Picrotoxane Sesquiterpene Glycosides and a Coumarin Derivative from Coriaria Nepalensis and Their Neurotrophic Activity

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Article Picrotoxane Sesquiterpene Glycosides and a Coumarin Derivative from Coriaria nepalensis and Their Neurotrophic Activity

Yuan-Yuan Wang, Jun-Mian Tian, Cheng-Chen Zhang, Bo Luo and Jin-Ming Gao *

Shaanxi Key Laboratory of Natural Products & Chemical Biology, College of Science, Northwest A&F University, Yangling 712100, Shaanxi, China; [email protected] (Y.-Y.W.); [email protected] (J.-M.T.); [email protected] (C.-C.Z.); [email protected] (B.L.) * Correspondence: [email protected]; Tel.: +86-29-8709-2335; Fax: +86-29-8709-2226

Academic Editor: Isabel C. F. R. Ferreira Received: 10 September 2016; Accepted: 9 October 2016; Published: 12 October 2016

Abstract: Two picrotoxane sesquiterpene lactone glycosides, nepalactones A (1) and B (2), and one new coumarin, nepalarin (3), were isolated from the root barks of the poisonous plant Coriaria nepalensis. Their structures were elucidated via HRESIMS and 1D and 2D NMR spectroscopic analyses, and further verified via transformation methods. In addition, compounds 1–3 and five semisynthetic congeners (1a–e) were assayed for the activity to induce neurite outgrowth in rat pheochromocytoma (PC12) cells. As a result, nepalactone A derivative 1c and nepalarin (3) significantly enhanced nerve growth factor (NGF)-mediated neurite outgrowth in PC12 cells.

Keywords: Coriaria nepalensis; Coriariaceae; natural products; picrotoxane; sesquiterpene; neurite outgrowth-potentiating activity; neurotrophic activity

1. Introduction Coriaria nepalensis Wall (Coriaria sinica Maxim) (Coriariaceae) is a Chinese medicine herb mainly distributed in the southwest of China. Traditionally, this herb is used to treat numbness, toothache, traumatic injury, and acute conjunctivitis [1]. Sesquiterpene lactones are the characteristic bioactive constituents from family Coriariaceae [2–9]. A series of sesquiterpenes [6–9] and prenyled coumarins [10,11] have been previously identiﬁed from C. nepalensis. Among these sesquiterpene lactones, coriamytin and tutin have been suggested as main bioactive substances for the treatment of schizophrenia [8,12], and as insecticidal agents [9]. In our recent effort to search for neurotrophic natural products [13–16], we employed rat dopaminergic PC12 cells as an in vitro cell model to screen various natural compounds for the effect on nerve growth factor (NGF)-induced neurite outgrowth. The active compounds are expected to be potentially useful for the treatment of Alzheimer’s disease and other neurological disorders [17,18]. Here, two new picrotoxane-type sesquiterpene glycosides, nepalactones A (1) and B (2), and one new polyprenyled coumarin, nepalarin (3) (Figure1), were isolated from the root barks of this plant. We describe herein the isolation and structure elucidation of the three new compounds as well as the assay for the ability to stimulate NGF-mediated neurite outgrowth from PC12 cells.

Molecules 2016, 21, 1344; doi:10.3390/molecules21101344 www.mdpi.com/journal/molecules Molecules 2016, 21, 1344 2 of 9 Molecules 2016, 21, 1356 2 of 9

FigureFigure 1. 1.Chemical Chemical structuresstructures of compounds 11––33 andand 1a1a–e–.e .

