|Year : 2019 | Volume
| Issue : 4 | Page : 396-399
Two Lycopodium alkaloids from the aerial parts of Huperzia phlegmaria
Dang Kim Thu1, Dao Thi Vui2, Bui Thanh Tung1
1 Department of Pharmacology and Clinical Pharmacy, VNU School of Medicine and Pharmacy, Vietnam National University, Hanoi, Vietnam
2 Department of Pharmacology, Hanoi University of Pharmacy, Hanoi, Vietnam
|Date of Web Publication||22-Nov-2019|
Dr. Bui Thanh Tung
School of Medicine and Pharmacy, Vietnam National University, 144 Xuan Thuy, Cau Giay, Hanoi
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Huperzia phlegmaria has been used to enhancing memory and alleviate brain disorders. It contains high amount of alkaloids, which are potent acetylcholinesterase (AChE) inhibitor. Materials and Methods: Lycopodium alkaloids from aerial parts of H. phlegmaria were isolated by chromatographic methods. Their structures were elucidated by spectroscopic methods, including mass spectrometry and nuclear magnetic resonance. AChE inhibitory effect of isolated compounds in vitro was evaluated using Ellman's assay. Results: These compounds were identified as fawcettidine and 12-epilycodoline N-oxide. Two compounds showed moderately AChE inhibitory effects with IC50values of 33.11 μg/mL and 64.56 μg/mL, respectively. Conclusion:These isolated compounds could be promising drugs for the treatment of Alzheimer's disease.
Keywords: 12-epilycodoline N-oxide, acetylcholinesterase, fawcettidine, Huperzia phlegmaria, lycopodium alkaloids
|How to cite this article:|
Thu DK, Vui DT, Tung BT. Two Lycopodium alkaloids from the aerial parts of Huperzia phlegmaria. Phcog Res 2019;11:396-9
- Two Lycopodium alkaloids were isolated from aerial parts of Huperzia phlegmaria by chromatographic methods. These compounds were identified as fawcettidine and 12.epilycodoline N.oxide. Two compounds showed moderately acetylcholinesterase inhibitory effects with IC50 values of 33.11 and 64.56 μg/mL, respectively.
Abbreviations Used:AChE: Acetylcholinesterase, Ach: Acetylcholine, AD: Alzheimer's disease.
| Introduction|| |
Alzheimer's disease (AD) is an age-related progressive neurodegenerative disease, which impairs memory and cognitive function. According to the World Health Organization report in 2018, 50 million people worldwide have dementia and out of this population, 60%–70% is AD patients. The number of people with dementia is predicted to nearly triple to 152 million by 2050, whereby with the rise of dementia patients, the AD patients' cases is also expected to increase. Cholinergic hypothesis explains that AD is caused by decreased the neurotransmitter acetylcholine (Ach). Many drugs used in the treatment of AD are based on cholinergic hypothesis. Many studies showed lower level of neurotransmitters in cholinergic system is responsible for cognitive decline and memory loss in Alzheimer patients.,,, Acetylcholinesterase (AChE) is the main enzyme, which degrades the neurotransmitter Ach. Drugs such as galantamine, tacrine, donepezil, metrifonate, or rivastigmine are inhibitors of AChE, augment the level of ACh, and thereby improving cholinergic transmission. These drugs have been used to alleviate the symptoms of Alzheimer which are caused by degeneration of cholinergic neurons and injured transmission. However, AChE inhibitors have many side effects such as nausea, vomiting, diarrhea, abdominal pain, dyspepsia, and skin rash. Therefore, it is important to find new AChE inhibitors with less adverse effects, which may be found in medicinal plant resources.
Medicinal plants may decrease the progress and symptoms of AD. From the genus Huperzia, several alkaloids have been isolated. Thorroad et al. have isolated eleven Lycopodium alkaloids from the whole plants of Huperzia carinata and Huperzia squarrosa and these Lycopodium alkaloids compounds could moderate AChE inhibitory activity. Nilsu et al. have isolated two Lycopodium alkaloids, squarrosine A, and pyrrolhuperzine A from the H. squarrosa. In Vietnam, Chuong et al. have isolated six Lycopodium alkaloids, namely lycosquarosine a, acetylaposerratinine, huperzine A, huperzine B, 8α-hydrophlemariurine B and huperzinine, from Vietnamese H. squarrosa. These compounds lycosquarosine A and acetylaposerratinine have showed strong AChE inhibitory activity. Among isolated compounds from the genus Huperzia, Huperzine A is the most important sesquiterpene alkaloid compounds found in Huperzia serrata. Huperzine A has been shown to strongly inhibit AChE and its mechanism was similar to rivastigmine, donepezil, and galantamine which have been used as drugs for the treatment of AD. Hirasawa et al. have isolated huperminone A, a novel C16N-type Lycopodium alkaloid consisting of a decahydroquinoline and a cyclohexanone, from the club moss of Huperzia phlegmaria and hupermine A, novel C16N2-type Lycopodium alkaloid. In this study, we report the two compounds isolated from H. phlegmaria and their AChE inhibitory activity.
