Schekwanglupaside C, a new lupane saponin from Schefflera kwangsiensis, is a potent activator of sarcoplasmic reticulum Ca2+-ATPase

Fitoterapia 137 (2019) 104150

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Schekwanglupaside C, a new lupane saponin from Schefflera kwangsiensis, is a potent activator of sarcoplasmic reticulum Ca2+-ATPase

T

Guolin Yanga,1, Yan Wangb,c,1, Yiyi Yua, Jing Zhenga, Juan Chena, Shaoheng Lia, Ruoyun Chenc, ⁎ ⁎ Chunlei Zhanga, C. Benjamin Namand, Dequan Yuc, , Zhengyu Caoa, a

State Key Laboratory of Natural Medicines and Department of Pharmacology, School of Traditional Chinese Pharmacy, China Pharmaceutical University, Nanjing 211198, PR China b Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences, Beijing 100193, PR China c State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100193, PR China d College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo 315800, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: S. kwangsiensis Lupane saponin Schekwanglupaside C Ca2+ Sarcoplasmic reticulum Ca2+-ATPase

Schefflera kwangsiensis Merr. ex H.L. Li (Araliaceae) is a widely used traditional Chinese medicine for pain management in the clinic. In the present study, we isolated a previously undescribed lupane saponin, designated as schekwanglupaside C (Sch C) from the ethanolic extract of S. kwangsiensis. The structure of Sch C was determined by comprehensive spectroscopic and spectrometric analyses and chemical degradation. In primary cultured cortical neurons, Sch C altered the pattern of spontaneous Ca2+ oscillation (SCO) with a slight increase in the frequency of SCO right after addition and a gradual decrease in the frequency and amplitude of SCO, that dynamic change mimicked by an activator of sarcoplasmic reticulum Ca2+-ATPase (SERCA). The IC50 values for Sch C suppression of the frequency and amplitude of SCO were 1.75 and 2.51 μM, respectively. Furthermore, we demonstrated that Sch C is a potent SERCA activator (EC50 = 1.20 μM). Given the pivotal role of SERCA in the progression of neuropathic pain and neurodegenerative diseases, Sch C represents a new drug lead compound to develop the treatment of neuropathic pain and Alzheimer's disease.

1. Introduction Schefflera kwangsiensis Merr. ex H.L. Li (Araliaceae), a traditional Chinese medicine, is widely distributed in the Guangxi and Yunnan provinces of China and has been extensively used in the clinic for treating many diseases such as pain, seizure, spasm and stroke [1]. Due to its effectiveness on the pain relief, S. kwangsiensis has been developed into a licensed drug, called “HanTaoYe”, for treating trigeminal neuralgia, sciatica, rheumatism and headache [1]. Early chemical investigations on this plant revealed the presence of organic acids [2], triterpenes and triterpenoid glycosides [3]. More recently, triterpenoid saponins were reported to be the major components of S. kwangsiensis [4]. Until now, over 60 triterpenoid saponins have been isolated from S. kwangsiensis [5], and these compounds can be categorized as oleananes [5–10], ursanes [8–10], and lupanes [8,11]. Despite extensive

investigation of chemical constitutes of S. kwangsiensis [5–11], the knowledge of active compounds and molecular mechanisms of how S. kwangsiensis exerts its pharmacological activity are limited. Several triterpenoid saponins purified from this plant display moderate hepatoprotective activities [8–11]. A fraction with three triterpenoid saponins has also been shown to relax smooth muscle contraction in isolated guinea pig tracheal chains, suggesting an anti-spasmodic activity [3]. Ca2+ signaling is involved in multiple physiological and pathological processes both in excitable and non-excitable cells. The orchestrated movement of Ca2+ ions is responsible for muscle contraction [12] and neurotransmitter release [13]. Studies have shown that hippocampal neurons derived from rats with status epilepsy displayed higher resting Ca2+ levels than normal neurons [14]. Dysregulated resting Ca2+ levels were also observed in sensory neurons acutely isolated from rats with spinal nerve ligation (SNL)-induced neuropathic

Abbreviations: ER, Endoplasmic reticulum; JSR, Junctional sarcoplasmic reticulum; NADH, Nicotinamide adenine dinucleotide; NBC, Neurobasal Complete medium; Sch C, Schekwanglupaside C; SERCA, Sarcoplasmic reticulum Ca2 + -ATPase; SCOs, Spontaneous calcium oscillations; TG, Thapsigargin ⁎ Corresponding authors. E-mail addresses: [email protected] (D. Yu), [email protected] (Z. Cao). 1 Equally contributed. https://doi.org/10.1016/j.fitote.2019.04.005 Received 24 February 2019; Received in revised form 6 April 2019; Accepted 13 April 2019 Available online 14 April 2019 0367-326X/ © 2019 Published by Elsevier B.V.

