Chemical composition and acetylcholinesterase inhibitory activity of essential oil from rhizomes of distichochlamys benenica

Hue University Journal of Science: Natural Science Vol. 129, No. 1D, 43–49, 2020 pISSN 1859-1388 eISSN 2615-9678 DOI: 10.26459/hueuni-jns.v129i1D.5804 43 CHEMICAL COMPOSITION AND ACETYLCHOLINESTERASE INHIBITORY ACTIVITY OF ESSENTIAL OIL FROM RHIZOMES OF DISTICHOCHLAMYS BENENICA Hanh Thi Nhu Hoang1, Thanh Thu Thi Dinh1, Ty Viet Pham2, Hien Bich Thi Le3, Duc Viet Ho3* 1 University of Agriculture and Forestry, Hue University, 102 Phung Hung St., Hue, Vietnam 2 University of Education, Hue University, 34 Le Loi St., Hue, Vietnam 3 University of Medicine and Pharmacy, Hue University, 6 Ngo Quyen St., Hue, Vietnam * Correspondence to Duc Viet Ho (Received: 03 May 2020; Accepted: 08 September 2020) Abstract. Twenty-seven constituents were identified by using GC/MS, representing 99.57% of the rhizome oil of Distichochlamys benenica. The major constituents of the essential oil are 1,8-cineole (54.39%), β-pinene (7.50%), (E)-citral (7.26%), and (Z)-citral (6.79%). The rhizome essential oil has anti- acetylcholinesterase activity with an IC50 value of 136.63  2.70 g/mL. Keywords: Distichochlamys benenica, essential oil, acetylcholinesterase, 1,8-cineole 1 Introduction Essential oils, which are complex mixtures of volatile compounds, mainly terpenes, are extracted from plants by using steam distillation and various solvents [1, 2]. All over the world, around 3000 essential oils have been extracted from at least 2000 plant species, out of which approximately 300 essential oils are considered important in commerce [1]. Since ancient times, essential oils have been used in traditional medicines to treat inflammatory disease, pain relief, gastrointestinal disease, or the reduction of stress. Modern pharmacological studies have shown that essential oils exhibit biological activity such as antifungal, antibacterial [3-6], anti-inflammatory [7-9], cytotoxicity, cancer chemoprotective [10, 11], cardiovascular effects [12, 13], anticonvulsant [14], and anti-insect [15, 16]. With a broad spectrum of biological activity and aromatic properties, essential oils are increasingly popular, especially in cosmetics, food products and pharmaceuticals. Distichochlamys, a genus belonging to Zingiberaceae family, was first discovered in 1995 by Newman [17]. Up to now, only four species of this genus have been identified, all endemic to Vietnam. These include D. benenica Q.B. Nguyen & Skornick [18], D. citrea M. F. Newman [17], D. orlowii K. Larsen & M. F. Newman [19], and D. rubrostriata W. J. Kress & Rehse [20]. The composition of rhizome essential oils from D. rubrostriata, D. citrea, and D. orlowii has been reported. 1,8-Cineole (13.2–22.0%), -citral (18.5– 22.1%), β-citral (14.2–22.3%), trans-geraniol (12.5– 12.8%), and geranyl acetate (6.6–14.9%) are the main constituents in D. rubrostriata [21]. The phytochemical investigation of the rhizome essential oil of D. citrea indicates that 1,8-cineole is the main component (30.71–43.67%) [22], while high contents of geranyl acetate (16.5%), β-elemene (9.2%), β-pinene (9.0%), and β-caryophyllene (7.9%) are present in the rhizome essential oil of D. orlowii [23]. To the best of our knowledge, the phytochemical analysis and biological activity of Hanh Thi Nhu Hoang et al. 44 D. benenica have not been performed yet. This article aims to report the chemical composition from the rhizome essential oil of D. benenica as well as its acetylcholinesterase (AChE) inhibitory activity. 