Home Neuroprotective effects of Cubebin and Hinokinin lignan fractions of Piper cubeba fruit in Alzheimer’s disease in vitro model
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Neuroprotective effects of Cubebin and Hinokinin lignan fractions of Piper cubeba fruit in Alzheimer’s disease in vitro model

  • Shirin Tarbiat ORCID logo EMAIL logo , Demet Unver ORCID logo , Salih Tuncay ORCID logo , Sevim Isik ORCID logo , Kiyak Bercem Yeman ORCID logo and Ali Reza Mohseni ORCID logo
Published/Copyright: June 1, 2023
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Turkish Journal of Biochemistry
From the journal Turkish Journal of Biochemistry Volume 48 Issue 3

Abstract

Objectives

The current research examines the protective effects of the Piper cubeba ethanolic extract and its isolated lignans; Cubebin and Hinokinin fractions against Alzheimer’s Disease (AD) in vitro model.

Methods

Dried and powdered fruit of P. cubeba were extracted in ethanol and fractionated using silica gel column chromatography. Of the 15 eluted fractions, two fractions indicated presence of targeted Lignans; Hinokinin and Cubebin. They were monitored by thin layered chromatography and their structures were confirmed by LC-HRMS spectrometry and NMR analysis. Antioxidant activity of the crude extract and isolated lignan fractions were analyzed using FRAP, DPPH and ABTS assays. Anti-acetylcholinesterase activity was investigated in vitro and β-amyloid (Aβ) cytotoxicity on SHSY-5Y human neuroblastoma cell lines was studied using MTT assay.

Results

The crude extract showed similar if not significantly stronger antioxidant capacity compared to ascorbic acid in FRAP and DPPH assays. Both lignans exerted weaker yet potent activity. The crude extract yielded the strongest acetylcholinesterase inhibitory potential compared to the lignan fractions however, there was no significant difference (p<0.05) between IC50 values of lignan fractions. Significant neuroprotective effects against 50 μM Aβ at p<0.05 was observed for selected fractions compared to Aβ treated control. The crude extract was highly protective against Aβ at both 5 and 10 μg/mL. Cubebin and Hinokinin-containing fractions significantly improved the viability of the SH-SY5Y cells against Aβ cytotoxicity both only at the concentration of 100 μg/mL.

Conclusions

Results from our studies suggest that these phytoconstituents might be good candidates in prevention and treatment of AD.

Keywords: acetylcholinesterase; amyloid beta; Cubebin; Hinokinin; lignans; Piper cubeba

Introduction

Alzheimer’s disease (AD) is defined as a chronic neurodegenerative disease presenting with progressive decline in memory that is accompanied by cognitive disorders and psychiatric symptoms and accounts for a majority of dementia-related cases [1]. The rise in the prevalence of AD with age is remarkable and the projected number of AD patients is estimated to quadruple by the year 2050 [2]. The process of AD is principally initiated by the formation of intraneuronal aggregations of the hyperphosphorylated protein tau accompanied by extracellular amyloid-beta (Aβ) plaque depositions, both of which consist of highly insoluble, densely packed filaments, which terminally lead to the loss of cholinergic neurons and therefore contributing to the cognitive dysfunctions [3].

The regulation of cholinergic neurotransmission by acetylcholinesterase (AChE) has been one of the main pharmacological targets to address symptoms brought about by cholinergic imbalances in AD [4]. Recently, plant-based inhibitors of AChE have attracted particular attention in AD research. Moreover, numerous studies have also demonstrated protective properties of plant extracts against Aβ-induced neurotoxicity [5]. Their remarkable impact on targeting various pathways, having fewer side effects, and extended period of therapeutic effects highlighted by recent studies, suggest that the use of active molecules from natural sources are promising approaches in the treatment of AD [6]. Piper cubeba, also known as cubeb or java pepper, is part of the Piperaceae family of over 700 species throughout the world. Native to tropical South-Asian countries like Indonesia and India, P. cubeba is mainly cultivated for its fruit and essential oil, with its fruit available commercially in the form of dried berries and utilized in culinary preparations [7].

Numerous species of the genus Piper have shown insecticidal, antibacterial, anthelmintic, and anticancer properties and are put to use in traditional herbal drugs and medicine [8, 9]. They are traditionally applied in the treatment of cough, flu, and rheumatism [10]. A variety of bioactive compounds have been obtained from P. cubeba and indicated to exhibit anti-tumor actions as well as antileukemic and antibiotic activities [11, 12]. These properties are attributed to the wide range of phytoconstituents and the plant’s essential oil. The qualitative phytochemical screening of its ethanolic extract have uncovered the existence of phenolic compounds, flavonoids, lignans, alkaloids, diterpens etc., and all other secondary metabolites [13]. Lignans, a group of dimeric phenylpropanoids, are common metabolites present in P. cubeba [14]. Protection against cardiovascular diseases, diabetes, and cancer have been cited as the main advantages of this class of polyphenols [14, 15].

Our study’s objective is to assess the potential antioxidant, AChE inhibitory and neuroprotective effects of the crude extract and isolated lignan fractions from the P. cubeba fruit in order to understand their therapeutic value in line with novel phytotherapic strategies for prevention and treatment of AD.

Materials and methods

Chemicals and reagents

All the chemical compounds and kits utilized in this research were purchased from Sigma-Aldrich Co. (Merck KGaA, Darmstadt, Germany). Silica Gel (60 F254) for Thin-Layer Chromatography (TLC) and Silica gel (0.063–0.200 mesh, Merck Co.,) was used in column chromatography. P. cubeba fruits in Turkey known as Kebabiye were commercially obtained from the ‘Aktar marka’ spices company in Turkey (https://aktarmarka.com. Accessed 30 October 2020) in dry form. The liquid chromatography-mass spectrometry (LC-HRMS) analyses of the prepared samples were performed by Thermo Orbitrap Q-Exactive mass spectrometer and NMR data of samples were attained using Bruker Magnet System 500′54 Ascend 500 NMR spectrometer (1H NMR 500 MHz, 13C NMR 125 MHz) at Drug Application and Research Center, Bezmialem Vakif University, Istanbul.