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pain [15,16]. This dysregulated level of resting Ca2+ may derive from the depletion of Ca2+ stores in the endoplasmic reticulum (ER) as a consequence of activity loss of sarco-endoplasmic reticulum Ca2+- ATP (SERCA) [16–19]. Herein reported a new lupane saponin, schekwanglupaside C (Sch C), from the ethanolic extract of S. kwangsiensis. Sch C alters the pattern of spontaneous Ca2+ oscillations (SCOs) in primary cultured cortical neurons similar to that of SERCA activator, CDN1163. Moreover, we demonstrate that Sch C is a potent SERCA activator (EC50 = 1.20 μM).

2 h and dried. After addition of 0.2 mL of N-trimethylsilylimidazole, the mixture was kept at 60 °C for another 2 h and then extracted with 2 mL of n-hexane. This extraction was analyzed by gas chromatography using the following condition: capillary column, HP-5 (30 m × 0.25 mm, Dikma, Beijing, China); detection, flame ionization detector; detector temperature, 280 °C; injection temperature, 250 °C; column initial temperature 160 °C, then increased to 280 °C at 5 °C/min, and maintained for an additional 10 min; carrier, N2 gas. The presence of Dglucuronic acid, D-glucose, and L-rhamnose in Sch C were confirmed by comparing the retention time of the derivatives with those of authentic sugars reacted by the same method, which peaked at 28.79, 27.96, and 22.31 min respectively.

2. Materials and methods 2.1. Materials and chemicals

2.5. Primary cortical neuron culture The leaves and stems of S. kwangsiensis were collected in Fusui, Guangxi, China in September 2011. These were authenticated by Prof. Lin Ma from the Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, and a voucher specimen (ID-22162) was deposited at the same institute. Cytosine β-Darabinofuraboside, DNase I, thapsigargin (TG), MgATP and nicotinamide adenine dinucleotide (NADH) were purchased from Sigma-Aldrich (St. Louis, MO). Fetal bovine serum, trypsin and soybean trypsin inhibitor, neurobasal medium, Fluo-4/AM, and Gluta-Max were obtained from Life Technology (Grand Island, NY). Poly- L-lysine was purchased from Peptides International Inc. (Louisville, KY).

All animal experiment protocols were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications No. 8023, revised 1978) approved by the China Pharmaceutical University Institutional Animal Care and Use Committee. The procedure used for culturing cortical neurons from postnatal day 0–1 (both male and female) C57BL/6 J mice was described previously [20]. In brief, dissociated cells were suspended in pre-warmed Neurobasal Complete medium [Neurobasal medium supplemented with NS21 (2%, v/v), Gluta-Max (1%, v/v), HEPES (10 mM)] containing 5% FBS. Suspended cells were plated onto poly-L-lysine (0.5 mg/mL in borate buffer) pre-coated 96-well imaging plate (Corning Life Sciences, Acton, MA) at a density of 100,000 cells/ well for measuring SCOs. A final concentration of 5 μM of cytosine β-Darabinofuranoside dissolved in FBS-free Neurobasal Complete medium was added at 24–30 h after plating. The neurons were maintained at 37 °C with 5% CO2 and 95% humidity until use at 9–10 days in vitro (DIV).