2 Material and methods 2.1 Plant collection and extraction of essential oil D. benenica Q.B.Nguyen & Škorničk species was collected in Tay Giang district, Quang Nam province, Vietnam in February 2020. A voucher specimen (B.En.01) was deposited at the Faculty of Fundamental Sciences, University of Agriculture and Forestry, Hue University, Vietnam. Fresh rhizomes of D. benenica (0.2 kg) were shredded and their essential oil obtained was by hydrodistillation for 6 hours. The oil was dried with Na2SO4 and kept under refrigeration (4 °C) until analysis. The experiments were performed in triplicates. 2.2 Analysis of essential oil A Shimadzu Technologies GCMS-QP2010 Plus chromatograph fitted with a fused silica Equity-5 capillary column (30 m × 0.25 mm, film thickness 0.25 µm, Supelco, USA) and coupled with a mass spectrometer (MSD QP2010 Plus) was used for GC- MS analysis. The analytical conditions are as follows: carrier helium (1.78 mL/min), injector temperature of 250 °C, interface temperature of 250 °C, and a column temperature programmed from 40 °C (1 min hold) to 285 °C (5 min hold) at 3 °C/min. Samples were injected using a split ratio of 30:1. The injected volume is 1.0 µL, and the inlet pressure is 100 kPa. The MS conditions are as follows: ionization voltage 70 eV, detector voltage 0.82 kV, and acquisition scan mass range 40–350 amu at a sampling rate of 0.5 scans/s. The MS fragmentation patterns were checked against those of other essential oils of known compositions by using NIST 11 and WILEY 7 Libraries (on ChemStation HP) and by comparison of mass spectra of the separated constituents with the data reported in the literature [24]. The relative percentage of particular components in the essential oils was calculated from the area percent report (Uncalibrated calculation procedure) generated in the GC software. 2.3 Acetylcholinesterase inhibition assay The AChE inhibition assay was determined with a modified version of the Ellman colorimetric method [25]. Each of the reaction mixtures contains 140 µL of Tris-HCl buffer (pH 8.0), 20 µL of the tested sample solution, and 20 µL of the AChE solution (0.25 units/mL). After incubation for 15 min, the reaction was initiated by adding 10 µL of 0.24 mM 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and 10 µL of 0.24 mM acetylthiocholine iodide (ACTI). The final mixture then was incubated at ambient temperature for 15 min. The same reaction mixture without sample was used as a negative control. The optical density was measured at 405 nm on an ELISA microplate reader (EMR-500, Labomed Inc.) and the percentage inhibition was calculated. Galanthamine was used as a positive control. All tested samples and the positive control, galanthamine, were dissolved in 10% DMSO (analytical grade). The reaction was performed in triplicates in 96-well microplates. The percentage inhibition (I%) was calculated according to the following equation: 𝐼% = 𝐴control − 𝐴sample 𝐴control × 100 where Asample is the absorbance of the sample solution; Acontrol is the absorbance of the negative control. Each sample was assayed at 5 Hue University Journal of Science: Natural Science Vol. 129, No. 1D, 43–49, 2020 pISSN 1859-1388 eISSN 2615-9678 DOI: 10.26459/hueuni-jns.v129i1D.5804 45 concentrations (60, 80, 100, 120, and 140 g/mL) so that the IC50 value could be calculated from the logarithmic dose-inhibition curve. 