Extraction

50 g of dry fruit was powdered in a mortar and was stored in an airtight container. The powdered plant material was weighed and 200 mL of ethanol was added to the plant powder at room temperature and was subsequently left in a 130 RPM shaker (Stuart SSL1-Shaker) for 24 h. The filtration of the plant extract, was performed using the filter paper method. Afterwards, the residues were homogenized with 150 mL of ethanol again and left in the shaker at 130 rpm for another 24 h. Each filtered filtrate was combined and stored at +4 °C. Following each extraction, the solvent was filtered and the residues were soaked again with ethanol. The filtrates were pooled together and concentrated using a rotary vacuum evaporator, and 10.64 g crude extract was obtained.

Column chromatography

The P. cubeba extract (5 g) was adsorbed on 150 gm silica gel (0.063–0.200 mm, Merck Co.,) and placed on a filled glass column (2.5 × 40 cm). Mobile phase elution based on increasing polarity was started with 5 % ethyl acetate-hexane (5–95 mL). A gradient of ethyl acetate was added with 5 % increments into reaching 100 % ethyl acetate. It was followed by 100 % ethanol and 100 % methanol. All the collected fractions were subjected to TLC on silica gel plate using 30 % ethyl acetate-hexane mixture as the developed solvent system. Fractions with similar Rf values were pooled together. All pooled fractions were concentrated and further purification was carried out by crystallization.

LC-HRMS and NMR analysis

Preparation of test Solution for LC-HRMS: Internal standard solution was added to the extract samples prepared with 100 mg/L methanol, with a final concentration of 3 ppm. The sample was passed through a 0.45 m filter and taken into a vial to make it suitable for the device [16].

Equipment and chromatography: Experiments were carried out by a Thermo ORBİTRAP Q-EXACTIVE mass spectrometry (Bremen, Germany) equipped with a Troyasil C18 HS column (150 × 3 mm i.d., 3 μm particle size). The composition of the mobile phase consisted of water (A, 0.1 % formic acid) and methanol (B, 0.1 % formic acid), with a gradient program that started with 50 % A and 50 % B for the first minute, then transitioned to 100 % B for the next 2 min, and finally returned to a composition of 50 % A and 50 % B for the last minute. The mobile phase was maintained at a flow rate of 0.35 mL/min, with the column temperature maintained at 30 °C. The volume of injection was 1 μL. The setting for the environment was set to a temperature of 22.0 ± 5.0 °C and relative humidity of (50 ± 15) % rh.

Interpretation of NMR analysis

3,4-bis(benzo[d] [1,3]dioxol-5-ylmethyl)dihydrofuran-2(3H)-one (1): white solid substance, 159 mg, Rf: 0.42, 1H NMR (500 MHz, CDCl3): δ=2.44–2.59 (m, 4H, OCH2CHCH2, OCH2CHCH2, OCCHCH2), 2.84 (dd, J=14.0, 7.3 Hz, 1H, OCCHCHH), 2.98 (dd, J=14.1, 5.02 Hz, 1H, OCCHCHH), 3.86 (dd, J=9.1, 7.1 Hz, 1H, OCHHCH), 4.12 (dd, J=9.0, 6.9 Hz, 1H, OCHHCH), 5.93 (s, 4H, 2 × OCH2O), 6.46 (m, 2H, arom. CH), 6.60 (dd, J=7.9, 1.6 Hz, 1H, arom. CH), 6.62 (d, J=1.6 Hz, 1H, arom. CH), 6.69 (d, J=8.2 Hz, 1H, arom. CH), 6.73 (d, J=7.8 Hz, 1H, arom. CH) ppm. 13C NMR (125 MHz, CDCl3): δ=34.76 (CH2CHCO), 38.29 (CH2CHCH2O), 41.22 (CH2CHCH2), 46.42 (CH2CHCO), 71.09 (CHCH2O), 100.96 (OCH2O), 108.22, 108.28, 108.75, 109.38, 121.48, 122.17 (arom. CH), 131.28, 131.55, 146.28, 146.40, 147.80, 147.83 (arom. C), 178.39 (CO) ppm. LC-HRMS m/z: calcd for C20H18O6 354.36; Found 355.11 [M + H]+ (Figure 1).

Figure 1: Chemical structures of Hinokinin (1) and Cubebin (2).
Figure 1:

Chemical structures of Hinokinin (1) and Cubebin (2).

3,4-bis(benzo[d] [1,3]dioxol-5-ylmethyl)tetrahydrofuran-2-ol (2): white solid substance, 130 mg, Rf: 0.28, 1H NMR (500 MHz, CDCl3): δ=1,99 (m, 1H, 2 × CH2CHCH2), 2,42–2.78 (m, 5H), 3.68 (dt, J=8.1 Hz, 1H, 2 × CHCH2O), 4.05 (dt, J=8.1 Hz, 1H, 2 × CHCH2O), 5.22 (s, 6H, OH), 5.91 (s, 4H, 2 × OCH2O), 6.51–6.73 (m, 6H, arom. CH) ppm. 13C NMR (125 MHz, CDCl3): δ=33.65 (CH2CHCH2), 38.92 (CH2CHCH), 42.90 (Ar-CH2), 45.91 (Ar-CH2), 52.08 (CH2-O), 72.27 (OCHOH), 100.88 (OCH2O), 103.39, 108.12, 108.95, 109.33, 121.40, 121.76 (Arom. CH), 133.32, 134.51, 145.75, 145.93, 147.72 (arom. C) ppm. LC-HRMS m/z: calcd for C20H20O6 356.37; Found 379.11 [M + Na]+ (Figure 1).

Antioxidant activity

Three supplementary assays, namely, ferric reducing antioxidant power (FRAP) assay and 1,1-Diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay and 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical scavenging assay were employed to estimate the antioxidant capacity of P. cubeba fruit’s ethanolic extract and its Cubebin and Hinokinin containing fractions following the methodology described in previous studies [17, 18].