| Materials and Methods|| |
The aerial parts of H. phlegmaria collected in Tam Dao, Vietnam, during 9/2018 and authenticated by the School of Medicine and Pharmacy (SMP), Vietnam National University, Hanoi, Vietnam. A voucher specimen has been deposited in the SMP.
General experimental procedures
The nuclear magnetic resonance (NMR) (1 H [500 MHz],13 C [125 MHz] and Distortionless Enhancement by Polarization Transfer [DEPT]-90 and 135 MHz) spectra were recorded on an AVANCE spectrometer AV 500 (Brucker, Germany) in the Institute of Chemistry, Vietnam Academy of Science and Technology. Chemical shifts were reported in ppm downfield from Tetramethylsilane (TMS) with J in Hz. Electrospray Ionization Mass Spectra (ESI-MS) were recorded on a Varian Agilent 1100 liquid chromatography mass spectrometry (MS) D mass spectrometer. Column chromatography (CC) was performed on silica gel (70–230 and 230–400 mesh, Merck). Organic solvents were of analytical grade.
Extraction and isolation
The aerial dried plants of H. phlegmaria (1 kg) were pulverized and defatted with n-hexane using a Soxhlet extractor for 1 day. The plants were subsequently extracted with EtOH three times by reflux. The solvent was removed at reduced pressure to give a residue (52.6 g). This crude extract was suspended in 5% HCl solution and washed with CH2 Cl2 the aqueous were obtained, basified with NH4 OH (pH = 11) and partitioned with CH2 Cl2, to obtain the crude alkaloid extract after filtration under Na2 SO4. The alkaloid extract was then subjected to CC over silica gel with CH2 Cl2–MeOH (5:1–0:1) to yield nice fractions (F1-F9). Fraction F5 (565 mg) was chromatographed on C-18 silica gel with a MeOH–H2O (0:1-1:0) to give compound 1 (21.3 mg) and compound 2 (19.6 mg) [Figure 1].
Acetylcholinesterase inhibitory activity assay
AChE inhibitory activity of isolated compounds was assayed by the spectrophotometric method developed by Ellman et al. with slightly modification. Samples were dissolved in dimethyl sulfoxide. Reaction mixture consisted of 140 μL of 0.1 M sodium phosphate buffer (pH 8.0), 20 μL of samples, and 20 μL of AChE 0.25 IU/mL. Incubated the mixture for 15 min at 25°C. Added 10 μL of 5-5'-dithiobis-2-nitrobenzoic acid 2.5 mM và 10 μL acetylthiocholine iodide 2.0 mM and mixed well. Then incubate the mixture for 10 min at 25°C. The absorbance was measured at 412 nm. Each assay was repeated three times. Donepezil was used as positive control.
Percentage of AChE inhibition (% I) was calculated by followed formula:
Where I% is the percentage of AChE inhibition
Ac: Absorbance of control (without 20 μL sample)
At: Absorbance of sample
Ao: Absorbance of blank (200 μl of 0.1 M sodium phosphate buffer)−
Value IC50 was calculated using the graph of log (dose) versus % I.
| Results and Discussion|| |
Chemical structure elucidation
Compound 1: Fawcettidine
ESI-MS (positive) m/z: 246.36 [M + H] + (calcd. 246.18 for C16H23 NO).