2. Results and Discussion 2. Results and Discussion Compound 1 was isolated as a white, amorphous powder. The molecular formula was Compound 1 was isolated as a white, amorphous powder. The molecular formula was established established as C21H32O8 via HRESIMS (m/z [M + Na]+ 435.1991; calcd. 435.1995) in combination with m/z + 13 asthe C21 H13C-NMR32O8 via spectrum, HRESIMS requiring ( [M +six Na] degrees435.1991; of unsatura calcd.tion. 435.1995) Its IR inspectrum combination indicated with the the presenceC-NMR spectrum,of hydroxy requiring (3563 cm six−1) degreesand γ-lactone of unsaturation. carbonyl (1765 Its cm IR−1 spectrum) groups. The indicated 13C and the DEPT presence NMR spectra of hydroxy of −1 −1 13 (35631 (Table cm 1)) exhibited and γ-lactone 21 carbon carbonyl signals (1765 correspondi cm )ng groups. to three ThemethylC groups, and DEPT four methylene NMR spectra groups of 1 (Table(including1) exhibited one olefinic 21 carbon carbon signals at δ 111.5 corresponding and one oxygenated to three methyl carbon groups, at δ 61.6), four 11 methylene methine groups groups (including(seven oxygenated one olefinic carbons carbon including at δ 111.5 five and carbon one oxygenateds of sugar), carbonand three at δquaternary61.6), 11methine carbons groups(one (sevenlactone oxygenated carbonyl at carbons δ 180.6 includingand one olefinic five carbons carbon at of δ sugar), 158.2). andThe three1H and quaternary 13C-NMR spectra carbons of (one1 lactonedisplayed carbonyl signals at forδ 180.6 tertiary and methyl one olefinic(δ 1.25, s, carbon 3H), isopropyl at δ 158.2). (δ 0.99, The 1.01,1H andd, each13C-NMR 3H, and spectraδ 1.83, m, of 1 displayed1H), and signals exocyclic for olefinic tertiary bond methyl (δH( δ5.21,1.25, s, s,1H, 3H), 5.42, isopropyl s, 1H) groups. (δ 0.99, A 1.01, series d, of each sugar 3H, signals and δ 1.83,were m, 1 1H),also and detected exocyclic in the olefinic H-NMR bond spectrum (δH 5.21, (Table s, 1H, 1) between 5.42, s, 1H)δH 3.22–3.87, groups. with A series an anomeric of sugar proton signals at wereδH 1 13 also4.35 detected (d, 1H, J in= 7.5 the Hz,H-NMR H-1′). The spectrum C-NMR (Table spectrum1) between indicatedδH the3.22–3.87, presence with of a anglucose anomeric unit due proton to a at 0 13 δHset4.35 of carbon (d, 1H, signalsJ= 7.5 at Hz, δC H-1101.5,). 73.5, The 76.2,C-NMR 70.2, 76.6, spectrum and 61.6. indicated These data the suggested presence the of presence a glucose of unita glucopyranosyl moiety in 1. The glucose in nepalactone A derivative 1a appeared to be tetraacetylated, due to a set of carbon signals at δC 101.5, 73.5, 76.2, 70.2, 76.6, and 61.6. These data suggested the + 1 presencebased on of aNMR glucopyranosyl spectra (Table moiety 1) and in HRESIMS1. The glucose (m/z 603.2416 in nepalactone [M+Na] A). derivative Furthermore,1a appeared the H and to be 13 tetraacetylated,C-NMR data based of 1 were on NMR similar spectra to those (Table of 1the) and known HRESIMS picrotoxane ( m/z 603.2416 sesquiterpenoid, [M + Na] corialactone+). Furthermore, D (1b), which has been previously identified from the same plant [8], except for the presence of the the 1H and 13C-NMR data of 1 were similar to those of the known picrotoxane sesquiterpenoid, glucose unit at C-12 in 1. The attachment of the glucose moiety to C-12 of the aglycone 1b was corialactone D (1b), which has been previously identified from the same plant [8], except for the deduced from the HMBC correlations from H-1′ (δH 4.35) to C-12 (δC 79.4). These spectroscopic data presence of the glucose unit at C-12 in 1. The attachment1 of1 the glucose moiety to C-12 of the aglycone suggested that 1 is a glucoside of corialactone D. The 0H- H COSY and HSQC correlations indicated 1b was deduced from the HMBC correlations from H-1 (δH 4.35) to C-12 (δC 79.4). These spectroscopic the presence of two substructures–CH2(2)–CH(3)–CH(4)–CH(5)–CH(6)–CH(11)–CH(12)–, and –CH(4)– data suggested that 1 is a glucoside of corialactone D. The 1H-1H COSY and HSQC correlations CH(8)[CH3(9)]–CH3(10)–units, whose connectivity was defined by the key HMBC correlations (Figure 2). indicatedThe the relative presence configuration of two substructures–CH of the aglycone2 (2)–CH(3)–CH(4)–CH(5)–CH(6)–CH(11)–CH(12)–,(1b) of 1 was defined by a NOESY experiment and(Figure –CH(4)–CH(8)[CH 2). The NOE correlations3(9)]–CH3(10)–units, of H3-7/H-6, whose H3-9/H-5, connectivity and H-12/H-1 was′ definedindicated by that the CH key3-7, HMBCH-5, correlationsH-6, H-12, (Figure and the2). isopropyl group were at the same orientation (α-configuration). Additionally, enzymaticThe relative hydrolysis configuration of 1 afforded of the aglycone the known (1b )intact of 1 was aglycone defined 1b by, which a NOESY was experimentidentified from (Figure 1H 2). 0 TheNMR, NOE and correlations its absolute of H 3configuration-7/H-6, H3-9/H-5, (1R,3 andS,4S,5 H-12/H-1R,6S,12S) indicatedwas previously that CH determined3-7, H-5, H-6, from H-12, CD and thespectrum isopropyl [8]. group In addition, were at thethe samecoupling orientation constant (α (-configuration).J = 7.5 Hz) of the Additionally, anomeric proton enzymatic (δH 4.35, hydrolysis H-1′) of suggested1 afforded a the β-configuration known intact for aglycone the glucose1b, which unit. wasIndeed, identified compound from 11H was NMR, hydrolyzable and its absolute via configurationβ-glucocidase. (1R GC,3S ,4analysisS,5R,6S suggested,12S) was previouslythat acid hydrolyzate determined of from1 contained CD spectrum the glucose [8]. In moiety addition, with the 0 couplingthe D-configuration. constant (J = 7.5Hence, Hz) ofthe the structure anomeric of proton1 was (determinedδH 4.35, H-1 unambiguously) suggested a β -configurationas shown, and for thenamed glucose nepalactone unit. Indeed, A. compound 1 was hydrolyzable via β-glucocidase. GC analysis suggested that acid hydrolyzate of 1 contained the glucose moiety with the D-configuration. Hence, the structure of 1 was determined unambiguously as shown, and named nepalactone A.