AChE inhibition assay

The Ellman’s method was performed in order to assess the extracts’ capacity to inhibit AChE. This assay was carried out for P. cubeba ethanolic crude extract as well as Cubebin and Hinokinin containing fractions according to the instructions given in our previous study. Galantamine hydrobromide was used as positive control [17].

Cell culture

Human neuroblastoma cell line, SH-SY5Y cells were acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA) cultured in DMEM with 15 % FBS and 100 μg/mL primocin, in a humidified incubator at 37 °C under 5 % CO2. Experiments were conducted when the cells at their exponential growth phase exponential phase.

Aβ (25–35) aggregates preparation

A large quantity of abnormally accumulated Aβ peptide in the brain has a huge impact in AD development, predominantly due to the highly toxic nature of the insoluble Aβ oligomers in contrast to the soluble monomeric Aβ or fibrils [19]. Thus, for the purpose of this research, Aβ (25–35) aggregations were prepared before use. For our experiment, prepared 1 mM Aβ (25–35) stock solution was kept at 37 °C for 3 days before treatment.

MTT(3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazoliumbromide) assay

SH-SY5Y cells were grown for 24 h in a 96-well plate containing 104 cells/well. The cells were exposed to Aβ (25–35) at concentrations of 5, 10, 20, 30, 40, and 50 μM for 24 h in order to establish the effective dosage. Additionally, to ascertain the impact of various concentrations of the crude extract (10, 5, 3, and 2 μg/mL), the Cubebin fraction (200, 100, 70, and 40 μg/mL), and the Hinokinin fraction (100, 40, 30, 15 μg/mL), cells were once again treated after 24 h of seeding as explained previously. Next, MTT cell proliferation and cytotoxicity assay kit (Boster, AR1156) was utilized according to the manufacturer’s instructions. In accordance with this, MTT (0.5 mg/mL) was included and kept in the dark for 4 h. Subsequently, 100 μL of solubilization buffer was transferred and the plate was incubated at 37 °C overnight until the formazan crystals were entirely dissolved. An automated microplate analyzer determined the absorbance at 570 nm. Following is the formula that was used to determine the rate of proliferation of SH-SY5Y cells: cell survival rate ð%Þ=experimental group OD570/control group OD570 × 100 % [20].

Neuroprotective effect of extracts against Aβ(25–35) cytotoxicity

SH-SY5Y cells were grown for 24 h in a 96-well plate containing 104 cells per well. To determine the neuroprotective effect of extracts against Aβ (25–35) cytotoxicity, before Aβ treatment, cells were pre-treated with two different doses of each extract for 2 h. Maximum non-toxic doses (10 and 5 μg/mL for crude extract, 100 and 70 μg/mL for Cubebin fraction, 100 and 40 μg/mL for Hinokinin fraction) were chosen for this assay. Cells were cultured for 24 h after each well received 50 μM Aβ. The MTT assay as previously mentioned was used to identify cell proliferation [20].

Statistical analysis

The mean ± standard deviation is used to represent all data. In the IBM SPSS Statistics (Version 27) software, multi-group comparisons were analyzed using one-way ANOVA followed by a post hoc Tukey test. At p<0.05, differences were accepted statistically significant.

Results

Extraction and characterization of two lignans from P. cubeba

Fifteen different fractions were obtained through the silica jell column. Fractions in the range of 70–75 and 85–89 were determined as to be nearly pure. These two fractions in solid phase were subjected to extra purification through the crystallization method. The characterization and structural determination of Cubebin [21] and Hinokinin [22] were proved by analyzing with NMR and they were confirmed by following the LC-HRMS procedure [13]. By comparing our NMR data with literature, it has been confirmed that these two fractions were rich mixture of Hinokinin (70–75) and Cubebin (85–89) Lignans, with 1.21 and 1.22 % yield respectively [14, 23], [24], [25]. Spectra of NMR and LC-HRMS analyzes are provided in the supplemental material.

Antioxidant activity of the P. cubeba fruit ethanolic extracts and isolated lignans

In the present research, the antioxidant capacities of the crude extract and two separated lignan fractions of P. cubeba were assessed. The numerical values of the results are portrayed in Table 1. The results obtained from FRAP assay, DPPH and ABTS radical scavenging assays indicated that the crude P. cubeba exhibited the strongest antioxidant capacity among the samples in relation to the standard ascorbic acid. The crude extract was as effective as ascorbic acid (difference not statistically significant) in the DPPH assay and stronger in the FRAP assay. However, according to the ABTS assay, ascorbic acid yet had significantly higher effectiveness than all three samples. Neither of the lignans were as effective as standard ascorbic acid bar Cubebin in the FRAP assay. Cubebin also proved to be the stronger lignan according to FRAP and DPPH assays. The difference between the antioxidant capacity of the two lignan fractions was not significant in the ABTS assay (Table 1).

Table 1:

Antioxidant activity of P. cubeba ethanolic extract and its isolated Cubebin and Hinokinin fractions.

Samples FRAP (µg/µ mol AA) DPPH (IC50 µg/mL) ABTS (IC50 µg/mL)
Ascorbic acid 176,12 (molecular weight) 27.71 ± 1.1a 28.4 ± 0.5
Crude extract 128.77 ± 0.99 25.02 ± 3.69a 35.29 ± 2.5
Cubebin 140.71 ± 0.76 38.49 ± 2.62 44.71 ± 2.89b
Hinokinin 185.24 ± 0.47 48.50 ± 4.81 45.72 ± 1.28b

  1. Values are mean ± standard deviation, n=3. Values in each column were compared with each other. Difference among the pairs marked with identical superscript alphabets a,b were not significant (p<0.05). AA, ascorbic acid; FRAP, ferric reducing antioxidant power; DPPH, 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay; ABTS, 2,2-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid radical scavenging assay.

Anti-AChE activity of P. cubeba fruit ethanolic extracts and isolated lignans

Here, we demonstrated the anti-AChE activities of the crude extract and the isolated Cubebin and Hinokinin lignan fractions. It was evident that the crude extract displayed the most potent anti-AChE activity indicating that crude extract possesses the highest amount of AChE inhibitors. In addition, Cubebin and Hinokinin fractions exhibited anti-AChE activities as well but no significant superiority was evident against one another. However, they yielded significantly weaker activities than that of the crude extract and the positive control Galantamine hydrobromide (p<0.05) (Figure 2).

