Planta Med 2018; 84(16): 1165-1173
DOI: 10.1055/a-0619-5710
Biological and Pharmacological Activity
Original Papers
Georg Thieme Verlag KG Stuttgart · New York

Differences in Neuritogenic Activity and Signaling Activation of Madecassoside, Asiaticoside, and Their Aglycones in Neuro-2a cells

Nonthaneth Nalinratana
1   Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand
,
Duangdeun Meksuriyen
2   Faculty of Pharmacy, Rangsit University, Pathum Thani, Thailand
,
Boonsri Ongpipattanakul
1   Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand
3   Chulalongkorn University Drugs and Health Products Innovation and Promotion Center, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, Thailand
› Author Affiliations
Further Information

Correspondence

Asst. Prof. Dr. Boonsri Ongpipattanakul
Department of Biochemistry and Microbiology
Faculty of Pharmaceutical Sciences
Chulalongkorn University
254 Phyathai road, Pathumwan
10330 Bangkok
Thailand   
Phone: + 66 22 18 83 66   
Fax: + 66 22 18 83 75   

Publication History

received 15 January 2018
revised 06 April 2018

accepted 18 April 2018

Publication Date:
02 May 2018 (online)

 

Abstract

Madecassoside (MS) and asiaticoside (AS) along with their aglycones, madecassic acid (MA) and asiatic acid (AA), are considered the major neuroactive triterpenoid constituents of Centella asiatica. In this study, we aimed to compare MS, AS, MA, and AA for their neurite outgrowth activities and mechanisms in Neuro-2a cells. Immunofluorescent cell staining showed MS and AS significantly increased the percentage of neurite-bearing cells (%NBC) and the neurite length with higher potency than MA and AA. The triterpenoid glycosides induced sustained extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) phosphorylation, while their aglycones activated only transient signaling of ERK1/2. Suppression of ERK1/2 activation significantly abolished not only cAMP response element-binding protein (CREB) phosphorylation but also the increment of %NBC and neurite length in MS- and AS-treated cells. Inhibition of ERK phosphorylation did not produce similar blockage of CREB activation and neurite outgrowth in MA- and AA-treated cells. On the other hand, inactivation of protein kinase B (Akt) resulted in a suppression of neurite lengthening in all studied triterpenoids. This is the first study discerning the different signaling pathways of neurite outgrowth activity induced by C. asiatica triterpenoid glycosides and aglycones. Neurite outgrowth activity of the glycosides MS and AS was found to involve the activation of sustained ERK phosphorylation leading to CREB activation, while ERK activation was not associated with MA- and AA-induced neurite outgrowth. In addition, Akt activation was evident to be more involved in neurite elongation process.


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Abbreviations

%NBC: percentage of neurite-bearing cells
AA: asiatic acid
Akt: protein kinase B
AS: asiaticoside
CREB: cAMP response element-binding protein
EGF: epidermal growth factor
ERK1/2: extracellular signal-regulated protein kinases 1 and 2
GAPDH: glyceraldehyde 3-phosphate dehydrogenase
HRP: horseradish peroxidase
Log P: octanol-water partition coefficient
LSD: least significant difference
MA: madecassic acid
MEK: mitogen-activated protein kinase
MS: madecassoside
NGF: nerve growth factor
PI3K: phosphoinositide 3-kinase
PVDF: polyvinylidene fluoride
Trk: tropomyosin receptor kinase
 

Introduction

Extension and remodeling of neurite processes is critical for neuronal development, regeneration, differentiation, and response to injury. In aged brains, the progressive deterioration of neural network such as neurite degeneration, neuronal atrophy, and loss of synapses usually occurs, leading to neurodegenerative conditions and cognitive impairment in the elderly. Increasing dendritic complexity such as dendrite length and dendritic spine density in cortex and hippocampus was observed in animals that exhibited improvement of performance in memory-related behavioral tests [1], [2]. An association between neurite formation and memory is therefore often suggested. As a result, the measurement of neuronal morphological changes is adopted as a possible primary tool to examine the ability of various compounds to modulate neuronal degeneration and memory impairment [3].

Centella asiatica (L.) Urban (Apiaceae) is traditionally used to promote brain health and function in India, China, and Southeast Asian countries. The major chemicals found in this plant are pentacyclic triterpenoid saponins, which mainly are MS and AS, and their aglycone derivatives (i.e., MA and AA) [4]. C. asiatica extracts were shown in various animal models to exert memory improvement and neuroprotection effects [5]. One such C. asiatica extract is a standardized extract ECa233 that contains 80% mixture of MS and AS at a ratio of 1.5 (± 0.5) to 1 respectively. The extract was established from an activity-guided separation based on anti-amnesic effect in memory-impairment mice [6]. ECa233 was shown to possess both neuritogenic effect in human neuroblastoma IMR-32 cells [7] and anxiolytic effect in mice [8]. Other C. asiatica extracts constituting different triterpenoid contents and combinations such as AA alone, mixture of MA and AA, and mixture of MA, AA, and MS could also induce similar neuronal effects in both in vitro and in vivo assays [9], [10], [11].

At the molecular level of the neurite outgrowth regulation, the activation of signaling pathways such as MEK/ERK1/2 and PI3K/Akt pathways including activation of CREB were suggested to be involved [12], [13]. C. asiatica extracts including AA alone or a combination of MA and AA were able to induce neurite outgrowth by modulating MEK/ERK1/2, PI3K/Akt, or CREB signaling [7], [9], [10], [14]. However, the identification of major neuroactive triterpenoids in C. asiatica extracts was still controversial by some findings that a combination of MA, AA, and MS was able to increase the expression of neurofilament 68 kDa, a biomarker for neurite outgrowth, in PC12 cells, while individual compound was unable [15]. Therefore, the present study was aimed to comparatively evaluate the neuritogenic effect between triterpenoid glycosides (MS and AS) and aglycones (MA and AA) from C. asiatica by using neurite outgrowth assay in mouse neuroblastoma Neuro-2a cells. Furthermore, the signaling mechanism of each compound was also investigated.


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Results

To determine the nontoxic concentration against Neuro-2a cells, cell viability after exposure to each studied triterpenoid was examined using MTT assay. MS and AS at concentrations up to 100 µM caused neither cytotoxicity nor cell proliferation at all incubation time points, 6, 12, or 24 h ([Fig. 1]). In contrast, 100 µM AA but not MA significantly decreased cell viability after 6 h of incubation ([Fig. 1 A]). Moreover, cell viability was dramatically decreased by treatment with 100 µM of either MA or AA at 12 or 24 h ([Fig. 1 B] and [C]). Therefore, concentrations at 50 µM and below were selected as the condition for further investigation of neurite outgrowth.

Zoom Image
Fig. 1 Effects of individual C. asiatica triterpenoid on Neuro-2a cell viability by MTT assay. Cells were treated with various concentrations (5 – 100 µM) of MS, AS, MA, and AA for (A) 6, (B) 12, and (C) 24 h. Data are presented as mean ± standard error estimated using Gaussian error propagation (n = 3). * p < 0.05 vs. control.

