全 文 ： 2012 年 3 月 第 10 卷 第 2 期 Chin J Nat Med Mar. 2012 Vol. 10 No. 2 125

Chinese Journal of Natural Medicines 2012, 10(2): 0125−0128
doi: 10.3724/SP.J.1009.2012.00125
Chinese
Journal of
Natural
Medicines







Variations of huperzine A content in Lycopodiaceae
species from tropics
NorShahidah Sahidan1, Chee Yan Choo1*, A. Latiff2, Razali Jaman2
1MedChem Herbal Research Group, Faculty of Pharmacy, Universiti Teknologi MARA, 42300 Selangor, Malaysia;
2Faculty of Sciences and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia
Available online 20 Mar. 2012
[ABSTRACT] (−) Huperzine A is a bioactive alkaloid from Huperzia serrata (Lycopodiaceae) used for the treatment of Alzheimer’s
disease. High yielding (−) huperzine A species is of interest for mass propagation since it grows slowly in temperate countries. The
content of (−) huperzine A from temperate countries was reported but none reported from tropical species. The aim of this study was to
evaluate the content of (−) huperzine A from tropical club mosses and further identify a high yielding species. Club mosses from
Lycopodiaceae family were collected from Peninsular Malaysia. The collected club mosses were dried, pulverized and extracted with
methanol. A gradient reverse phase HPLC-photodiode array detector method with increasing amount of methanol in 0.01 %
trifluoroacetic acid was developed. The calibration curve was linear from 5 to 100 μg·mL-1 with correlation coefficient, r2, of 0.998 1.
The precision for both intra- and inter-day peak area were between 0.48% to 1.24 % and 0.95% to 3.85 %, respectively. The recovery of
the method was between 99.8% to 103.8 %. Though geographically segregated, (−) huperzine A content in Huperzia phlegmaria and
H.carinata found in the tropics was highest and similar to species in Australasia. Both species may provide a good source of (−)
huperzine A.
[KEY WORDS] Lycopodiaceae; (−) Huperzine A; HPLC; Tropics
[CLC Number] R917, R931 [Document code] A [Article ID] 1672-3651(2012)02-0125-04

1 Introduction
(−) Huperzine A is an alkaloid originally isolated from
Huperzia serrata and has been found to be a potent,
reversible and selective acetylcholinesterase inhibitor [1-2].
Clinically (−) huperzine A was proven useful for Alzheimer’s
disease in China, marketed in the United States as a dietary
supplement [3] and is in clinical trials in the United States [4].
A survey in China has shown Huperzia species grows at a
slow rate [5]. Due to its potency, it has a high demand both as
dietary supplement or source of pure (−) huperzine A.
Most of the (−) huperzine A determined with high per-
formance liquid chromatography (HPLC) methods were
based on isocratic mobile phase system. These methods were

[Received on] 30-May-2011
[Research funding] This project was supported by the eScience (No.
UiTM 02-01-01-SF105) grant from Ministry of Science, Technology
and Innovation.
[*Corresponding author] Chee Yan Choo: E-mail: choo715@
puncakalam.uitm.edu.my
These authors have no any conflict of interest to declare.
developed for the determination of (−) huperzine A in plas-
ma [3-4, 6-9], serum [10], plant shoots [11] or club mosses ex-
tracts [12-14]. Only a gradient method was reported by Yang et
al [15] for the determination of (−) huperzine A in formulated
products but was not suitable for crude club mosses extracts.
Thus, a gradient HPLC method was developed to avoid
interfering peaks from the complex extract matrix. The objec-
tive of this study was to evaluate the content of (−) huperzine
A from various Lycopodiaceae species found in the tropics,
since all reported studies are from the temperate countries,
namely Europe [16], China [5, 12, 17] and Australasia [13].
2 Experimental
2.1 Chemicals
(−) Huperzine A (95 %, Sigma Aldrich, USA), methanol
(Merck, Germany), trifluoroacetic acid (Sigma Aldrich, USA)
were used. Deionized water was collected from a water
purification system (Elga, UK).
2.2 Club moss collection
The club mosses were collected in Peninsular Malaysia.
Eleven species were collected and authenticated by Prof. Dr.
Abdul Latiff Mohamad from Universiti Kebangsaan
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126 Chin J Nat Med Mar. 2012 Vol. 10 No. 2 2012 年 3 月 第 10 卷 第 2 期