2.2. General experimental procedures The optical rotation and molecular mass were determined using a JASCO P-2000 polarimeter and an Agilent 1100 LC/MSD ion trap mass spectrometer, respectively. NMR spectra were obtained from a Varian VNS-600 spectrometer. Preparative reversed-phase HPLC (RP-HPLC) was conducted on a Shimadzu HPLC system equipped with two LC-6AD pumps, a YMC-Pack ODS-A column (250 × 20 mm, 5 μm) and an SPD20A detector. TLC was performed on GF254 plates (Qingdao Marine Chemical Factory, Qingdao, China). Diaion HP-20 (Mitsubishi Chemical Industries Ltd., Tokyo, Japan), silica gel (100–200 mesh and 200–300 mesh, Qingdao Marine Chemical Factory), ODS (50 μm, YMC Co. Ltd., Kyoto, Japan), and Sephadex LH-20 (GE healthcare, Boston, MA) were used for column chromatography.

2.6. Measurement of SCOs in primary cultured neurons Intracellular Ca2+ dynamics in cultured neurons were measured as previously described [21]. Briefly, after incubation for 1 h with the Ca2+ indicator, Fluo-4/AM (4 μM), neurons were gently rinsed with pre-warmed Locke's buffer (in mM: HEPES 8.6, KCl 5.6, NaCl 154, Dglucose 5.6, MgCl2 1.0, CaCl2 2.3, and glycine 0.1, adjust pH to 7.4 with NaOH) leaving 175 μL in each well. The plate was then loaded onto the imaging chamber of a fluorescent imaging plate reader (FLIPRtetra, Molecular Devices, Sunnyvale, CA). After a 4-min baseline recording, a 25 μL of Sch C (8 x final concentration dissolved in Locke's buffer with 0.1% DMSO) was added and the fluorescent signals were recorded for an additional 8 min. The emitted fluorescence signals were recorded at 515–575 nm with 488 nm excitation. Data were presented as F/F0, where F is the fluorescent signal at different time points and F0 is the basal fluorescence. To determine the response of compounds on SCOs, the SCO frequency and amplitude from an epoch of 8 min after addition was automatically counted by Origin 9.0 (Origin Lab Corporation). An event having F/F0 > 0.05 units was considered to be an SCO and was included in the analysis of frequency and amplitude. All the data were presented as % vehicle.

2.3. Extraction, isolation and purification of Sch C Dried stems and leaves of S. kwangsiensis (15.4 kg) were extracted under reflux, and the combined solution was concentrated to a residue (940.0 g) that was suspended in water and extracted with petroleum ether, EtOAc and n-BuOH, subsequently. The n-BuOH partition (285.4 g) was loaded onto an HP-20 column eluted by step-gradient with 30%, 50%, 70%, and 95% EtOH-H2O. The fraction eluted with 70% EtOH-H2O (67.3 g) was separated by silica gel chromatography eluted with CH2Cl2-MeOH (30:1 → 1:1) to obtain 11 fractions. Fraction 10 (3.2 g) was submitted to MPLC and eluted with 40% → 100% MeOH in H2O to get 20 subfractions. The subfraction of 10−17 (0.3 g) was further fractionated by Sephadex LH-20 eluted with 70%–80% MeOHH2O to afford three new subfractions. Fraction of 10−17−2 (44.4 mg) was further applied to preparative HPLC and eluted with isocratic 76% MeOH-H2O (5 mL/min) to obtain Sch C (12.4 mg, tR = 46 min; 0.08% extraction yield from dry weight).

2.7. Preparation of junctional sarcoplasmic reticulum (JSR) vesicles Junctional sarcoplasmic reticulum (JSR) enriched sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) was isolated from fasttwitch skeletal muscles of 1-year old male (2–2.5 kg) New Zealand White rabbits, as previously described [22]. Briefly, 250 g of muscle was grounded and homogenized by a blender in buffer containing (in mM): sucrose 300, imidazole 5, phenylmethylsulfonyl fluoride 0.1 and 5 μg/mL leupeptin, pH 7.4. The fraction enriched with JSR was then isolated through a discontinuous sucrose gradient. The JSR preparation was re-suspended in storage buffer containing 300 mM sucrose and

2.4. Acid hydrolysis and sugar analysis Acid hydrolysis was performed by dissolving Sch C (2 mg) in 2 mL of 2 M HCl and heating for 15 h at 85 °C. After extraction with EtOAc, the aqueous layer was dried by rotary evaporation, and the residue was dissolved in a solvent with anhydrous pyridine (1 mL) and L-cysteine methyl ester hydrochloride (2 mg). The mixture was stirred at 60 °C for 2

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10 mM HEPES, pH 7.4 and flash frozen in liquid nitrogen followed by transferring to a − 80 °C freezer until use. Protein concentrations were determined using BCA assay.

