Variations of Huperzine a Content in Lycopodiaceae Species from Tropics Norshahidah Sahidan1, Chee Yan Choo1*, A

Chinese Journal of Natural Chinese Journal of Natural Medicines 2012, 10(2): 0125−0128 Medicines

doi: 10.3724/SP.J.1009.2012.00125

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

developed for the determination of (−) huperzine A in plas- 1 Introduction ma [3-4, 6-9], serum [10], plant shoots [11] or club mosses ex- [12-14] (−) Huperzine A is an alkaloid originally isolated from tracts . Only a gradient method was reported by Yang et [15] Huperzia serrata and has been found to be a potent, al for the determination of (−) huperzine A in formulated reversible and selective acetylcholinesterase inhibitor [1-2]. products but was not suitable for crude club mosses extracts. Clinically (−) huperzine A was proven useful for Alzheimer’s Thus, a gradient HPLC method was developed to avoid disease in China, marketed in the United States as a dietary interfering peaks from the complex extract matrix. The objec- supplement [3] and is in clinical trials in the United States [4]. tive of this study was to evaluate the content of (−) huperzine A survey in China has shown Huperzia species grows at a A from various Lycopodiaceae species found in the tropics, slow rate [5]. Due to its potency, it has a high demand both as since all reported studies are from the temperate countries, [16] [5, 12, 17] [13] dietary supplement or source of pure (−) huperzine A. namely Europe , China and Australasia . Most of the (−) huperzine A determined with high per- 2 Experimental formance liquid chromatography (HPLC) methods were based on isocratic mobile phase system. These methods were 2.1 Chemicals (−) Huperzine A (95 %, Sigma Aldrich, USA), methanol (Merck, Germany), trifluoroacetic acid (Sigma Aldrich, USA) [Received on] 30-May-2011 were used. Deionized water was collected from a water [Research funding] This project was supported by the eScience (No. purification system (Elga, UK). UiTM 02-01-01-SF105) grant from Ministry of Science, Technology 2.2 Club moss collection and Innovation. The club mosses were collected in Peninsular Malaysia. [*Corresponding author] Chee Yan Choo: E-mail: choo715@ Eleven species were collected and authenticated by Prof. Dr. puncakalam.uitm.edu.my Abdul Latiff Mohamad from Universiti Kebangsaan These authors have no any conflict of interest to declare.

