Virescenosides from the Holothurian-Associated Fungus Acremonium striatisporum Kmm 4401

Abstract

Ten new diterpene glycosides virescenosides Z₉-Z₁₈ (1–10) together with three known analogues (11–13) and aglycon of virescenoside A (14) were isolated from the marine-derived fungus Acremonium striatisporum KMM 4401. These compounds were obtained by cultivating fungus on wort agar medium with the addition of potassium bromide. Structures of the isolated metabolites were established based on spectroscopic methods. The effects of some isolated glycosides and aglycons 15–18 on urease activity and regulation of Reactive Oxygen Species (ROS) and Nitric Oxide (NO) production in macrophages stimulated with lipopolysaccharide (LPC) were evaluated.

1. Introduction

Marine fungi are promising and prolific sources of new biologically active compounds. At the same time, glycosylated secondary metabolites of marine fungi such as ribofuranosides, containing as aglycon moieties anthraquinones [1,2,3], diphenyl ethers [4,5], isocoumarin [6] and naphthyl derivatives [7] are relatively rare. Recently, two steroid glycosides with β-D-mannose as sugar part were isolated from ascomycete Dichotomomyces cejpii [8] and new triterpene glycoside auxarthonoside bearing rare sugar N-acetyl-6-methoxy-glucosamine was described from sponge-derived fungus Auxarthron reticulatum [9]. Some of these glycosides exhibited cytotoxic [5], radical scavenging [3,4], and neurotropic [8] activities.

During our ongoing search for new natural compounds from marine-derived fungi, we have investigated the strain Acremonium striatisporum KMM 4401 associated with the holothurian Eupentacta fraudatrix. Twenty-one new diterpene glycosides, virescenosides have previously been isolated from this strain under cultivation on solid rice medium and wort agar medium [10,11,12]. Virescenosides Z₅ and Z₇ exhibited an unusual 16-chloro-15-hydroxyethyl group as their side chains in aglycones [12]. So, we attempted directed biosynthesis for the production of other halogenated compounds by culturing the fungus Acremonium striatisporum KMM 4401 in media containing potassium bromide. Unfortunately, we were unable to obtain glycoside derivatives with the incorporation of a bromine atom in a molecule structure. Chromatographic separation of the CHCl₃-EtOH extract of the culture of fungus has now led to the isolation of ten undescribed diterpene glycosides virescenosides Z₉-Z₁₈ (1‒10) (Figure 1) together with known virescenosides F (11) and G (12), lactone of virescenoside G (13) and aglycon of virescenoside A (14) (Figure S1).

2. Results and Discussion

The CHCl₃-EtOH (2:1, v/v) extract of the culture of A. striatisporum was separated by low-pressure reversed-phase column chromatography on Teflon powder Polycrome-1 followed by Si gel flash column chromatography and then by RP HPLC to yield individual compounds 1‒14 as colorless, amorphous solids.

The molecular formula of virescenoside Z₉ (1) was determined as C₂₆H₄₂O₁₁ based on the analysis of HRESIMS (m/z 529.2656 [M–H]⁻, calcd for C₂₆H₄₁O₁₁, 529.2654) and NMR data. A close inspection of the ¹H and ¹³C NMR data (

Table 1. ¹³C NMR data (δ in ppm) for virescenosides Z₉-Z₁₈ (1–10).

Position	1a	2b	3b	4c	5b	6c	7b	8c	9b	10d
1	46.9, CH2	35.9, CH2	44.1, CH2	40.0, CH2	48.0, CH2	46.6, CH2	44.1, CH2	40.3, CH2	48.1, CH2	38.5, CH2
2	69.1, CH	29.0, CH2	69.6, CH	29.4, CH2	69.5, CH	69.3, CH	69.4, CH	37.3, CH2	69.5, CH	28.6, CH2
3	84.7, CH	80.0, CH	84.6, CH	81.5, CH	85.6, CH	84.7, CH	84.4, CH	218.4, C	85.6, CH	79.0, CH
4	43.8, C	41.3, C	44.7, C	43.8, C	44.6, C	44.9, C	44.7, C	54.1, C	44.6, C	43.0, C
5	55.9, CH	51.8, CH	51.7, CH	53.5, CH	53.2, CH	51.9 CH	51.5, CH	55.5, CH	53.2, CH	51.5 CH
6	57.3, CH	37.3, CH2	37.6, CH2	25.2, CH2	25.3, CH2	39.3, CH2	37.8, CH2	25.8, CH2	25.3, CH2	24.6, CH2
7	178.0, C	202.9, C	202.7, C	123.1, CH	123.2, CH	203.1, C	202.8, C	123.1, CH	123.2, CH	122.8, CH
8	80.2, C	130.3, C	130.3, C	137.2, C	137.1, C	136.8, C	130.2, C	137.7, C	136.9, C	134.9, C
9	60.5, CH	168.7, C	167.8, C	54.1, CH	54.1, CH	52.6, CH	167.9, C	53.1, CH	54.1, CH	52.3, CH
10	43.8, C	43.9, C	42.2, C	36.8, C	37.9, C	38.4, C	42.2, C	37.0, C	37.9, C	35.5, C
11	18.4, CH2	24.9, CH2	25.0, CH2	22.1, CH2	22.2, CH2	21.0, CH2	25.0, CH2	22.1, CH2	22.2, CH2	20.5, CH2
12	34.3, CH2	35.3, CH2	35.2, CH2	37.9, CH2	37.8, CH2	35.6, CH2	35.2, CH2	37.8, CH2	37.8, CH2	36.4, CH2
13	35.8, C	36.0, C	36.1, C	38.4, C	38.4, C	40.4, C	36.1, C	38.4, C	38.4, C	37.1, C
14	49.8, CH2	34.9, CH2	34.9, CH2	47.7, CH2	47.6, CH2	146.5, CH	34.9, CH2	47.6, CH2	47.6, CH2	46.3, CH2
15	151.0, CH	147.0, CH	147.2, CH	152.0, CH	151.9, CH	148.6, CH	146.9, CH	151.9, CH	151.9, CH	150.6, CH
16	108.4, CH2	112.7, CH2	112.7, CH2	110.4, CH2	110.4, CH2	112.9, CH2	112.7, CH2	110.5, CH2	110.4, CH2	109.6, CH2
17	28.5, CH3	29.2, CH3	29.1, CH3	22.6, CH3	22.6, CH3	26.8, CH3	29,2, CH3	22.6, CH3	22.6, CH3	21.7, CH3
18	25.8, CH3	22.8, CH3	23.7, CH3	23.9, CH3	24.6, CH3	23.6, CH3	23.7, CH3	22.0, CH3	24.6, CH3	23.9, CH3
19	74.0, CH2	73.3, CH2	73.8, CH2	73.9, CH2	74.1, CH2	73.1, CH2	73,6, CH2	75.1, CH2	73.9, CH2	72.1, CH2
20	17.7, CH3	18.7, CH3	19.7, CH3	16.9, CH3	17.6, CH3	16.1, CH3	19,7, CH3	16.7, CH3	17.6, CH3	15.7, CH3
1′	101.2, CH	103.5, CH	103.3, CH	103.7, CH	103.3, CH	102.8, CH	102.8, CH	101.9, CH	103.3, CH	103.5, CH
2′	72.7, CH	71.1, CH	71.3, CH	70.9, CH	71.2, CH	71.6 CH	71.7, CH	72.1, CH	71.3, CH	71.6 CH
3′	72.3, CH	70.8, CH	71.1, CH	70.6, CH	71.1, CH	71.8, CH	72.0, CH	72.0, CH	71.2, CH	75.1, CH
4′	67.2, CH	69.9, CH	69.7 CH	69.9, CH	69.6, CH	69.1, CH	69.0, CH	68.7, CH	69.7, CH	70.0, CH
5′	75.7, CH	76.5, CH	76.6, CH	76.6, CH	76.4, CH	76.4, CH	76.1, CH	76.0, CH	76.4, CH	77.8, CH
6′	64.0, CH2	174.0, C	174.0, C	172.7, C	172.9, C	173.2, C	172.8, C	172.6, C	172.9, C	170.7, C
7′				53.3, CH3	53.3, CH3	53.4 CH3	53.3, CH3	53.3, CH3	66.8, CH2	51.8 CH3
8′									32.3, CH2
9′									20.7, CH2
10′									14.7, CH3

^a Chemical shifts were measured at 176.04 in Pyr-d₅. ^b Chemical shifts were measured at 176.04 in CD₃OD. ^c Chemical shifts were measured at 125.77 in CD₃OD. ^d Chemical shifts were measured at 125.77 in Pyr-d₅.

and

Table 2. ¹H NMR data (δ in ppm, J in Hz) for virescenosides Z₉-Z₁₃ (1–5).

