Hericium erinaceus, a well-known edible and medicinal mushroom, has a long history of being used as healthy food in China and Japan [1]. The fruiting bodies or mycelia of H. erinaceus have been demonstrated with a wide range of beneficial activities, such as hypolipidemic, anticancer, antihypertensive, and neuronal disease-protecting activities [2, 3]. Previous chemical researches on H. erinaceus have revealed a number of bioactive secondary metabolites with diverse chemical skeletons, such as pyrones (erinapyrones A and B) [4], sesterterpene (erinacine S) [5], meroterpenoid (herienones C–E) [6], and cyathae-type diterpenes (erinacines A–K and P–R) [7,8,9,10,11].

Cyathane diterpenes represent a group of secondary metabolites with an unusual 5/6/7 tricyclic skeleton from basidiomycota. A number of cyathane diterpenes have been reported from mushrooms in the genera of Cyathus, Hericium, and Sarcodon, and proven to exhibit a wide range of interesting and significant biological activities [12, 13]. Cyathins Q and R from C. africanus exhibited potent apoptosis-inducing effects on HCT116 cell line and further in vivo anticancer activities in rats [14, 15]. Erinacines from H. erinaceus and scabronines from S. scabrosus displayed stimulating effect on the synthesis of nerve growth factor (NGF) in human nerve cells [7,8,9,10,11, 16]. Striatoids A–F from C. striatus dose-dependently enhanced NGF-mediated neurite outgrowth in rat pheochromocytoma (PC12) cells [17]. Recently, erinacine A, a cyathane-type diterpene isolated from the mycelia of H. erinaceus, was confirmed to possess a beneficial effect on increasing the content of catecholamine and NGF in the central nervous system of rats, which implicates its potential as therapeutic agents to treat neurodegenerative ailments such as Alzheimer’s or Parkinson’s disease [18].

In our searching for neurotrophic natural products from the medicinal mushrooms, chemical investigations on the liquid cultures extract from H. erinaceus led to the isolation of three new cyathane diterpenes (1–3) and two known compounds (4 and 5). In this paper, we report the isolation, structure determination, and neurotrophic effects on PC12 cells of 1–5 (Fig. 1).

Fig. 1
figure 1

Structures of compounds 1–5

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The ethyl acetate extract of H. erinaceus fermented on HY culture medium was isolated to afford five cyathane diterpenes by comprehensive chromatography methods. Two known compounds were identified as erinacine A (4) [9] and erinacine P (5) [11] by comparing their nuclear magnetic resonance (NMR) and mass spectrometry (MS) data with previously reported data. The structures of new compounds erinacines T–V (1–3) were assigned by extensive spectroscopic analysis.

Erinacine T (1) was obtained as white powder. Its molecular formula of C25H38O7 was established on the basis of the electron spray ionization (ESI) high-resolution MS (HRMS) ion at m/z 473.2511 [M + Na]+ (calcd for C25H38O7Na, 473.2510). The 1H, 13C NMR (Table 1), and heteronuclear single quantum coherence spectroscopy data displayed four methyls, six methylenes containing one oxygenated methylene, eight methines including six oxygenated methines, two pairs of olefinic carbons, one aldehyde group, two sp3 quaternary carbons, and four hydroxyl protons. These data suggested that 1 has a similar structure as that of erinacine P (5). The key HMBC correlations from H2-1 to C-3, C-4, C-8, C-9, and C-17; from H2-2 to C-3, C-4, and C-9; from H-5 to C-3, C-4, C-6, C-7, and C-11; from H2-7 to C-6, C-9, and C-16; from H2-8 to C-4, C-6, C-9, and C-17; from H2-10 to C-6 and C-12; from H-11 to C-12, C-13, and C-15; from H-13 to C-11, C-12, C-6, and C-15; from H-14 to C-6 and C-12; from H-15 to C-11, C-12, and C-13; from H3-16 to C-6, C-7, and C-14; from H3-17 to C-1, C-4, C-8, and C-9; from H-18 to C-2, C-3, and C-4; from H3-19(20) to C-3 and C-18, along with the 1H-1H COSY correlations of H2-1/H2-2, H2-7/H2-8, H2-10/H-11, H-11/OH-11, H-13/H-14, and H-18/H3-19(20) indicated the cyathane diterpene unit in 1 (Figure S1). The 1H-1H COSY correlations of H-1′/H-2′/H-3′/H-4′/H2-5′, H-2′/OH-2′, H-3′/OH-3′, and H-4′/OH-4′, and key HMBC correlation from H-1′ to C-5′ disclosed a pentose moiety in 1. Further acid hydrolysis of 1 indicated the presence of d-xylose in 1 by thin-layer chromatography comparison with the authentic product and measurement of the specific rotation (xylose from 1, [α]D25 + 19°; authentic d-xylose: [α]D25 + 20°). The β configuration of the sugar in 1 was determined by comparing the chemical shift due to the anomeric carbon with literature data (α configuration, 98–103 ppm; β configuration, 103–106 ppm) [19]. The linkage between cyathane diterpene unit and xylose moiety was confirmed by HMBC correlations from H-1′ to C-14.