Malaysia. Voucher specimens of the species are kept at
Universiti Kebangsaan Malaysia.
2.3 Sample preparation
The club mosses were dried at (40 ± 1) °C until constant
weight. After drying, the club mosses were reduced to
powder form using a cutter mill (Taiwan). Approximately 5.0
g of the ground sample was extracted with 350 mL of
methanol at 40 °C for 1 h. The methanolic extract was
filtered and collected. The filtrate was again extracted in
fresh 350 mL of methanol at 40 °C for 1 h. The extraction
procedure was repeated five cycles. (−) Huperzine A was
found to be stable at 40 °C for the duration of the extraction.
The pooled methanol extract was dried under reduced
pressure with a rotary evaporator (Buchi, Switzerland). All
the pooled methanol extract were finally lyophilized with a
freeze dryer (Labconco, USA) at −80 °C, 0.015 mBar for 48
h. All dried samples were weighed and stored at −20 °C until
use. Recovery was calculated from spiking (−) huperzine A
into the ground L. clavatum dried sample which does not
contain (−) huperzine A. Dried methanol extract weighing
200 mg was dissolved in a 10 mL volumetric flask and mark
up to volume with methanol. The flask was sonicated for 10
min. An aliquout was filtered through a 0.45 μm PTFE
syringe filter (Whatman, UK). Duplicate samples from each
plant were extracted. Each sample was analysed in triplicates.
2.4 Method validation
An HPLC (Waters, USA) method with an autoinjector,
600 pump, 2998 photodiode detector monitored from
200−500 nm was developed. The separation of the (−)
huperzine A was conducted with a 250 mm × 4.6 mm,
ODS-3, 3 μm column (Inertsil, Japan) thermostated at 35 °C.
The mobile phase consisted of 20% to 70% methanol in
0.01% trifluoroacetic acid in deionized water with a flow rate
of 1 mL·min-1 eluting for 30 min. Stock solutions of (−)
huperzine A (100 μg·mL-1) in methanol was used to prepare a
series of six standard solutions of 5, 10, 30, 50, 80 and 100
μg·mL-1 for the standard curve. The stock solution was stored
at 4 °C. Each concentration were analysed five times for a
period of five days. The recovery was calculated from
spiking 5, 10 and 30 μg·mL-1 of (−) huperzine A into the
methanol extracts of L. clavatum. Quantitation was based on
peaks integrated at 308 nm.
3 Results and Discussion
In Peninsular Malaysia, the collected Lycopodiaceae
families are from the Huperzia, Lycopodiella and Lycopo-
dium genus (Table 1). Lycopodiella species are found mainly
on poor soil, terrestrial lowland or montane forests. The
Lycopodium species are found on the mountain-top in mode-
rately exposed areas. Huperzia species are found as epiphytes
on mossy trees or moist boulders in various habitats
distributing from lowland to montane forest. Amongst the
Huperzia species, Huperzia phlegmaria is the most common
epiphyte found on trees in the forest. Other Huperzia species,
Table 1 Content of (−) huperzine A in Lycopodiaceae species from tropics (n = 3)
No. Name Above sea level/m Location Methanol yield/% Hup. A/%
1 Huperzia phlegmaria 600 Perak/Pahang Border 0.079 ± 0.001
2 H. phlegmaria 300 Cameron Highlands 0.069 ± 0.001
3 H phlegmaria 600 Pahang Border
21.6 ± 2.4
38.7 ± 1.8
28.1 ± 4.2 0.030 ± 0.001
4 H pinifolia 250 Taman Negara. 0.041 ± 0.001
5 H.cf.pinifolia Trevis. 250 Taman Negara 0.020 ± 0.001
6 H. pinifolia 600 Perak/Pahang Border 0.020 ± 0.001
7 H pinifolia 600 Cameron Highlands
14.3 ± 4.2
20.8 ± 2.5
26.9 ± 3.1
11.8 ± 3.6 0.013 ± 0.001
8 H phyllanth 200 Kelantan 18.3 ± 5.1 0.008 ± 0.001
9 H. carinata 600 Cameron Highlands 27.5 ± 4.5 0.087 ± 0.001
10 H. nummulariifolia 500 Pahang 24.1 ± 3.1 0.007 ± 0.001
11 H. nummulariifolia 500 Perak/Pahang Border 18.3 ± 1.2 0.050 ± 0.002
12 H. tetrasticha 700 Pahang 31.9 ± 6.7 0.053 ± 0.009
13 H. squarrosa 200 Perlis 26.7 ± 5.4 0.029 ± 0.001
14 Lycopodiella cernua 600 Langkawi 19.8 ± 3.8 -
15 L. cernua 100 Selangor 12.8 ± 2.3 -
16 L. cernua 800 Frasers Hill 13.5 ± 1.8 -
17 L. platyrhizoma 1600 Cameron Highlands 20.5 ± 3.8 -
18 L. platyrhizoma 1400 Frasers Hill 22.3 ± 2.5 -
19 L. casuarinoides 2000 Cameron Highlands 18.7 ± 3.4 -
20 L. casuarinoides 1700 Pahang 14.5 ± 2.6 -
21 L. casuarinoides 1400 Frasers Hill 8.4 ± 4.4 -
22 L. clavatum 1700 Pahang 22.0 ± 3.8 -
23 L. clavatum 1600 Cameron Highlands 16.9 ± 2.9 -