Position	1a	2b	3b	4c	5b
1	α: 1.54 t (11.5) β: 2.34 dd (4.6, 12.2)	α: 1.35 m β: 1.94 m	α: 1.23 m β: 2.17 dd (4.5, 12.8)	α: 1.22 dt (4.6, 13.5) β: 1.90 dd (3.5, 13.5)	α: 1.11 β: 2.11 dd (4.2, 12.6)
2	4.28 ddd (4.5, 9.3, 11.5)	α: 1.82 dd (3.5, 11.9) β: 1.75 dd (4.0, 13.6)	3.82 m	α: 1.74 dd (3.4, 11.8) β: 1.65 dd (3.0, 13.4)	3.76 m
3	3.61 d (9.3)	3.26 dd (4.0, 11.9)	2.99 d (9.8)	3.24 dd (4.1, 11.8)	2.98 d (9.8)
5	2.41 d (13.2)	1. 67 dd (3.6, 14.4)	1. 76 dd (3.5, 14.7)	1. 26 t (8.2)	1.34 dd (3.9, 11.4)
6	3.70 d (13.2)	α: 2.54 dd (3.6, 18.0) β: 2.64 dd (14.4, 18.0)	α: 2.56 dd (3.3, 18.2) β: 2.70 dd (14.7, 18.2)	2.03 m	2.03 m
7				5.38 brs	5.39 brs
9	1.93 t (7.5)			1.66 dd (3.9, 7.8)	1.74 m
11	α: 1.38 m β: 1.69 m	α: 2.23 m β: 2.27 m	α: 2.26 m β: 2.32 m	α: 1.38 m β: 1.58 m	α: 1.41 m β: 1.61 m
12	α: 1.87 β: 1.36	α: 1.35 m β: 1.62 m	α: 1.64 m β: 1.36 m	α: 1.37 m β: 1.48 dt (2.7, 8.9)	α: 1.50 dd (2.8, 12.1) β: 1.39 td (2.8, 11.5)
14	α: 2.37 d (14.0) β: 1.48 d (14.0)	α: 2.30 d (17.5) β: 1.93 d (17.5)	α: 2.31 m β: 1.94 dt (2.6, 17.9)	α: 1.97 brd (14.1) β: 1.91 dd (2.6, 14.1)	α: 1.99 m β: 1.92 dd (2.6, 14.1)
15	6.64 dd (10.8, 17.6)	5.70 dd (10.6, 17.5)	5.70 dd (10.8, 17.5)	5.80 dd (10.7, 17.5)	5.81 dd (10.8, 17.6)
16	a: 4.85 dd (1.8, 10.8) b: 4.96 dd (1.8, 17.6)	a: 4.82 dd (1.4, 17.5) b: 4.93 dd (1.4, 10.6)	a: 4.82 dd (1.5, 17.6) b: 4.93 dd (1.5, 10.8)	a: 4.84 dd (1.3, 10.7) b: 4.92 dd (1.3, 17.5)	a: 4.85 dd (1.4, 10.8) b: 4.93 dd (1.4, 17.6)
17	0.95 s	1.00 s	1.01 s	0.86 s	0.86 s
18	1.81 s	1.13 s	1.16 s	1.10 s	1.11 s
19	a: 4.23 d (9.9) b: 4.98 d (9.9)	a: 3.73 d (10.2) b: 4.17 d (10.2)	a: 3.67 d (10.4) b: 4.14 d (10.4)	a: 3.83 d (10.2) b: 4.04 d (10.2)	a: 3.72 d (10.3) b: 4.03 d (10.3)
20	1.28 s	1.14 s	1.21 s	0.87 s	0.95 s
1	5.43 d (1.2)	4.84 d (2.9)	4.82 d (2.5)	4.85 d (2.5)	4.85 d (2.7)
2′	4.56 dd (1.2, 3.9)	3.77 dd (2.7, 7.8)	3.77 dd (2.5, 7.4)	3.77 dd (2.8, 7.9)	3.77 m
3′	4.74 t (3.7)	3.93 dd (2.9, 7.7)	3.94 dd (3.0, 7.4)	3.89 dd (3.0, 7.9)	3.90 dd (3.3, 7.3)
4′	4.50 m	4.23 t (4.8)	4.20 dd (3.0, 5.7)	4.26 dd (3.0, 4.9)	4.23 dd (3.3, 5.6)
5′	4.69 d (3.2, 12.2)	4.24 d (4.8)	4.22 d (5.7)	4.28 d (4.9)	4.28 d (5.6)
6′	a: 4.40 dd (6.5, 12.3) b: 4.51 m
7′				3.78 s	3.78 s

^a Chemical shifts were measured at 700.13 in Pyr-d₅. ^b Chemical shifts were measured at 700.13 in CD₃OD. ^c Chemical shifts were measured at 500.13 in CD₃OD.

; Figures S3‒S8) of 1 by DEPT and HSQC revealed the presence of three quaternary methyls (δ_H 0,95, 1.28, 1.81; δ_C 28.5, 17.7, 25.8), six methylenes (δ_C 18.4, 34.3, 46.9, 49.8, 64.0 and 74.0), including two oxygen-bearing, eight oxygenated methines (δ_H 3.61, 3.70, 4.28, 4.50, 4.56, 4.69, 4.74, 5.43; δ_C 84.7, 57.3, 69.1, 75.7, 72.7, 67.2, 72.3, 101.2) including one methine linked to an anomeric carbon, two tertiary (δ_H 1.93, 2.41; δ_C 60.5, 55.9), four saturated quaternary carbons (δ_C 35.8, 43.8 (2C) and 80.2), including one oxygen-bearing, one monosubstituted double bond (δ_C 108.4, 151.0) and one carbonyl or carboxyl carbon (δ_C 178.0). HMBC correlations from H₃-20 (δ_H 1.28) to C-1 (δ_C 46.9), C-5 (δ_C 55.9), C-9 (δ_C 60.5) and C-10 (δ_C 43.8), from H₃-18 (δ_H 1.81) to C-3 (δ_C 84.7), C-4 (δ_C 43.8), C-5 (δ_C 55.9) and C-19 (δ_C 74.0), from H-3 (δ_H 3.61) to C-2 (δ_C 69.1), C-4 and C-19, from H-1β (δ_H 2.34) to C-3, from H-6 (δ_H 3.70) to C-4, C-5, C-7 (δ_C 178.0) and C-8 (δ_C 80.2), from H-9 (δ_H 1.93) to C-8 and C-10 established the structures of the A and B rings and the location of hydroxy groups at C-2, C-3, C-6, C-8 and carbonyl function at C-7. The correlations observed in the COSY and HSQC spectra of 1 indicated the presence of the isolated spin system: >CH‒CH₂‒CH₂‒ (C-9‒C-11‒C-12). These data and HMBC correlations from H₃-17 (δ_H 0.95) to C-12 (δ_C 34.3), C-13 (δ_C 35.8), C-14 (δ_C 49.8), C-15 (δ_C 151.0) and from H-14β (δ_H 1.48) to C-8, C-9 and C-12 established the structure of the C ring in 1.