Figure 2: Anti-AChE activities of crude ethanolic extract, Cubebin fraction, Hinokinin fraction and galantamine hydrobromide in vitro. Values are mean ± standard deviation, n=3. The difference between the bar pair marked with ns was not significant (p<0.05).
Figure 2:

Anti-AChE activities of crude ethanolic extract, Cubebin fraction, Hinokinin fraction and galantamine hydrobromide in vitro. Values are mean ± standard deviation, n=3. The difference between the bar pair marked with ns was not significant (p<0.05).

The cytotoxic effect of crude extracts and isolated lignans from P. cubeba on human SH-SY5Y cell lines

The general use of nutraceuticals by the common population warrants the need to study and evaluate the cytotoxicity of these natural compounds. Regarding this objective, it was essential to measure the cytotoxicity of each concentration of all samples in order to determine safe but effective doses through the MTT method performed on human SHSY5Y cell line.

According to the results, the crude extract presented a cytotoxic effect with an IC50 value of 66.88 ± 3.5 μg/mL and isolated Cubebin and Hinokinin containing fractions showed cytotoxicity with values of IC50=332 ± 4.3 μg/mL (0.93 ± 0.01 µM) and IC50=688.38 ± 5.6 μg/mL (1.94 ± 0.2 µM) respectively, when compared to the control.

Neuroprotective effect of extracts against Aβ25-35 cytotoxicity

This research was designed to assess the potential neuroprotective effect of P. cubeba extracts and in line with that, we assessed their ability against Aβ-mediated toxicity. Given that about 74 % of cells were deemed to be surviving cells at 50 μM of Aβ, the Aβ concentration for all subsequent assays was set at this concentration. The crude extract was highly protective against Aβ at both of the treated concentrations (5 and 10 μg/mL). It was evident that Cubebin and Hinokinin containing fractions significantly improve the viability of the SH-SY5Y cells against 50 μM Aβ toxicity both only at the concentration of 100 μg/mL compared to the control. However, the cell viability was not significantly different between the two given concentrations for each of the samples under study (Figure 3).

Figure 3: The cells were subjected to pretreatment with different doses of the crude extract, Cubebin and Hinokinin containing fractions for 2 h prior to treatment with 50 μM Aβ (25–35). The findings of three independent experiments, each carried out in triplicate, are shown as mean ± standard deviation. Excluding the untreated sample, all samples were compared with each other. The difference between bars pairs with *(p<0.05) and **(p<0.01) was significant.
Figure 3:

The cells were subjected to pretreatment with different doses of the crude extract, Cubebin and Hinokinin containing fractions for 2 h prior to treatment with 50 μM Aβ (25–35). The findings of three independent experiments, each carried out in triplicate, are shown as mean ± standard deviation. Excluding the untreated sample, all samples were compared with each other. The difference between bars pairs with *(p<0.05) and **(p<0.01) was significant.

Discussion

Emerging evidence demonstrates that free radical over-production impairs mitochondrial function and inflicts damage upon the central nervous system together and the rest of the body leading to neurodegeneration in aging and age-associated diseases like AD and Parkinson’s disease [23], [24], [25], [26], [27]. In the elderly population, cases of AD patients are on the rise, making it urgent to find effective therapies for age-associated oxidative stress as a chief culprit of AD [28].

Endogenous biomolecules in the living organisms are known to exist and act as free radical scavengers [29]. Acetyl-L-carnitine and R-alpha lipoic acid, examples for mitochondria specific antioxidants, are proven to be potent in overcoming oxidative imbalance and amyloid beta oxidation [30]. Exogenously, dietary supplements, particularly consisting of vitamins, polyphenols, or flavones are also of considerable importance in this regard [31]. Moreover, several herbal drugs, having free radical scavenging capabilities, have come to the fore in the treatment of such chronic diseases [32]. Considering the outcomes of three antioxidant experiments in relation to the standard ascorbic acid, the crude P. Cubeba, Cubebin, and Hinokinin fractions exhibited potent antioxidant activity. These findings are consistent with different studies demonstrating that P. cubeba aqueous methanolic and ethanolic extracts exhibit significant antioxidant activity [10, 11, 33]. Gayatri Nahak investigated the antioxidant activity of P. cubeba fruit extract, obtained using three different solvents: ethanol, methanol, and water [34]. They reported that the highest DPPH radical scavenging activity was observed in the ethanol extract, with a IC50 value of 77.61 ± 0.02. Another study by Medola et al. evaluated the antioxidant activity of hinokinin, which was obtained through partial synthesis of cubebin isolated from the dry seeds of P. cubeba [35]. The researchers measured the production of H2O2 in LLC-MK2 cell lines and found that different concentrations of hinokinin inhibited H2O2 production in the oxidation-induced cell lines. Zahin et al. also assessed the antioxidant potential of P. cubeba fruit extracts using different solvents, and found that the ethanolic extract exhibited the highest antioxidant activity in all four tests. Based on this, ethanol was preferred for extraction in our study [36]. The crude extract exerted an antioxidant effect stronger than that of the standard ascorbic acid in the FRAP and DPPH assays but not the ABTS assay. This is consistent with the phytocomponents present in P. cubeba, such as polyphenolic acids (phenolic acids, flavonoids, and lignans), alkaloids, and terpenoids [13, 37]. According to our results from the FRAP and DPPH assays, Cubebin exerts higher antioxidant activity compared to Hinokinin, which may be attributed to the difference in their polarity. This could be due to the fact that the fractions obtained from the silica gel column chromatography were eluted based on a non-polar to polar solvent system. Cubebin and Hinokinin differ in structure due to the presence of a hydroxyl group in Cubebin and a ketone group in Hinokinin on the tetrahydrofuran ring (Figure 1). The presence of a hydroxyl group enhances the polarity of molecules and provides a strong electron-donating ability to the molecule, which in turn, imparts higher antioxidant capacity [38]. Cubebin’s higher antioxidant activity may be attributed to the presence of the hydroxyl group.