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Molecules 2016Table, 21, 1344 1. 1H-NMR (500 MHz) and 13C-NMR (125 MHz) data for 1, 1a, 1b, and 2 (δ ppm). 3 of 9 1 a 1a b 2 a No. Tableδ 1.H 1(JH-NMR in Hz) (500 MHz)δC and 13δHC-NMR(J in Hz) (125 MHz) dataδC for 1, 1aδ,H1b(J,in and Hz)2 (δ ppm).δC 1 42.5 46.1 42.5 2α 1.74 (d, 15.5)1 a 34.0 1.63 (d, 15.0) 1a b 33.6 1.64 (d, 15.5) 2 a 33.3 No.2β 2.45 (dd, 2.5, 15.5) 2.40 (dd, 3.5, 15.0) 2.46 (dd, 3.5, 15.5) δ (J in Hz) δ δ (J in Hz) δ δ (J in Hz) δ 3 4.72H (m) 80.2 C 4.63H (m) 79.6 C 4.68H (m) 79.7C 14 2.02 (m) 51.5 42.5 1.96 (m) 51.5 46.1 2.02 (m) 51.4 42.5 α 25 1.74 2.35 (d, (m) 15.5) 45.5 34.0 2.27 1.63 (m) (d, 15.0) 45.5 33.6 1.642.50 (d,(m) 15.5) 45.0 33.3 2β 2.45 (dd, 2.5, 15.5) 2.40 (dd, 3.5, 15.0) 2.46 (dd, 3.5, 15.5) 6 2.36 (m) 41.7 2.26 (m) 41.3 2.39 (d, 3.0) 49.1 3 4.72 (m) 80.2 4.63 (m) 79.6 4.68 (m) 79.7 47 1.25 2.02 (s) (m) 31.9 51.5 1.15 1.96 (s) (m) 31.7 51.5 1.28 2.02 (s) (m) 32.7 51.4 58 1.83 2.35 (m) (m) 25.0 45.5 1.76 2.27(m) (m) 24.8 45.5 1.81 2.50 (m) (m) 25.3 45.0 69 0.99 2.36 (d, (m)6.0) 19.3 41.7 0.96 (d, 2.26 6.0) (m) 19.6 41.3 0.99 2.39 (d, (d, 6.5) 3.0) 19.8 49.1 107 1.01 1.25 (d, (s)6.0) 20.0 31.9 0.98 (d, 1.15 6.0) (s) 20.7 31.7 1.03(d, 1.28 6.5) (s) 20.9 32.7 118α 2.31 1.83 (m) (m) 34.2 25.0 2.31 1.76(m) (m) 34.7 24.8 4.45 1.81 (m) (m) 81.9 25.3 9 0.99 (d, 6.0) 19.3 0.96 (d, 6.0) 19.6 0.99 (d, 6.5) 19.8 11β 2.23 (m) 2.02 (m) 10 1.01 (d, 6.0) 20.0 0.98 (d, 6.0) 20.7 1.03(d, 6.5) 20.9 1112α 4.902.31 (t, (m)7.5) 79.4 34.2 4.77 (t, 2.31 7.4) (m) 81.7 34.7 2.75 4.45 (m) (m) 39.4 81.9 1112ββ 2.23 (m) 2.02 (m) 2.92 (dd, 8.0, 18.5) 1213α 4.90 (t, 7.5)158.2 79.4 4.77 (t, 7.4) 156.4 81.7 2.75 (m) 156.3 39.4 1214aβ 5.21 (d, 1.5) 111.5 5.19 (d, 1.5) 112.9 2.92 4.91 (dd, (d, 8.0,8.0) 18.5) 106.1 14b13 5.42 (d, 1.5) 158.2 5.44 (d, 1.5) 156.4 4.88 (d, 8.0) 156.3 14a 5.21 (d, 1.5) 111.5 5.19 (d, 1.5) 112.9 4.91 (d, 8.0) 106.1 15 180.6 179.0 178.6 14b 5.42 (d, 1.5) 5.44 (d, 1.5) 4.88 (d, 8.0) 151′ 4.35 (d, 7.5) 101.5 180.6 4.56 (d, 8.0) 100.4 179.0 4.33 (d, 7.5) 102.4 178.6 120′ 4.35 3.22 (d,(m) 7.5) 73.5 101.5 4.96 4.56 (m) (d, 8.0) 75.1 100.4 4.33 3.27 (d,(s) 7.5) 73.5 102.4 230′ 3.403.22 (m) (m) 76.6 73.5 5.18 (t, 4.96 10.0) (m) 73.2 75.1 3.44 3.27 (m) (s) 76.4 73.5 0 34′ 3.393.40 (m) (m) 70.2 76.6 5.05 5.18(t, 10.0) (t, 10.0) 68.5 73.2 3.44 3.44 (m) (m) 69.7 76.4 0 45′ 3.293.39 (m) (m) 76.2 70.2 3.68 5.05 (m) (t, 10.0) 71.9 68.5 3.29 3.44 (m) (m) 75.7 69.7 50 3.29 (m) 76.2 3.68 (m) 71.9 3.29 (m) 75.7 6′a 3.87 (dd, 2.5, 12.0) 61.6 4.26 (dd, 5.0, 12.0) 62.2 3.87 (dd, 3.0,12.0) 61.6 60a 3.87 (dd, 2.5, 12.0) 61.6 4.26 (dd, 5.0, 12.0) 62.2 3.87 (dd, 3.0,12.0) 61.6 60′b 3.753.75 (dd, (dd, 5.5, 5.5, 12.0) 12.0) 4.12 4.12(dd, (dd,2.0, 2.0,12.0) 12.0) 3.783.78 (dd, (dd, 3.0,12.0) 3.0,12.0) 20′-Ac 2.03 2.03(s) (s) 169.0/20.7169.0/20.7 30′-Ac 1.99 1.99(s) (s) 170.8/20.8170.8/20.8 40′-Ac 2.01 2.01(s) (s) 169.3/20.7169.3/20.7 0 6′-Ac 2.08 2.08(s) (s) 170.6/20.6170.6/20.6 a,b a,b: Data: Data were were recorded recorded in in MeOH- MeOH-dd44,, andand CDCl CDCl3,3 respectively., respectively.