The proton signals of a typical ABX system of a vinyl group at δ_H 6.64 (1H, dd, 10.8, 17.6 Hz), 4.96 (1H, dd, 1.8, 17.6) and 4.85 (1H, dd, 1.8, 10.8) indicated the C-15, C-16 position of this double bond [13,14,15,16]. NOE correlations (Figure 2) H₃-20 (δ_H 1.28)/H-2 (δ_H 4.28), H-6 (δ_H 3.70), H-19b (δ_H 4.96) and H-5 (δ_H 2.41)/H-3 (δ_H 3.62), H₃-18 (δ_H 1.81) indicated a trans-ring fusion of the A and B rings, as well as the stereochemistry of the methyl and hydroxymethyl groups at C-4, methyl group at C-10 and hydroxy groups at C-2, C-3 and C-6. NOE cross-peaks H-9 (δ_H 1.93)/H-5 and H-14β (δ_H 1.48)/H₃-20, H₃-17 (δ_H 0.95), H-6 showed the stereochemistry of the methyl group at C-13 and suggested the β-orientation of hydroxy group at C-8.

Interpretation of the COSY data gave rise to spin systems for monosaccharide involving one anomeric proton, four oxymethines and protons of a hydroxymethyl group. A comparison of the ¹³C NMR spectrum of 1 with the data published for α-D-altropyranoses and β-D-altropyranoses as well as a good coincidence of carbon signals due to the glycosidic moiety with those of virescenosides O, T, W [10] together with magnitudes of ¹H-¹H spin coupling constants in ¹H NMR spectra of 1 elucidated the presence of a β-D-altropyranoside unit of ⁴C₁ form in 1. A long-range correlation H-1′ (δ_H 5.43)/C-19 as well as the NOESY cross-peak between H-1′ and H-19a and downfield chemical shift of C-19 (δ_C 74.0) revealed a linkage between the altrose and aglycon. Thus, the structure of virescenoside Z₉ (1) was represented as 19-O-β-D-altropyranosyl-7-oxo-isopimara-15-en-2α,3β,6α,8β-tetraol.

In HRESIMS virescenoside Z₁₀ (2) gave a quasimolecular ion at m/z 493.2446 [M–H]⁻. These data, coupled with ¹³C NMR spectral data (DEPT), established the molecular formula of 2 as C₂₆H₃₈O₉. ¹H and ¹³C NMR spectra of 2 (Table 1 and Table 2; Figures S9‒S13) indicated the presence of a ∆¹⁵-pimarene-type aglycon possessing primary alcohol on a quaternary carbon (AB system, coupling at 3.73 d, 10.2 Hz and 4.17 d, 10.2 Hz) and one secondary alcohol function at δ_C 80.0. The remaining functionality, corresponding to the carbon signals at δ 202.9 (C), 168.7 (C) and 130.3 (C), suggested the presence of the tetrasubstituted enone chromophore. The structure of the aglycon part of 2 was found by extensive NMR spectroscopy to be the same as that of virescenoside P [17].

The HRESIMS of virescenosides Z₁₁ (3) showed the quasimolecular ion at m/z 509.2408 [M–H]⁻. These data, coupled with ¹³C NMR spectral data (DEPT), established the molecular formula of 3 as C₂₆H₃₈O₁₀. The structure of the aglycon moiety of 3 was found by extensive NMR spectroscopy (¹H, ¹³C, HSQC, HMBC and NOESY) (Table 1 and Table 2; Figures S14‒S18) to be the same as those of virescenoside M [18].

The ¹³C and ¹H NMR spectra of the sugar moieties of virescenoside Z₁₀ (2) and Z₁₁ (3) showed a close similarity of all proton and carbon chemical shifts with those of virescenosides Z₇ and Z₈ [12]. The 7.7-,7.4-Hz splitting between H-2 and H-3 indicated that both were axial, whereas the 4.8-, 5.7-Hz splitting between H-4 and H-5 showed that these protons in equatorial position. These data and HMBC correlations between anomeric protons and C-5′-methine groups and between H-5′ (δ_H 4.24, 4.22) and C-6′ (δ_C 174.0) suggested the presence of a β-altruronopyranoside unit of ¹C₄ conformation in 2 and 3. The long-range correlations H-1′ (δ_H 4.84, 4.82)/C-19 as well as the NOESY cross-peak between H-1′ and H-19a and downfield chemical shifts of C-19 (δ_C 73.3, 73.8) indicated that sugar moieties were linked at C-19. Earlier in result of reduction of the sum of virescenosides Z₄‒Z₈ with LiAlH₄ and the acid hydrolysis of obtained products was isolated D-altrose as the only sugar that was identified by GLC of the corresponding acetylated (+)- and (‒)-2-octyl glycosides using authentic samples prepared from D-altrose [12]. Thus, the structure of virescenoside Z₁₀ (2) was determined as 19-O-β-D-altruronopyranosyl-7-oxo-isopimara-8(9),15-dien-3β-ol, and the structure of virescenoside Z₁₁ (3) was established as 19-O-β-D-altruronopyranosyl-7-oxo-isopimara-8(9),15-dien-2α,3β-diol.

The HRESIMS of virescenosides Z₁₂ (4) and Z₁₃ (5) showed the quasimolecular ions at m/z 517.2770 [M + Na]⁺ and m/z 533.2718 [M + Na]⁺, respectively. These data, coupled with ¹³C NMR spectral data (DEPT), established the molecular formula of 4 and 5 as C₂₇H₄₂O₈ and C₂₇H₄₂O₉, respectively. A close inspection of the ¹H and ¹³C NMR data of 4 (Table 1 and Table 2; Figures S19‒S23) revealed that virescenoside Z₁₂ (4) was structurally identical to virescenosides B [13] and G [19] (See Extraction and Isolation) with respect to the aglycon. The structure of the aglycon moiety of 5 was found by extensive NMR spectroscopy (Table 1 and Table 2; Figures S24‒S28) to be the same as that of virescenosides A [13,20] and F [19] (See Extraction and Isolation).

The NMR spectra of glycosides 4 and 5 indicated that both compounds contained closed carbohydrate moieties (Table 1 and Table 2). Initial examination of the 1-D proton and one bond correlation NMR data suggested the presence of one sugar (anomeric signals at δ_H 4.85, δ_C 103.7 for 4 and δ_H 4.85, δ_C 103.3 for 5). The ¹H and ¹³C NMR spectra of the sugar parts of 4 and 5 indicated the presence of the methoxy groups (both, δ_H 3.78, δ_C 53.3). HMBC correlations from anomeric protons to C5′-methine groups and from H-5′ (δ_H 4.28) to C-6′ (δ_C 172.7, 172.9) and from H₃-7′ (δ_H 3.78) to C-6′ together with magnitudes of ¹H-¹H spin coupling constants suggested the presence of the methyl ester of a β-altruronopyranoside unit of ¹C₄ form in 4 and 5. A long-range correlations H-1′ (δ_H 4.85)/C-19 (δ_C 73.9, 74.1) as well as the NOESY cross-peaks between H-1′ and H-19a (δ_H 3.83, 3.72) and downfield chemical shifts of C-19 indicated that sugar moieties were linked at C-19. Thus, the structure of virescenoside Z₁₂ (4) was determined as 19-O-[(methyl-β-D-altruronopyranosyl)-uronat]-isopimara-7,15-dien-3β-ol, and the structure of virescenoside Z₁₃ (5) was established as 19-O-[(methyl-β-D-altruronopyranosyl)-uronat]-isopimara-7,15-dien-2α,3β-diol.