Numerous studies have shown that cholinergic dysfunctions are linked to oxidative stress and the reducing breakdown of acetylcholine by inhibition of AChE is the most promising symptomatic treatments for AD [39]. AChE may also have non-cholinergic functions involving protein-protein interactions in the extracellular matrix, where it binds to Aβ and the AChE-amyloid complexes are formed resulting in the neuronal loss observed in Alzheimer’s brain [40].

As a result of the research conducted by Murata et al. on the AChE inhibitory properties of the Hinokinin isolated from the Chamaecyparis obtusa extract, the anti-AChE activity was found to have an IC50 value of 176 µM [41]. Moreover, Somani et al. reported the IC50 value of Cubebin as 992 μM for AChE inhibition in vitro, their further ex vivo AChE assay showed that (25 and 50 g/kg) Cubebin significantly inhibited brain AchE activity. They concluded that antioxidant properties of Cubebin was proven to be the source for the high inhibitory effects of this extract [42]. In the present study, Cubebin and Hinokinin fractions showed AChE inhibitory activities with IC50 values; 72.72 ± 8.7 and 84.54 ± 9.86 μg/mL respectively. While our study corroborates the activity of Hinokinin described in the literature, we observed a substantial difference in the Cubebin IC50 values, which were up to 5 times higher than those reported in a previous study. These results suggest that both of the lignan containing fractions and crude extract could be effective in developing novel strategies against AD.

In our attempt to assess the contribution of antioxidant properties in neuroprotection against AD we also evaluated the Cubebin and Hinokinin fractions’ influence against Aβ beta cytotoxicity on human SH-SY5Y cell line. Since reactive oxygen species (ROS) play a crucial part in Aβ-induced neurotoxicity, our observed neuroprotective effect may be the result of antioxidant properties of extracts we study herein. Chemical structure of both Cubebin and Hinokinin demonstrate that the presence of oxygenated functional groups [14] in their carbon skeleton may contribute to their neuroprotective potency in our study [43]. The crude extract exerted similar neuroprotectivity compared to the samples but in 10-and 20-fold lower concentrations. It is important to mention that Aβ-induced inflammation is also an important factor in the pathogenesis of AD [44]. Therefore, the presence of metabolites having antioxidant as well as anti-inflammatory activities alongside the two lignans within the crude extract, could also play an additional role in its greater efficacy against Aβ-induced neurotoxicity [45].

This study marks, to the extent of our knowledge, the first exploration of P. cubeba and its lignans fractions for anti-Aβ activity. The present study is limited by financial constraints that restricted the research design and materials utilized. The limited materials and sample size may also limit the statistical power and generalizability of the findings. However, our results demonstrated that the crude extract and isolated lignans were nevertheless impactful, in terms of neuroprotective properties. The collective effect of highly enriched secondary metabolites like phenolic acids and flavonoids present alongside the two studied lignans within the crude extract could explain its observed significantly greater activity compared to the individual lignans across all properties. However, a possible synergistic interaction among the two lignans and also with the aforementioned metabolites could also be an important factor in this superior potency. Hence, it could be argued that the P. cubeba crude extract and isolated lignan-containing fractions having inhibitory actions against acetylcholinesterase (AChE) and Aβ toxicity, has potential therapeutics for AD as remarkable efficiency is achieved by the development of multitargeted pharmaceuticals against this disease [46].

Although molecular docking studies have elucidated the binding mechanisms of AChE and Hinokinin [47], no such studies have been performed to date on Cubebin, leaving its molecular interaction with AChE unknown. Furthermore, dual binding capacity is a crucial attribute of AChE inhibitors that effectively prevents β-amyloid deposition associated with AChE [48]. Hence, future studies should focus on Cubebin-AChE molecular docking and possible dual binding properties in both lignans.

Conclusions

Our analyses of the brain AChE inhibition, antioxidant activity and cytotoxic effects shown by the extracts establish that the plant constituents present in the crude extract exert significantly higher effects than isolated cubebin and isolated hinokinin fractions by either an additional impact of other phytochemicals or possible synergistic interaction among all the metabolites altogether. According to the data revealed from our study crude extract possessed a promising cholinesterase inhibitory and neuroprotective effect against Aβ25-35 cytotoxicity, in comparison to the Hinokinin and Cubebin lignan containing fractions. We therefore suggest that the combination of lignans along with other phytochemicals in crude extract, synergistically may improve their action in prevention and treatment of AD. Our findings could form the basis for the future development of novel neuroprotective pharmacological agents for the treatment of AD.

Corresponding author: Assis. Prof. Dr. Shirin Tarbiat, Department of Molecular Biology and Genetics (English), Faculty of Engineering and Natural Sciences, Uskudar University, Altunizade, Haluk Türksoy Street. No: 14, 34662 Üsküdar, Istanbul, Türkiye, Phone: +90 543 776 15 87, E-mail:

Acknowledgments

Extraction and characterization of lignans were carried out by Salih Tuncay and Demet Unver. The research on antioxidant and enzyme activities were performed by Shirin Tarbiat. Neuroprotective effects were studied by Sevim Isik and Bercem Yeman Kiyak. Ali Reza Mohseni carried out the statistical analyses and helped edit the manuscript. We would like to thank Uskudar University and Bezmialem Vakif University for providing the necessary means to conduct this research. We also like to thank Ahmet Balci and Sule Yalcin for their expertise and technical assistance in NMR and LC-HRMS.

  1. Research funding: None declared.

  2. Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.