In addition,addition, inin order order to to enhance enhance molecular molecular diversity diversity of sesquiterpene of sesquiterpene compounds, compounds, additional additional three threederivatives derivatives of 1. The of 1 conversion. The conversion of the aglyconeof the aglycone1b using 1b Dess-Martin using Dess-Martin periodinane periodinane (DMP) in(DMP) CH2Cl in2 ◦ CHat room2Cl2 at temperature room temperature afforded afforded1c; and treatment 1c; and treatment of 1 with 4of N 1 HCl with in 4 dioxane N HCl atin 100dioxaneC yielded at 100 two °C yieldedcompounds two 1dcompoundsand 1e (Scheme 1d and1). 1e These (Scheme three 1). new These congeners three new were congeners characterized were bycharacterized an application by an of applicationspectroscopic of dataspectroscopic and HRESIMS. data and HRESIMS.

Scheme 1. Conversion of 1 into picrotoxane sesquiterpenes 1a–e. Reagents and conditions: ( a) acetic anhydride, pyridine,pyridine, r.t.;r.t.; ( b()b citrate) citrate buffer, buffer,β-glucocidase, β-glucocidase, rt; (crt;) Dess–Martin(c) Dess–Martin periodinane periodinane (DMP), (DMP), DCM, DCM,r.t., 40 min;r.t., 40 (d min;) 4 N (d HCl-dioxane) 4 N HCl-dioxane (1:1), 100 (1:1),◦C, 100 2 h. °C, 2 h.

Molecules 2016, 21, 1344 4 of 9 Molecules 2016, 21, 1356 4 of 9

Figure 2. Selected 2D NMR correlations for compounds 1–3. Figure 2. Selected 2D NMR correlations for compounds 1–3.

Compound 2 was obtained as white powder with a molecular formula C21H32O8, as established viaCompound HRESIMS (m2/wasz [M obtained+ Na]+ 435.1987; as white calcd. powder 435.2417). with aIts molecular molecular formula formula Cwas21H identical32O8, as to established that of + via1, HRESIMS indicating ( mthat/z [M2 was + Na] a positional435.1987; calcd.isomer 435.2417). of 1. TheIts similar molecular IR spectrum formula wasof 2 identicalcompared to to that 1 of 1, indicatingindicated the that presence2 was a of positional similar functional isomer of groups.1. The The similar 1H and IR spectrum 13C-NMR ofspectral2 compared data (Table to 1 indicated 1) of 2 thewere presence highly of similar similar to functional those of nepalactone groups. The A1 H(1). and Compound13C-NMR 1 spectralsignificantly data (Tablediffered1) offrom2 were 2 by highlythe similarsignals to for those the ofglucopyranosyl nepalactone A group, (1). Compound attached at 1C-12significantly for 1 while differed at C-11 from(δC 81.9)2 by for the 2. signalsThe HMBC for the glucopyranosylcorrelations from group, the attachedanomeric atproton C-12 forH-11′ atwhile δH 4.33 at C-11 (d, J (=δ 7.5C 81.9) Hz) forto C-112. The (δC HMBC 81.9) implied correlations that the from 0 theglucose anomeric group proton is attached H-1 at toδH C-114.33 (d,(FigureJ = 7.5 2). Hz)The to stereochemistry C-11 (δC 81.9) impliedof 2 was that established the glucose from group a is attachedNOESY experiment to C-11 (Figure (Figure2). The2). The stereochemistry most significant of 2NOEwas establishedcorrelations fromobserved a NOESY were experimentbetween H-6/H3-7, H-5/H3-9, and H-1′/H-11, indicating that H-5, H-6, H-6, the methyl group at C-1, and the (Figure2). The most significant NOE correlations observed were between H-6/H 3-7, H-5/H3-9, and H-1isopropyl0/H-11, indicatinggroup at C-4 that were H-5, allH-6, on the H-6, same the α methyl-face of groupthe molecule. at C-1, andThe NMR the isopropyl data of 2 groupwere fully at C-4 1 1 wereassigned all on by the analysis same α -faceof the of 2D-NMR the molecule. data including The NMR its dataHSQC, of 2HMBC,were fully and assignedH- H COSY by analysisspectra. of Thus, the structure of 2 was established as shown for nepalactone B. the 2D-NMR data including its HSQC, HMBC, and 1H-1H COSY spectra. Thus, the structure of 2 was Compound 3, yellowish crystalline needles, was assigned the molecular formula C25H32O4 from established as shown for nepalactone B. [M−H]− at m/z 395.2226 in HRESIMS, requiring 10 degrees of unsaturation. The IR spectrum Compound 3, yellowish crystalline needles, was assigned the molecular formula C H O from displayed the presence of hydroxy (3319 cm−1), C=O (1691 cm−1) groups and a benzene25 ring32 (1627,4 [M − H]− at m/z 395.2226 in HRESIMS, requiring 10 degrees of unsaturation. The IR spectrum 1583, and 1458 cm−1). The 13C-NMR and DEPT data showed 25 carbon signals for seven methyls, displayed the presence of hydroxy (3319 cm−1), C=O (1691 cm−1) groups and a benzene ring (1627, three methylenes, four methines, and 11 quaternary carbons. The 13C-NMR data suggested the 1583, and 1458 cm−1). The 13C-NMR and DEPT data showed 25 carbon signals for seven methyls, presence of three isopentenyl groups (δC 119.8, δH 5.34, δC 135.2; and δC 121.1, δH 5.33, δC 133.1; and δC 13 three122.6, methylenes, δH 5.11, δC 132.8), four methines, one methoxy and group 11 quaternary (δC 62.2). carbons.Besides three The isopentenylC-NMR and data one suggested methoxy the presencesubstituent, of three the NMR isopentenyl characteristic groups signals (δC 119.8, and UVδH spectra5.34, δ Cwere135.2; indicative and δC of121.1, a coumarinδH 5.33, skeletonδC 133.1; and[11].δC One122.6, downfieldδH 5.11, δsingletC 132.8), (δHone 7.51, methoxy s) in the group 1H-NMR (δC 62.2).spectrum Besides of 3 threesuggested isopentenyl that 3 was and a one methoxypentasubstituted substituent, coumarin. the NMR Furthermore, characteristic the 1 signalsH and 13 andC-NMR UV spectradata of 3 were were indicative similar to ofthose a coumarin of the 1 skeletonknown [11coumarin,]. One downfield 7-hydroxy-6-methoxy-3,8-bis(3-me singlet (δH 7.51, s) in thethyl-2-butenyl)coumarin,H-NMR spectrum ofwhich3 suggested was previously that 3 was 1 13 a pentasubstitutedidentified from the coumarin. same plant Furthermore, [11], except the forH th ande presenceC-NMR of an data additional of 3 were isopentenyl similar tothose unit [ ofδH the known5.11 (1H, coumarin, m, H-2 7-hydroxy-6-methoxy-3,8-bis(3-methyl-2-butenyl)coumarin,′′), 3.56 (2H, d, J = 6.7 Hz, H-1′′), 1.86 (s, H-5′′), 1.75 (s, H-4′′); δ whichC 132.8 was(C-3 previously′′), 122.6 identified(C-2′′), 25.6 from (C-4 the′′), same 24.7 (C-1 plant′′), [18.111], (C-5 except′′)] at for C-5 the in presence 3. The location of an additional of the isopentenyl isopentenyl unit at unit C-5 [δ wasH 5.11 00 00 00 00 00 (1H,supported m, H-2 by), 3.56the HMBC (2H, d, correlationsJ = 6.7 Hz, of H-1 H-2′′), to 1.86 C-5, (s, and H-5 H-1),′′ to 1.75 C-6 (s, and H-4 C-10.); δ FullC 132.8 analyses (C-3 of), the 122.6 (C-21H00 ),and 25.6 13C-NMR (C-400), 24.7spectral (C-1 data00), 18.1 of 3 (C-5, supported00)] at C-5 by in the 3. The1H-1H location COSY, ofDEPT, the isopentenyl HSQC, and unit HMBC at C-5 wasexperiments supported by(Figure the HMBC 2), permitted correlations the of assignme H-200 to C-5,nt of and all H-1 proton00 to C-6 and and carbon C-10. Fullresonances. analyses of theConsequently,1H and 13C-NMR the structure spectral of 3 data was ofestablished3, supported as 7-hydroxy-6-methoxy-3,5,8-tri(3-methyl-2-butenyl) by the 1H-1H COSY, DEPT, HSQC, and HMBC experimentscoumarin and (Figure named2), permittednepalin C. theTo assignmentour knowledge, of all this proton is a rare and coumarin carbon resonances. with three Consequently,isopentenyl units in natural source. the structure of 3 was established as 7-hydroxy-6-methoxy-3,5,8-tri(3-methyl-2-butenyl) coumarin and named nepalin C. To our knowledge, this is a rare coumarin with three isopentenyl units in natural source.