The NMR data (Table 1 and

Table 3. ¹H NMR data (δ in ppm, J in Hz) for virescenosides Z₁₄-Z₁₈ (6–10)

Position	6c	7b	8c	9b	10d
1	α: 1.22 m β: 2.07 dd (4.3, 12.7)	α: 1.23 m β: 2.18 dd (4.5, 12.8)	α: 1.50 m β: 2.19 m	α: 1.11 m β: 2.11 dd (4.2, 12.5)	α: 1.15 dt β: 1.78 brd (3.9, 13.1)
2	3.79 m	3.80 dd (9.8, 13.9)	α: 2.84 dt (5.4, 14.2) β: 2.23 m	3.76 m	α: 1.85 m β: 1.92 m
3	3.04 d (9.9)	3.00 d (9.8)		2.98 d (9.8)	3.55 dd (4.0, 11.9)
5	1.73 dd (5.0, 13.8)	1. 75 dd (3.4, 14.7)	1.63 dd (4.1, 12.3)	1.34 dd (4.5, 11.8)	1.27 m
6	α: 2.59 dd (5.0, 19.0)	α: 2.53 dd (3.4, 18.2) β: 2.80 dd (14.7, 18.2)	α: 2.04 m β: 2.11 m	α: 2.01 m β: 2.06 m	α: 2.06 m β: 2.40 m
7			5.41 brs	5.38 m	5.30 m
9	2.13 m		1.76 m	1.74 m	1.60 m
11	α: 1.79 m β: 1.54 m	α: 2.26 m β: 2.31 m	α: 1.64 m β: 1.47 m	α: 1.41 m β: 1.61 m	α: 1.46 m β: 1.32 m
12	α: 1.54 m β: 1.67 m	α: 1.64 m β: 1.36 m	α: 1.51 β: 1.44	α: 1.50 m β: 1.39 m	α: 1.32 m β: 1.45 m
14	6.68 t (2.1)	α: 2.32 m β: 1.95 d (17.8)	α: 2.00 m β: 1.94 d (2.6, 14.0)	α: 1.99 m β: 1.92 dd (2.6, 14.1)	α: 2.03 brd (14.0) β: 1.94 brd (14.0)
15	5.83 dd (10.7, 17.5)	5.71 dd (10.8, 17.5)	5.81 dd (10.7, 17.5)	5.81 dd (10.8, 17.4)	5.87 dd (10.6, 17.4)
16	5.00 m	a: 4.83 dd (1.2, 17.5) b: 4.93 dd (1.2, 10.8)	a: 4.86 dd (1.4, 10.7) b: 4.94 dd (1.4, 17.5)	a: 4.85 dd (1.4, 10.8) b: 4.93 dd (1.4, 17.4)	a: 4.95 d (10.6) b: 5.02 d (17.4)
17	1.12 s	1,01 s	0.89 s	0.86 s	0.90 s
18	1.13 s	1.15 s	1.11 s	1.11 s	1.41 s
19	a: 3.68 d (10.3) b: 4.09 d (10.3)	a: 3.65 d (10,4) b: 4.11 d (10,4)	a: 3.90 d (9.8) b: 3.96 d (9.8)	a: 3.71 d (10.5) b: 4.04 d (1053)	a: 4.26 d (10.3) b: 4.59 d (10.3)
20	0.95 s	1.22 s	1.17 s	0.95 s	0.93 s
1′	4.78 brs	4.79 d (2.0)	4.75 d (1.9)	4.84 d (2.1)	4.97 brs
2′	3.76 m	3.76 m	3.66 dd (1.9, 5.6)	3.77 dd (2.5, 7.4)	4.55 d (3.2)
3′	3.91 dd (3.3, 6.3)	3.92 dd (3.3, 6.0)	3.89 dd (3.0, 5.6)	3.92 dd (3.0, 7.4)	4.14 dd (3.3, 9.4)
4′	4.15 dd (3.3, 7.0)	4.14 dd (3.3, 7.3)	4.08 dd (3.2, 8.0)	4.22 m	4.87 t (9.3)
5′	4.24 d (7.0)	4.24 d (7.3)	4.23 d (8.0)	4.25 d (5.6)	4.40 d (9.3)
7′	3.76 s	3.76 s	3.77 s	a: 4.15 dt (6.6, 10.7) b: 4.19 m	3.64 s
8′				a,b: 1.68 m
9′				a,b: 1.45 m
10′				0.96 t (7.5)

^a Chemical shifts were measured at 700.13 in Pyr-d₅. ^b Chemical shifts were measured at 700.13 in CD₃OD. ^c Chemical shifts were measured at 500.13 in CD₃OD. ^d Chemical shifts were measured at 500.13 in Pyr-d₅.

) of virescenosides Z₁₄ (6), Z₁₅ (7) and Z₁₆ (8) suggested the presence of one sugar (anomeric signals at δ_H 4.78, δ_C 102.8, δ_H 4.79, δ_C 102.8, δ_H 4.75, δ_C 101.9). The ¹H and ¹³C NMR spectra of the sugar moieties of 6, 7 and 8 showed a close similarity of all proton and carbon chemical shifts and proton multiplicities. These data and HMBC correlations from anomeric protons to C-5′ methine groups and from H-5′ (δ_H 4.24, 4.24, 4.23) to C-6′ (δ_C 173.2, 172.8, 172.6) and from H₃-7′ (δ_H 3.76, 3.76, 3.77) to C-6′ suggested the presence of the methyl ester of a β-altruronopyranoside unit in 6, 7 and 8. The 7.0-, 7.3-, 8.0-Hz splitting between H-4 and H-5 indicated that both were axial and conformation of sugar parts in 6, 7 and 8 is ⁴C₁.

The HRESIMS of virescenosides Z₁₄ (6) showed the quasimolecular ion at m/z 547.2508 [M + Na]⁺. These data, coupled with ¹³C NMR spectral data (DEPT), established the molecular formula of 6 as C₂₇H₄₀O₁₀. The structure of the aglycon moiety of 6 was found by extensive NMR spectroscopy (¹H, ¹³C, HSQC, HMBC and NOESY) (Table 1 and Table 3; Figures S29‒S33) to be the same as those of virescenoside V [21].

The molecular formula of virescenoside Z₁₅ (7) was determined as C₂₇H₄₀O₁₀ based on the analysis of HRESIMS (m/z 523.2550 [M-H]⁻, calcd for C₂₇H₃₉O₁₀, 523.2549) and NMR data. The ¹H and ¹³C NMR data (Table 1 and Table 3; Figures S34‒S38) observed for the aglycon part of 7 closely resembled those obtained for virescenoside Z₁₀ (2) with the exception of the C-1‒C-4 carbon and proton signals of ring A. The HMBC correlations from H-5 (δ_H 1.75) to C-3 (δ_C 84.4), H-3 (δ_H 3.00) and from H₂-1 (δ_H 1.23, 2.18) to C-2 (δ_C 69.2) and downfield chemical shifts of C-2 placed an additional hydroxy group at C-2 of ring A. The relative stereochemistry of protons on C-2 and C-3 was defined based on the ¹H-¹H coupling constant (J=9.8) and assigned as axial. Previously, a similar aglycon has been described for virescenoside M [10].