1 H NMR (400 MHz, CDCl3): δH5.71 (1H, d, J = 5.0 Hz, H-14); 2.72 (1H, dd, J = 7.5; 17.0 Hz); 1.04 (3H, d, J = 7.0 Hz, H-16); 3.14–2.98 (m, 4H), 2.34–2.24 (m, 2H), 2.20–2.05 (m, 3H), 1.79–1.59 (m, 3H), 1.41–1.34 (m, 2H), 1.28–1.21 (m, 2H), 1.99–1.93 (m, 1H), 1.91–1.83 (m, 1H);13 C NMR (125 MHz, CDCl3): δC218.8 (C-5); 145.7 (C-13); 127.3 (C-14); 60.3 (C-1); 56.2 (C-4); 51.9 (C-9); 46.1 (C-12); 44.0 (C-6); 39.1 (C-11); 37.3 (C-7); 34.1 (C-8); 31.2 (C-3); 29.1 (C-2); 27.7 (C-15); 23.7 (C-10); 20.8 (C-16).
Compound 1 was obtained as pale yellow oil. Its molecular formula was deduced to be C16H23 NO by high-resolution electrospray ionization-MS (HRESI-MS) data in conjunction with NMR data analysis, which contains eight degrees of unsaturation. The1 H NMR spectrum of compound 1 measured in CDCl3 showed typical one methyl group (δ H 1.04 [3H, d, J = 7.0 Hz]). The characteristic of two signals of methine proton at (δH5.71 [1H, d, J = 5.0 Hz, H-14]) and (δH2.72 [1H, dd, J = 7.5; 17.0 Hz]) were observed. Moreover, the characteristic of multiplet signals at 3.14–2.98 (m, 4H), 2.34–2.24 (m, 2H), 2.20–2.05 (m, 3H), 1.79–1.59 (m, 3H), 1.41–1.34 (m, 2H), 1.28–1.21 (m, 2H), 1.99–1.93 (m, 1H), and 1.91–1.83 (m, 1H) were observed.
Analysis of the13 C NMR (composite pulse decouplingand DEPT) spectra of compound 1 revealed sixty signals for one methyl, eight methylenes, four methines, and three non-hydrogenated carbons. Furthermore, the13 C NMR spectrum contained signals corresponding to one ketone carbon δC218,8 (C-5); one non-hydrogenated carbons δC145.7 (C-13); and one methine nitrogenated carbon δC127.3 (C-14); two nitrogenated carbon (δ C 60.3 [C-1] and δC56.2 [C-4]). By comparison with previously reported literature, the structure of compound 1 was deduced as fawcettidine [Figure 2].
Compound 2: 12-epilycodoline N-oxide
ESI-MS m/z: 280.37 (M + H) + (calcd. 280.18 for C16H25 NO3).
1 H NMR (400 MHz, CDCl3): δH2.95–3.62 (2H, m, H-1); 1.83–1.90 (2H, m, H-2); 1.68–2.21 (2H, m, H-3); 2.84 (1H, dd, H-4); 2.43–2.62 (2H, dd, H-6); 2.09 (1H, overlap, H-7); 1.33–2.09 (2H, m, H-8); 3.07–4.05 (2H, m, H-9); 1.78–3.05 (2H, m, H-10); 1.65–2.25 (2H, m, H-11); 1.89–2.78 (2H, m, H-14); 1.49 (1H, m, H-15); 0.96 (3H, d, H-16).
13 C NMR (125 MHz, CDCl3): δC63.2 (C-1); 21.5 (C-2); 17.8 (C-3); 50.0(C-4); 207.0 (C-5); 44.1(C-6); 41.2(C-7); 35.2 (C-8); 59.7 (C-9); 16.4 (C-10); 29.6 (C-11); 71.0 (C-12); 72.8 (C-13); 29.6 (C-14); 24.8 (C-15); 22.5 (C-16).
Compound 2 was obtained as white oil. Its molecular formula was deduced to be C16H25 NO3 by HRESI-MS data in conjunction with NMR data analysis, which contains five degrees of unsaturation. The1 H NMR spectrum of compound 2 measured in CDCl3 showed typical one methyl group (δ H 0.96 [3H, d, J = 6.0 Hz], H-16]. The characteristic of three signals of methine proton at 2.84 (1H, dd, H-4); 2.09 (1H, overlap, H-7); and 1.49 (1H, m, H-15) were observed. Moreover, the characteristic of multiplet signals at δH2.95–3.62 (2H, m, H-1); 1.83–1.90 (2H, m, H-2); 1.68–2.21 (2H, m, H-3); 2; 2;43-2;62 (2H, dd, H-6); 1.33–2.09 (2H, m, H-8); 3.07–4.05 (2H, m, H-9); 1.78–3.05 (2H, m, H-10); 1.65–2.25 (2H, m, H-11); 1.89–2.78 (2H, m, H-14) were observed. The13 C-NMR (DEPT) spectrum of compound 2 displayed 16 signals: One Me, nine CH2, and three CH groups and three quaternary C-atoms. Furthermore, the13 C NMR spectrum contained signals corresponding to one ketone carbon δC207.0 (C-5); one non-hydrogenated carbons δC71.0 (C-12); and three nitrogenated carbon (δ C 63.2 [C-1]; δ C 59.7 [C-9]; and δC72.8 [C-13]). By comparison with previously reported literature,, the structure of compound 2 was deduced as 12-epilycodoline N-oxide [Figure 3].