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Molecules 2016, 21, 1356 5 of 9 NeuriteOutgrowth-Promoting Activity NeuriteOutgrowth-PromotingCompounds (1–3, and 1a Activity–1e) were evaluated for the effect on NGF-induced neurite outgrowth in rat pheochromocytomaCompounds (1–3, and PC12 1a– cells1e) were according evaluated toour for the previously effect on reportedNGF-induced procedures neurite outgrowth [17,18]. in ratAll pheochromocytoma these compounds PC12 at the cells concentrations according to ofour 1, previously 5, 10, and 20reportedµM failed procedures to induce [17,18]. neurite outgrowth from PC12All these cells incompounds the absence at the of NGF.concentrations However, of in 1, the 5, presence10, and 20 of μ 2M ng/mL failed NGF,to induce compounds neurite 2, 3, andoutgrowth1c–1e at afrom concentration PC12 cells ofin 10theµ Mabsence markedly of NG increasedF. However, the NGF-inducedin the presence neurite of 2 ng/mL outgrowth NGF, based oncompounds the numbers 2, of3, neurite-bearingand 1c–1e at a concentration cells by 21.67% of 10± μ2.64%M markedly to 24.40% increased± 2.04%, the greaterNGF-induced than those (17.60%neurite± outgrowth0.24%) of controlbased on NGF the (2numbers ng/mL). of Inneurit particular,e-bearing compounds cells by 21.67%1c and ± 2.64%3 were to selected 24.40% for± the 2.04%, greater than those (17.60% ± 0.24%) of control NGF (2 ng/mL).In particular, compounds 1c assay of neurotrophic activity. As shown in Figure3, compounds 1c and 3 at concentrations ranging and 3 were selected for the assay of neurotrophic activity. As shown in Figure 3, compounds 1c and3 from 1 to 10 µM increased the numbers of neurite-bearing cells in a concentration dependent manner. at concentrations ranging from 1 to 10 μM increased the numbers of neurite-bearing cells in a Compared with 21.11% ± 1.62% for 10 ng/mL NGF alone, compounds 1c and 3 at a concentration concentration dependent manner. Compared with 21.11% ± 1.62% for 10 ng/mL NGF alone, ofcompounds 10 µM increased 1c and the3 at numbersa concentration of neurite-bearing of 10 μM increased cells the by numbers 29.89% ± of1.86% neurite-bearing and 27.81% cells± by1.48%, respectively.29.89% ± 1.86% Nevertheless, and 27.81% compounds ± 1.48%, respectively.1c and 3 at Nevertheless, 20 µM showed compounds somewhat 1c toxicityand 3 at to 20 PC12 μM cells. Theseshowed results somewhat suggested toxicity that theto PC12 attachment cells. These of the results sugar suggested unit to the that picrotoxane the attachment skeleton of the might sugar reduce theunit neurotrophic to the picrotoxane activity. skeleton might reduce the neurotrophic activity.