  3. Competing interests: Authors state no competing of interest.

  4. Informed consent: Not applicable.

  5. Ethical approval: Not applicable.

References

1. El Haj, M, Kessels, RPC. Context memory in Alzheimer’s disease. Dement Geriatr Cogn Dis Extra 2013;3:342–50. https://doi.org/10.1159/000354187.Search in Google Scholar PubMed PubMed Central

2. Kern, A, Behl, C. The unsolved relationship of brain aging and late-onset Alzheimer disease. Biochim Biophys Acta Gen Subj 2009;1790:1124–32. https://doi.org/10.1016/j.bbagen.2009.07.016.Search in Google Scholar PubMed

3. DeTure, MA, Dickson, DW. The neuropathological diagnosis of Alzheimer’s disease. Mol Neurodegener 2019;14:1–18. https://doi.org/10.1186/s13024-019-0333-5.Search in Google Scholar PubMed PubMed Central

4. Walczak-Nowicka, LJ, Herbet, M. Acetylcholinesterase inhibitors in the treatment of neurodegenerative diseases and the role of acetylcholinesterase in their pathogenesis. Int J Mol Sci 2021;22:1–63. https://doi.org/10.3390/ijms22179290.Search in Google Scholar PubMed PubMed Central

5. Park, SY, Kim, HS, Hong, SS, Sul, D, Hwang, KW, Lee, D. The neuroprotective effects of traditional oriental herbal medicines against β-amyloid-induced toxicity. Pharm Biol 2009;47:976–81. https://doi.org/10.1080/13880200902967987.Search in Google Scholar

6. Khan, H, Marya, AS, Kamal, MA, Patel, S. Flavonoids as acetylcholinesterase inhibitors: current therapeutic standing and future prospects. Biomed Pharmacother 2018;101:860–70. https://doi.org/10.1016/j.biopha.2018.03.007.Search in Google Scholar PubMed

7. Andriana, Y, Xuan, TD, Quy, TN, Tran, HD, Le, QT. Biological activities and chemical constituents of essential oils from piper cubeba bojer and piper nigrum L. Molecules 2019;24:1–16. https://doi.org/10.3390/molecules24101876.Search in Google Scholar PubMed PubMed Central

8. Mgbeahuruike, EE, Yrjonen, T, Vuorela, H, Holm, Y. Bioactive compounds from medicinal plants: focus on piper species. South Afr J Bot 2017;112:54–69. https://doi.org/10.1016/j.sajb.2017.05.007.Search in Google Scholar

9. Carpinella, MC, Andrione, DG, Ruiz, G, Palacios, SM. Screening for acetylcholinesterase inhibitory activity in plant extracts from Argentina. Phytother Res 2010;24:259–63. https://doi.org/10.1002/ptr.2923.Search in Google Scholar PubMed

10. Alminderej, F, Bakari, S, Almundarij, TI, Snoussi, M, Aouadi, K, Kadri, A. Antioxidant activities of a new chemotype of piper cubeba L. Fruit essential oil (Methyleugenol/Eugenol): in silico molecular docking and ADMET studies. Plants 2020;9:1–18. https://doi.org/10.3390/plants9111534.Search in Google Scholar PubMed PubMed Central

11. Alqadeeri, F, Rukayadi, Y, Abbas, F, Shaari, K. Antibacterial and antispore activities of isolated compounds from piper cubeba L. Molecules 2019;24:1–15. https://doi.org/10.3390/molecules24173095.Search in Google Scholar PubMed PubMed Central

12. Graidist, P, Martla, M, Sukpondma, Y. Cytotoxic activity of piper cubeba extract in breast cancer cell lines. Nutrients 2015;7:2707–18. https://doi.org/10.3390/nu7042707.Search in Google Scholar PubMed PubMed Central

13. Drissi, B, Mahdi, I, Yassir, M, Ben Bakrim, W, Bouissane, L, Sobeh, M. Cubeb (Piper cubeba L.f.): a comprehensive review of its botany, phytochemistry, traditional uses, and pharmacological properties. Front Nutr 2022;9:1048520. https://doi.org/10.3389/fnut.2022.1048520.Search in Google Scholar PubMed PubMed Central

14. de Lima, RG, Barros, MT, Laurentiz, RD. Medicinal attributes of lignans extracted from piper Cubeba: current developments. ChemistryOpen 2018;7:180–91. https://doi.org/10.1002/open.201700182.Search in Google Scholar PubMed PubMed Central

15. Gusson-Zanetoni, JP, da Silva, J, Picao, TB, Cardin, LT, Prates, J, Sousa, SO, et al.. Effect of Piper cubeba total extract and isolated lignans on head and neck cancer cell lines and normal fibroblasts. J Pharmacol Sci 2022;148:93–102. https://doi.org/10.1016/j.jphs.2021.09.002.Search in Google Scholar PubMed

16. Kiziltas, H, Bingol, Z, Goren, AC, Kose, LP, Durmaz, L, Topal, F, et al.. LC-HRMS profiling and antidiabetic, anticholinergic, and antioxidant activities of aerial parts of kinkor (ferulago stellata). Molecules 2021;26:1–18. https://doi.org/10.3390/molecules26092469.Search in Google Scholar PubMed PubMed Central

17. Tarbiat, S, Turutoglu, AS, Ekingen, M. Acetylcholinesterase inhibitory potential and antioxidant activities of five cultivars of rosa damascena mill. From isparta, Turkey. Curr Top Nutraceutical Res 2020;18:354–9. https://doi.org/10.37290/ctnr2641-452x.18:354-359.10.37290/ctnr2641-452X.18:354-359Search in Google Scholar

18. Re, R, Pellegrini, N, Proteggente, A, Pannala, A, Yang, M, Rice-Evans, C. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med 1999;26:1231–7. https://doi.org/10.1016/s0891-5849(98)00315-3.Search in Google Scholar PubMed

19. Kelly, BL, Ferreira, A. β-amyloid-induced dynamin 1 degradation is mediated by N-methyl-D-aspartate receptors in hippocampal neurons. J Biol Chem 2006;281:28079–89. https://doi.org/10.1074/jbc.m605081200.Search in Google Scholar PubMed

20. Terzioglu-Usak, S, Negis, Y, Karabulut, DS, Zaim, M, Isik, S. Cellular model of Alzheimer’s disease: a beta(1−42) peptide induces amyloid deposition and a decrease in topo isomerase II beta and Nurr1 expression. Curr Alzheimer Res 2017;14:636–44. https://doi.org/10.2174/1567205014666170117103217.Search in Google Scholar PubMed

