd. for C₁₉H₁₉O₆: 343.1195).

Teucrin A (2)

Colorless powder. TLC: R_f 0.40 (EtOAc). ¹H and ¹³C NMR: see Tables 1 and Table 2s in Supplemental Material 1.

6-Ketoteuscordin (3)

Colorless powder. TLC: Rf 0.34 (EtOAc). IR ν_max (KBr): 3436, 2959, 2928, 1786, 1760, 1504, 1469, 1182, 1165, 1105, 975, 875, 730, 599 cm⁻¹.¹H and ¹³C NMR: see Tables 1 and Table 3s in the Supplemental Material 1. Positive ESI-MS (70 eV, direct inlet) m/z (rel. int. in %): 381 [M + Na]⁺ (95.2), 301 (15.2), 243 (18.3). HR-ESI-MS m/z 381.1308 [M + Na]⁺ (calcd. for C₂₀H₂₂O₆Na: 381.1314).

Antimicrobial Testing

The antimicrobial effects of the studied compounds were tested against the Gram-positive bacteria S. aureus ATCC 1805, S. pyogenes ATCC 12344, and L. monocytogenes ATCC 8632, as well as the following Gram-negative bacteria E. coli ATCC 3397 and S. abony ATCC 6017. Additionally, antimicrobial testing against 2 species of C. albicans ATCC 10231 and C. glabrata ATCC 90030 was performed. The bacterial strains were stored on nutritional agar (HiMedia Ltd., India) and the yeast strains on Sabouraud dextrose agar with chloramphenicol (HiMedia Ltd.). Stock solutions of the samples for antimicrobial testing were prepared by dissolving the respective compound in dimethylsulfoxide (DMSO; Sigma-Aldrich Co.). Antibacterial activity of the studied compounds was performed according to the Clinical Laboratory Standard Institute (CLSI) M7-A7 reference method for dilution antimicrobial susceptibility tests for bacteria that grow aerobically.¹⁵ Anticandidal activity of the studied compounds was performed according to CSLI M27-A3 reference method for broth dilution antifungal susceptibility testing of yeasts.¹⁶ Briefly, stock solutions were added to the Roswell Park Memorial Institute 1640 broth medium buffered to pH 7.0 with 0.165 mol/L 3-N-morpholino-propanesulfonic acid buffer (Sigma-Aldrich, Co) to reach dilutions with final sample concentrations, after inoculation with microbial test suspension, between 2000 and 125 µg/mL. Controls consisting of inoculated medium without tested sample and DMSO, as well as with DMSO were also prepared. Antimicrobial activity determined by broth microdilution tests was expressed as a minimal inhibitory concentration (MIC) in μg/mL. MIC was defined as the lowest concentration of the tested compound at which total inhibition of microbial growth was detected. All tests were performed in triplicate.

Supplemental Material

Supplementary Material 1 - Supplemental material for Diterpenoid Constituents of Teucrium scordium L. Subsp. scordioides (Shreb.) Maire Et Petitmengin

Supplemental material, Supplementary Material 1, for Diterpenoid Constituents of Teucrium scordium L. Subsp. scordioides (Shreb.) Maire Et Petitmengin by Petko I. Bozov, Plamen N. Penchev, Tania D. Girova and Velizar K. Gochev in Natural Product Communications

Footnotes

Acknowledgments

We thank Bulgarian Ministry of Education and Sciences for the funds Grant, National Scientific Program “Innovative low toxic bioactive compounds for precise medicine – Bio Activ Med” D01-217/30.11.2018.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Petko I. Bozov

Supplemental Material

Supplemental material for this article is available online.

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