Acetylcholinesterase inhibitory activity
AD is a neurodegenerative disease characterized by progressive loss of neurons. The pathogenesis of AD is still not completely understood. Cholinergic deficits have been considered play an important role in the process of AD., The cholinergic hypothesis of AD proposed that ACh deficits level induced AD is widely accepted. AChE enzyme hydrolyze ACh to choline and decrease its level in the brain. Then, many intentions for increasing ACh levels in the brain have been studied for the treatment of AD by inhibiting AChE. We have evaluated the AChE inhibitory effect of two isolated compounds using Ellman's assay.
[Table 1] summarizes IC50 values of isolated compound 1, 2 and donepezil. [Figure 4] presents the relationship between log (concentration) versus % inhibition of AChE activity. The AChE inhibitory activity of both compounds was dose-dependent manner. Compound 1 and compound 2 have activities with IC50 was 33.11 and 64.56 μg/mL, respectively, as compared with donepezil 2.57 μg/mL.
|Table 1: Acetylcholinesterase inhibitory activity of of isolated compound 1 and compound 2 and donezepil|
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|Figure 4: Acetylcholinesterase inhibitory activity on Ellman's assay. (a) Donezepil; (b) Compound 1 and compound 2|
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Several members from Huperzia species have been studied about the phytochemicals and its AChE inhibitory. Chuong et al. have isolated two compounds lycosquarosine A and acetylaposerratinine from Vietnamese H. squarrosa and showed that lycosquarosine A and acetylaposerratinine inhibit AChE with IC50 values of 54.3 and 15.2 μg/mL, respectively. Hirasawa et al. showed the Hupercumines A and B, two Lycopodium alkaloids from Huperzia cunninghamioides inhibited AChE with IC50, 41.9 and 92.3 μM, respectively. Ohba et al. have showed that huperzine A, the main compound from H. serrata, has inhibited AChE with IC50 87.17 nM. Nguyen et al. have isolated two novel Lycopodium alkaloids, huperphlegmines A and B from the aerial parts of H. phlegmaria and showed they inhibited moderately AChE activity with IC50 values of 25.95 ± 0.67 and 29.14 ± 0.77 μg/mL, respectively. In this study, we first time reported that fawcettidine and 12-epilycodoline N-oxide compounds were isolated from H. phlegmaria, and they showed moderately AChE inhibitory effects with IC50 values of 33.11 μg/mL and 64.56 μg/mL, respectively.
| Conclusion|| |
From the aerial parts of H. phlegmaria collected in Vietnam, two Lycopodium alkaloids were isolated by chromatographic methods. On the basis of spectroscopic analyses and by spectral comparison with the published literature, the isolated compounds were identified as fawcettidine and 12-epilycodoline N-oxide. Two compounds showed moderately AChE inhibitory effects, with IC50 values of 33.11 and 64.56 μg/mL, respectively.
Financial support and sponsorship
The research was supported by has been financed by Vietnam National University, Hanoi, with grants number: QG.19.57.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Essa MM, Vijayan RK, Castellano-Gonzalez G, Memon MA, Braidy N, Guillemin GJ. Neuroprotective effect of natural products against Alzheimer's disease. Neurochem Res 2012;37:1829-42.
Patterson C. World Alzheimer Report 2018 - The State of the art of Dementia Research: New Frontiers. London, UK: Alzheimer's Disease International; 2018.
Francis PT, Palmer AM, Snape M, Wilcock GK. The cholinergic hypothesis of Alzheimer's disease: A review of progress. J Neurol Neurosurg Psychiatry 1999;66:137-47.