Table 1 1 H NMR and No. 1a 1b 2a 2b 3

2.8. Measurement of SERCA activity Activity of SERCA from skeletal JSR was measured using a coupled enzyme assay that monitored the rate of oxidation of NADH at 340 nm as described previously [23]. In brief, each cuvette containing 1.5 mL of assay buffer that consisted of 25 μg/mL JSR protein, 7 mM HEPES, 143 mM KCl, 7 mM MgCl2, 0.085 mM EGTA, 0.048 mM free calcium, 0.43 mM sucrose, pH 7.0 (KOH), 1 mM phosphoenolpyruvate and 15 μL of coupling enzyme mixture (600–1000 units/mL pyruvate kinase, 900–1400 units/mL lactic dehydrogenase). After recording the baseline for ~3 min, a final concentration of 0.3 mM NADH was added to each cuvette followed by addition of a final concentration of 0.4 mM MgATP to initiate the oxidative reaction. Compounds were introduced before baseline establishment. TG (10 μM) was used as a positive control to determine SERCA-independent ATPase activity (non-specific oxidation) and to verify the successfulness of the assay system. SERCA activity was obtained by subtracting SERCA-independent ATPase activity from each group.

5 6a 6b 7a 7b 9 11a 11b 12a 12b 13 15a 15b 16a 16b 18 19

2.9. Data analysis

21a 21b 22a 22b 23 24 25 26 27

All data were presented as mean ± SEM and graphed using Prism GraphPad software (version 6.0, GraphPad Software Inc., San Diego, CA). The concentration-response curves were fitted using a non-linear regression equation with Prism GraphPad software (version 6.0). 3. Results and discussion

29a 29b 30

3.1. Structure determination of Sch C Sch C (Fig. 1) was obtained as a white amorphous powder. The molecular formula of Sch C was determined to be C64H104O27 according to the molecular ion peak at m/z 1303.6665 in the HRESIMS [M – H]− (calcd 1303.6695) (Fig. S1). The 1D NMR data of Sch C (Table 1) displayed the proton resonances of six methyls at δH 1.69 (H3-30), 1.30 (H3-23), 1.03 (H326), 1.02 (H3-24), 1.02 (H3-27), and 0.67 (H3-25), two olefinic protons at δH 4.68 (br s) and 4.84 (br s), and the carbon resonances of two olefinic carbons at δC 150.8 and 110.1, a carboxylic carbon at δC 174.9, altogether characteristics consistent with a skeleton of lup-20(29)-en-28-oic acid. 1H NMR showed an oxymethine proton resonance at δH 3.26 (dd, J = 12.0, 4.8 Hz), assigned to H-3 of the aglycone moiety. The α-orientation of H-3 was confirmed by a ROESY

1 2

13

C NMR data obtained for Sch C a.

δH

No. b

δH b

δC

No.

δC

1 2 3 4 5

39.0 28.3 90.1 39.8 56.0

1′ 2′ 3′ 4′

65.0 30.9 19.3 13.8 Glc I 102.0 78.5 79.4 72.7 77.9 63.4 Rha I 102.0 72.4 72.7 74.3 69.5 19.1 Glc II 95.4 74.3 77.1

1.52 m 0.84 mb 2.08 mb 1.24 mb 3.26 dd (12.0, 4.8) 0.71 d (11.4) 1.44 mb 1.28 mb 1.56 mb 1.27 mb 1.30 mb 1.31 mb 1.11 mb 1.84 mb 1.10 mb 2.61 mb 1.97 mb 1.24 mb 2.61 mb 1.50 mb 1.70 mb 3.36 td (10.8, 4.8) 2.05 mb 1.38 mb 2.10 mb 1.48 mb 1.30 s 1.02 s 0.67 s 1.03 s 1.02 s

3 4 5 1′ 2′

4.55 4.32 4.48 4.23 1.57

3′ 4′

1.30 mb 0.75 t (7.8) Glc I 5.83 d (7.2) 4.27 mb 4.22 mb 4.05 mb 3.83 m 4.47 mb 4.28 mb Rha I 6.40 br s 4.75 br s 4.66 mb 4.33 mb 5.01 m 1.78 d (6.6)