For the effects of C. asiatica triterpenoids on neurite outgrowth, the morphological changes of Neuro-2a cells were evaluated using immunofluorescent staining against βIII-tubulin. Neurite was identified as a filopodia-like protrusion with its length equal to or greater than a cell body diameter [16]. The untreated cells mostly appeared in round shape ([Fig. 2 A]; control panel), with 10 – 15% of the total untreated cells showing spontaneous neurites of average 40 µm in length. In the treatment groups, an increase in the %NBC compared to the untreated cells was observed at various effective concentrations after 6, 12, or 24 h of incubation. For the glycosides, the effective concentration was seen varying from 0.1 to 50 µM for MS and 1 to 50 µM for AS ([Fig. 2 B] and [D]). The aglycone MA significantly induced neurite-bearing cells at 10 and 50 µM ([Fig. 2 F]), while AA appeared consistently effective at 10 µM for all incubation times ([Fig. 2 H]).

Zoom Image
Fig. 2 Effects of individual C. asiatica triterpenoid on neurite outgrowth in Neuro-2a cell. A Representative immunofluorescent images of cells treated with 10 µM of MS, AS, MA, and AA for 6, 12, and 24 h. Cells were stained with primary antibody against βIII-tubulin followed by fluorescent AlexaFluor 488 secondary antibody (green fluorescence), and nuclei were also stained with Hoechst33342 (blue fluorescence). Images were merged and analyzed by using ImageJ software. Histograms show the %NBC and average neurite length in Neuro-2a cells treated with 0.01 – 50 µM of (B, C) MS, (D, E) AS, (F, G) MA, and (H, I) AA. Data are presented as mean ± SEM (n = 4). * p < 0.05 vs. control.

For neurite elongation effect, MS and AS at 1, 10, and 50 µM significantly increased average neurite length (50 – 80 µm), particularly at 12 or 24 h of incubation when compared to those of untreated cells (~ 40 µm) ([Fig. 2 C] and [E]). Incubating cells with AA at 1, 10, and 50 µM for 6 h also significantly increased average neurite length ([Fig. 2 I]). Interestingly, longer incubation with AA (12 or 24 h) could increase average neurite length (~ 50 µm) at a concentration as low as 0.01 µM, whereas a higher concentration of AA at 50 µM had virtually no effect on neurite lengthening ([Fig. 2 I]). In contrast, MA showed minimal effect on neurite length, and the only noticeable changes were observed at 10 µM (24 h) and 50 µM (12 or 24 h) ([Fig. 2 G]). Therefore, the neurite outgrowth assay indicated that when C. asiatica triterpenoids were compared at similar concentrations, the glycoside derivatives significantly showed higher potency in enhancing both %NBC and average neurite length than their aglycones (MS versus MA and AS versus AA). As for the comparison of MS against AS and MA against AA, the 6β-hydroxylated derivatives (MS and MA) appeared to be more potent in the induction of neurite-bearing cells. However, for promoting neurite length, the nonhydroxylated derivatives were found to be significantly better. Moreover, we found that most triterpenoids in the study clearly exhibited neurite outgrowth activity at 10 µM. Subsequent mechanistic comparison was thus performed at this concentration.

Phosphorylation of ERK1/2 and Akt were known to be part of the signal transduction regulating neurite outgrowth [17]. All four triterpenoids in our study were found to increase ERK1/2 phosphorylation (p-ERK1/2) after 30 min of incubation. Nonetheless, MS and AS induced a sustained activation of ERK1/2 with the level of p-ERK1/2 significantly elevated from 30 min to 12 h of incubation ([Fig. 3 A, B] and [E]). Treatment with MA and AA, on the other hand, showed a more transient activation with elevated p-ERK1/2 observed beginning from approximately 30 – 60 min of incubation and restoring to the normal level after 4 h ([Fig. 3 C – E]). When compared at 30 min incubation, AA could increase the level of p-ERK1/2 at a greater magnitude than MA. No significant difference was found between MS and AS. For the phosphorylation of Akt (p-Akt), all studied triterpenoids significantly increased p-Akt at the onset of 1 h of incubation and restored to the baseline level within 4 h ([Fig. 3 F]). It was noted that MA, among the studied triterpenoids, induced p-Akt with the strongest magnitude. Activation of CREB by phosphorylation (p-CREB) at Ser133 could be modulated by several signaling pathways, including ERK1/2 and Akt. All studied triterpenoids significantly induced p-CREB at the onset of 30 – 60 min of incubation, which peaked at around 1 – 2 h and subsided to the normal level by 4 h ([Fig. 3 G]). MS and AS showed higher magnitudes of p-CREB induction compared to MA and AA.

Zoom Image
Fig. 3 Time-dependent studies on phosphorylation of ERK1/2, Akt, and CREB in Neuro-2a cells after treatment with individual C. asiatica triterpenoid. Representative immunoblots of cells treated with 10 µM of (A) MS, (B) AS, (C) MA, and (D) AA at indicated time points (0.5 – 12 h). The intensity of GAPDH was used for normalization as a loading control. Histograms show densitometric analysis of (E) ERK1/2, (F) Akt, and (G) CREB phosphorylation. Data are presented as mean ± standard error estimated using Gaussian error propagation (n = 3). * p < 0.05 vs. control.

To substantiate that ERK1/2 and Akt were involved in initiating CREB activation, MEK1/2 inhibitor PD098059 and PI3K inhibitor LY294002 were used to inhibit ERK1/2 and Akt signaling pathways, respectively. Pretreatment with PD098059 or LY294002 for 30 min prior to incubation with individual triterpenoid successfully inhibited the formation of p-ERK1/2 and p-Akt accordingly ([Fig. 4 A – D]). However, inhibition of ERK1/2 signaling by PD098059 abolished only p-CREB in MS- and AS-treated cells ([Fig. 4 E]) with no observable changes of p-CREB for MA- or AA-treated groups ([Fig. 4 F]). Furthermore, inhibition of Akt did not suppress p-CREB induced by all studied triterpenoids, suggesting that Akt phosphorylation might not be involved in the activation of CREB ([Fig. 4 E] and [F]).

Zoom Image
Fig. 4 Effects of ERK1/2 and Akt inhibitions on CREB phosphorylation in MS- and AS-treated Neuro-2a cells. A PD098059 pretreatment effect on MS- and AS-induced ERK phosphorylation at 1 h. B LY294002 pretreatment effect on MS- and AS-induced Akt phosphorylation at 1 h. C PD098059 pretreatment effect on MA- and AA-induced ERK phosphorylation at 1 h. D LY294002 pretreatment effect on MA- and AA-induced Akt phosphorylation at 1 h. E MS- and AS-induced CREB phosphorylation in the presence and absence of PD098059 or LY294002. F MA- and AA-induced CREB phosphorylation in the presence and absence of PD098059 or LY294002. Data are presented as mean ± standard error estimated using Gaussian error propagation (n = 3). * p < 0.05 vs. control.