Pappas BA, Bayley PJ, Bui BK, Hansen LA, Thal LJ. Choline acetyltransferase activity and cognitive domain scores of Alzheimer's patients. Neurobiol Aging 2000;21:11-7.
McGleenon BM, Dynan KB, Passmore AP. Acetylcholinesterase inhibitors in Alzheimer's disease. Br J Clin Pharmacol 1999;48:471-80.
Upadhyaya P, Seth V, Ahmad M. Therapy of Alzheimer's disease: An update. Afr J Pharm Pharmacol 2010;4:408-21.
Howes MJ, Perry NS, Houghton PJ. Plants with traditional uses and activities, relevant to the management of Alzheimer's disease and other cognitive disorders. Phytother Res 2003;17:1-8.
Thorroad S, Worawittayanont P, Khunnawutmanotham N, Chimnoi N, Jumruksa A, Ruchirawat S, et al
. Three new Lycopodium
alkaloids from Huperzia carinata
and Huperzia squarrosa
. Tetrahedron 2014;70:8017-22.
Nilsu T, Thorroad S, Ruchirawat S, Thasana N. Squarrosine A and pyrrolhuperzine A, new Lycopodium
alkaloids from thai and philippine Huperzia squarrosa
. Planta Med 2016;82:1046-50.
Chuong NN, Huong NT, Hung TM, Luan TC. Anti-cholinesterase activity of Lycoipodium
alkaloids from vietnamese Huperzia squarrosa
(Forst) trevis. Molecules 2014;19:19172-9.
Hirasawa Y, Kato Y, Wong CP, Uchiyama N, Goda Y, Hadi AH, et al
. Huperminone A, a novel C16N-type Lycopodium
alkaloid from Huiperzia phlegmaria
. Tetrahedron Lett 2013;54:1593-5.
Hirasawa Y, Kato Y, Wong CP, Uchiyama N, Goda Y, Hadi AH, et al
. Hupermine A, a novel C16N2-type Lycopodium
alkaloid from Huperzia phlegmaria
. Tetrahedron Lett 2014;55:1902-4.
Ellman GL, Courtney KD, Andres V Jr., Feather-Stone RM. A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 1961;7:88-95.
Li P, Huang W, Zhuo J, Guo Z, Cao W, Xu L, et al.
Seven new Lycopodium
alkaloids from the aerial parts of Phlegmariurus squarrosus
. Tetrahedron. 2015;71:5308-14.
Tan CH, Zhu DY. Lycopodine-ÉType Lycopodium
alkaloids from Huperzia serrata
. Helv chim acta 2004;87:1963-7.
Zhao XH, Zhang Q, Du JY, Lu XY, Cao YX, Deng YH, et al
. Total synthesis of (±)-lycojaponicumin D and lycodoline-type Lycopodium
alkaloids. J Am Chem Soc 2017;139:7095-103.
Adalier N, Parker H. Vitamin E, turmeric and saffron in treatment of Alzheimer's disease. Antioxidants (Basel) 2016;5. pii: E40.
Zhao Y, Zhao B. Oxidative stress and the pathogenesis of Alzheimers disease. Oxid Med Cell Longev 2013;2013:316523, Page 1-10.
Siegfried K. The cholinergic hypothesis of Alzheimer's disease. Eur Neuropsychopharmacol 1993;3:170-1.
Kamkwalala AR, Newhouse PA. Beyond acetylcholinesterase inhibitors: Novel cholinergic treatments for Alzheimer's disease. Curr Alzheimer Res 2017;14:377-92.
Hirasawa Y, Mitsui C, Uchiyama N, Hakamatsuka T, Morita H. Hupercumines A and B, Lycopodium
alkaloids from Huperzia cunninghamioides
, inhibiting acetylcholinesterase. Org Lett 2018;20:1384-7.
Ohba T, Yoshino Y, Ishisaka M, Abe N, Tsuruma K, Shimazawa M, et al.
Japanese Huperzia serrata
extract and the constituent, huperzine A, ameliorate the scopolamine-induced cognitive impairment in mice. Biosci Biotechnol Biochem 2015;79:1838-44.
Nguyen HT, Doan HT, Ho DV, Pham KT, Raal A, Morita H. Huperphlegmines A and B, two novel lycopodium alkaloids with an unprecedented skeleton from Huperzia phlegmaria
, and their acetylcholinesterase inhibitory activities. Fitoterapia 2018;129:267-71.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]