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

18.5 34.6 41.2 50.8 37.1 21.1 26.0 38.4 42.8 30.2 32.2 57.0 49.8 47.5 150.8 30.9 36.9

1 2 3 4 5 6

1 2 3 4 5 6a 6b

Glc II 6.31 d (8.4) 4.12 t (8.4) 4.23 mb 4.53 mb 3.81 m 4.17 mb 4.06 mb Rha II

23 24 25 26 27 28 29 30

4 5 6

4.84 br s 4.68 br s 1.69 s 6-O-n-butyl-Glu A 4.98 d (7.2) 4.48 (o)

1 2 3 4

5.90 4.67 4.54 4.31

1 2 3 4

28.3 16.6 16.3 16.3 14.8 174.9 110.1 19.4 6-O-n-butyl-Glu A 105.5 78.3 78.7 73.3

5 6

4.95 m 1.68 d (6.0)

5 6

76.9 170.2

1 2 3 4 5 6a 6b 1 2 3 4 5 6

m mb mb mb mb

No.

br s br s mb mb

1 2 3 4 5 6 1 2 3

1 2 3 4 5

77.8 77.9 61.1 Rha II 102.7 72.6 72.7 73.9 70.4

6

18.6

a1 H NMR data (δH) were measured in pyridine‑d5 at 600 MHz; 13C NMR data (δC) were measured in pyridine‑d5 at 150 MHz. bsignals partially overlapped, assignments were made using 1D and 2D NMR spectra together.

correlation (Fig. 2) between H-3 and H-5 (δH 0.71, d, J = 11.4 Hz). Thus, the aglycone of Sch C was determined to be 3β-hydroxylup20(29)-en-28-oic acid (betulinic acid) [24]. Analysis of hydrolytic trimethylsilyl L-cysteine derivatives revealed that schekwanglupaside C

Fig. 1. The structure of Sch C. 3

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Veh/Sch C

A

Ctrl Sch C (0.12 µM)

F/F0=2

Sch C (0.38 µM) Sch C (1.15 µM) Sch C (3.83 µM) Sch C (11.51 µM) 0

100

200

B

300

400

500

600

700

Time(s)

SCO frequenc y (% Ctrl)

150 Fig. 2. The key HMBC and ROESY correlations of Sch C.

contained D-glucuronic acid, D-glucose and L-rhamnose moieties with a proportion of 1:2:2. In addition to resonances of the aglycone, the 1H NMR spectrum displayed the resonances for five anomeric protons at δH 4.98 (d, J = 7.2 Hz), 5.83 (d, J = 7.2 Hz), 6.40 (br s), 6.31 (d, J = 8.4 Hz), and 5.90 (br s), which correlated to δC 105.5, 102.0, 102.0, 95.4 and 102.7 in the HSQC spectrum, respectively. The 3JH1, H2 value of 7.2 Hz indicated β-anomeric configurations for the glucuronic acid unit and two glycose units, whereas the two rhamnose units were deduced to be α-anomers by associated 3JH1, H2 values < 1.5 Hz. The HMBC correlations observed (Fig. 2) between Glu A-H-1 (δH 4.98) and C-3 (δC 90.1), Glc I-H-1 (δH 5.83) and Glu A-C-2 (δC 78.3), Rha-H-1 (δH 6.40) and Glc I-C-2 (δC 78.5) allowed for the assignment of a sugar chain as being α-L-rhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-β-D-glucuronopyranoside. The 1H and 13C NMR spectrum also displayed the resonances for a butyl group [δH 4.23 (H-1′), 1.57 (H-2′), 1.30 (H-3′), and 0.75 (H-4′); δC 65.0 (C-1′), 30.9 (C-2′), 19.3 (C-3′), and 13.8 (C-4′)]. The position of this group was assigned at Glu A-C-6 (δC 170.2) based on the HMBC correlation between H-1′ and this carbon. In addition, the HMBC spectrum showed correlations between Glc II-H-1 (δH 6.31) and C-28 (δC 174.9), Rha-H-1 (δH 5.90) and Glc II-C-4 (δC 77.8), which allowed for the assignment of another sugar chain as being α-L-rhamnopyranosyl-(1 → 4)-β-D-glucopyranoside at C-28. Accordingly, the total structure of Sch C was determined to be 3β-O-[α-Lrhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-β-D-(6-n-butylglucuronopyranosyl)] betulinic acid 28-O-[α-L-rhamnopyranosyl-(1 → 4)-β-D-glucopyranoside] (Fig. 1). The assignment of NMR data presented in Table 1 was achieved based on complete analysis of the 1D and 2D NMR spectra (Fig. S2-S8). We previously demonstrated that the ester-containing saponins were derived artificially during the extraction and isolation procedure but not naturally occurred by using LC-HR-ESIMS [9]. Accordingly, Sch C is likely the n-butanol ester of 3β-O-[α-Lrhamnopyranosyl-(1 → 2)-β-D-glucopyranosyl-(1 → 2)-β-D-glucuronopyranosyl] betulinic acid 28-O-[α-L-rhamnopyranosyl-(1 → 4)-β-Dglucopyranoside] which is not reported previously.