Figure 3. Neurite outgrowth-promoting activity of 1c and 3 in PC12 cells. (A) Morphology of PC12 Figure 3. Neurite outgrowth-promoting activity of 1c and 3 in PC12 cells. (A) Morphology of PC12 cells; (B) Quantitative analysis of neurite outgrowth promoted by 1c and 4. Compounds 1c and 3 (1, cells;5, 10, (B )and Quantitative 20 μM) + NGF analysis (10 ofng/mL). neurite Data outgrowth are expressed promoted as the by means1c and ± SD4. Compounds(n = 3) (** p < 1c0.01and vs.3 (1, 5, 10,NGF; and 20Dunnett’sµM) + NGFt test). (10 In control ng/mL). experiments, Data are expressed the percentages as the of means neurite-bearing± SD (n = cells 3) (** werep < 21.11% 0.01 vs. ± NGF; Dunnett’s1.62% followingt test). Inincubation control experiments, with 10 ng/mL the of percentagesNGF after 48 ofh. neurite-bearing cells were 21.11% ± 1.62% following incubation with 10 ng/mL of NGF after 48 h. Molecules 2016, 21, 1344 6 of 9

3. Experimental Section

3.1. General Procedures Optical rotations were recorded on an Autopol III automatic polarimeter. UV spectra were obtained using a Thermo Scientific Evolution 300 UV-VIS Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). 1H and 13C-NMR spectra were recorded on 400 MHz and 500 MHz NMR instruments. Chemical shifts were reported using the solvent residual peak as the internal standard. ESI-MS spectra were performed on a Thermo Fisher LTQ Fleet instrument spectrometer (Thermo Fisher Scientiﬁc Inc.). HRESIMS data were obtained on an API QSTAR time-ofﬂight spectrometer. Column chromatography (CC) was performed on silica gel (90–150 µm) (Qingdao Marine Chemical Inc., Qingdao, China), Sephadex LH-20 (40–70 µm), and Lichroprep RP-18 gel (40–63 µm; Merck, Darmstadt, Germany). GF254 plates were used for thin-layer chromatography (TLC). Preparative TLC (PTLC) was carried out on silica gel 60 GF254; semi-preparative HPLC was performed via a Thermo BDS Hypersil column (250 mm× 4.6 mm and 250 mm× 10 mm, 5 µm).

3.2. Plant Materials The dried root barks of Coriaria nepalensis were collected from the Qinling mountains, in the Shaanxi province of China in November 2013. The plant was authenticated by Professor Wu. A voucher specimen (0023466) was deposited at the Herbarium of College of Life Sciences, Northwest A&F University, P. R. China.

3.3. Extraction, Isolation, and Purification The dried and powdered root-bark of C. nepalensis (4.2 kg) were extracted by 95% EtOH at room temperature for three times. After removal of the solvent, the residue (1.9 kg) obtained was suspended in water and then extracted successively with petroleum ether (PE), EtOAc, and n-BuOH. The EtOAc portion (911 g) was chromatographed on silica gel over CC eluting with PE/EtOAc mixtures of increasing polarity (9:1, 8:2, 7:3, 6:4, 4:6) to yield five fractions (Frs. 1–5). Fr. 2 was separated by repeated CC over silica gel (EtOAc/MeOH 9:1 to 6:4) to yield five fractions (2-1–2-5). Fraction 2-2 (2 g) was submitted to a RP-18 column (MeOH/H2O, 10:90 to 100:0) to yield two further fractions (2-2-1 and 2-2-2), and Fraction 2-2-1 was separated by Sephadex LH-20 (CHCl3/MeOH 1:1) and silica gel CC (CHCl3/MeOH 8:2) to yield 1 (216 mg). Fraction 2-3 was separated by silica gel CC (PE/acetone 20:1) to afford four fractions (2-3-1–2-3-4). Fraction 2-3-3 was separated by CC on LH-20 (CHCl3/MeOH 1:1), RP-18 CC (70% MeOH), and then silica gel with (PE/acetone, 50:1 to 40:1) to afford 3 (1.23 g). The n-butanol fraction (242 g) was separated by silica gel CC using EtOAc-MeOH (30:1–15:1) as eluent to afford seven fractions (Frs. A–G). Fr. C was separated by CC over silica gel (CHCl3/MeOH, 12:1 to 5:1), Sephadex LH-20 (MeOH), and then RP-18 to yield 1 (500 mg). Purification of Fr. C-3 by LH-20 CC (MeOH) followed by HPLC (40% EtOH, flow rate = 2 mL/min) provided 2 (2 mg, tR = 17.8 min). 20 Nepalactone A (1). White, amorphous powder; [α]D −26.3 (c 0.24, MeOH); IR (KBr)νmax 3402, 1765, 1076, 1025 cm−1; 1H and 13C-NMR data, see Table1, Figures S1–S9; ESIMS m/z 435.17 [M + Na]+, + + 846.62 [2M + Na] ; HRESIMS m/z 435.1991 [M + Na] (calcd. for C21H32O8Na, 435.1995). 20 −1 Nepalactone B (2). White powder; [α]D + 6.9 (c 0.24, MeOH); IR (KBr) νmax 3390, 1769, 1076, 1028 cm ; 1H and 13C-NMR data, see Table1, Figures S19–27; ESIMS m/z 823.15 [2M − H]−; HRESIMS m/z + 435.1987 [M + Na] (calcd. for C21H32O8Na, 435. 1995).