To further confirm that the activation of ERK1/2 and Akt was associated with morphological change, cells were pretreated with either PD098059 or LY294002 followed by triterpenoid incubation and immunofluorescent staining to monitor neurite outgrowth. In inhibited MS- and AS-treated cells, suppression of ERK1/2 phosphorylation strongly negated the increment of both %NBC and average neurite length when compared to noninhibited cells ([Fig. 5 A]). In contrast, inactivation of Akt affected only the neurite length but not %NBC ([Fig. 5 A]). In inhibited MA- and AA-treated cells, inhibition of ERK1/2 phosphorylation did not suppress the increment of both %NBC and average neurite length, while inhibition of Akt suppressed only neurite lengthening ([Fig. 5 B]). These suggested that ERK1/2 activation was involved in the induction of neurite outgrowth activity by C. asiatica triterpenoid glycosides but not aglycones, while Akt activation was involved in regulation of neurite length for both C. asiatica triterpenoid glycosides and aglycones.

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Fig. 5 Effects of ERK1/2 and Akt inhibitions on neurite outgrowth in MA and AA treated Neuro-2a cells. Histograms represent the %NBC and average neurite length observed in the presence and absence of inhibitor pretreatment in cells subjected to (A) MS and AS and (B) MA and AA. Data are presented as mean ± SEM (n = 3). * p < 0.05 vs. control.

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Discussion

Several pharmacological studies suggested the potential of C. asiatica extract to mitigate symptoms of various neurodegenerative diseases such as memory impairment, anxiety, and locomotor dysfunction [6], [8], [14], [18]. Neurite formation is considered one of the key processes in neuro-regeneration, especially for memory improvement [2]. Therefore, an in vitro assessment of neurite outgrowth capacity has been adopted for screening of neuroactive compounds as well as for elucidating the regulation pathways of neuro-regeneration. MS, AS, MA, and AA, the pentacyclic triterpenoids found in C. asiatica, were among various natural products that showed positive neuroactivity. However, due to the variations of plant cultivation and extraction, the composition of C. asiatica extracts in many reports might be differed. These included extracts composed of the following combinations: MS and AS, MA and AA, together with purified triterpenoid such as AA [8], [9], [10]. All were demonstrated to be neuroactive using various in vitro and in vivo models, but there is not any one study that directly evaluates individual C. asiatica triterpenoids against each other. Given that the absorption and the cell permeability of triterpenoid glycosides and aglycones could be differed, it would be of interest to clarify and compare the potency and the mechanism of two major triterpenoid derivatives in C. asiatica, MA/AA and their glycosides in a control setup for neurite outgrowth evaluation.

Early neurite outgrowth consists of two main steps: neurite initiation and neurite elongation. Neurite initiation is a process that the neuronal sphere is broken, and neurites start to sprout out from the cell body. The neurite elongation is a step that neurites extend to increase their length [19]. These two steps are known to be regulated by different pathways. Neurite initiation mostly involves transcription factors that control the neuronal differentiation such as CREB, while neurite elongation is mainly regulated through the reorganization of cytoskeletal proteins [19], [20]. Therefore, the ability to increase the proportion of neurite-processing cells is evaluated by %NBC parameter, and the ability to promote the neurite length is determined by average neurite length measurement in neuronal-related cells such as Neuro-2a cells.

All studied triterpenoids, MS, AS, MA, and AA, showed the ability to induce neurite outgrowth by increasing %NBC and neurite length. However, we found the different potency on neurite outgrowth induction between glycosides and aglycones. MS and AS exhibited higher potency in increasing both %NBC and neurite length than MA and AA. C. asiatica and some of its triterpenoids were previously reported to modulate the activity of ERK1/2, Akt, or CREB, signaling proteins found to be associated with the initiation of neurite formation [7], [9], [10], [14]. In addition, CREB signaling was suggested to be regulated by the activation of ERK1/2 or Akt [14], [20], [21]. We thus hypothesized that the neurite outgrowth activity exerted by all studied C. asiatica triterpenoids might involve CREB activation, which could be regulated by either ERK1/2 or Akt signaling or both pathways. Monitoring the phosphorylation of postulated signaling proteins was then performed in either glycoside- and aglycone-treated cells.

All investigated triterpenoids clearly increased phosphorylation of ERK1/2, Akt, and CREB. However, our time-course study revealed that triterpenoid glycosides and aglycones induced different kinetics of ERK phosphorylation in Neuro-2a cells. Sustained ERK1/2 phosphorylation was observed with either MS or AS treatment, while transient ERK1/2 activation was triggered by MA and AA. Different longevity of ERK phosphorylation may consequently precipitate different cell responses [22]. Sustained ERK phosphorylation is one of the main transducing signals regulating neuronal differentiation and can be induced by NGF through TrkA receptor in PC12 cells [23]. Activation of TrkA receptor by NGF can trigger both Ras and Rap1, small GTPases acting as intracellular effectors and regulating the temporal specificity of Raf-MEK-ERK signals. A rapid and transient ERK activation can be initially stimulated by NGF through Ras, followed by a sustained activation through Rap1, which is possibly enabled by a stable FRS2-Crk-C3G-Rap1-B-Raf signaling complex [24]. Meanwhile, in other growth factor receptors such as EGF receptor, EGF can stimulate transient signal through Ras and also through a dominant path, Rap1. Activation by EGF through Rap1 has been explained to be due to the unstable signaling Crk-C3G-Rap1-B-Raf complex, resulting in temporary activation despite the continuous presence of EGF [24], [25]. Sustained ERK phosphorylation is required for CREB activation as evidenced from in vitro and in vivo investigations of neuroplasticity-related gene expression and cortical neuronal development [26], [27]. This was in accordance with our results that sustained ERK1/2 induced by MS and AS was associated with CREB phosphorylation while transient ERK1/2 induced by MA and AA did not involve in CREB phosphorylation. Moreover, the increments of both neurite-bearing cells and neurite length were notably subdued in MS- and AS-treated cells when ERK1/2 was inhibited. Several reports have suggested the possibility of triterpenoid and steroidal glycosides activating TrkA receptor and subsequently stimulating ERK1/2 signal transduction in neuronal cells [28], [29], [30].