The HRESIMS of virescenoside Z₁₆ (8) showed the quasimolecular at m/z 515.2617 [M + Na]⁺. These data, coupled with ¹³C NMR spectral data (DEPT), established the molecular formula of 8 as C₂₇H₄₀O₈ (Table 1 and Table 3). The structure of the aglycon moiety of 8 was found by 2D NMR experiments (Figures S39‒S43) to be the same as that of virescenoside Z₄ [12].

The attachment of a carbohydrate chains at C-19 of aglycon moieties of 6, 7 and 8 was confirmed by cross-peaks H-1′ (δ_H 4.78, 4.79, 4.75)/H-19a (δ_H 3.68, 3.65, 3.90) and H-1′/C-19 (δ_C 73.1, 73.6, 75.1) in the NOESY and HMBC spectra, respectively. From all these data, virescenoside Z₁₄ (6) was structurally identified as 19-O-[(methyl-β-D-altruronopyranosyl)-uronat]-7-oxo-isopimara-8(14),15-dien-2α,3β-diol, virescenoside Z₁₅ (7) as 19-O-[(methyl-β-D-altruronopyranosyl)-uronat]-7-oxo-isopimara-8(9),15-dien-2α,3β-diol and virescenoside Z₁₆ (8) as 19-O-[(methyl-β-D-altruronopyranosyl)-uronat]-3-oxo-isopimara-7,15-dien.

The HRESIMS of virescenoside Z₁₇ (9) showed the quasimolecular ion at m/z 575.3194 [M + Na]⁺. These data, coupled with ¹³C NMR spectral data (DEPT), established the molecular formula of 9 as C₃₀H₄₈O₉. The ¹H and ¹³C NMR data observed for aglycon and sugar (C-1′‒C-6′) parts of 9 (Table 1 and Table 3; Figures S44‒S48) matched those reported for virescenoside Z₁₃ (5). The correlations observed in the COSY and HSQC spectra of 9 indicated the presence of the isolated spin system: ‒CH₂‒CH₂‒CH₂‒CH₃ (C-7′‒C-10′). These data and HMBC correlations from H₃-10′ (δ_H 0.96) to C-8′ (δ_C 32.3), C-9′ (δ_C 20.7) and from Ha-7′ (δ_H 4.15) to C-6′ (δ_C 172.9), C-8′ and C-9′ suggested the presence of the butyl ester of a β-altruronopyranoside unit of ¹C₄ form in 9. On the basis of all the data above, the structure of virescenosides Z₁₇ (9) was established as 19-O-[(butyl-β-D-altruronopyranosyl)-uronat]-isopimara-7,15-dien-2α,3β-diol.

The HRESIMS of virescenoside Z₁₈ (10) showed the quasimolecular at m/z 517.2773 [M + Na]⁺. These data, coupled with ¹³C NMR spectral data (DEPT), established the molecular formula of 10 as C₂₇H₄₂O₈. The ¹H and ¹³C NMR data observed for the aglycon part of 10 (Table 1 and Table 3; Figures S49‒S54) matched those reported for virescenoside Q [17]. Initial examination of the 1-D proton and one bond correlation NMR data suggested the presence of one sugar (anomeric signal at δ_H 4.97, δ_C 103.5). The ¹H and ¹³C NMR spectra of the sugar part of 10 indicated the presence of the methoxycarbonyl group (δ_H 3.64, δ_C 51.8, 170.7). A comparison of the ¹³C NMR spectrum with the data published for α- and β-D-mannopyranoses as well as a good coincidence of carbon signals C-1′‒C-4′ with those of virescenoside Q together with magnitudes of ¹H-¹H spin coupling constants in ¹H NMR spectrum of 10 elucidated the presence of β-D-mannouronopyranoside unit of ⁴C₁ form in 10 [17,22,23]. A long-range correlation H-1′ (δ_H 4.97)/C-19 (δ_C 72.1) as well as the NOESY cross-peak between H-1′ and H-19a (δ_H 4.26) and downfield chemical shifts of C-19 indicated that sugar moiety was linked at C-19. Thus, the structure of virescenoside Z₁₈ (10) was determined as 19-O-[(methyl-β-D-mannopyrananosyl)-uronat]-isopimara-7,15-dien-3β-ol.

Since methanol is used in the isolation procedure of virescenosides, it is possible that the methyl esters of the sugar units may be obtained during the course of isolation. Therefore, we separated the part of subfraction II by RP-HPLC using acetonitrile instead of methanol and obtain virescenosides Z₁₂ (4) and Z₁₃ (5) which were characterized by ¹H and ¹³C NMR spectra. Furthermore, we observed compounds 4-8 and 10 in subfraction II by HPLC-MS method (See Supplementary Figure S2).

The structures of known compounds virescenosides F (11) and G (12), lactone of virescenoside G (13) [19] and aglycon of virescenoside A (14) [13] (See Supplementary Figure S1) were determined based on HRESIMS and NMR data and comparison with literature. The aglycons of virescenosides B (15, 16), C (17) and M (18) (See Supplementary Figure S1, Experimental Section) were prepared as a result of acid hydrolysis of the corresponding glycosides for examination of their biological activity.

Next, we investigated the effects of some isolated compounds and aglycones 15‒18 on urease activity and regulation of ROS and NO production in macrophages stimulated with lipopolysaccharide (LPS).

The development of urease inhibitors, usually considered as antiulcer agents, carries a significant interest for medicinal chemists. Urease is an enzyme that is clinically used as diagnostic to determine the presence of pathogens in the gastrointestinal and urinary tracts. It has been described that the bacterial urease causes many clinically harmful infections, like stomach cancer, infectious stones and peptic ulcer formation in human and animal health [24]. Urease is also involved in the pathogenesis of hepatic coma, urolithiasis, urinary catheter encrustation and oral cavity infections by hydrolyzing the salivary urea [25].

Aglycons 14 and 15 inhibit urease activity with an IC₅₀ of 138.8 and 125.0 μM, respectively. Thiourea used as positive control inhibited urease activity with IC₅₀ of 23.0 μM.

Compounds 1, 2, 5, 15‒18 at a concentration of 10 μM induced a significant down-regulation of ROS production in macrophages stimulated with lipopolysaccharide (LPS) (Figure 3). Virescenoside Z₁₀ (2) decreased the ROS content in macrophages by 45%.

Compounds 2, 5, 16 and 17 induced a moderate down-regulation of NO production in LPS-stimulated macrophages at concentration of 1 μM (Figure 4).

3. Materials and Methods

3.1. General Experimental Procedures

Optical rotations were measured on a Perkin-Elmer 343 polarimeter (Perkin Elmer, Waltham, MA, USA). UV spectra were recorded on a Shimadzu UV-1601PC spectrometer (Shimadzu Corporation, Kyoto, Japan) in methanol. NMR spectra were recorded in CD₃OD, CDCl₃, DMSO-d₆ and C₅D₅N on a Bruker DPX-500 (Bruker BioSpin GmbH, Rheinstetten, Germany) and a Bruker DRX-700 (Bruker BioSpin GmbH, Rheinstetten, Germany) spectrometer, using TMS as an internal standard. The Bruker Impact II Q-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) was used to record the MS and MS/MS spectra within m/z range 50–1500. The capillary voltage was set to 1300 V, and the drying gas was heated to 150 °C at the flow rate 3 L/min. Collision-induced dissociation (CID) product ion mass spectra were obtained using nitrogen as the collision gas. The instrument was operated using the program otofControl (ver. 4.0, Bruker Daltonics, Bremen, Germany) and the data were analyzed using the DataAnalysis Software (ver. 4.3, Bruker Daltonics, Bremen, Germany).