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Malaysia. Voucher specimens of the species are kept at 600 pump, 2998 photodiode detector monitored from Universiti Kebangsaan Malaysia. 200−500 nm was developed. The separation of the (−) 2.3 Sample preparation huperzine A was conducted with a 250 mm × 4.6 mm, The club mosses were dried at (40 ± 1) °C until constant ODS-3, 3 μm column (Inertsil, Japan) thermostated at 35 °C. weight. After drying, the club mosses were reduced to The mobile phase consisted of 20% to 70% methanol in powder form using a cutter mill (Taiwan). Approximately 5.0 0.01% trifluoroacetic acid in deionized water with a flow rate g of the ground sample was extracted with 350 mL of of 1 mL·min-1 eluting for 30 min. Stock solutions of (−) methanol at 40 °C for 1 h. The methanolic extract was huperzine A (100 μg·mL-1) in methanol was used to prepare a filtered and collected. The filtrate was again extracted in series of six standard solutions of 5, 10, 30, 50, 80 and 100 fresh 350 mL of methanol at 40 °C for 1 h. The extraction μg·mL-1 for the standard curve. The stock solution was stored procedure was repeated five cycles. (−) Huperzine A was at 4 °C. Each concentration were analysed five times for a found to be stable at 40 °C for the duration of the extraction. period of five days. The recovery was calculated from The pooled methanol extract was dried under reduced spiking 5, 10 and 30 μg·mL-1 of (−) huperzine A into the pressure with a rotary evaporator (Buchi, Switzerland). All methanol extracts of L. clavatum. Quantitation was based on the pooled methanol extract were finally lyophilized with a peaks integrated at 308 nm. freeze dryer (Labconco, USA) at −80 °C, 0.015 mBar for 48 3 Results and Discussion h. All dried samples were weighed and stored at −20 °C until use. Recovery was calculated from spiking (−) huperzine A In Peninsular Malaysia, the collected Lycopodiaceae into the ground L. clavatum dried sample which does not families are from the Huperzia, Lycopodiella and Lycopo- contain (−) huperzine A. Dried methanol extract weighing dium genus (Table 1). Lycopodiella species are found mainly 200 mg was dissolved in a 10 mL volumetric flask and mark on poor soil, terrestrial lowland or montane forests. The up to volume with methanol. The flask was sonicated for 10 Lycopodium species are found on the mountain-top in mode- min. An aliquout was filtered through a 0.45 μm PTFE rately exposed areas. Huperzia species are found as epiphytes syringe filter (Whatman, UK). Duplicate samples from each on mossy trees or moist boulders in various habitats plant were extracted. Each sample was analysed in triplicates. distributing from lowland to montane forest. Amongst the 2.4 Method validation Huperzia species, Huperzia phlegmaria is the most common An HPLC (Waters, USA) method with an autoinjector, 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 21.6 ± 2.4 0.079 ± 0.001 2 H. phlegmaria 300 Cameron Highlands 38.7 ± 1.8 0.069 ± 0.001 3 H phlegmaria 600 Pahang Border 28.1 ± 4.2 0.030 ± 0.001 4 H pinifolia 250 Taman Negara. 14.3 ± 4.2 0.041 ± 0.001 5 H.cf.pinifolia Trevis. 250 Taman Negara 20.8 ± 2.5 0.020 ± 0.001 6 H. pinifolia 600 Perak/Pahang Border 26.9 ± 3.1 0.020 ± 0.001 7 H pinifolia 600 Cameron Highlands 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 Fraser's Hill 13.5 ± 1.8 - 17 L. platyrhizoma 1600 Cameron Highlands 20.5 ± 3.8 - 18 L. platyrhizoma 1400 Fraser's 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 Fraser's 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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NorShahidah Sahidan, et al. /Chinese Journal of Natural Medicines 2012, 10(2): 125−128 namely H. pinifolia, H. phyllantha, H. carinata, H. nummu- addition of TFA into the mobile phase improved the peak larifolia, H. tetrasticha and H. squarrosa are rare species. symmetry and reduced the retention time of (−) huperzine A An HPLC photodiode array method provided a useful peak. The calibration curve of peak area versus concentration tool for the analysis of (−) huperzine A content in crude for (−) huperzine A was linear over the concentration range of methanolic extract. The spectra of peaks provided additional 5 to 100 μg·mL-1. The linear regression equation was y = information for the confirmation on the peak of interest. Fine 10.133 x − 13.011 with a correlation coefficient, r2, of 0.998 particle size (3 μm) of the column’s packing material 1. The limit of detection (LOD) was 1 μg·mL−1 in the spiked provided higher separation efficiency and increased the plate samples with a signal to noise ratio of less than three. The count and resolution of the peaks from the complex matrix. LOQ of the method was 5 μg·mL-1 with a signal to noise ratio Though the small particle size increased peak resolution, it of ten within five replicate injections. The intra- and inter-day also increased the back pressure of the pump. 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. 1 Chromatogram of (A) methanolic extract of L. clavatum 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 Fig. 2 HPLC Profiles of Huperzia species (i) H. nummu- complex methanol matrix. The separation of (−) huperzine A lariifolia; (ii) H. phyllantha; (iii) H. pinifolia; (iv) H. peak without other interfering peaks was achieved with 20% tetrasticha; (v) H. phlegmaria; (vi) H. carinata; (vii) H. to 70 % methanol in 0.01 % TFA water (Figs. 1−2). The squarrosa

Table 2 Precision measurements of (−) huperzine A analysis ( x ± s, n = 5)

