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ORIGINAL ARTICLE
Year : 2019  |  Volume : 11  |  Issue : 4  |  Page : 396-399  

Two Lycopodium alkaloids from the aerial parts of Huperzia phlegmaria


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 Publication22-Nov-2019

Correspondence Address:
Dr. Bui Thanh Tung
School of Medicine and Pharmacy, Vietnam National University, 144 Xuan Thuy, Cau Giay, Hanoi
Vietnam
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/pr.pr_82_19

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   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

How to cite this URL:
Thu DK, Vui DT, Tung BT. Two Lycopodium alkaloids from the aerial parts of Huperzia phlegmaria. Phcog Res [serial online] 2019 [cited 2019 Dec 14];11:396-9. Available from: http://www.phcogres.com/text.asp?2019/11/4/396/271445



SUMMARY

  • 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 Top


Alzheimer's disease (AD) is an age-related progressive neurodegenerative disease, which impairs memory and cognitive function.[1] 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.[2] 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.[3] Many studies showed lower level of neurotransmitters in cholinergic system is responsible for cognitive decline and memory loss in Alzheimer patients.[3],[4],[5],[6] 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.[5] 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.[7] 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.[8] Nilsu et al. have isolated two Lycopodium alkaloids, squarrosine A, and pyrrolhuperzine A from the H. squarrosa.[9] 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.[10] 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[11] and hupermine A, novel C16N2-type Lycopodium alkaloid.[12] In this study, we report the two compounds isolated from H. phlegmaria and their AChE inhibitory activity.


   Materials and Methods Top


Plant material

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].
Figure 1: Scheme for extraction of alcaloid from H. phlegmaria

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Acetylcholinesterase inhibitory activity assay

AChE inhibitory activity of isolated compounds was assayed by the spectrophotometric method developed by Ellman et al. with slightly modification.[13] 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 Top


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,[14] the structure of compound 1 was deduced as fawcettidine [Figure 2].
Figure 2: Compound 1

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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,[15],[16] the structure of compound 2 was deduced as 12-epilycodoline N-oxide [Figure 3].
Figure 3: Compound 2

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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.[17],[18] The cholinergic hypothesis of AD proposed that ACh deficits level induced AD is widely accepted.[19] 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.[20] 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.[10] 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.[21] Ohba et al. have showed that huperzine A, the main compound from H. serrata, has inhibited AChE with IC50 87.17 nM.[22] 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.[23] 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 Top


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.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
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