Table 1 1H (500 MHz) and 13C (125 MHz) NMR data of 1–3 in DMSO-d6
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The relative configuration of 1 was confirmed by analysis of the ROESY spectrum. The NOE correlations of H-14 with H3-16 and H-10α indicated that H-14 and H3-16 were on the same side (Figure S2). The NOE correlations of H-5 with H-10β, H-11, and H3-17 placed them on the opposite face. The absolute configurations of C-5, C-6, C-9, C-11, and C-14 in 1 were assigned as 5R, 6R, 9R, 11R, and 14S by the similar negative Cotton effect around 210 nm and positive Cotton effect around 239 nm with those of 5 (Figure S3).

Erinacine U (2) possess a molecular formula of C26H40O7, as determined by ESI HRMS ion at m/z 487.2667 [M + Na]+ (calcd for C26H40O7Na, 487.2666). The 1H and 13C NMR spectra of 2 (Table 1) were similar to those of 1, except for an additional methoxyl group at δH 3.11 (3 H, s, H-21). The HMBC correlation from δH 3.11 to C-11 (δC 72.0) indicated 2 was a methylation product of 1 at the 11-OH. The β configurations of H-5, H-11, and H3-17, and the α configuration of H-14 and H3-16 were confirmed from the NOE cross peaks of H-5 with H-10β, H-11, and H3-17, and H-16 with H-10α and H-14 (Figure S2). The identical negative Cotton effect at 212 nm and positive Cotton effect at 240 nm between 2 and 1 implied the same absolute configuration of 5R, 6R, 9R, 11R, and 14S (Figure S3).

Erinacine V (3) has a molecular formula of C26H40O7, as determined on the basis of the ESI HRMS ion at m/z 487.2669 [M + H]+ (calcd for C26H40O7Na, 487.2666). Interpretation of the 1H, 13C NMR and two-dimensional NMR spectra of 3 (Table 1) suggested the same planar structure as that of 2. The NOE correlations of H-5 with H3-17 and H-10β, H3-16 with H-10α and H-14, and H-14 with H-11 (Figure S2) assigned the β configuration for H-5 and H3-17 and the α configuration for H-11, H-14, and H3-16 in 3. Compound 3 is different from 2 in the stereochemistry of C-11.

Compounds 2 and 3 are O-methylated natural products. To confirm the origin of compounds 2 and 3, the product of 1 under treatment with EtOAc and MeOH-water as used for the separation of 2 and 3 was analyzed by TLC. The fact that compound 1 was not transformed into 2 and 3 in the above test supports the natural origin of 2 and 3. O-methylated cyathane diterpenes have been repeatedly isolated from the culture of H. erinaceus, which further supported that 2 and 3 are natural products.

The cytotoxicity and neurite-promoting activities of compounds 1−5 were evaluated using PC12 cell line. The cytotoxicities against PC12 cell line of 1–5 were tested by using CCK8 method. Only compound 4 showed weak cytotoxicity against PC12 cells with IC50 of 73.7 μM. The effects of compounds 1–5 on the neurite outgrowth of undifferentiated PC12 cells were evaluated by morphological observations and a quantitative analysis of neurite-bearing cells and neurite length. Compounds 1–3 and 5 showed significant neurotrophic effects in the range of 2.5–10 μM (Fig. 2, Figure S4−S6), as compared with control group. The percentage of neurite-bearing cells for cells treated with compounds 1–3 and 5 at 10 μM reached 43.7 ± 1.6%, 76.31 ± 1.7%, 65.3 ± 1.3%, and 48.3 ± 1.3%, respectively. NGF is used as positive control with neurite-bearing cells of 40.3 ± 0.7% at the concentration of 40 ng/mL.

Fig. 2
figure 2

Neurite outgrowth of PC12 cells after 24 h treatment with NGF and compound 1, (a) Control, (b) NGF 40 ng/mL (c) compound 1 10 μM. Cell with one or more neurites whose lengths were at least twice the diameter of the cell body were scored as positive. The percentage of positive neurite-bearing cells was determined from at least three different regions of interest in three independent experiments (d). Values were considered significant at ***P < 0.001, **P < 0.01 versus control group

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In summary, three new compounds erinacines T–V (1–3) and two known compounds erinacine A (4) and erinacine P (5) were isolated from the liquid cultures of H. erinaceus. Compounds 1–3 and 5 exhibited significant neurite outgrowth-promoting effects on PC12 cells in the range of 2.5–10 μM, providing novel leading compounds for neurotrophic agents to treat neurodegenerative diseases. Our research for these neurotrophic compounds further confirms the medicinal value of H. erinaceus.