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2012 年 3 月 第 10 卷 第 2 期 Chin J Nat Med Mar. 2012 Vol. 10 No. 2 127

namely H. pinifolia, H. phyllantha, H. carinata, H. nummu-
larifolia, H. tetrasticha and H. squarrosa are rare species.
An HPLC photodiode array method provided a useful
tool for the analysis of (−) huperzine A content in crude
methanolic extract. The spectra of peaks provided additional
information for the confirmation on the peak of interest. Fine
particle size (3 μm) of the column’s packing material
provided higher separation efficiency and increased the plate
count and resolution of the peaks from the complex matrix.
Though the small particle size increased peak resolution, it
also increased the back pressure of the pump.

Fig. 1 Chromatogram of (A) methanolic extract of L. clava-
tum and (B) methanolic extract of L. clavatum spiked with 10
μg·mL−1 huperzine A monitored at 308 nm
Various ratios of methanol in water were evaluated over
time for the separation of (−) huperzine A peak from the
complex methanol matrix. The separation of (−) huperzine A
peak without other interfering peaks was achieved with 20%
to 70 % methanol in 0.01 % TFA water (Figs. 1−2). The
addition of TFA into the mobile phase improved the peak
symmetry and reduced the retention time of (−) huperzine A
peak. The calibration curve of peak area versus concentration
for (−) huperzine A was linear over the concentration range of
5 to 100 μg·mL-1. The linear regression equation was y =
10.133 x − 13.011 with a correlation coefficient, r2, of 0.998
1. The limit of detection (LOD) was 1 μg·mL−1 in the spiked
samples with a signal to noise ratio of less than three. The
LOQ of the method was 5 μg·mL-1 with a signal to noise ratio
of ten within five replicate injections. The intra- and inter-day
precision for the measurement of (−) huperzine A
concentration and retention times are shown in Table 2. The
precision for both intra- and inter-day peak area were
between 0.48% to 1.24 % and 0.93% to 3.85 %, respectively.
The retention time precision of both intra- and inter-day
range from 0.18% to 1.44% indicating small variability of it’s
retention time. The recovery of all the spiked samples was
between 99.8% to 103.8% (Table 3).

