Three new pyrrole alkaloids from the endophytic fungus Albifimbria viridis

Three new pyrrole alkaloids albifipyrrols A–C (1–3), were isolated from the endophytic fungus Albifimbria viridis collected from the Chinese medicinal plant. Their structures were elucidated by extensive NMR and HRESIMS spectrometric analyses. All compounds were evaluated for immunosuppressive activity. Fortunately, compound 2 exhibits certain inhibition specifically against the LPS-induced proliferation of B lymphocyte cells with IC50 value 16.16 μM. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s13659-022-00327-2.


Introduction
The human immune system is a complex network of defensing against foreign invaders. Autoimmune diseases arise when the immune system fails to distinguish between self and non-self [1,2]. Immunosuppressants are often used to prevent and treat the immune rejection of organs and tissues of transplant patients and play an important role in the treatment of various autoimmune diseases [3][4][5][6][7][8]. Nevertheless, some common immunomodulatory drugs such as mycophenolate mofetil (MMF) and cyclosporin A (CsA) have low efficacy, toxicity, and serious side effects in transplant patients [9][10][11][12]. Therefore, it is necessary to find more efficient, safe and novel immunosuppressants to improve rejection.
In recent years, endophytic fungi from plants have been widely regarded as a significant source of drugs [13]. A great quantity of compounds with novel structures and multiple bioactivities are constantly isolated [14][15][16]. For instance, the well-known anticancer drug paclitaxel can be produced from Pacific yew by the endophytic fungus Taxomyces andreance [17]. Coptis chinensis Franch is a famous Chinese medicine in China. Modern pharmacological and clinical studies have indicated that it has anti-tumor, anti-inflammatory, antibacterial, hypoglycemic and other pharmacological activities [18][19][20] During the past few years, we had the aim of finding new potential immunosuppressive agents from endophytic fungus of C.chinensis Franch. Fortunately, we obtained a pyrrole alkaloid with immunosuppressive activity from Albifimbria viridis. Herein, we report the details of the isolation, structure elucidation, and bioactivities of three pyrrole alkaloids albifipyrrols A-C (1-3) (Fig. 1).

Results and discussion
Compound 1 was obtained as yellow oil. The molecular ion peak of HR-ESI-MS was at m/z 266.1150 [M + Na] + (calcd for 266.1157), which indicated that the molecular formula of compound 1 is C 15 H 17 NO 2 , with eight degrees of unsaturation. In the 1 H-NMR spectrum (Table 1) (Table 1) of 1 showed the presence of fifteen carbons, including one methyl, three methylenes [including two heteroatom-bearing methylenes at δ C 58.5 (C-6), 52.5 (C-8′)], seven aromatic or olefinic methines and four nonprotonated carbons [including one ketone carbonyl at δ C 197.4 (C-7)]. Among them, one benzene ring, an acetyl group and four olefinic carbons occupied seven degrees of unsaturation. Hence, the remaining one degree of unsaturation can only be due to the presence of one ring. The key HMBC correlations (Fig. 2) from H-2 to C-3/C-4/C-5 and from H-5 to C-2/C-3/C-4 and from H-8′ to C-2/C-5 demonstrated the existence of a pyrrole   2) between H 2 -7′ and H 2 -8′ and the key HMBC correlations from H-7′ to C-1′/C-2′/C-6′, H-8′ to C-2/C-5/C-7′ showed the phenylethyl was attached to the nitrogen atom. In addition, the acetyl can be confirmed by the key HMBC correlation from H-8 to C-7. Finally, the locations of the two substituents (an acetyl group and an ethoxy group) on the pyrrole nucleus were also confirmed at C-3, C-4 based on the HMBC correlations from H-8 to C-3 and from H-6 to C-3/C-4/C-5. Compound 1 was, therefore, established as albifipyrrol A, as depicted.
Compound 2 was obtained as yellow oil. The molecular ion peak of HR-ESI-MS was at m/z 280.1306 [M + Na] + (calcd for 280.1313), which deduced that the molecular formula of compound 2 was C 16 H 19 NO 2 , with eight degrees of unsaturation. The 1 H-NMR and 13 C-NMR data (Table 1) suggested 2 was similar to 1 and the only observed difference was that the hydroxy group in 1 was replaced by a methoxy group in 2. This change can be confirmed by the key HMBC correlations (Fig. 2) from H 3 -OMe to C-6. Compound 2 was, therefore, established as albifipyrrol B, as depicted.
Compound 3 was obtained as yellow oil. The molecular ion peak of HR-ESI-MS is at m/z 262.1046 [M + Na] + (calcd for 262.1055), which indicated that the molecular formula of compound 3 is C 12 H 17 NO 4 , with five degrees of unsaturation. The 1 H-NMR (Table 1) and HSQC spectrum of 3 revealed 3 has the same pyrrole ring as 1 and the major difference was the substituents on nitrogen.  (Fig. 2) from H-5′ to C-4′ and from H-3′ to C-4′. Finally, the attachment position of the methyl butyrate residue to the pyrrole ring was defined on the basis of HMBC correlations between H-1′and C-2/C-5. Compound 3 was, therefore, established as albifipyrrol C, as depicted.
All new compounds were evaluated for their in vitro inhibition activities on concanavalin A (Con A) induced T cell proliferation and lipopolysaccharide (LPS) induced B cell proliferation. Compound 2 exhibited certain inhibition specifically against the LPS-induced proliferation of B lymphocyte cells with IC 50 value 16.6 μM ( Table 2).

Fungal material
The strain were isolated from the roots of C. chinensis collected from Enshi, Hubei province, and was identified as Albifimbria viridis via 18S rDNA sequences and deposited at South-Central University for Nationalities, China. The sequence data for this strain had been

Extraction and isolation
The fungus Albifimbria viridis was fermented on solid rice medium (100 g of rice, 100 mL of water, in each 500 mL culture flask) and was cultured at 30 °C for one month. The fermented material was soaked in absolute methanol (20 L × 4). The combined extracts were evaporated under reduced pressure to afford an crude extract, which was further dissolved in water and extracted three times with EtOAc (10 L × 4) to yield 110 g of the extract.

Immunosuppressive activities assay
Fresh spleen cells were obtained from female BALB/c mice (6-8 weeks old). Spleen cells (1 × 10 6 cells) were cultured in triplicate on a 96-well plate for 48 h at 37 °C in a humidified incubator containing 5% CO 2 (with or without different concentrations of compounds). During the last 8 h of culture, a certain amount of CCK-8 was added to each well. At the end of culture, the OD values at 450 nm was measured by a bio-RAD 650 microplate reader. Cells with viability above 85% were further screened for their inhibitory activity against T and B lymphocytes. The 5 × 10 5 spleen cells were cultured at the same conditions as those mentioned above. T cell or B cell proliferation was induced with 10 µg ml −1 of LPS or 5 µg ml −1 of ConA, respectively. Proliferation was assessed in terms of uptake of [ 3 H]-thymidine during 8 h of pulsing with 25 µL/well of [ 3 H]-thymidine, and then cells will be harvested onto glass fiber filters. The incorporated radioactivity was counted using a Beta scintillation counter (MicroBeta Trilux, PerkinElmer Life Sciences). Cells treated without any stimuli were used as negative control. The immunosuppressive activity of each compound was expressed as the concentration of compound that inhibited ConA induced T cell proliferation or LPS-induced B cell proliferation to 50% (IC 50 ) of the control value. Both the cytotoxicity and proliferation assessment repeated twice. Cyclosporin A (CsA) an immunosuppressive agent, was used as a positive control (Table 2; Additional file 1: Figs. S1-S24).