Nepalarin (3). Pale yellow needles; UV(MeOH): λmax (logε) nm: 221 (4.28), 336 (4.22); IR (KBr) νmax −1 −1 1 3355, 1731, cm ; IR (KBr) νmax 3319, 1692, 1583, 1458, 1394, 1306, 1067, 1041, 1015 cm ; H-NMR 0 000 00 (500 MHz, CDCl3) δ 7.51 (1H, s, H-4), 5.34 (1H, m, H-2 ), 5.33 (1H, m, H-2 ), 5.11 (1H, m, H-2 ), 3.84 000 00 (3H, s, 6-OCH3), 3.58 (2H , d, J = 7.5 Hz, H-1 ), 3.56 (2H, d, J = 6.7 Hz, H-1 ), 3.26 (2H, d, J = 7.0 Hz, H-10), 1.89 (3H, s, H-4000), 1.86 (3H, s, H-500), 1.83 (3H, s, H-40), 1.75(3H, s, H-400), 1.72 (3H, s, H-5000), 1.71 Molecules 2016, 21, 1344 7 of 9

0 13 (3H, s, H-5 ); C-NMR (125 MHz, CDCl3) δ 162.3 (C-2), 149.5 (C-7), 149.3 (C-9), 142.0 (C-6), 135.9 (C-4), 135.2 (C-30), 133.1 (C-3000), 132.8 (C-300), 128.2 (C-10), 124.6 (C-3), 122.6 (C-200), 121.1 (C-2000), 119.8 (C-20), 0 0 000 00 00 114.3 (C-8), 111.8 (C-5), 62.2 (6-OCH3), 28.5 (C-1 ), 25.9 (C-4 ), 25.8 (C-5 ), 25.6 (C-4 ), 24.7 (C-1 ), 22.4 (C-1000), 18.1 (C-500), 18.0 (C-4000), 17.8 (C-50); ESI-MS m/z 419.49 [M + Na]+, 815.18 [2M + Na]+, + − − 341.19 [M − C4H7] ; ESI-MS m/z 395.41 [M − H] ; HRESIMS m/z 395.2226 [M − H] (calcd. for C25H31O4, 395.2222).

3.4. Nepalactones A Tetraacetate 1a To a solution of 1 (3.2 mg, 0.03 mmol) in pyridine (1 mL), acetic anhydride (1 mL) was added, and the mixture stood at RT for 24 h. The reaction mixture was concentrated to dryness, and the residue were puriﬁed on silica gel CC using EtOAc, affording tetraacetate 1a (ca. 3 mg) as an off-white 20 1 13 gum. [α]D −20.9 (c 0.2, MeOH); H and C-NMR data, see Table1, Figures S10–18; HRESIMS m/z + [M + Na] 603.2416 (calcd. for C29H40O12Na, 603.2417).

3.5. Acid Hydrolysis of 1 and theDetermination of Sugar Compound 1 (2 mg) was hydrolyzed with 2N HCl-dioxane (1:1, 1 mL) at 100 ◦C for 2 h. The reaction mixture was partitioned between chloroform and H2O three times. The aqueous layer was neutralized with 2 M Ag2CO3 and evaporated in vacuo. The residue was dissolved in pyridine (0.5 mL), to which L-cysteine methyl ester hydrochloride in pyridine (0.1 M, 0.5 mL) was added. After reacting at 60 ◦C for 1 h, trimethylsilylimidazole (0.05 mL) was added to the reaction mixture and kept at 4 ◦C for another 8 h. The mixture was analyzed via GC–MS. By comparison of the retention time of the authentic sample, the monosaccharide of 1 was determined to be D-glucose (tR 11.85 min) (tR 12.15 min of L-glucose).

3.6. Enzymatic Hydrolysis of 1 To a solution of 1 (20 mg) in a mixture of a citrate buffer (pH = 5, 1 mL) and water (1 mL), β-gluosidase (1.3 mg, Sigma-Aldrich, St. Louis, MO, USA) was added. After the reaction mixture was incubated at 30 ◦C for 5 days, it was extracted with EtOAc, dried over anhydrous sodium sulfate, concentrated, and puriﬁed via column chromatography using MeOH/CHCl3 (1:15) to yield aglycon 1b as a white solid (8.2 mg, 68%), which was identiﬁed as a sesquiterpenoid, corialactone D, via 1 1 comparison with the H-NMR spectrum. H-NMR (500 MHz, CDCl3) δ 1.73 (d, J = 15.0 Hz, H-2α), 2.46 (dd, J = 3.5, 15.0 Hz, H-2β), 4.68 (m, H-3), 2.02 (m, H-4), 2.36 (m, H-5), 2.36 (m, H-6), 1.30 (s, 3H, H-7), 1.83 (m, 1H, H-8), 1.02 (d, J = 6.9 Hz, 3H, H-9), 1.00 (d, J = 6.9 Hz, 3H, H-10), 2.37 (m, 1H, H-11α), 2.10 (m, 1H, H-11β), 4.86 (m, 1H, H-12), 5.33 (s, 1H, H-14a), 5.18 (s, 1H, H-14b).