Besides ERK1/2 phosphorylation, our studied triterpenoids also induced Akt phosphorylation, which did not appear cascading to phosphorylate CREB. Furthermore, inhibition of Akt opposed neurite elongation in either glycoside- or aglycone-treated cells. It has been suggested that activation of Akt through TrkB receptor, a brain-derived neurotrophic factor receptor, could trigger downstream signaling proteins involved in regulating cytoskeleton rearrangement in neuronal elongation such as Ras-PI3K-Akt-mTOR, glycogen synthase kinase 3β (GSK3β), and Rho-associated coiled-coil forming protein kinases [17], [31], [32], [33]. Taken together, triterpenoid glycosides, MS and AS, might activate TrkA receptor followed by sustained ERK1/2-CREB phosphorylation, which could induce neurite initiation and elongation ([Fig. 6 A]). In the case of aglycones, MA and AA possibly acted on other growth factor receptors such as EGF receptor ([Fig. 6 B]) to initiate a transient activation. Furthermore, MS, AS, MA, and AA might interact with TrkB receptor to trigger PI3K-Akt induced neurite elongation.

Zoom Image
Fig. 6 Schematic representation of possible signaling proteins/pathways related to the proposed mechanisms of C. asiatica triterpenoids on inducing neurite outgrowth in Neuro-2a cells. A Proposed pathways of MS and AS in induction of sustained ERK phosphorylation and resultant CREB activation through TrkA receptor and induction of Akt phosphorylation through TrkB receptor. B Proposed pathways of MA and AA in induction of transient ERK phosphorylation through other growth factor receptor and induction of Akt phosphorylation through TrkB receptor. ERK-independent CREB activation is proposed to be induced by cell-permeating MA and AA.

Another possibility to explain the neuroactivity of triterpenoids, particularly aglycones, is their structure similarity to steroid. As such, triterpenoid aglycones might be able to diffuse across cell membrane to interact with intracellular targets [34], [35], [36]. The Log P of all four triterpenoids are as follows: 5.8 (AA), 3.0 (MA), 0.1 (AS), and − 1.2 (MS) [37]. MA and AA, to some extent, could permeate cells and trigger intracellular signaling pathway ([Fig. 6 B]). MS and AS, on the other hand, are more polar and would preferentially act on cell surface receptors. Our cytotoxicity study also agreed with the postulation that the higher the cell permeation, the greater their cytotoxicity. AA provoked the greatest cytotoxicity followed by MA, while MS and AS did not show any effect on cell viability at the concentration up to 1000 µM (data not shown). The structure-cytotoxicity relationship study in rat hepatic cells also reported that AA exhibited high cytotoxicity (IC50 = 60.97 µM), which was greatly reduced when sugar moiety was attached to AA to become AS (IC50 > 2000 µM) [38].

Several recent studies have demonstrated pharmacological effects of C. asiatica triterpenoids, particularly AA and AS, in various rodent models including those related to memory and learning assessment, where bioactive compound needs to reach brain tissue [18], [39], [40]. As a preliminary estimation of potency to relate our results to in vivo neurological responses, the apparently bioactive doses of AA or AS from memory and learning behavioral studies using similar animal species, oral administration of test compound, and purified test compounds from commercial source were compared. The effective doses of AA and AS were shown to be 30 mg/kg/d and between 5 and 45 mg/kg/d, respectively, indicating that AS could be slightly more potent (about two-fold) when the molecular weights of both compounds are accounted [39], [40]. Based on bioavailability data from two independent reports, the bioavailability of AA (16.25%, 20 mg/kg administered orally) appeared approximately ten times higher than that of AS (1.86%, 38 mg/kg administered orally), implying a much higher potency of AS than AA if bioactivities are only originated from intact molecules [41], [42]. The observation of unchanged MS and AS present in brain tissues after oral administration of ECa233 extract comprising mainly MS and AS supports the possibility of the glycosides transporting to the brain and modulating neurological activities [43]. Although the systemic hydrolysis of triterpenoid glycosides to aglycones is not confirmed, a more recent finding of a possible existence of in vivo MS-AS interconversion emphasizes the need for further investigation to understand the biotransformation and to verify the in vivo effect of individual intact C. asiatica triterpenoid and their possible synergism [42].

In conclusion, we firstly reported the different potency of triterpenoid glycosides (MS and AS) and aglycones (MA and AA) in exerting neuritogenic activity. Glycosides were found to exhibit greater potency than aglycones. Moreover, we observed some distinct phosphorylation and proposed preferentially different signaling pathways of glycosides and aglycones in stimulating neurite outgrowth. MS and AS might modulate some cell surface receptors to activate MEK1/2-ERK1/2-CREB as well as PI3K-Akt. MA and AA could interact with the same or different cell surface receptors to activate PI3K-Akt, in conjunction with entering Neuro-2a cells to induce CREB activation through ERK-independent pathway. Even though the information on bioavailability and the understanding of biotransformation of triterpenoids are not complete, our findings supported the possibility of bioactivity synergism from diverse triterpenoid structures in plant extracts and could aid in drug design.


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Materials and Methods

Chemicals, antibodies and reagents

MS (purity ≥ 90%), AS (purity ≥ 90%), MA (purity ≥ 95%), and AA (purity ≥ 95%) were purchased from LKT Laboratories Inc. MTT, Hoechst33342, protease, and phosphatase cocktail inhibitors and other chemicals were purchased from Sigma. Primary antibodies such as βIII-tubulin, CREB, and p-CREB (Ser133) were purchased from EMD Millipore. Antibodies for ERK1/2, p-ERK1/2 (Thr202/Tyr204), Akt, and p-Akt (Ser473) were purchased from Cell Signaling Technology. Antibodies for GAPDH and secondary antibody conjugated with horseradish peroxidase were purchased from Abcam. Secondary antibody against mouse IgG conjugated with AlexaFluor 488 was purchased from Life Technologies-Molecular Probes. BSA was purchased from Calbiochem.


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Cell culture and treatment

Neuro-2a mouse neuroblastoma cells (CCL-131) were purchased from ATCC. Cells were cultured in DMEM medium supplemented with 10% FBS (Hyclone) and 100 units/mL penicillin/streptomycin in a humidified atmosphere of 5% CO2 at 37°C. Neuro-2a cells of passage numbers between 10 and 20 were used in the experiments. All-trans retinoic acid (purity ≥ 98%, Sigma), a well-known cell differentiation inducer, was used as a positive control for verifying cell differentiation ability. All test compounds were dissolved in DMSO and diluted with DMEM medium (0.5% final concentration of DMSO).


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Cell viability assay

Cell viability was determined by MTT assay. Briefly, cells (5000 cells/mL) were seeded into each well of 96-well plate (500 cells/well). After 24 h, cells were treated with test compounds (5 – 100 µM) for 6, 12, or 24 h. The culture media were later replaced by MTT solution (0.4 mg/mL) and incubated for 4 h. After discarding MTT solution, DMSO was added to dissolve purple formazan crystals. The absorbance was read by a microplate reader (Perkin Elmer) at 570 nm.