Low-pressure liquid column chromatography was performed using silica gel (50/100 μm, Imid, Russia) and Polychrome-1 (powder Teflon, Biolar, Latvia). Plates precoated with silica gel (5–17 μm, 4.5 × 6.0 cm, Imid) and silica gel 60 RP-18 F₂₅₄S (20 × 20 cm, Merck KGaA, Germany) were used for thin-layer chromatography. Preparative HPLC was carried out on a Agilent 1100 chromatography (Agilent Technologies, USA) using a YMC ODS-AM (YMC Co., Ishikawa, Japan) (5 µm, 10 × 250 mm) and YMC ODS-A (YMC Co., Ishikawa, Japan) (5 µm, 4.6 × 250 mm) columns with a Agilent 1100 refractometer (Agilent Technologies, USA).

3.2. Cultivation of Fungus

The fungus was grown stationary at 22 °C for 14 days on 6 flasks (1 L) (medium: wort-200 mL, sodium tartrate-0.05 g/L, agar-20 g/L, potassium bromide-30 g/L, seawater-800 mL).

3.3. Extraction and Isolation

At the end of the incubation period, the mycelium and medium were homogenized and extracted three times with a mixture of CHCl₃–EtOH (2:1, v/v, 2.5 L). The combined extracts (4.5 g) were concentrated to dryness and separated by low pressure RP CC (the column 20 × 8 cm) on Polychrome-1 Teflon powder in H₂O and 50% EtOH. After elution of inorganic salts and highly polar compounds by H₂O, 50% EtOH was used to obtain the fraction of amphiphilic compounds, including the virescenosides. After evaporation of the solvent, the residual material (2.6 g) was subjected to Si gel flash CC (7 × 13 cm) chromatography with a solvent gradient system of increasing polarity from 10 to 60% EtOH in CHCl₃ (total volume 8 L). Fractions of 20 mL were collected and combined by TLC examination to obtain two subfractions. Subfraction I (CHCl₃−EtOH 5:1, 3:1, 180 mg) was purified and separated by RP HPLC on a YMC ODS-A column eluting with MeOH−H₂O‒TFA (85:15:0.1) to yield 8 (2.4 mg), 9 (3.6 mg), 13 (2.4 mg) and 14 (4.0 mg). Subfraction II (CHCl₃−EtOH 2:1, 840 mg) was purified by RP HPLC on a YMC ODS-AM column eluting at first with MeOH−H₂O‒TFA (80:20:0.1) and then with MeOH−H₂O‒TFA (70:30:0.1) to yield 1 (2.5 mg), 2 (2.5 mg), 3 (7.5 mg), 4 (15.5 mg), 5 (71 mg), 6 (1.4 mg), 7 (6.6 mg) 10 (1.4 mg), 11 (98 mg) and 12 (63 mg).

The part of subfraction II (35 mg) was purified by RP HPLC on a YMC ODS-A column eluting with CH₃CN−H₂O‒TFA (50:50:0.1) to yield 4 (1.1 mg), 5 (4.5 mg), 11 (6 mg) and 12 (2 mg).

3.4. Spectral Data

Virescenoside Z₉ (1): amorphous solids; [α]${}_{\mathrm{D}}^{20}$ +1.5 (c 0.15, MeOH); ¹H and ¹³C NMR data, see Table 1 and Table 2, Supplementary Figures S3–S8; HRESIMS m/z 553.2618 [M + Na]⁺ (calcd. for C₂₆H₄₂O₁₁Na, 553.2619, Δ + 0.2 ppm).

Virescenoside Z₁₀ (2): amorphous solids; [α]${}_{\mathrm{D}}^{20}$ +10.0 (c 0.09, MeOH); UV (MeOH) λ_max (log ε) 248 (3.91) nm; ¹H and ¹³C NMR data, see Table 1 and Table 2, Supplementary Figures S9–S13; HRESIMS m/z 493.2446 [M–H]⁻ (calcd. for C₂₆H₃₇O₉, 493.2443, Δ–0.6 ppm).

Virescenoside Z₁₁ (3): amorphous solids; [α]${}_{\mathrm{D}}^{20}$ + 7.5 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 248 (3.64) nm; ¹H and ¹³C NMR data, see Table 1 and Table 2, Supplementary Figures S14–S18; HRESIMS m/z 509.2403 [M–H]⁻ (calcd. for C₂₆H₃₇O₁₀, 509.2392, Δ−2.0 ppm).

Virescenoside Z₁₂ (4): amorphous solids; [α]${}_{\mathrm{D}}^{20}$ −50.0 (c 0.10, MeOH); ¹H and ¹³C NMR data, see Table 1 and Table 2, Supplementary Figures S19–S23; HRESIMS m/z 517.2770 [M + Na]⁺ (calcd. for C₂₇H₄₂O₈Na, 517.2772, Δ + 0.4 ppm).

Virescenoside Z₁₃ (5): amorphous solids; [α]${}_{\mathrm{D}}^{20}$ −69.2 (c 0.13, MeOH); ¹H and ¹³C NMR data, see Table 1 and Table 2, Supplementary Figures S24–S28; HRESIMS m/z 533.2718 [M + Na]⁺ (calcd. for C₂₇H₄₂O₉Na, 533.2721, Δ + 0.6 ppm).

Virescenoside Z₁₄ (6): amorphous solids; [α]${}_{\mathrm{D}}^{20}$ −44.0 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 249 (3.81) nm; ¹H and ¹³C NMR data, see Table 1 and Table 3, Supplementary Figures S29–S33; HRESIMS m/z 547.2508 [M + Na]⁺ (calcd. for C₂₇H₄₀O₁₀Na, 547.2514, Δ +1.0 ppm).

Virescenoside Z₁₅ (7): amorphous solids; [α]${}_{\mathrm{D}}^{20}$ + 17.3 (c 0.15, MeOH); UV (MeOH) λ_max (log ε) 248 (3.99) nm; ¹H and ¹³C NMR data, see Table 1 and Table 3, Supplementary Figures S34–S38; HRESIMS m/z 547.2515 [M + Na]⁺ (calcd. for C₂₇H₄₀O₁₀Na, 547.2514, Δ −0.2 ppm).

Virescenoside Z₁₆ (8): amorphous solids; [α]${}_{\mathrm{D}}^{20}$ −78.0 (c 0.05, MeOH); ¹H and ¹³C NMR data, see Table 1 and Table 3, Supplementary Figures S39–S43; HRESIMS m/z 515.2617 [M + Na]⁺ (calcd. for C₂₇H₄₀O₈Na, 515.2615, Δ−0.4 ppm).

Virescenoside Z₁₇ (9): amorphous solids; [α]${}_{\mathrm{D}}^{20}$ −60.0 (c 0.10, MeOH); ¹H and ¹³C NMR data, see Table 1 and Table 3, Supplementary Figures S44–S48; HRESIMS m/z 575.3194 [M + Na]⁺ (calcd. for C₂₉H₄₈O₉Na, 575.3191, Δ‒0.5 ppm), m/z 551.3229 [M‒H]⁻ calcd. for C₂₉H₄₇O₉, 551.3226, Δ ‒0.6 ppm).

Virescenoside Z₁₈ (10): amorphous solids; [α]${}_{\mathrm{D}}^{20}$ ‒32.5 (c 0.12, MeOH); ¹H and ¹³C NMR data, see Table 1 and Table 3, Supplementary Figures S49–S54; HRESIMS m/z 517.2773 [M + Na]⁺ (calcd. for C₂₇H₄₂O₈Na, 517.2772, Δ‒0.3 ppm).

