Phytochemical profiling of the essential oils from three Curcuma species and their in vitro and in silico dengue protease inhibition activity

Abstract The chemical compositions, in vitro and in silico anti-dengue activity of the essential oils of the rhizomes of Curcuma longa Linn., C. aeruginosa Roxb., and C. xanthorrhiza Roxb. had been investigated. The C. longa oil was mainly composed of ar-turmerone (54.0%) and curlone (17.7%), while the C. aeruginosa oil was rich in curzerenone (23.4%), 1,8-cineole (21.2%), and camphor (7.1%). Xanthorrhizol (21.6%), β-curcumene (19.5%), ar-curcumene (14.2%), and camphor (9.2%) were the major compounds in the C. xanthorrhiza oil. Among the oils, the C. longa oil was found to be the most active NSB-NS3 protease inhibitor (IC50 1.98 μg/mL). PLS biplot disclosed that the essential oils were classified into three separated clusters based on their characteristic chemical compositions, with C. longa positioned closest to the in vitro anti-dengue activity. Four compounds from the C. longa oil have both hydrogen and hydrophobic bonds that could be responsible for the DENV-2 NS2B-NS3 inhibitory effect. Graphical Abstract


Introduction
Curcuma longa Linn., C. aeruginosa Roxb., and C. xanthorrhiza Roxb.are among the economically important Curcuma species belonging to the family Zingiberaceae (Jantan et al. 1999).The genus Curcuma comprises approximately 93-100 species of rhizomatous herbs occurring throughout the tropics of Asia, from India to South China, Southeast Asia, Papua New Guinea, and Northern Australia (Sirirugsa et al. 2007;Fitria et al. 2019).The members of this genus have been extensively used in traditional medicine to treat multiple illnesses and are greatly valued as flavouring and food colourants in Asian cuisine, spices, perfumes, cosmetics, and ornamental plants (Fitria et al. 2019).The Curcuma species produce various groups of bioactive compounds, such as essential oils, diarylheptanoids, phenolics, alkaloids, and terpenoids (Akarchariya et al. 2017;Kulyal et al. 2021).Their essential oils which are predominantly composed of terpene and their oxygenated derivatives were reported to exhibit a wide spectrum of biological effects including antitumor, anti-inflammatory, antioxidant, antimicrobial, gastroprotective, choleretic, insecticidal, and neuropharmacological activities (Jarikasem et al. 2005;Liu et al. 2012;oyemitan et al. 2017;Akinyemi and Adeniyi 2018;Septama et al. 2022).Even though there are many studies on the bioactivities of Curcuma essential oils, nevertheless, studies on the antiviral potential against DENV have not been widely carried out.To date, the antiviral effect has only been conducted on the extracts of C. longa and C. aeruginosa as well as on pure compounds from C. longa (Tan et al. 2006;Ichsyani et al. 2017;Deep et al. 2018;Ardebili et al. 2021;Ferreira et al. 2022).Hence, the purpose of this study was to characterize the essential oils extracted from the rhizomes of three Curcuma species (C.longa, C. aeruginosa, and C. xanthorrhiza) growing in Malaysia and correlate them with their DENV-2 NS2B-NS3 inhibitory potential.For this, we applied gas chromatography coupled with multivariate data analysis (MVDA).In parallel, molecular docking techniques were employed to investigate the binding interactions of selected compounds in the essential oils with NS2B-NS3 protease.

Chemical characterization of the essential oils
The fresh rhizomes of C. longa, C. aeruginosa, and C. xanthorrhiza yielded essential oils in 0.41%, 0.19%, and 0.60% based on the fresh weight, respectively.Table S1 displayed the identified compounds in the investigated oils in order of elution on a ZB-5 type column.The C. longa oil was characterized by the presence of 20 compounds, accounting for 82.9% of the total oil.This oil was rich in oxygenated sesquiterpenes (77.7%) with ar-turmerone (54.0%) and curlone (17.7%) being the main compounds.In line with our results, ar-turmerone and curlone were also previously identified in a remarkable amount in the C. longa rhizomes oils growing in different localities (Naz et al. 2010;Liju et al. 2011;Stanojević et al. 2015;Tanaka et al. 2015;oyemitan et al. 2017;Ferreira Guimares et al. 2020;Zheng et al. 2020).A high content of ar-turmerone in this species suggests the potentiality of C. longa to be utilised as a source for ar-turmerone.
Forty-three compounds were identified in the C. aeruginosa oil, representing 83.2% of the oil content.The oil was made up predominantly of oxygenated sesquiterpenes (32.4%) and oxygenated monoterpenes (32.1%).Sesquiterpenes and monoterpene hydrocarbons were also detected in a significant amount, constituting 11.6% and 7.0%, respectively.The major compounds in the oil were curzerenone (23.4%), 1,8-cineole (21.2%), and camphor (7.1%).Apart from terpenes, a ketone, identified as 2-undecanone (0.1%) also existed in the C. aeruginosa oil.This result was in agreement with prior studies, which characterized curzerenone, 1,8-cineole, and camphor as the principal compounds of essential oil from C. aeruginosa grown in Peninsular Malaysia (Sirat et al. 1998;Jantan et al. 1999;Jani et al. 2021).on the other hand, the current results were almost identical with the oil from Thailand, except for camphor which was absent in the oil (Jarikasem et al. 2005).on a contrary, curzerenone, 1,8-cineole, and camphor were not previously found in the rhizomes oil of C. aeruginosa from Vietnam (oanh et al. 2018).