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Immunofluorescent staining

Neuro-2a cells were seeded into each well of a 24-well plate at the density of 2500 cells/well. After 24 h, cells were treated with test compounds at various concentrations and time intervals. Cells were fixed with 4% paraformaldehyde for 20 min and permeabilized with 3% BSA/0.3% Triton-X100 in PBS for 1 h. Cells were further incubated with mouse primary antibody against βIII-tubulin (1 : 500) overnight at 4 °C to indicate the structure of cell body and neurites [16], followed by exposing to secondary antibody against mouse IgG conjugated with AlexaFluor 488. Nuclei were also stained by Hoechst33342 solution (10 µg/mL) to indicate the number of cells.


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Measurement of neurite extension

Images were obtained under Olympus IX51inverted fluorescence microscope (Olympus). Cell images were randomly captured in six to eight fields from each well and analyzed using Measure command and Cell counter tools in ImageJ 1.50c software (NIH). The %NBC was calculated from the ratio between the number of cells with neurites and the total number of cells. The average neurite length was estimated from the mean of the maximal neurite length possessed by each cell within an observed field [16].


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Western blot analysis

Cells were lysed by lysis buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton-X100, and 1% cocktail protease and phosphatase inhibitors). Proteins in the whole-cell lysates were quantitated using Bradford assay. An aliquot of the cell lysates containing 20 µg proteins were electrophoresed on 12.5% SDS-PAGE and subsequently electro-transferred onto PVDF membranes. The blots were blocked with 5% BSA and probed with primary antibodies against ERK1/2, p-ERK1/2, Akt, p-Akt, CREB, and p-CREB, followed by incubation with HRP-conjugated secondary antibody. The protein bands were detected by using chemiluminescence detection kit (GE Healthcare). Band images were acquired using ImageQuant LAS 4000 (GE Healthcare) and quantitatively analyzed by ImageQuant TL 7.0 software (GE Healthcare). Relative band intensity of each protein was normalized with GAPDH band intensity.


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Statistical analysis

All data are expressed as mean ± standard error estimated using Gaussian error propagation or standard error of the mean (SEM) from at least three independent experiments, and each experiment was performed in triplicate. The differences among the groups were evaluated by one-way analysis of variance (ANOVA), followed by LSDʼs post hoc test using SPSS version 22. Statistical significance was defined as p < 0.05 for all tests.


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Conflict of Interest

The authors declare no conflicts of interest.

Acknowledgements

This work was financially supported by the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund).