3 Results and discussion 3.1 Essential oils composition The rhizome essential oil of D. benenica is a pale yellow liquid with a characteristic aromatic odor and lighter than water. The yield of essential oil is 0.2% (v/w), calculated on a fresh weight basis. A total of 27 components are identified, representing 99.57% of the oil content. Oxygenated monoterpene derivatives are the major type of compounds present in the rhizome essential oil with 75.89%, followed by monoterpene hydrocarbons (19.97%), sesquiterpene hydrocarbons (0.5%), and other compounds (3.21%). Moreover, 1,8-cineole (54.39%), -pinene (7.50%), (E)-citral (7.26%), (Z)-citral (6.79%), and - pinene (4.51%) are found in this oil as main constituents (Table 1). Table 1. Chemical composition of the rhizome essential oil of Distichochlamys benenica No. Compound Class Percentage composition (%) 1 (Z)-3-Propylidenecyclopentene NT 0.04 2 -Thujene MH 0.12 3 -Pinene MH 4.51 4 Camphene MH 2.53 5 2,4,4-Trimethyl-2-penten-1-ol NT 0.04 6 Isopropyl glycolate NT 0.06 7 Sabinene MH 0.93 8 β-Pinene MH 7.50 9 6-Methylhept-5-ene-2-one NT 2.97 10 (5S,8R)-5-Isopropyl-8-methyl-2-methylene-3,9-decadien-1-ol NT 0.09 11 -Phellandrene MH 0.77 12 Limonene MH 2.93 13 1,8-Cineole OM 54.39 14 γ-Terpinene MH 0.59 15 Terpinolene MH 0.09 16 Linalool OM 2.10 17 Borneol OM 1.33 18 Terpinene-4-ol OM 1.68 19 Arthole OM 0.67 20 -Terpineol OM 0.73 21 Fenchyl acetate OM 0.56 22 (Z)-Geraniol OM 0.23 23 (Z)-Citral OM 6.79 24 β-Farnesene SH 0.50 Hanh Thi Nhu Hoang et al. 46 No. Compound Class Percentage composition (%) 25 (E)-Citral OM 7.26 26 Neryl acetate OM 0.14 27 Isovalerone NT 0.01 Total 99.57 MH (Monoterpene Hydrocarbons) 19.97 OM (Oxygenated Monoterpenes) 75.89 SH (Sesquiterpene Hydrocarbons) 0.5 NT (Non-Terpenes) 3.21 Out of 27 compounds identified in the D. benenica oil, 19 of them are also found in the rhizome essential oils of D. rubrostriata, D. citrea, and D. orlowii. The most abundant class of the rhizome essential oil of D. benenica is oxygenated monoterpenes (75.89%), similar to those of D. rubrostriata (64.92–94.06%) [21] and of D. citrea (79.47–90.73%) [22, 23]. However, this finding is very different from that of the essential oil of D. orlowii, which comprises oxygenated monoterpenes (29.4%), monoterpene hydrocarbons (23.9%), sesquiterpene hydrocarbons (33.7%), and oxygenated sesquiterpenes (11.2%) [23]. Furthermore, 1,8-cineole presented in rhizome essential oil of D. benenica, D. rubrostriata, and D. citrea as the major component with 54.39%, 13.2–22.0% and 23.00–43.67%, respectively [21-23]. Surprisingly, this compound is not identified in D. orlowii [23]. Similarly, a remarkable amount of (E)-citral is found in the rhizome essential oil of D. benenica, D. rubrostriata, and D. citrea but conspicuously absent in D. orlowii. Besides, a significant quantity of -pinene (7.50%), (Z)-citral (6.79%), and -pinene (4.51%) in D. benenica is previously reported on three other Distichochlamys species [21-23]. All of the data in the present and previous studies indicate a similarity in the chemical composition of the rhizome essential oil of D. rubrostriata, D. citrea, and D. benenica. 3.2 Acetylcholinesterase inhibition The essential oil is tested for AChE inhibitory activity at various concentrations. Galanthamine is used as a positive control. The essential oil exhibits moderate AChE inhibition with an IC50 value of 136.63  2.70 g/mL. However, this oil displays a much weaker activity compared with galanthamine (IC50 = 0.33 ± 0.01 g/mL) (Table 2). The potency of D. benenica essential oil is stronger than that of Lavandula officinalis (IC50 = 820 g/mL) and Ocimum sanctum oils (IC50 = 1600 g/mL) [26], but slightly weaker than that of Artemisia maderaspatana, Artemisia dracunculus, Pinus heldreichii subsp. leucodermis, and Pinus nigra subsp. nigra oils with IC50 values of 31.33, 58, 51.1, and 94.4 g/mL, respectively [26-28]. A literature survey indicates that 1,8-cineole, -pinene, and β-pinene possess a potent