Peak area Retention time Measured concentration c/(μg·mL−1) intra-day inter-day Retention time intra-day inter-day /(μg·mL−1) /% RSD /%RSD /min /% RSD /%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- found only in the Huperzia genera, namely, H. phlegmaria, H. perzine A (n=5) pinifolia, H. phyllantha, H. carinata, H. nummulariifolia, H. Spiked concentration Measured concen- Mean recovery tetrasticha and H. squarrosa (Table 1). Since the club mosses −1 −1 %RSD /(μg·mL ) tration /(μg·mL ) /% were collected wild, the age was not able to be determined. 5 5.19 103.8 2.35 Nonetheless, H. phlegmaria collected from three different 10 9.98 99.8 6.13 locations exhibited high content of (−) huperzine A ranging 30 30.94 103.1 6.49 from (0.030 ± 0.001)% to (0.079 ± 0.001)% dry weight. The

The percentage yield of methanol extracts ranged from content of (−) huperzine A in H. carinata was the highest (8.4 ± 4.4) % to (31.9 ± 6.7) in L. casuarinoides and H. with (0.087 ± 0.001)% in the dried club moss. Only a sample tetrasticha, respectively (Table 1). (−) Huperzine A was of H.carinata was able to be collected because this species is

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NorShahidah Sahidan, et al. /Chinese Journal of Natural Medicines 2012, 10(2): 125−128 rarely found in the forest. Similarly, H. carinata from [7] Wang Y, Chu D, Gu J, et al. Liquid chromatographic-tandem Australia also exhibited the highest yield of (−) huperzine A mass spectrometric method for the quantitation of huperzine A (0.103%) compared with other Huperzia species [13]. The in dog plasma [J]. J Chromatogr B, 2004, 803(2): 375-378. content of (−) huperzine A in another rare species, H. [8] Gordon RK, Haigh JR, Garcia GE, et al. Oral administration of pyridostigmine bromide and huperzine A protects human whole nummulariifolia was rather high with (0.050 ± 0.002)% dry blood cholinesterases from ex vivo exposure to soman [J]. weight. Most of the Huperzia species found in Peninsular Chem Biol Interact, 2005, 157-158, 239-246. Malaysia have higher (−) huperzine A content compared to H. [9] Wei G, Xial S, Lu R, et al. Simultaneous determination of ZT-1 serrata used in traditional Chinese medicine having an and its metabolite huperzine A in plasma by high-performance average content of 0.011 8 % dry weight [12]. Thus, Huperzia liquid chromatography with ultraviolet detection [J]. J species found in tropics may be a potential source of (−) Chromatogr B, 2006, 830(1): 120-125. huperzine A or provide genes with high yield of (−) huperzine [10] Ye J, Zeng S, Zhang W, et al. Ion-pair reverse-phase high A for mass propagation. Both Lycopodiella and Lycopodium performance liquid chromatography method for determination of huperzine A in beagle dog serum [J]. J Chromatogr B, 2005, genera evaluated do not contain (−) huperzine A. 817(2): 187-191. The content of (−) huperzine A found distributed from [11] Szypula W, Pietrosiuk A, Suchocki P, et al. Somatic the Huperzia species in Peninsular Malaysia correlated with embryogenesis and in vitro culture of Huperzia selago shoots species in Australia, Papua New Guinea, Fiji and a species as a potential source of huperzine A [J]. Plant Sci, 2005, 168(6): [13] from Borneo reported by Goodger et al . Based on 1443-1452. phylogenetic analysis, Huperzia species from Australia, New [12] Wu Q, Gu Y. Quantification of huperzine A in Huperzia serrata Zealand and Tasmania showed diverse relationship with by HPLC-UV and identification of the major constituents in its South East Asia groups [21]. Though this distinctly diverse alkaloid extracts by HPLC-DAD-MS-MS [J]. J Pharm Biomed Anal, 2006, 40(4): 993-998. relationship was observed in its phylogenetic analysis, it was [13] Goodger JQD, Whincup AL, Field AR, et al. Variation in not observed in the production of (−) huperzine A. 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