Abstract
The essential oil from rhizomes of Curcuma alismatifolia Gagnep. was extracted by hydrodistillation. Chemical analysis by gas chromatography-mass spectrometry identified 12 compounds, of which the most prominent were xanthorrhizol (82.2%), ar-curcumene (6.5%), α-cedrene (1.8%), and β-bisabolol (1.1%). The essential oil at 1 ng/mL showed neuroprotective and neuritogenic activity on P19-derived neurons by significantly enhancing cell viability, length, and branching numbers of the cultured P19-derived neurons. In addition, xanthorrhizol, the major compound of the essential oil, at 10 ng/mL also exhibited the protection of P19-derived neurons. However, the mechanism of their neuroprotective activity was not correlated with their antioxidant activity.
Alzheimer’s disease (AD) is the most common neurodegenerative disorder. AD is a progressive disease that causes the irreversible loss of neurons in the brain. 1,2 The number of Asia Pacific people with dementia including AD is estimated to increase from 23 million people in 2015 to 71 million people by the year 2050. 3 For this reason, multifunctional substances from various sources have been developed as a potential drug for AD treatment.
Curcuma alismatifolia Gagnep. or Siam tulip is a member of family Zingiberaceae found in Thailand, Laos, and Cambodia. It is used as a pot plant and cut flower because of its attractive pink bracts. 4,5 Recently, antioxidant activity has been reported from the roots and rhizomes essential oil of C. alismatifolia Gagnep (EOCA). 6
The neuroprotective activities have been frequently reported in plant extracts. 7 -9 Nevertheless, the activities of essential oil have not been mentioned much. Therefore, this study aimed to investigate the neurological activities including neuroprotective and neuritogenic activities of EOCA which have not been reported before.
The EOCA was extracted by water distillation. The chromatogram from different polarities of gas chromatography (GC) columns showed that xanthorrhizol is the major component of EOCA (Table 1). Xanthorrhizol is a bisabolane-type sesquiterpenoid mostly found in the rhizome of Curcuma xanthorrhiza Roxb. This study showed that EOCA is a new rich source of xanthorrhizol which has a wide range of activities including neuroprotection. 10 -15 Although xanthorrhizol was found to be the major component of EOCA in this study, its ratio is different from previous report. 6 The variation of chemical components of essential oil may come from different cultivated areas.
Chemical Components of the Essential Oil From Curcuma alismatifolia Gagnep. Rhizomes.
LRI = Linear retention indices of the peaks on the INNOWAX column.
The 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) assay was performed to reveal that EOCA at 1 ng/mL had no toxicity with the P19-derived neuron. It was found that EOCA at 1 ng/mL has neuroprotective activity by significantly increasing the percentage of cell viability of the cultures (% cell viability = 36.22% compared to the serum-deprived culture) and no statistically significant differences between EOCA and positive control (1 nM of quercetin) as shown in Table 2. The neuroprotective activity of EOCA may be caused by the synergistic effect of xanthorrhizol and other components because the concentration of xanthorrhizol is higher than EOCA to exhibit the similar neuroprotective effect.
Neuroprotective Activity of the Essential Oil From Curcuma alismatifolia Gagnep. Rhizomes on P19-Derived Neurons Against Oxidative Stress Induced by Serum Deprivation.
P <0.05 versus serum-supplemented culture.
P <0.05 versus serum-deprived culture.
Moreover, the superoxide anion scavenging assay was performed to investigate the mechanism of EOCA neuroprotective activity. Superoxide anion is one of the important reactive oxygen species that can become a strong oxidant and cause cellular damage. 16,17 This study found that EOCA showed superoxide anion scavenging ability with an IC50 value of 376 µg/mL, which was higher than its neuroprotection concentration. This result indicated that the mechanism of neuroprotective activity was not correlated with antioxidant activity.
In addition, the results of neuritogenic activity showed that EOCA at 1 ng/mL significantly enhanced the neurite outgrowth of P19-derived neurons of branching numbers and length of cultured neurons when compared to the control group, as presented in Table 3. This study shows that EOCA possesses neuroprotective and neuritogenic activities. According to these activities, EOCA may have a potential to develop as drug for AD prevention or treatment.
Neuritogenic Activity of the Essential Oil From Curcuma alismatifolia Gagnep. Rhizomes on P19-Derived Neurons.
P <0.05 versus control group.
Experimental
Plant Material
The rhizomes of C. alismatifolia were collected from Amphoe Mae Rim, Chiang Mai, Thailand, in March 2016. A voucher specimen was deposited at Queen Sirikit Botanical Garden, Thailand. The essential oil was extracted by water distillation with Clevenger apparatus from the small pieces of plant materials. It was kept at 2°C to 8°C until being used.
Gas Chromatography-Mass Spectrometry
The chemical constituents of the essential oil were analyzed by gas chromatography-mass spectrometry (GC-MS) with 2 different polarities of columns. One was analyzed on a Finnigan Trace GC ultra (Thermo Electron Corporation, USA), DSQ Quadrupole detector fitted with a fused silica column BPX5 (30 m × 0.25 mm i.d.; 0.25 µM film thickness) under condition as reported earlier. 18 Mass spectra library and the percentage composition were computed from GC peak areas. 19 The other was utilized on an Agilent Technologies Model 6890 N, Quadrupole Mass Selective Detector Model 5973 fitted with INNOWAX column (30 m × 0.25 mm i.d.; 0.25 µM film thickness); column temperature was 50°C for 1 minute, then ramped up to 240°C with the rate of 3°C/min; sample volume injected was 1 µL; split ratio was 1:100; and the carrier gas was He (1 mL/min). MS was performed by EI-positive mode at 70 eV ionization voltage. Compounds of the essential oil were identified by linear retention indices and calculated using linear interpolation relative to retention times of C8 to C24 of n-alkanes, compared with standard and reference data. Mass spectra were compared with data of reference from Wiley7n library.
Cell Culture
P19 cells (American Type Culture Collection, USA) were grown and differentiated into P19-derived neurons as described in Tadtong et al. 7
Cell Viability Assay
The cell viability assay was investigated by XTT reduction method. The procedure was described by Tadtong et al. 8
Neuroprotective Activity Assay
The assay was carried out on P19-derived neurons cultured in 96-well plate as described by Tadtong et al. Quercetin (Fluka, Switzerland) at concentration of 1 nM was used as positive control. The neuroprotective activity of xanthorrhizol (Cayman, USA) was also evaluated. 2,8
Neuritogenic Activity Assay
The assay was elevated by measuring the length and branching numbers of neurons. It was carried out on P19-derived neurons cultured in 96-well plate as described by Tadtong et al. Quercetin (Fluka, Switzerland) at concentration of 1 nM was used as positive control. 2,8
Superoxide Anion (O2∙−) Scavenging Activity Assay
The reduction of NBT to blue formazan was measured to estimate the O2∙− radical scavenging activity. The assay was performed as described by Supasuteekul et al. L-Ascorbic acid (Sigma, USA) was used as a positive control. 16
Statistical Analysis
One-way analysis of variance test from IBM SPSS Statistics version 21.0 was used to analyze the significant difference of average % cell viability, length, and branching number of the neurons. Data are presented in the form of mean with standard deviation and considering P-value < 0.05 as significant.
Footnotes
Acknowledgments
The authors would like to thank Asst Prof Wattanaporn Phattanaphukdee for statistical analysis.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by grant 181/2560 from Srinakharinwirot University.