Virescenoside F (11): amorphous solids; ¹H NMR (700 MHz, CD₃OD) δ: 5.80 (1H, dd, J = 10.8, 17.4 Hz, H-15), 5.38 (1H, m, H-7), 4.92 (1H, dd, J = 1.4, 17.4 Hz, H-16b), 4.86 (1H, d, J = 3.0 Hz, H-1′), 4.85 (1H, dd, J = 1.4, 10.8 Hz, H-16a), 4.27 (1H, t, J = 3.7 Hz, H-4′),4.24 (1H, d, J = 4.3 Hz, H-5′), 4.12 (1H, d, J = 9.8 Hz, H-19b), 3.93 (1H, dd, J = 3.3, 8.3 Hz, H-3′), 3.78 (1H, dd, J = 3.0, 8.0 Hz, H-2′), 3.76 (1H, m, H-2), 3.73 (1H, d, J = 9.8 Hz, H-19a), 2.98 (1H, d, J = 9.8 Hz, H-3), 2.11 (1H, dd, J = 4.2, 12.5 Hz, H-1β), 2.04 (1H, m, H2-6), 1.99 (1H, m, H-14α), 1.92 (1H, dd, J = 2.8, 14.4 Hz, H-14β), 1.73 (1H, m, H-9), 1.60 (1H, m, H-11α), 1.50 (1H, m, H-12α), 1.39 (1H, m, H-12β), 1.39 (1H, m, H-11β), 1.34 (1H, dd, J = 5.8, 10.7 Hz, H-5), 1.14 (3H, s, Me-18), 1.11 (1H, d, J = 12.3 Hz, H-1α), 0.92 (3H, s, Me-20), 0.86 (3H, s, Me-17). ¹³C NMR (176 MHz, CD₃OD) δ: 174.0 (C-6′), 151.9 (C-15), 136.9 (C-8), 123.2 (C-7), 110.4 (C-16), 103.5 (C-1′), 85.8 (C-3), 76.6 (C-5′), 74.3 (C-19), 70.9 (C-2′), 70.4 (C-3′), 70.2 (C-4′), 69.7 (C-2), 54.1 (C-9), 53.4 (C-5), 48.0 (C-1), 47.6 (C-14), 44.4 (C-4), 38.4 (C-13), 37.9 (C-10), 37.9 (C-12), 25.1 (C-6), 24.2 (C-18), 22.6 (C-17), 22.1 (C-11), 17.7 (C-20); Supplementary Figures S55–S59; HRESIMS m/z 519.2563 [M + Na]⁺ (calcd. for C₂₆H₄₀O₉Na, 519.2565, Δ + 0.3 ppm).

Virescenoside G (12): amorphous solids; ¹H NMR (700 MHz, CD₃OD) δ: 5.80 (1H, dd, J = 10.8, 17.5 Hz, H-15), 5.37 (1H, m, H-7), 4.92 (1H, dd, J = 1.6, 17.5 Hz, H-16b), 4.86 (1H, d, J = 3.3 Hz, H-1′), 4.84 (1H, dd, J = 1.6, 10.8 Hz, H-16a), 4.30 (1H, t, J = 3.5 Hz, H-4′),4.24 (1H, d, J = 3.7 Hz, H-5′), 4.10 (1H, d, J = 9.9 Hz, H-19b), 3.93 (1H, dd, J = 3.3, 8.7 Hz, H-3′), 3.84 (1H, d, J = 9.9 Hz, H-19a), 3.78 (1H, dd, J = 3.3, 8.7 Hz, H-2′), 3.24 (1H, dd, J = 4.0, 11.8 Hz, H-3), 2.07 (1H, m, H-6α), 2.01 (1H, m, H-6β), 1.97 (1H, m, H-14α), 1.91 (1H, dd, J = 2.7, 14.0 Hz, H-14β), 1.90 (1H, dd, J = 3.4, 13.4 Hz, H-1β), 1.74 (1H, dd, J = 3.5, 11.8 Hz, H-2β),1.68 (1H, dd, J = 3.5, 7.5 Hz, H-2α),1.66 (1H, dd, J = 3.7, 7.7 Hz, H-9), 1.57 (1H, m, H-11α), 1.47 (1H, td, J = 2.9, 9.1 Hz, H-12β), 1.37 (1H, m, H-12α), 1.38 (1H, m, H-11β), 1.26 (1H, dd, J = 4.5, 12.1 Hz, H-5), 1.23 (1H, m, H-1α), 1.12 (3H, s, Me-18), 0.86 (3H, s, Me-17), 0.85 (3H, s, Me-20). ¹³C NMR (176 MHz, CD₃OD) δ: 173.7 (C-6′), 152.0 (C-15), 137.1 (C-8), 123.2 (C-7), 110.4 (C-16), 103.9 (C-1′), 81.7 (C-3), 76.7 (C-5′), 73.9 (C-19), 70.6 (C-4′), 70.4 (C-3′), 70.1 (C-2′), 54.1 (C-9), 53.7 (C-5), 47.7 (C-14), 43.7 (C-4), 40.0 (C-1), 38.4 (C-13), 37.9 (C-12), 36.8 (C-10), 29.4 (C-2), 25.0 (C-6), 23.6 (C-18), 22.6 (C-17), 22.1 (C-11), 17.0 (C-20); Supplementary Figures S60–S64; HRESIMS m/z 503.2617 [M + Na]⁺ (calcd. for C₂₆H₄₀O₈Na, 503.2615, Δ−0.3 ppm).

Lactone of virescenoside G (13): amorphous solids; ¹H NMR (700 MHz, DMSO-d₆) δ: 5.80 (1H, dd, J = 10.8, 17.6 Hz, H-15), 5.66 (1H, d, J = 7.8 Hz, 5′-OH), 5.56 (1H, d, J = 5.6 Hz, 2′-OH), 5.37 (1H, m, H-7), 5.29 (1H, d, J = 3.4 Hz, 2′-OH), 4.93 (1H, dd, J = 1.7, 17.6 Hz, H-16b), 4.85 (1H, dd, J = 1.7, 10.7 Hz, H-16a), 4.68 (1H, d, J = 6,8 Hz, H-1′), 4.41 (1H, dd, J = 5.6, 7.8 Hz, H-5′), 4.39 (1H, brs, H-3′), 4.14 (1H, d, J = 11.0 Hz, H-19b), 4.12 (1H, dd, J = 3.4, 5.8 Hz, H-4′), 3.55 (1H, ddd, J = 1.5, 5.4, 6.8 Hz, H-2′), 3.49 (1H, dd, J = 4.0, 11.9 Hz, H-3), 3.48 (1H, d, J = 11.0 Hz, H-19a), 2.22 (1H, dd, J = 3.1, 12.4 Hz, H-2b), 1.93 (1H, m, H-6β), 1.91 (1H, m, H-14α), 1.88 (1H, m, H-1β), 1.87 (1H, m, H-14β), 1.71 (1H, m, H-6α), 1.64 (1H, m, H-9), 1.55 (1H, m, H-11b), 1.46 (1H, m, H-2a), 1.44 (1H, m, H-12b), 1.31 (2H, m, H-11a, H-12a), 1.25 (3H, s, Me-18), 1.22 (1H, dd, J = 2.5, 9.3 Hz, H-5), 1.12 (1H, dt, J = 3.1, 12.5 Hz, H-1α), 0.89 (3H, s, Me-20), 0.82 (3H, s, Me-17). ¹³C NMR (176 MHz, DMSO-d₆) δ: 174.0 (C-6′), 149.9 (C-15), 135.5 (C-8), 121.1 (C-7), 109.8 (C-16), 93.8 (C-1′), 84.0 (C-3′), 80.1 (C-3), 71.4 (C-2′), 70.1 (C-4′), 68.5 (C-5′), 68.4 (C-19), 50.8 (C-9), 49.9 (C-5), 45.4 (C-14), 36.5 (C-13), 36.1 (C-4), 35.9 (C-1), 35.5 (C-12), 34.7 (C-10), 25.5 (C-18), 21.7 (C-6), 21.3 (C-17), 21.2 (C-2), 19.7 (C-11), 15.7 (C-20); Supplementary Figures S65–S68; HRESIMS m/z 485.2508 [M + Na]⁺ (calcd. for C₂₆H₃₈O₇Na, 485.2510, Δ + 0.4 ppm).