21. Davidson, SJ, Pearce, AN, Copp, BR, Barker, D. Total synthesis of (−)-Bicubebin A, B, (+)-Bicubebin C and structural reassignment of (−)-cis-Cubebin. Org Lett 2017;19:5368–71. https://doi.org/10.1021/acs.orglett.7b02644.Search in Google Scholar PubMed

22. Enders, D, Milovanović, M. Asymmetric synthesis of (+)-Hinokinin, (+)-Dihydrocubebin and cubebin dimethyl ether, a new lignan from Phyllanthus niruri. Z Naturforsch B Chem Sci 2007;62:117–20. https://doi.org/10.1515/znb-2007-0117.Search in Google Scholar

23. Kayama, Y, Raaz, U, Jagger, A, Adam, M, Schellinger, IN, Sakamoto, M, et al.. Diabetic cardiovascular disease induced by oxidative stress. Int J Mol Sci 2015;16:25234–63. https://doi.org/10.3390/ijms161025234.Search in Google Scholar PubMed PubMed Central

24. Losada-Barreiro, S, Bravo-Diaz, C. Free radicals and polyphenols: the redox chemistry of neurodegenerative diseases. Eur J Med Chem 2017;133:379–402. https://doi.org/10.1016/j.ejmech.2017.03.061.Search in Google Scholar PubMed

25. Pavolic, D, Dordevic, V, Kocic, G. Signal transduction-free radical modulation. Jugosl Med Biohemija 2002;21:69–84. https://doi.org/10.2298/jmh0202069p.Search in Google Scholar

26. Han, ES, Muller, FL, Perez, VI, Qi, WB, Liang, HY, Xi, L, et al.. The in vivo gene expression signature of oxidative stress. Physiol Genom 2008;34:112–26. https://doi.org/10.1152/physiolgenomics.00239.2007.Search in Google Scholar PubMed PubMed Central

27. Polimeni, L, Del Ben, M, Baratta, F, Perri, L, Albanese, F, Pastori, D, et al.. Oxidative stress: new insights on the association of non-alcoholic fatty liver disease and atherosclerosis. World J Hepatol 2015;7:1325–36. https://doi.org/10.4254/wjh.v7.i10.1325.Search in Google Scholar PubMed PubMed Central

28. Ionescu-Tucker, A, Cotman, CW. Emerging roles of oxidative stress in brain aging and Alzheimer’s disease. Neurobiol Aging 2021;107:86–95. https://doi.org/10.1016/j.neurobiolaging.2021.07.014.Search in Google Scholar PubMed

29. Jadon, N, Jain, R, Aribam, NG, Chauhan, P. Review-monitoring of endogenous antioxidants: an electroanalytical approach. J Electrochem Soc 2017;164:H266–77. https://doi.org/10.1149/2.1311704jes.Search in Google Scholar

30. Palacios, HH, Yendluri, BB, Parvathaneni, K, Shadlinski, VB, Obrenovich, ME, Leszek, J, et al.. Mitochondrion-specific antioxidants as drug treatments for Alzheimer disease. CNS Neurol Disord: Drug Targets 2011;10:149–62. https://doi.org/10.2174/187152711794480474.Search in Google Scholar PubMed

31. Ardelean, F, Vlase, L, Mocan, AM, Gheldiu, AM, Antal, DS, Trandafirescu, C, et al.. Dietary supplements with resveratrol, flavonoids and phenolic acids: in-depth HPLC profiling and antioxidant capacity as quality markers. Rev Chim 2017;68:401–7. https://doi.org/10.37358/rc.17.2.5463.Search in Google Scholar

32. Leite, KCD, Garcia, LF, Lobon, GS, Thomaz, DV, Moreno, EKG, de Carvalho, MF, et al.. Antioxidant activity evaluation of dried herbal extracts: an electroanalytical approach. Rev Bras Farmacogn 2018;28:325–32. https://doi.org/10.1016/j.bjp.2018.04.004.Search in Google Scholar

33. Ahmed, AS, Ahmed, QU, Saxena, AK, Jamal, P. Evaluation of in vitro antidiabetic and antioxidant characterizations of Elettaria cardamomum (L.) Maton (Zingiberaceae), Piper cubeba L. f. (Piperaceae), and Plumeria rubra L. (Apocynaceae). Pak J Pharm Sci 2017;30:113–26.Search in Google Scholar

34. Nahak, G, Sahu, RK. Phytochemical evaluation and antioxidant activity of Piper cubeba and Piper nigrum. J Appl Pharm Sci 2011;1:153–7.Search in Google Scholar

35. Medola, JF, Cintra, VP, Pesqueira, ESEP, de Andrade Royo, V, da Silva, R, Saraiva, J, et al.. (-)-Hinokinin causes antigenotoxicity but not genotoxicity in peripheral blood of Wistar rats. Food Chem Toxicol 2007;45:638–42. https://doi.org/10.1016/j.fct.2006.10.012.Search in Google Scholar PubMed

36. Zahin, M, Khan, MS, Qais, FA, Abulreesh, HH, Ahmad, I. Antioxidant properties and anti-mutagenic potential of Piper Cubeba fruit extract and molecular docking of certain bioactive compounds. Drug Chem Toxicol 2018;41:358–67. https://doi.org/10.1080/01480545.2018.1429459.Search in Google Scholar PubMed

37. Abdul-Jalil, TZ, Zahra, AN. Piper cubeba: phytochemical and pharmacological review of a routinely used spices. Int J Pharmaceut Res 2020;12:761–8.10.31838/ijpr.2020.SP1.119Search in Google Scholar

38. Chen, J, Yang, J, Ma, L, Li, J, Shahzad, N, Kim, CK. Structure-antioxidant activity relationship of methoxy, phenolic hydroxyl, and carboxylic acid groups of phenolic acids. Sci Rep 2020;10:2611. https://doi.org/10.1038/s41598-020-59451-z.Search in Google Scholar PubMed PubMed Central

39. Talesa, VN. Acetylcholinesterase in Alzheimer’s disease. Mech Ageing Dev 2001;122:1961–9. https://doi.org/10.1016/s0047-6374(01)00309-8.Search in Google Scholar PubMed