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  • 6 Tantisira M, Tantisira B, Patarapanich C, Suttisri R, Luangcholatan S, Mingmalailak S, Wanasuntronwong A, Saifah E. Effects of standardized extract of Centella asiatica ECa 233 on learning and memory impairment induced by transient bilateral common carotid artery occlusion in mice. Thai J Pharmacol 2010; 32: 22-33
  • 7 Wanakhachornkrai O, Pongrakhananon V, Chunhacha P, Wanasuntronwong A, Vattanajun A, Tantisira B, Chanvorachote P, Tantisira MH. Neuritogenic effect of standardized extract of Centella asiatica ECa233 on human neuroblastoma cells. BMC Complement Altern Med 2013; 13: 204
  • 8 Wanasuntronwong A, Tantisira MH, Tantisira B, Watanabe H. Anxiolytic effects of standardized extract of Centella asiatica (ECa 233) after chronic immobilization stress in mice. J Ethnopharmacol 2012; 143: 579-585
  • 9 Soumyanath A, Zhong YP, Gold SA, Yu X, Koop DR, Bourdette D, Gold BG. Centella asiatica accelerates nerve regeneration upon oral administration and contains multiple active fractions increasing neurite elongation in-vitro . J Pharm Pharmacol 2005; 9: 1221-1229
  • 10 Jiang H, Zheng G, Lv J, Chen H, Lin J, Li Y, Fan G, Ding X. Identification of Centella asiatica s effective ingredients for inducing the neuronal differentiation. Evid Based Complement Alternat Med 2016; 2016: 9634750
  • 11 Gray NE, Zweig JA, Murchison C, Caruso M, Matthews DG, Kawamoto C, Harris CJ, Quinn JF, Soumyanath A. Centella asiatica attenuates Aβ-induced neurodegenerative spine loss and dendritic simplification. Neurosci Lett 2017; 646: 24-29
  • 12 Perron JC, Bixby JL. Distinct neurite outgrowth signaling pathways converge on ERK activation. Mol Cell Neurosci 1999; 13: 362-378
  • 13 Read D, Gorman A. Involvement of Akt in neurite outgrowth. Cell Mol Life Sci 2009; 66: 2975-2984
  • 14 Xu Y, Cao Z, Khan I, Luo Y. Gotu Kola (Centella asiatica) extract enhances phosphorylation of cyclic AMP response element binding protein in neuroblastoma cells expressing amyloid beta peptide. J Alzheimers Dis 2008; 13: 341-349
  • 15 Lin J, Jiang H, Ding X. Synergistic combinations of five single drugs from Centella asiatica for neuronal differentiation. Neuroreport 2017; 1: 23-27
  • 16 Harrill JA, Mundy WR. Quantitative assessment of neurite outgrowth in PC12 cells. Methods Mol Biol 2011; 758: 331-348
  • 17 Kumar V, Zhang MX, Swank MW, Kunz J, Wu GY. Regulation of dendritic morphogenesis by Ras-PI3K-Akt-mTOR and Ras-MAPK signaling pathways. J Neurosci 2005; 25: 11288-11299
  • 18 Lin X, Huang R, Zhang S, Wei L, Zhuo L, Wu X, Tang A, Huang Q. Beneficial effects of asiaticoside on cognitive deficits in senescence-accelerated mice. Fitoterapia 2013; 87: 69-77
  • 19 da Silva JS, Dotti CG. Breaking the neuronal sphere: regulation of the actin cytoskeleton in neuritogenesis. Nat Rev Neurosci 2002; 9: 694-704
  • 20 Lonze BE, Ginty DD. Function and regulation of CREB family transcription factors in the nervous system. Neuron 2002; 4: 605-623
  • 21 Xiao J, Liu Y. Differential roles of ERK and JNK in early and late stages of neuritogenesis: a study in a novel PC12 model system. J Neurochem 2003; 6: 1516-1523
  • 22 Ebisuya M, Kondoh K, Nishida E. The duration, magnitude and compartmentalization of ERK MAP kinase activity: mechanisms for providing signaling specificity. J Cell Sci 2005; 118: 2997-3002
  • 23 von Kriegsheim A, Baiocchi D, Birtwistle M, Sumpton D, Bienvenut W, Morrice N, Yamada K, Lamond A, Kalna G, Orton R, Gilbert D, Kolch W. Cell fate decisions are specified by the dynamic ERK interactome. Nat Cell Biol 2009; 11: 1458-1464
  • 24 Kao S, Jaiswal RK, Kolch W, Landreth GE. Identification of the mechanisms regulating the differential activation of the MAPK cascade by epidermal growth factor and nerve growth factor in PC12 cells. J Biol Chem 2001; 21: 18169-18177
  • 25 Schoeberl B, Eichler-Jonsson C, Gilles ED, Müller G. Computational modeling of the dynamics of the MAP kinase cascade activated by surface and internalized EGF receptors. Nat Biotechnol 2002; 20: 370-375
  • 26 Papadeas ST, Blake BL, Knapp DJ, Breese GR. Sustained extracellular signal-regulated kinase 1/2 phosphorylation in neonate 6-hydroxydopamine-lesioned rats after repeated D1-dopamine receptor agonist administration: implications for NMDA receptor involvement. J Neurosci 2004; 26: 5863-5876
  • 27 Ha S, Redmond L. ERK mediates activity dependent neuronal complexity via sustained activity and CREB-mediated signaling. Dev Neurobiol 2008; 68: 1565-1579
  • 28 Hur J, Lee P, Moon E, Kang I, Kim SH, Oh MS, Kim SY. Neurite outgrowth induced by spicatoside A, a steroidal saponin, via the tyrosine kinase A receptor pathway. Eur J Pharmacol 2009; 620: 9-15
  • 29 Kim MS, Yu JM, Kim HJ, Kim HB, Kim ST, Jang SK, Choi YW, Lee DI, Joo SS. Ginsenoside Re and Rd enhance the expression of cholinergic markers and neuronal differentiation in Neuro-2a cells. Biol Pharm Bull 2014; 37: 826-833
  • 30 Zhou H, Xue W, Chu SF, Wang ZZ, Li CJ, Jiang YN, Luo LM, Luo P, Li G, Zhang DM, Chen NH. Polygalasaponin XXXII, a triterpenoid saponin from Polygalae Radix, attenuates scopolamine-induced cognitive impairments in mice. Acta Pharmacol Sin 2016; 37: 1045-1053
  • 31 Gu X, Meng S, Liu S, Jia C, Fang Y, Li S, Fu C, Song Q, Lin L, Wang X. miR-124 represses ROCK1 expression to promote neurite elongation through activation of the PI3K/Akt signal pathway. J Mol Neurosci 2014; 1: 156-165
  • 32 Hur EM, Zhou FQ. GSK3 signalling in neural development. Nat Rev Neurosci 2010; 11: 539-551
  • 33 Mullen LM, Pak KK, Chavez E, Kondo K, Brand Y, Ryan AF. Ras/p38 and PI3K/Akt but not Mek/Erk signaling mediate BDNF-induced neurite formation on neonatal cochlear spiral ganglion explants. Brain Res 2012; 1430: 25-34
  • 34 Liu J, Shimizu K, Tanaka A, Shinobu W, Ohnuki K, Nakamura T, Kondo R. Target proteins of ganoderic acid DM provides clues to various pharmacological mechanisms. Sci Rep 2012; 2: 905
  • 35 Chen YC, Liu YL, Li FY, Chang CI, Wang SY, Lee KY, Li SL, Chen YP, Jinn TR, Tzen JT. Antcin A, a steroid-like compound from Antrodia camphorata, exerts anti-inflammatory effect via mimicking glucocorticoids. Acta Pharmacol Sin 2011; 32: 904-911
  • 36 Honda T, Rounds BV, Bore L, Finlay HJ, Favaloro jr. FG, Suh N, Wang Y, Sporn MB, Gribble GW. Synthetic oleanane and ursane triterpenoids with modified rings A and C: a series of highly active inhibitors of nitric oxide production in mouse macrophages. J Med Chem 2000; 43: 4233-4246
  • 37 Rafat M, Fong KW, Goldsipe A, Stephenson BC, Coradetti ST, Sambandan TG, Sinskey AJ, Rha C. Association (micellization) and partitioning of aglycon triterpenoids. J Colloid Interface Sci 2008; 2: 324-330
  • 38 Dong MS, Jung SH, Kim HJ, Kim JR, Zhao LX, Lee ES, Lee EJ, Yi JB, Lee N, Cho YB, Kwak WJ, Park YI. Structure-related cytotoxicity and anti-hepatofibric effect of asiatic acid derivatives in rat hepatic stellate cell-line, HSC-T6. Arch Pharm Res 2004; 27: 512-517
  • 39 Zhang Z, Li X, Li D, Luo M, Li Y, Song L, Jiang X. Asiaticoside ameliorates beta-amyloid-induced learning and memory deficits in rats by inhibiting mitochondrial apoptosis and reducing inflammatory factors. Exp Ther Med 2017; 13: 413-420
  • 40 Welbat JU, Sirichoat A, Chaijaroonkhanarak W, Prachaney P, Pannangrong W, Pakdeechote P, Sripanidkulchai B, Wigmore P. Asiatic acid prevents the deleterious effects of valproic acid on cognition and hippocampal cell proliferation and survival. Nutrients 2016; 8: 1-11
  • 41 Yuan Y, Zhang H, Sun F, Sun S, Zhu Z, Chai Y. Biopharmaceutical and pharmacokinetic characterization of asiatic acid in Centella asiatica as determined by a sensitive and robust HPLC-MS method. J Ethnopharmacol 2015; 163: 31-38
  • 42 Hengjumrut P, Anukunwithaya T, Tantisira MH, Tantisira B, Khemawoot P. Comparative pharmacokinetics between madecassoside and asiaticoside presented in a standardised extract of Centella asiatica, ECa 233 and their respective pure compound given separately in rats. Xenobiotica 2018; 48: 18-27
  • 43 Anukunwithaya T, Tantisira MH, Tantisira B, Khemawoot P. Pharmacokinetics of a standardized extract of Centella asiatica ECa 233 in rats. Planta Med 2016; 83: 710-717

Correspondence

Asst. Prof. Dr. Boonsri Ongpipattanakul
Department of Biochemistry and Microbiology
Faculty of Pharmaceutical Sciences
Chulalongkorn University
254 Phyathai road, Pathumwan
10330 Bangkok
Thailand   
Phone: + 66 22 18 83 66   
Fax: + 66 22 18 83 75   