125 100 75 50 25 0 Ctrl

C

-7.0

-6.5

-6.0

-5.5

-5.0

-5.5

-5.0

Log [Sch C] M

SCO amplitude (% Ctrl)

150 125 100 75 50 25 0 Ctrl

-7.0

-6.5

-6.0

Log [Sch C] M Fig. 3. Sch C suppresses SCO in primary cultured cortical neurons. (A) Representative traces of neuronal SCOs in the absence and presence of different concentrations of Sch C as a function of time. The arrowhead indicates the addition of Veh (0.1% DMSO) or Sch C. (B) Concentration-response relationships for Sch C suppression of SCO frequency. (C) Concentration-response relationship for Sch C suppression of SCO amplitude. Each data point represents mean ± SEM (n = 3). This experiment was repeated three time each in triplicate with similar results.

of 2–3 min, followed by a gradual decrease on the amplitude of SCO as a function of time (Fig. 3A). Higher concentration (11.51 μM) of Sch C produced a gradual decrease in the amplitude of SCO (Fig. 3A). The overall response of Sch C on the frequency and amplitude of SCO was inhibitory and was concentration-dependent (Fig. 3). The IC50 values for Sch C suppression of the frequency and amplitude of SCO were 1.7 μM [1.22–2.50 μM, 95% Confidence Interval (95% CI)] and 2.5 μM (1.22–5.17 μM, 95% CI), respectively (Fig. 3B&C). Many channels affecting membrane excitability and Ca2+ permeation can alter the spatiotemporal pattern of SCOs [21,26]. The spatiotemporal pattern of Sch C response on SCOs is distinct to that of tetrodotoxin, a voltage-gated sodium channel (VGSC) blocker, which, upon addition, immediately abolishes SCOs [26]. L-type channel blocker, nifedipine, and the Na+Ca2+ exchanger inhibitor, KBR7943, have no effect on the frequency and amplitude of SCO in mice cortical neuronal cultures [26,28]. Moreover, although activities of inotropic glutamate receptors and GABAA receptors are all able to modulate the SCO in primary cultured neuronal network, modulation of these channels is not able to completely abolish the SCOs [26,28,29]. Considered together, these data

3.2. Sch C modulates SCOs in primary cultured neocortical neurons Cultured neocortical neurons form neuronal network connectivity and display SCOs [25]. These SCOs are dependent on action potential firing, and the frequency and amplitude of SCOs are determined by the balance of excitatory ionotropic glutamatergic and inhibitory GABAergic neurotransmission [26,27], therefore representing the neuronal excitability which is essential for nociception. S. kwangsiensis is a medicinal plant that has been used clinically to treat pain [1]. We therefore determined whether Sch C could modulate Ca2+ dynamics in primarily cultured cortical neurons. Addition of Sch C altered the SCO dynamics. An intermediate concentration of Sch C (1.15 μM) produced an increase on the frequency and amplitude of SCO in the initial period 4

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A

Veh/TG

Veh

'F/F 0 =1

TG (10 µM)

400 S

B

Veh/CDN1163

Veh

'F/F 0 =1

30 PM

CDN163

10 PM

100 PM 200 S

Fig. 4. Representative traces of SERCA inhibitor, thapsigargin (TG, 10 μM) (A), and SERCA activator, CDN 1163 (B) on SCOs in primary cultured cortical neurons.

suggest that Sch C response on SCO is not likely derived from modulation of VGSC, L-type Ca2+ channels, Na+-Ca2+ exchangers, inotropic glutamate receptors and GABAA receptors.