40. Alvarez, A, Alarcón, R, Opazo, C, Campos, EO, Muñoz, FJ, Calderón, FH, et al.. Stable complexes involving acetylcholinesterase and amyloid-beta peptide change the biochemical properties of the enzyme and increase the neurotoxicity of Alzheimer’s fibrils. J Neurosci 1998;18:3213–23. https://doi.org/10.1523/jneurosci.18-09-03213.1998.Search in Google Scholar PubMed PubMed Central

41. Murata, K, Tanaka, K, Akiyama, R, Noro, I, Nishio, A, Nakagawa, S, et al.. Anti-cholinesterase activity of crude drugs selected from the ingredients of incense sticks and heartwood of chamaecyparis obtusa. Nat Prod Commun 2018;13:803–6. https://doi.org/10.1177/1934578x1801300704.Search in Google Scholar

42. Somani, GS, Nahire, MS, Parikh, AD, Mulik, MB, Ghumatkar, PJ, Laddha, KS, et al.. Neuroprotective effect of Cubebin: a dibenzylbutyrolactone lignan on scopolamine-induced amnesia in mice. Indian J Med Res 2017;146:255–9. https://doi.org/10.4103/ijmr.IJMR_156_14.Search in Google Scholar PubMed PubMed Central

43. Sung, YJ, Cheng, CL, Chen, CS, Huang, HB, Huang, FL, Wu, PC, et al.. Distinct mechanisms account for beta-amyloid toxicity in PC12 and differentiated PC12 neuronal cells. J Biomed Sci 2003;10:379–88. https://doi.org/10.1159/000071157.Search in Google Scholar

44. Serpente, M, Bonsi, R, Scarpini, E, Galimberti, D. Innate immune system and inflammation in Alzheimer’s disease: from pathogenesis to treatment. Neuroimmunomodulation 2014;21:79–87. https://doi.org/10.1159/000356529.Search in Google Scholar PubMed

45. Mothana, RA, Al-Said, MS, Raish, M, Khaled, JM, Alharbi, NS, Alatar, A, et al.. Chemical composition, anti-inflammatory and antioxidant activities of the essential oil of Piper cubeba L. Rom Biotechnol Lett 2017;22:12366–76.10.3390/molecules22030367Search in Google Scholar PubMed PubMed Central

46. Manzoor, S, Gabr, MT, Rasool, B, Pal, K, Hoda, N. Dual targeting of acetylcholinesterase and tau aggregation: design, synthesis and evaluation of multifunctional deoxyvasicinone analogues for Alzheimer’s disease. Bioorg Chem 2021;116:1–13. https://doi.org/10.1016/j.bioorg.2021.105354.Search in Google Scholar PubMed

47. Faloye, KO, Mahmud, S, Fakola, EG, Oyetunde, YM, Fajobi, SJ, Ugwo, JP, et al.. Revealing the acetylcholinesterase inhibitory potential of Phyllanthus amarus and its phytoconstituents: in vitro and in silico approach. Bioinf Biol Insights 2022;16:1–11. https://doi.org/10.1177/11779322221118330.Search in Google Scholar PubMed PubMed Central

48. Galdeano, C, Viayna, E, Arroyo, P, Bidon-Chanal, A, Blas, JR, Muñoz-Torrero, D, et al.. Structural determinants of the multifunctional profile of dual binding site acetylcholinesterase inhibitors as anti-Alzheimer agents. Curr Pharmaceut Des 2010;16:2818–36. https://doi.org/10.2174/138161210793176536.Search in Google Scholar PubMed

Supplementary Material

This article contains supplementary material (https://doi.org/10.1515/tjb-2023-0032).

Received: 2023-02-13
Accepted: 2023-05-09
Published Online: 2023-06-01

© 2023 the author(s), published by De Gruyter, Berlin/Boston

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

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  2. Review
  3. Molecular mechanisms and genetics of Alzheimer’s disease
  4. Short Communication
  5. A simple PCR-SSP method for detection of HLA-B*15:02, *15:13, and *15:21
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  16. Neuroprotective effects of Cubebin and Hinokinin lignan fractions of Piper cubeba fruit in Alzheimer’s disease in vitro model
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https://doi.org/10.1515/tjb-2023-0032
Keywords for this article
acetylcholinesterase; amyloid beta; Cubebin; Hinokinin; lignans; Piper cubeba
Creative Commons
BY 4.0

Articles in the same Issue

  1. Frontmatter
  2. Review
  3. Molecular mechanisms and genetics of Alzheimer’s disease
  4. Short Communication
  5. A simple PCR-SSP method for detection of HLA-B*15:02, *15:13, and *15:21
  6. Research Articles
  7. The influence of CASP8 D302H gene variant in colorectal cancer risk and prognosis
  8. Silencing TCAB1 suppresses proliferation of hepatocellular carcinoma cells by inducing apoptosis
  9. Association of a haplotype in the NRG1 gene with schizophrenia: a case-control study
  10. Investigation of the roles of TGFβ1, CUG2, TGFBI genes, and thiol-disulfide balance on prostate cancer and metastasis
  11. The effect of krill oil on Wnt/β-catenin signaling pathway in acetaminophen-induced acute liver injury in mice
  12. Antiproliferative activity of Malus sylvestris Miller against HepG2 cell line with their antioxidant properties and phenolic composition
  13. Assessment of the effects of CNR1, FAAH and MGLL gene variations on the synthetic cannabinoid use disorder
  14. Screening of medicinal mushroom strains with antimicrobial activity and polysaccharides production
  15. The effects of Hericium erinaceus extracts on cell viability and telomerase activity in MCF-7 cells
  16. Neuroprotective effects of Cubebin and Hinokinin lignan fractions of Piper cubeba fruit in Alzheimer’s disease in vitro model
  17. Effects of kynurenic acid and choline on lipopolysaccharide-induced cyclooxygenase pathway
  18. Effects of PON1 QR192 genetic polymorphism and paraoxonase, arylesterase activities on deep vein thrombosis
  19. Evaluation of calcium/magnesium ratio in patients with type 2 diabetes mellitus
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