Expression and purification of a recombinant NS2B-NS3 protease complex
Previously, we have developed a recombinant NS2B-NS3 protease complex by fusing the DENV-2 NS2B viral protein and a novel NS3 protease enzyme (Choon et al. 2013).The recombinant enzyme complex was expressed in Escherichia coli BL21(DE3).In this work, the recombinant NS2B-NS3 protease complex was purified to homogeneity by a single-step purification using a Ni-NTA affinity chromatography column.Purified NS2B-NS3 protease complex exhibited a molecular mass of 40 kDa, as observed on SDS-PAGE (Figure S1).

DENV-2 NS2B-NS3 protease inhibitory activity
Initial screening of the rhizomes essential oils of three Curcuma species using in vitro DENV-2 NS2B-NS3 protease inhibitory assay showed modest inhibition of protease activity.The C. longa, C. aeruginosa, and C. xanthorrhiza oils displayed moderate inhibitory activities of 72.30%, 55.26%, and 32.69%, respectively at 200 μg/mL against DENV-2 NS2B-NS3 (Table S2).Amongst the oils, the C. longa oil demonstrated the highest anti-dengue potential against DENV-2 NS2B-NS3 with an IC 50 value of 1.98 µg/ mL.This was followed by the C. aeruginosa oil with an IC 50 of 14.28 µg/mL.Nevertheless, the IC 50 value of the C. xanthorrhiza oil was not determined since its percentage of inhibition was less than 50%.The present results are comparable with previous studies whereby the methanol fraction and extract of the rhizomes of C. longa displayed antiviral activity against DENV-2 ( Tan et al. 2006;Ichsyani et al. 2017).

Multivariate data analysis
In this study, multivariate data analysis (MVDA) was utilized to integrate chemical compositions profiled from GC-MS data produced by the essential oils of three Curcuma species, i.e. C. longa, C. aeruginosa, and C. xanthorrhiza to their DENV-2 NS2B-NS3 protease inhibitory activity.The correlation of both sets of data was determined using supervised analysis of the MVDA method known as partial least square (PLS).Figure S2 is a PLS biplot that shows the correlation between chemical compounds and DENV-2 NS2B-NS3 protease inhibitory activity.This biplot is a combination of score and loading plots showing the species clustering and variables that contribute to the separation and inhibitory activity.Due to the differences in chemical profiles, all essential oils were positioned in three different quadrants.The closest species to the activity is C. longa with ten compounds serving as chemical markers for the activity.The markers were listed in Table S3 with the variable importance for the projection (VIP) score generated from the model.

Molecular docking
In total, 66 first confirmations, one for each ligand bound to the receptor, were viewed and analysed.The values of the binding affinity range from −7.40 to −4.20 kcal/mol.By comparing each confirmation with the control drug, quercetin (CID-5280343), the results showed that only seven compounds interacted at the same binding site as quercetin, and the remaining compounds interacted at a different binding site of the receptor.The control drug has the value of binding affinity of −7.7 kcal/mol with three hydrogen bonds found between three hydroxy groups and amino acids Lys73 (3.29 Å), Asn152 (2.97 Å), and Leu149 (3.21 Å) and eight hydrophobic bonds (with Lys74, Leu76, Ile123, Gly148, Leu149, Ile165, Ala166, and Asn167).only molecular interactions of the 10 compounds in Table S3 were analysed as their presence correlated to the inhibitory activities of DENV2 NSB-NS3 based on GC-MS features.All 10 compounds were present in C. longa.The binding affinity values and their molecular interactions can be found in Table S4.
The results (Figure S3) indicated that compounds from oxygenated sesquiterpene except for ar-turmerol, form both hydrogen bonds and hydrophobic interactions.The compounds (E)-α-atlantone, (6S,7R)-bisabolene (CID-91753614) and ar-turmerone (CID-160512) were observed to interact with Thr118 and/or Thr120 although ar-turmerone was observed to interact at a different binding site as of quercetin.It was suggested that the hydrogen bonds were formed due to the presence of carbonyl group in the compounds.It was demonstrated that curlone (CID-196216) also formed hydrogen bonds due to the existence of carbonyl group.In addition, this group also formed hydrogen bonds with two amino acids in the catalytic triad, which could contribute to the disruption of the functional activity of the NS2B-NS3 complex.In brief, the molecular interactions that contribute to the inhibitory activity of the essential oils from C. longa could be observed, from ten selected compounds, from four amino acids, of which two amino acids were in the catalytic triad.

Conclusion
A total of 66 different volatile compounds were identified in the essential oils of the rhizomes of C. longa, C. aeruginosa, and C. xanthorrhiza.The phytochemical profiling of these essential oils conducted by a combination of chromatographic fingerprint data and multivariate analysis revealed clearly different profiles.The in vitro assay disclosed that the most potent anti-DENV2 NS2B-NS3 activity was observed by the C. longa essential oil.A good correlation between the major compounds, ar-turmerone, and curlone and inhibitory activity can be seen from their significant VIP scores and abilities to form both hydrogen and hydrophobic bonds with certain amino acids.Thus, it can be concluded that the essential oil of the rhizomes of C. longa could serve as a promising source of a natural therapeutic agent for the treatment of dengue infection.