  • References

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  • 2 Wartman BC, Holahan MR. The impact of multiple memory formation on dendritic complexity in the hippocampus and anterior cingulate cortex assessed at recent and remote time points. Front Behav Neurosci 2014; 8: 128
  • 3 Ramm P, Alexandrov Y, Cholewinski A, Cybuch Y, Nadon R, Soltys BJ. Automated screening of neurite outgrowth. J Biomol Screen 2003; 1: 7-18
  • 4 Rumalla CS, Ali Z, Weerasooriya AD, Smillie TJ, Khan IA. Two new triterpene glycosides from Centella asiatica . Planta Med 2010; 76: 1018-1021
  • 5 Lokanathan Y, Omar N, Ahmad Puzi NN, Saim A, Hj Idrus R. Recent updates in neuroprotective and neuroregenerative potential of Centella asiatica . Malays J Med Sci 2016; 23: 4-14
  • 6 Tantisira M, Tantisira B, Patarapanich C, Suttisri R, Luangcholatan S, Mingmalailak S, Wanasuntronwong A, Saifah E. Effects of standardized extract of Centella asiatica ECa 233 on learning and memory impairment induced by transient bilateral common carotid artery occlusion in mice. Thai J Pharmacol 2010; 32: 22-33
  • 7 Wanakhachornkrai O, Pongrakhananon V, Chunhacha P, Wanasuntronwong A, Vattanajun A, Tantisira B, Chanvorachote P, Tantisira MH. Neuritogenic effect of standardized extract of Centella asiatica ECa233 on human neuroblastoma cells. BMC Complement Altern Med 2013; 13: 204
  • 8 Wanasuntronwong A, Tantisira MH, Tantisira B, Watanabe H. Anxiolytic effects of standardized extract of Centella asiatica (ECa 233) after chronic immobilization stress in mice. J Ethnopharmacol 2012; 143: 579-585
  • 9 Soumyanath A, Zhong YP, Gold SA, Yu X, Koop DR, Bourdette D, Gold BG. Centella asiatica accelerates nerve regeneration upon oral administration and contains multiple active fractions increasing neurite elongation in-vitro . J Pharm Pharmacol 2005; 9: 1221-1229
  • 10 Jiang H, Zheng G, Lv J, Chen H, Lin J, Li Y, Fan G, Ding X. Identification of Centella asiatica s effective ingredients for inducing the neuronal differentiation. Evid Based Complement Alternat Med 2016; 2016: 9634750
  • 11 Gray NE, Zweig JA, Murchison C, Caruso M, Matthews DG, Kawamoto C, Harris CJ, Quinn JF, Soumyanath A. Centella asiatica attenuates Aβ-induced neurodegenerative spine loss and dendritic simplification. Neurosci Lett 2017; 646: 24-29
  • 12 Perron JC, Bixby JL. Distinct neurite outgrowth signaling pathways converge on ERK activation. Mol Cell Neurosci 1999; 13: 362-378
  • 13 Read D, Gorman A. Involvement of Akt in neurite outgrowth. Cell Mol Life Sci 2009; 66: 2975-2984
  • 14 Xu Y, Cao Z, Khan I, Luo Y. Gotu Kola (Centella asiatica) extract enhances phosphorylation of cyclic AMP response element binding protein in neuroblastoma cells expressing amyloid beta peptide. J Alzheimers Dis 2008; 13: 341-349
  • 15 Lin J, Jiang H, Ding X. Synergistic combinations of five single drugs from Centella asiatica for neuronal differentiation. Neuroreport 2017; 1: 23-27
  • 16 Harrill JA, Mundy WR. Quantitative assessment of neurite outgrowth in PC12 cells. Methods Mol Biol 2011; 758: 331-348
  • 17 Kumar V, Zhang MX, Swank MW, Kunz J, Wu GY. Regulation of dendritic morphogenesis by Ras-PI3K-Akt-mTOR and Ras-MAPK signaling pathways. J Neurosci 2005; 25: 11288-11299
  • 18 Lin X, Huang R, Zhang S, Wei L, Zhuo L, Wu X, Tang A, Huang Q. Beneficial effects of asiaticoside on cognitive deficits in senescence-accelerated mice. Fitoterapia 2013; 87: 69-77
  • 19 da Silva JS, Dotti CG. Breaking the neuronal sphere: regulation of the actin cytoskeleton in neuritogenesis. Nat Rev Neurosci 2002; 9: 694-704
  • 20 Lonze BE, Ginty DD. Function and regulation of CREB family transcription factors in the nervous system. Neuron 2002; 4: 605-623
  • 21 Xiao J, Liu Y. Differential roles of ERK and JNK in early and late stages of neuritogenesis: a study in a novel PC12 model system. J Neurochem 2003; 6: 1516-1523
  • 22 Ebisuya M, Kondoh K, Nishida E. The duration, magnitude and compartmentalization of ERK MAP kinase activity: mechanisms for providing signaling specificity. J Cell Sci 2005; 118: 2997-3002
  • 23 von Kriegsheim A, Baiocchi D, Birtwistle M, Sumpton D, Bienvenut W, Morrice N, Yamada K, Lamond A, Kalna G, Orton R, Gilbert D, Kolch W. Cell fate decisions are specified by the dynamic ERK interactome. Nat Cell Biol 2009; 11: 1458-1464
  • 24 Kao S, Jaiswal RK, Kolch W, Landreth GE. Identification of the mechanisms regulating the differential activation of the MAPK cascade by epidermal growth factor and nerve growth factor in PC12 cells. J Biol Chem 2001; 21: 18169-18177
  • 25 Schoeberl B, Eichler-Jonsson C, Gilles ED, Müller G. Computational modeling of the dynamics of the MAP kinase cascade activated by surface and internalized EGF receptors. Nat Biotechnol 2002; 20: 370-375
  • 26 Papadeas ST, Blake BL, Knapp DJ, Breese GR. Sustained extracellular signal-regulated kinase 1/2 phosphorylation in neonate 6-hydroxydopamine-lesioned rats after repeated D1-dopamine receptor agonist administration: implications for NMDA receptor involvement. J Neurosci 2004; 26: 5863-5876
  • 27 Ha S, Redmond L. ERK mediates activity dependent neuronal complexity via sustained activity and CREB-mediated signaling. Dev Neurobiol 2008; 68: 1565-1579
  • 28 Hur J, Lee P, Moon E, Kang I, Kim SH, Oh MS, Kim SY. Neurite outgrowth induced by spicatoside A, a steroidal saponin, via the tyrosine kinase A receptor pathway. Eur J Pharmacol 2009; 620: 9-15
  • 29 Kim MS, Yu JM, Kim HJ, Kim HB, Kim ST, Jang SK, Choi YW, Lee DI, Joo SS. Ginsenoside Re and Rd enhance the expression of cholinergic markers and neuronal differentiation in Neuro-2a cells. Biol Pharm Bull 2014; 37: 826-833
  • 30 Zhou H, Xue W, Chu SF, Wang ZZ, Li CJ, Jiang YN, Luo LM, Luo P, Li G, Zhang DM, Chen NH. Polygalasaponin XXXII, a triterpenoid saponin from Polygalae Radix, attenuates scopolamine-induced cognitive impairments in mice. Acta Pharmacol Sin 2016; 37: 1045-1053
  • 31 Gu X, Meng S, Liu S, Jia C, Fang Y, Li S, Fu C, Song Q, Lin L, Wang X. miR-124 represses ROCK1 expression to promote neurite elongation through activation of the PI3K/Akt signal pathway. J Mol Neurosci 2014; 1: 156-165
  • 32 Hur EM, Zhou FQ. GSK3 signalling in neural development. Nat Rev Neurosci 2010; 11: 539-551
  • 33 Mullen LM, Pak KK, Chavez E, Kondo K, Brand Y, Ryan AF. Ras/p38 and PI3K/Akt but not Mek/Erk signaling mediate BDNF-induced neurite formation on neonatal cochlear spiral ganglion explants. Brain Res 2012; 1430: 25-34
  • 34 Liu J, Shimizu K, Tanaka A, Shinobu W, Ohnuki K, Nakamura T, Kondo R. Target proteins of ganoderic acid DM provides clues to various pharmacological mechanisms. Sci Rep 2012; 2: 905
  • 35 Chen YC, Liu YL, Li FY, Chang CI, Wang SY, Lee KY, Li SL, Chen YP, Jinn TR, Tzen JT. Antcin A, a steroid-like compound from Antrodia camphorata, exerts anti-inflammatory effect via mimicking glucocorticoids. Acta Pharmacol Sin 2011; 32: 904-911
  • 36 Honda T, Rounds BV, Bore L, Finlay HJ, Favaloro jr. FG, Suh N, Wang Y, Sporn MB, Gribble GW. Synthetic oleanane and ursane triterpenoids with modified rings A and C: a series of highly active inhibitors of nitric oxide production in mouse macrophages. J Med Chem 2000; 43: 4233-4246
  • 37 Rafat M, Fong KW, Goldsipe A, Stephenson BC, Coradetti ST, Sambandan TG, Sinskey AJ, Rha C. Association (micellization) and partitioning of aglycon triterpenoids. J Colloid Interface Sci 2008; 2: 324-330
  • 38 Dong MS, Jung SH, Kim HJ, Kim JR, Zhao LX, Lee ES, Lee EJ, Yi JB, Lee N, Cho YB, Kwak WJ, Park YI. Structure-related cytotoxicity and anti-hepatofibric effect of asiatic acid derivatives in rat hepatic stellate cell-line, HSC-T6. Arch Pharm Res 2004; 27: 512-517
  • 39 Zhang Z, Li X, Li D, Luo M, Li Y, Song L, Jiang X. Asiaticoside ameliorates beta-amyloid-induced learning and memory deficits in rats by inhibiting mitochondrial apoptosis and reducing inflammatory factors. Exp Ther Med 2017; 13: 413-420
  • 40 Welbat JU, Sirichoat A, Chaijaroonkhanarak W, Prachaney P, Pannangrong W, Pakdeechote P, Sripanidkulchai B, Wigmore P. Asiatic acid prevents the deleterious effects of valproic acid on cognition and hippocampal cell proliferation and survival. Nutrients 2016; 8: 1-11
  • 41 Yuan Y, Zhang H, Sun F, Sun S, Zhu Z, Chai Y. Biopharmaceutical and pharmacokinetic characterization of asiatic acid in Centella asiatica as determined by a sensitive and robust HPLC-MS method. J Ethnopharmacol 2015; 163: 31-38
  • 42 Hengjumrut P, Anukunwithaya T, Tantisira MH, Tantisira B, Khemawoot P. Comparative pharmacokinetics between madecassoside and asiaticoside presented in a standardised extract of Centella asiatica, ECa 233 and their respective pure compound given separately in rats. Xenobiotica 2018; 48: 18-27
  • 43 Anukunwithaya T, Tantisira MH, Tantisira B, Khemawoot P. Pharmacokinetics of a standardized extract of Centella asiatica ECa 233 in rats. Planta Med 2016; 83: 710-717