Fig. 2 HPLC Profiles of Huperzia species (i) H. nummu-
lariifolia; (ii) H. phyllantha; (iii) H. pinifolia; (iv) H.
tetrasticha; (v) H. phlegmaria; (vi) H. carinata; (vii) H.
squarrosa
Table 2 Precision measurements of (−) huperzine A analysis ( x ± s, n = 5)
Peak area Retention time
c/(μg·mL−1) Measured concentration
/(μg·mL−1) intra-day
/% RSD
inter-day
/%RSD
Retention time
/min
intra-day
/% RSD
inter-day
/%RSD
5 4.84 1.24 2.35 10.76 0.23 0.18
10 10.32 1.00 0.93 10.68 0.43 0.20
50 50.97 0.75 3.85 10.63 0.40 1.44
100 102.06 0.48 3.80 10.75 0.25 0.96

Table 3 Accuracy for the determination of spiked (−) hu-
perzine A (n=5)
Spiked concentration
/(μg·mL−1)
Measured concen-
tration /(μg·mL−1)
Mean recovery
/% %RSD
5 5.19 103.8 2.35
10 9.98 99.8 6.13
30 30.94 103.1 6.49

The percentage yield of methanol extracts ranged from
(8.4 ± 4.4) % to (31.9 ± 6.7) in L. casuarinoides and H.
tetrasticha, respectively (Table 1). (−) Huperzine A was
found only in the Huperzia genera, namely, H. phlegmaria, H.
pinifolia, H. phyllantha, H. carinata, H. nummulariifolia, H.
tetrasticha and H. squarrosa (Table 1). Since the club mosses
were collected wild, the age was not able to be determined.
Nonetheless, H. phlegmaria collected from three different
locations exhibited high content of (−) huperzine A ranging
from (0.030 ± 0.001)% to (0.079 ± 0.001)% dry weight. The
content of (−) huperzine A in H. carinata was the highest
with (0.087 ± 0.001)% in the dried club moss. Only a sample
of H.carinata was able to be collected because this species is
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128 Chin J Nat Med Mar. 2012 Vol. 10 No. 2 2012 年 3 月 第 10 卷 第 2 期

rarely found in the forest. Similarly, H. carinata from
Australia also exhibited the highest yield of (−) huperzine A
(0.103%) compared with other Huperzia species [13]. The
content of (−) huperzine A in another rare species, H.
nummulariifolia was rather high with (0.050 ± 0.002)% dry
weight. Most of the Huperzia species found in Peninsular
Malaysia have higher (−) huperzine A content compared to H.
serrata used in traditional Chinese medicine having an
average content of 0.011 8 % dry weight [12]. Thus, Huperzia
species found in tropics may be a potential source of (−)
huperzine A or provide genes with high yield of (−) huperzine
A for mass propagation. Both Lycopodiella and Lycopodium
genera evaluated do not contain (−) huperzine A.
The content of (−) huperzine A found distributed from
the Huperzia species in Peninsular Malaysia correlated with
species in Australia, Papua New Guinea, Fiji and a species
from Borneo reported by Goodger et al [13]. Based on
phylogenetic analysis, Huperzia species from Australia, New
Zealand and Tasmania showed diverse relationship with
South East Asia groups [21]. Though this distinctly diverse
relationship was observed in its phylogenetic analysis, it was
not observed in the production of (−) huperzine A. This may
be due to the expression of protein for the production of (−)
huperzine A was not coded at the 1.1 kb region of noncoding
(intron and spacer sequencer) plastid DNA located between
the trnL and trnF genes.
4 Conclusion
Both H. phlegmaria and H. carinata are high yielding (−)
huperzine A species and this further supports the postulate
that alkaloids content has a genetic basis. This is the first
report on the distribution of (−) huperzine A found in the
Lycopodiaceae family from the tropics.
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