Fig. 5. Sch C activates SERCA in JSR preparation. (A) Representative traces showing NADH oxidation in a coupled enzyme assay by measuring the absorbance at 340–400 nm. Thapsigargin (TG) was used as a positive control. Veh (trace a, 0.1% DMSO), TG (trace g, 10 μM) or different concentrations of Sch C (traces b-f) were introduced in the buffer before addition of NADH and MgATP to initiate the reaction. (B) Concentration response relationship curve for Sch C increasing NADH oxidation. Each data point represents the mean ± SEM (n = 4).

3.3. Influence of thapsigargin and CDN1163 on the SCOs in cortical neuronal cultures In addition to extracellular cellular Ca2+ influx, Ca2+ release from intracellular Ca2+ store and Ca2+ reuptake also influence SCO dynamics. Thapsigargin (TG), an irreversible inhibitor of SERCA pump, was shown to moderately decrease the frequency and amplitude of SCO [26]. We also found that application of TG gradually increased the intracellular Ca2+ concentration in primary cultured cortical neurons with concomitant suppression of both the frequency and amplitude of SCO (Fig. 4A). We next examined whether activation of SERCA pump was able to mimic the spatiotemporal pattern of Sch C on SCOs. CDN1163 increased the frequency and amplitude of SCO right after addition followed by a gradually decreased the amplitude of SCO as a function of time, eventually to a complete suppression of SCOs (Fig. 4B). The time to reach complete suppression of SCOs of CND1163 was much longer than that of Sch C. This is likely because of the distinct rates for Sch C and CDN1163 entering to the cells. It has been demonstrated that U73122, a phospholipase C inhibitor, gradually inhibits the amplitude of SCO in cortical neuronal cultures but without initial augmentation of SCOs [26]. However, due to the unavailability of selective antagonist of IP3 receptors, we are not able to test direct inhibition of IP3 receptors on the modulation of SCOs. Thus, we are not able to rule out the effect of Sch C on the IP3 receptors signaling. However, CDN1163, the SERCA activator, produced a similar spatiotemporal pattern on SCO to that of Sch C highly suggesting that activation of SERCA pump was sufficient to modulate the SCO in cortical neurons.

activity using a coupled enzyme assay. As shown in Fig. 5, Sch C increased SERCA activity in a concentration-dependent manner, with an EC50 value of 1.2 μM (0.56 μM–2.56 μM, 95% CI). The comparable potency of Sch C on both SERCA activation and suppression of SCOs suggests that the activation of SERCA pumps by Sch C may contribute to the modulation of SCO. Although many channels/receptors contribute to the generation of SCOs, it is generally agreed that SCO is a dynamic process between Ca2+-induced Ca2+ release from intracellular Ca2+ store and re-uptake through SERCA or efflux through PMCA [30–33]. Although in this study, we could not elucidate the mechanism of how SERCA activation modulates the SCOs. The initial increase of SCO is possible derived from the higher Ca2+ concentration in the store due to activation of SERCA pump which may increase the Ca2+ release from intracellular Ca2+ store. The suppression of SCO can be explained by that activation of SERCA pump can rapidly abolish the increase of intracellular Ca2+ which is required for Ca2+-induced Ca2+ release [30,31]. Additionally, since our recording measures the synchronous Ca2+ signals in a population of neuronal cells, these SCOs are dependent on the synaptic transmission [21,26]. It is also possible that activation of SERCA pumps counters the increase in intracellular Ca2+ which is required for Ca2+-dependent synaptic neurotransmission [34]. 4. Conclusions

3.4. Sch C activates SERCA activity in JSR preparation In this study, a new lupane saponin was isolated from the pain-relieving medicinal plant, S. kwangsiensis, and was designated as schekwanglupaside C (Sch C). It was further demonstrated that Sch C was a