Zoom Image
Fig. 1 Effects of individual C. asiatica triterpenoid on Neuro-2a cell viability by MTT assay. Cells were treated with various concentrations (5 – 100 µM) of MS, AS, MA, and AA for (A) 6, (B) 12, and (C) 24 h. Data are presented as mean ± standard error estimated using Gaussian error propagation (n = 3). * p < 0.05 vs. control.
Zoom Image
Fig. 2 Effects of individual C. asiatica triterpenoid on neurite outgrowth in Neuro-2a cell. A Representative immunofluorescent images of cells treated with 10 µM of MS, AS, MA, and AA for 6, 12, and 24 h. Cells were stained with primary antibody against βIII-tubulin followed by fluorescent AlexaFluor 488 secondary antibody (green fluorescence), and nuclei were also stained with Hoechst33342 (blue fluorescence). Images were merged and analyzed by using ImageJ software. Histograms show the %NBC and average neurite length in Neuro-2a cells treated with 0.01 – 50 µM of (B, C) MS, (D, E) AS, (F, G) MA, and (H, I) AA. Data are presented as mean ± SEM (n = 4). * p < 0.05 vs. control.
Zoom Image
Fig. 3 Time-dependent studies on phosphorylation of ERK1/2, Akt, and CREB in Neuro-2a cells after treatment with individual C. asiatica triterpenoid. Representative immunoblots of cells treated with 10 µM of (A) MS, (B) AS, (C) MA, and (D) AA at indicated time points (0.5 – 12 h). The intensity of GAPDH was used for normalization as a loading control. Histograms show densitometric analysis of (E) ERK1/2, (F) Akt, and (G) CREB phosphorylation. Data are presented as mean ± standard error estimated using Gaussian error propagation (n = 3). * p < 0.05 vs. control.
Zoom Image
Fig. 4 Effects of ERK1/2 and Akt inhibitions on CREB phosphorylation in MS- and AS-treated Neuro-2a cells. A PD098059 pretreatment effect on MS- and AS-induced ERK phosphorylation at 1 h. B LY294002 pretreatment effect on MS- and AS-induced Akt phosphorylation at 1 h. C PD098059 pretreatment effect on MA- and AA-induced ERK phosphorylation at 1 h. D LY294002 pretreatment effect on MA- and AA-induced Akt phosphorylation at 1 h. E MS- and AS-induced CREB phosphorylation in the presence and absence of PD098059 or LY294002. F MA- and AA-induced CREB phosphorylation in the presence and absence of PD098059 or LY294002. Data are presented as mean ± standard error estimated using Gaussian error propagation (n = 3). * p < 0.05 vs. control.
Zoom Image
Fig. 5 Effects of ERK1/2 and Akt inhibitions on neurite outgrowth in MA and AA treated Neuro-2a cells. Histograms represent the %NBC and average neurite length observed in the presence and absence of inhibitor pretreatment in cells subjected to (A) MS and AS and (B) MA and AA. Data are presented as mean ± SEM (n = 3). * p < 0.05 vs. control.
Zoom Image
Fig. 6 Schematic representation of possible signaling proteins/pathways related to the proposed mechanisms of C. asiatica triterpenoids on inducing neurite outgrowth in Neuro-2a cells. A Proposed pathways of MS and AS in induction of sustained ERK phosphorylation and resultant CREB activation through TrkA receptor and induction of Akt phosphorylation through TrkB receptor. B Proposed pathways of MA and AA in induction of transient ERK phosphorylation through other growth factor receptor and induction of Akt phosphorylation through TrkB receptor. ERK-independent CREB activation is proposed to be induced by cell-permeating MA and AA.