AChE inhibitory effect with IC50 values of 0.06 ± 0.01, 0.09 ± 0.005, and 0.2 ± 0.004 mg/mL, respectively [29]. These components are found in the essential oil of D. benenica with a high content (4.51–54.39%). Therefore, it is reasonable to believe that these compounds contribute significantly to the AChE inhibition of D. benenica oil. Hue University Journal of Science: Natural Science Vol. 129, No. 1D, 43–49, 2020 pISSN 1859-1388 eISSN 2615-9678 DOI: 10.26459/hueuni-jns.v129i1D.5804 47 Table 2. AChE inhibitory activity of rhizome essential oil of Distichochlamys benenica Samples Concentration (g/mL) Percentage of AChE inhibiton (%) IC50 (g/mL)  SD Essential oil 140 51.56  0.58 136.63  2.70 120 45.72  0.53 100 39.89  0.38 80 33.67  0.91 60 25.69  0.62 Galanthamine# 0.5 57.86  1.79 0.33 ± 0.01 0.4 52.88  0.75 0.3 47.61  0.34 0.2 42.44  1.09 0.1 28.37  1.23 #Positive control 4 Conclusion In this study, we report the phytochemical composition and AChE inhibitory activity of the rhizome essential oil of D. benenica for the first time. The oil is a pale yellow liquid with a characteristic aromatic odor. Twenty-seven constituents are present in the oil, in which 1,8- cineole (54.39%), -pinene (7.50%), (E)-citral (7.26%), (Z)-citral (6.79%), and -pinene (4.51%) are major compounds. In addition, the essential oil has moderate AChE inhibitory activity with an IC50 value of 136.63  2.70 g/mL. The obtained results contribute positively in the establishment of the database on Vietnamese endemic plants. Funding statement This work is financially supported by University of Agriculture and Forestry, Hue University (ID No. DHL2020-CB-02). Acknowledgments The authors would like to thank Mr. Cam Xuan Do, University of Agriculture and Forestry, Hue University for plant identification. References 1. Raut JS, Karuppayil SM. A status review on the medicinal properties of essential oils. Industrial Crops and Products. 2014;62:250-264. 2. Swamy MK, Akhtar MS, Sinniah UR. Antimicrobial properties of plant essential oils against human pathogens and their mode of action: An updated review. Evidence-Based Complementary and Alternative Medicine. 2016;2016:1-21. 3. Reichling J, Schnitzler P, Suschke U, Saller R. Essential oils of aromatic plants with antibacterial, antifungal, antiviral, and cytotoxic properties-an overview. Complementary Medicine Research. 2009;16(2):79-90. 4. Solórzano-Santos F, Miranda-Novales MG. Essential oils from aromatic herbs as antimicrobial agents. Current Opinion in Biotechnology. 2011;23(2):136- 141. Hanh Thi Nhu Hoang et al. 48 5. Bassolé IHN, Juliani HR. Essential oils in combination and their antimicrobial properties. Molecules. 2012;17(4):3989-4006. 6. Lakhdar L, Hmamouchi M, Rida S, Ennibi O. Antibacterial activity of essential oils against periodontal pathogens: A qualitative systematic review. Odontostomatol Trop. 2012;35(140):38-46. 7. Woollard AC, Tatham KC, Barker S. The influence of essential oils on the process of wound healing: A review of the current evidence. Journal of Wound Care. 2007;16(6):255-257. 8. Rakover Y, Ben-Arye E, Goldstein LH. The treatment of respiratory ailments with essential oils of some aromatic medicinal plants. Harefuah. 2008;147(10):783-838. 9. Taga I, Lan CQ, Altosaar I. Plant essential oils and mastitis disease: Their potential inhibitory effects on pro-inflammatory cytokine production in response to bacteria related inflammation. Natural Products Communication. 2012;75(5): 1934578X1200700. 10. Buchbauer G. On the biological properties of fragrance compounds and essential oils. Wiener Medizinische Wochenschrift. 2004;154(21-22):539- 547. 