3.7. Preparation of 1c

To a solution of 1b (8.2 mg, 0.03 mmol) in CH2Cl2 (1 mL) at room temperature, NaHCO3 (13.8 mg, 0.16 mmol, 5 equiv.) and Dess-Martin periodinane (DMP) (20.9 mg, 0.05 mmol, 1.5 equiv.) were added. After 40 min of stirring, the reaction mixture was poured into a mixture of a saturated aqueous solution of Na2S2O3 (5 mL), a saturated aqueous solution of NaHCO3 (5 mL), and water (5 mL). The layers were separated, and the aqueous phase was extracted with CH2Cl2. The combined organic extracts were washed with brine, dried over MgSO4, filtered, and concentrated under reduced pressure. The crude product was purified via flash chromatography on silica gel (PE/EtOAc 5/1 to 2/1), affording 1c 1 (7.8 mg, yield 98%) as a slightly yellow oil; H-NMR (500 MHz, CDCl3) δ 6.06 (s, 1H, H-14a), 5.25 (s, 1H, H-14b), 4.66 (t, J = 4.0 Hz, 1H, H-3), 2.65 (dd, J = 19.3, 8.8 Hz, 1H, H-11α), 2.46 (d, J = 19.3 Hz, 1H, H-11β), 2.38 (m, 1H, H-2β), 2.36 (m, 1H, H-5), 2.34 (m, 1H, H-6), 1.99 (m, 1H, H-4), 1.78 (m, 1H, H-8), 1.77 (m, 1H, H-2α), 1.15 (s, 3H, H-7), 0.96 (d, J = 7.0 Hz, 3H, H-10), 0.94 (d, J = 7.0 Hz, 3H, H-9); 13 C-NMR (125 MHz, CDCl3) δ 203.7 (C-12), 176.7 (C-15), 151.9 (C-13), 117.5 (C-14), 79.0 (C-3), 51.2 (C-4), 45.0 (C-5), 40.1 (C-1), 39.8 (C-11), 35.5 (C-6), 33.5 (C-2), 32.2 (C-7), 25.3 (C-8), 20.6 (C-10), 19.73 Molecules 2016, 21, 1344 8 of 9

(C-9); ESIMS m/z 249.08 [M + H]+, 271.15 [M + Na]+; HRESIMS m/z 271.1307 [M + Na]+ (calcd. for C15H20O3Na, 271.1305).

3.8. Preparation of 1d and 1e Compound 1 (30 mg) was hydrolyzed with 4 N HCl–dioxane (1:1, 15 mL) at 100 ◦C for 2 h. The reaction mixture was partitioned between chloroform and H2O three times. The organic layer was neutralized with 20 M Ag2CO3 and ﬁltered. The resulting solution was washed with brine (50 mL), dried over Na2SO4, and then ﬁltered and evaporated. The residue was subjected to column chromatography on silica gel using petroleum ether and ethyl acetate as eluent to yield 1d (11 mg, 56%) and 1e (7 mg, 38%) as a yellowish oil.

−1 1 1d: amorphous powder; IR (KBr)νmax 3402, 1765, 1076, 1025 cm ; H-NMR (500 MHz, CDCl3) δ 5.74 (s, 1H, H-12), 4.64 (s, 1H, H-3), 4.23 (d, J = 13.0 Hz, 1H, H-14a), 4.12 (d, J = 13.0 Hz, 1H, H-14b), 1.79 (m, 1H, H-8), 1.25 (s, 3H, H-7), 0.99 (d, J = 6.5 Hz, 3H, H-10), 0.96 (d, J = 6.5 Hz, 3H, H-9); 13C-NMR (125 MHz, CDCl3) δ 178.1 (C-15), 144.9 (C-13), 130.2 (C-12), 79.6 (C-3), 50.9 (C-4), 46.4 (C-1), 44.7 (C-5), 43.8 (C-6), 40.8 (C-14), 34.0 (C-11), 30.8 (C-2), 29.1 (C-7), 25.8 (C-8), 20.6 (C-10), 19.8 (C-9); HRESIMS + m/z 291.1128 [M + Na] (calcd. for C15H21O2ClNa, 291.1127). −1 1 1e: amorphous powder; IR (KBr)νmax 3402, 1765, 1076, 1025 cm ; H-NMR (500 MHz, CDCl3) δ 5.59 (s, 1H, H-12), 4.65 (s, 1H, H-3), 4.18 (s, 2H, H-14), 1.79 (m, 1H, H-8), 1.15 (s, 3H, H-7), 1.00 13 (d, J = 6.5 Hz, 3H, H-10), 0.97 (d, J = 6.5 Hz, 3H, H-9); C-NMR (125 MHz, CDCl3) δ 178.3 (C-15), 149.1 (C-13), 126.4 (C-12), 79.9 (C-3), 59.8 (C-14), 51.0 (C-4), 46.1 (C-1), 44.8 (C-5), 42.7 (C-6), 34.2 (C-11), 31.4 (C-2), 28.4 (C-7), 25.2 (C-8), 20.7 (C-10), 19.8 (C-9); HRESIMS m/z 273.1470 [M + Na]+ (calcd. for C15H22O3Na, 273.1471).

3.9. Neurite Outgrowth-Promoting Assay Neurite outgrowth-promoting activity of these isolated compounds was assessed as described previously by our group [17,18]. Experiments were repeated at least three times, and data are expressed as mean ± SD (** p < 0.01). Statistical analyses were performed using Dunnett’s t-test.

4. Conclusions In conclusion, the present study reported the identification of three new compounds, including two new picrotoxane sesquiterpene glycosides nepalactones A (1) and B (2) and one new coumarin nepalarin (3), from the root barks of C. nepalensis. In addition, five congeners 1a–e were synthesized from nepalactone A. We for the first time discovered that these compounds, especially compound 1c and 3, could potentiate NGF-induced neurite outgrowth in PC12 cells.

Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/ 10/1344/s1. Acknowledgments: This work was financially supported by the Natural Science Foundation of China (21572182, 31300283), and the Natural Science Foundation of Shaanxi Province (2014JZ2-001, 2015JM3111), as well as the Chinese Universities Scientific Fund (Z109021547, 2014YB025). Author Contributions: J.-M.G. designed the research; Y.-Y.W., J.-M.T., C.-C.Z. and B.L. performed the research and analyzed the data; J.-M.G. wrote the paper. All authors read and approved the final manuscript. Conflicts of Interest: The authors declare no conflict of interest.

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