Given the similar spatiotemporal patterns on the modulation of SCO by CDN1163 and Sch C, we next evaluated influence of Sch C on SERCA 5

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potent SERCA activator that modulated SCOs in cortical neuronal cultures, with spatiotemporal pattern similar to CDN1163, suggesting that activation of SERCA pump activity was sufficient to modulate SCOs in primary cultured cortical neurons. Diminished SERCA activity has already been demonstrated in the sensory neurons derived from SNL rats [15,18]. It has also been reported that CDN1163, the only currently available SERCA activator, improves memory and behaviors in neurodegenerative diseases [35,36], protects the liver from chemical-mediated damages [37], as well as attenuates diabetes and metabolic disorders [38] through reducing ER stress triggered by loss of SERCA activity. Therefore, Sch C may represent an important lead structure for developing new drugs for the treatment of neuropathic pain, Alzheimer's disease, diabetes and metabolic disorders [38].

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Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 81473539 and 21777192); the National Key R&D program of China (No. 2018YFF0215200); the National Science and Technology Major Projects for “Major New Drugs Innovation and Development” (No. 2018ZX09101003-004-002); the “Double firstClass” project of China Pharmaceutical University (No. CPU2018GY18); the Knowledge Innovation Program Funding of Institute of Food Science and Technology, Chinese Academy of Agricultural Sciences (No. 125161015000150013), and a grant from the China Agriculture Research System (No. CARS-21). Author contributions Experiment design: ZC, DY, RC. Experimentation: GY, YW, YY, JZ, JC, SL. Data analysis: JC, SL, CBN, CZ. Paper writing: GY, YW, JZ, CBN. Conflicts of interest The authors declare no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fitote.2019.04.005. References [1] Commission, C.P, Pharmacopoeia of the People's Republic of China, 1 China Medical Science Press, 1977, pp. 193–195. [2] A. Panthong, D. Kanjanapothi, W.C. Taylor, Ethnobotanical review of medicinal plants from Thai traditional books, part I: plants with anti-inflammatory, antiasthmatic and antihypertensive properties, J. Ethnopharmacol. 18 (1986) 213–228. [3] O. Pancharoen, P. Tuntiwachwuttikul, W.C. Taylor, K. Picker, Triterpenoid glycosides from Schefflera lucantha, Phytochemistry. 35 (1994) 987–992. [4] L. Zhang, Y. Wang, D.Q. Yu, Simultaneous quantification of six major triterpenoid saponins in Schefflera kwangsiensis using high-performance liquid chromatography coupled to orbitrap mass spectrometry, Nat. Prod. Res. 29 (2015) 1350–1357. [5] Q. Zhang, J.S, Y.W. Zhao, Z.Z. Wang, W. Xiao, Study on glycosides from stems of Schefflera kwangsiensis, Chin. Tradit. Herb. Drugs. 43 (2012) 2141–2145. [6] C.Q. Wang, Y. Wang, W.J. Wang, L. Wang, W.C. Ye, New oleanane saponins from Schefflera kwangsiensis, Phytochem. Lett. 10 (2014) 268–271. [7] Y. Wang, L. Wang, W.J. Wanga, X.Q. Zhang, H.Y. Tian, Q.W. Zhang, et al., New triterpenoid saponins from the aerial parts of Schefflera kwangsiensis, Carbohydr. Res. 385 (2014) 65–71. [8] Y. Wang, C.L. Zhang, Y.F. Liu, D. Liang, H. Luo, Z.Y. Hao, et al., Hepatoprotective Triterpenoids and Saponins of Schefflera kwangsiensis, Planta Med. 80 (2014) 215–222. [9] Y. Wang, L.L. Zhang, C.L. Zhang, Y.F. Liu, D. Liang, H. Luo, et al., Esters of new oleanane-type triterpenoid saponins from Schefflera kwangsiensis, Phytochem. Lett. 11 (2015) 95–101. [10] Y. Wang, Y.F. Liu, C.L. Zhang, D. Liang, H. Luo, Z.Y. Hao, et al., Four new triterpenoid saponins isolated from Schefflera kwangsiensis and their inhibitory activities against FBPase1, Phytochem. Lett. 15 (2016) 204–209.

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