Aglycon of virescenoside A (14): amorphous solids; ¹H NMR (500MHz, CD₃OD) δ: 5.80 (1H, dd, J = 10.8, 17.5 Hz, H-15), 5.37 (1H, brs, H-7), 4.93 (1H, dd, J = 1.4, 17.5 Hz, H-16b), 4.85 (1H, dd, J = 1.4, 10.8 Hz, H-16a), 4.14 (1H, d, J = 11.2 Hz, H-19b), 3.79 (1H, ddd, J = 4.3, 9.8, 11.7 Hz, H-2), 3.50 (1H, d, J = 11.2 Hz, H-19a), 3.09 (1H, d, J = 9.8 Hz, H-3), 2.11 (1H, dd, J = 4.3, 12.6 Hz, H-1β), 1.98 (1H, m, H-6β), 1.97 (1H, m, H-14α), 1.91 (1H, dd, J = 2.2, 13.7 Hz, H-14β), 1.92 (1H, m, H-6α), 1.73 (1H, m, H-9), 1.61 (1H, dt, J = 3.9, 10.0 Hz, H-11β), 1.50 (1H, d, J = 8.7 Hz, H-12α), 1.39 (2H, m, H-11α, H-12β), 1.35 (1H, dd, J = 4.2, 12.0 Hz, H-5), 1.21 (3H, s, Me-18), 1.12 (1H, t, J = 12.3 Hz, H-1α), 0.93 (3H, s, Me-20), 0.86 (3H, s, Me-17). ¹³C NMR (125 MHz, CD₃OD) δ: 151.9 (C-15), 137.1 (C-8), 123.1 (C-7), 110.4 (C-16), 86.5 (C-3), 69.7 (C-2), 66.6 (C-19), 54.0 (C-9), 53.0 (C-5), 47.7 (C-1), 47.6 (C-14), 44.4 (C-4), 38.4 (C-13), 37.9 (C-10), 37.8 (C-12), 24.8 (C-6), 24.5 (C-18), 22.6 (C-17), 22.1 (C-11), 17.9 (C-20), Supplementary Figures S69–S73; HRESIMS m/z 343.2241 [M + Na]⁺ (calcd. for C₂₀H₃₂O₃Na, 343.2244, Δ + 0.8 ppm).

3.5. Urease Inhibition Assay

The reaction mixture consisting of 25 µL enzyme solution (urease from Canavalia ensiformis, Sigma, 1U final concentration) and 5 µL of test compounds dissolved in water (10–300.0 µM final concentration) was preincubated at 37 °C for 60 min in 96-well plates. Then 55 µL of phosphate buffer solution with 100 µM urea was added to each well and incubated at 37 °C for 10 min. The urease inhibitory activity was estimated by determining of ammonia production using indophenol method [26]. Briefly, 45 µL of phenol reagent (1% w/v phenol and 0.005% w/v sodium nitroprusside) and 70 µL of alkali reagent (0.5% w/v NaOH and 0.1% active chloride NaOCl) were added to each well. The absorbance was measured after 50 min at 630 nm using a microplate reader Multiskan FC (Thermo Scientific, Canada). All the reactions were performed in triplicate in a final volume of 200 µL. The pH was maintained 7.3–7.5 in all assays. DMSO 5% was used as a positive control.

3.6. Reactive Oxygen Species (ROS) Level Analysis in LPS-Treated Cells

The suspension of macrophages on 96-well plates (2 × 104 cells/well) were washedwith the PBS and treated with 180 µL/well of the tested compounds (10 μM) for 1 h and 20 µL/well LPS from E. coli serotype 055:B5 (Sigma, 1.0 μg/mL), which were both dissolved in PBS and cultured at 37 °C in a CO2-incubator for one hour. For the ROS levels measurement, 200 μL of 2,7-dichlorodihydrofluorescein diacetate (DCF-DA, Sigma, final concentration 10 μM) fresh solution was added to each well, and the plates were incubated for 30 min at 37 °C. The intensity of DCF-DA fluorescence was measured at λex 485 n/λem 518 nm using the plate reader PHERAstar FS (BMG Labtech, Offenburg, Germany) [27].

3.7. Reactive Nitrogen Species (RNS) Level Analysis in LPS-Treated Cells

The suspension of macrophages on 96-well plates (2 × 104 cells/well) were washed withthe PBS and treated with 180 µL/well of the tested compounds (10 μM) for 1 h and 20 µL/well LPS from E. coli serotype 055:B5 (Sigma, 1.0 μg/mL), which were both dissolved in PBS and cultured at 37 °C in a CO2-incubator for one hour. For the RNS levels measurement, 200 μL Diaminofluorescein-FM diacetate (DAF FM-DA, Sigma, final concentration 10 μM) fresh solution was added to each well, and the plates were incubated for 40 min at 37 °C, then replaced with fresh PBS, and then incubated for an additional 30 min to allow complete de-esterification of the intracellular diacetates. The intensity of DAF FM-DA fluorescence was measured at λex 485 n/λem 520 nm using the plate reader PHERAstar FS (BMG Labtech, Offenburg, Germany).

3.8. Peritoneal Macrophage Isolation

Mice BALB/c were sacrificed by cervical dislocation. Peritoneal macrophages were isolated using standard procedures. For this purpose, 3 mL of PBS (pH 7.4) was injected into the peritoneal cavity and the body intensively palpated for 1–2 min. Then the peritoneal fluid was aspirated with a syringe. Mouse peritoneal macrophage suspension was applied to a 96-well plate left at 37 °C in an incubator for 2 h to facilitate attachment of peritoneal macrophages to the plate. Then a cell monolayer was triply flushed with PBS (pH 7.4) for deleting attendant lymphocytes, fibroblasts and erythrocytes and cells were used for further analysis.

All animal experiments were conducted in compliance with all rules and international recommendations of the European Convention for the Protection of Vertebrate Animals used for experimental and other scientific purposes. All procedures were approved by the Animal Ethics Committee at the G. B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences (Vladivostok, Russia), according to the Laboratory Animal Welfare guidelines.

3.9. Statistical Analysis

Average value, standard error, standard deviation and p-values in all experiments were calculated and plotted on the chart using SigmaPlot 3.02 (Jandel Scientific, San Rafael, CA, USA). Statistical difference was evaluated by t-test, and results were considered as statistically significant at p < 0.05.

4. Conclusions

Ten new diterpene glycosides, virescenosides Z₉‒Z₁₈ (1‒10) were isolated from a marine strain of Acremonium striatisporum KMM 4401 associated with the holothurian Eupentacta fraudatrix. Virescenoside Z₉ (1) is an altroside of a new 7-oxo-isopimara-15-en-2α,3β,6α,8β-tetraol aglycon. Virescenosides Z₁₂-Z₁₆ (4‒8) were determined as the monosides having unique methyl esters of altruronic acid as their sugar moieties. Carbohydrate chain of virescenoside Z₁₈ (10) was structurally identified as the methyl ester of mannuronic acid. The effects of some isolated glycosides and aglycons 15‒18 on urease activity and regulation of ROS and NO production in macrophages stimulated with lipopolysaccharide (LPC) were evaluated.