11. Edris AE. Pharmaceutical and therapeutic potentials of essential oils and their individual volatile constituents: A review. Phytotherapy Research. 2007;21(4):308-323. 12. Bester D, Esterhuyse AJ, Truter EJ, van Rooyen J. Cardiovascular effects of edible oils: a comparison between four popular edible oils. Nutrition Research Reviews. 2010;23(2):334-348. 13. Nóbrega de Almeida R, Agra MDF, Negromonte Souto Maior F, De Sousa DP. Essential oils and their constituents: anticonvulsant activity. Molecules. 2011;16(3):2726-2742. 14. Bagetta G, Morrone LA, Rombolà L, Amantea D, Russo R, Berliocchi L, et al. Neuropharmacology of the essential oil of bergamot. Fitoterapia. 2010; 81(6):453-461. 15. Fallatah SA, Khater EI. Potential of medicinal plants in mosquito control. Journal of the Egyptian Society of Parasitology. 2010;40(1):1-26. 16. Pohlit AM, Lopes NP, Gama RA, Tadei WP, Neto VF. Patent literature on mosquito repellent inventions which contain plant essential oils--a review. Planta Medica. 2011;77(6):598-617. 17. Newman MF. Distichochlamys, a new genus from Vietnam. Edinburgh Journal of Botany. 1995;52(1):65-69. 18. Binh NQ, Jana LŠ. Distichochlamys benenica (Zingiberaceae), a new species from Vietnam. The Gardens’ Bulletin Singapore. 2012;64(1):195-200. 19. Larsen K, Newman MF. A new species of Distichochlamys from Vietnam and some observations on generic limits in Hedychieae (Zingebraceae). Natural History Bulletin of the Siam Society. 2001;49(1):77-80. 20. Rehse T, Kress WJ. Distichochlamys rubrostriata (Zingiberaceae), a new species from northern Vietnam. Brittonia. 2003;55(3):205-208. 21. Tuyet NTB. Research on the chemical compositions of some Vietnamese Distichochlamys species of Distichochlamys rubrostriata W. J. Kress & Rehse and Pukdarak. Vietnam Journal of Material Medicine. 2012;17(2):112-116. Vietnamese 22. Ty PV, Duc HV, Thang LQ. Chemical composition of essential oil from the rhizomes of Distichochlamys citrea collected in Central Vietnam. Journal of Sciences, An Giang University. 2015;8(4): 60-65. Vietnamese 23. Huong LT, Chau DTM, Hung NV, Dai DN, Ogunwande IA. Volatile constituents of Distichochlamys citrea M. F. Newman and Distichochlamys orlowii K. Larsen & M. F. Newman (Zingiberaceae) from Vietnam. Journal of Medicinal Plants Research. 2017;11(9):188-193. 24. Adams RP. Identification of essential oil components by gas chromatography/mass spectrometry. 4th Edition. Illinois: Allured Publishing. 2017. 804 p. 25. Ellman GL, Courtne KD, Andres VJ, Feather-Stone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochemical Pharmacology. 1961;7(2):88-95. 26. Dohi S, Terasaki M, Makino M. Acetylcholinesterase inhibitory activity and chemical composition of commercial essential oils. Journal of Agricultural and Food Chemistry. 2009;57(10):4313-4318. 27. Srivastava N, Singh B, Chanda D, Shanker K. Chemical composition and acetylcholinesterase inhibitory activity of Artemisia maderaspatana essential oil. Pharmaceutical Biology. 2015;53(11): 1677-1683. 28. Bonesi M, Menichini F, Tundis R, Loizzo MR, Conforti F, Passalacqua NG, et al. Acetylcholinesterase and butyrylcholinesterase inhibitory activity of Pinus species Hue University Journal of Science: Natural Science Vol. 129, No. 1D, 43–49, 2020 pISSN 1859-1388 eISSN 2615-9678 DOI: 10.26459/hueuni-jns.v129i1D.5804 49 essential oils and their constituents. Journal of Enzyme Inhibition and Medicinal Chemistry. 2010;25(5):622-628. 29. Savelev S, Okello E, Perry NS, Wilkins RM, Perry EK. Synergistic and antagonistic interactions of anticholinesterase terpenoids in Salvia lavandulaefolia essential oil. Pharmacology Biochemistry and Behavior. 2003;75(3):661-668.