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Phlegmadine A, a Lycopodium Alkaloid with a Unique Cyclobutane Ring from Phlegmariurus phlegmaria Zhi-Jun Zhang, Chen Wang, Xing-De Wu, Yan Huang, Wen-Xia Zhou, and Qin-Shi Zhao J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b01723 • Publication Date (Web): 14 Aug 2019 Downloaded from pubs.acs.org on August 14, 2019
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The Journal of Organic Chemistry
Phlegmadine A, a Lycopodium Alkaloid with a Unique Cyclobutane Ring from Phlegmariurus phlegmaria Zhi-Jun Zhang,†,║ Chen Wang,‡,║ Xing-De Wu,† Yan Huang,‡ Wen-Xia Zhou*,‡, and Qin-Shi Zhao*,† †State
Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China ‡State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, People’s Republic of China Supporting Information Placeholder
H
OH
H
H
N HO
retrosynthetic
O N 3
H H Me
OH
H
OH
O
O
Phlegmadine A (1) Up to 60% yield
N
2
Me
Protective effects of LTP impairment
X-ray crystal structure of 1
ABSTRACT: Phlegmadine A (1), a Lycopodium alkaloid with a unique cyclobutane ring and featuring a complex tetracyclo[4.2.2.03,8.03,10]decane-bridged system, together with three biogenetically related known compounds, were isolated from the Phlegmariurus phlegmaria. The structures and absolute configurations of 1 and 2 were determined by NMR and single-crystal X-ray analysis. Among them, compound 2 exhibited noticeable protective effects for long-term potentiation (LTP) impairment by corticosterone induced in mice. Moreover, we succeeded in the efficient synthesis of 1 from 3 by biomimetic synthesis method. Natural products with cyclobutane unit were found in plants or microorganisms as a small but diverse family with various biological activities.1 They are mainly found in terrestrial plants, but also in a small number of marine species. Nowadays, the reported activities for those isolated have shown strong antimicrobial, antibacterial, antinociceptive, antifeedant, insecticidal, and others activities. The alkaloids, amino acids, and nucleosides with cyclobutane unit, as well as their derivatives, have proven to be promising candidates for the development of new drugs for several diseases.2 The Lycopodiaceae comprising 16 accepted genera and ca 400 known species, endemic to temperate and tropical climates, and particularly occurring in coniferous forests, mountainous areas, and marshlands.3 They are discovered all over the word, with a variety of species in the Guizhou province of China;4 the Northern Ontario, Canada;5 the Eastern Transvaal region of South Africa;6 the mountain range in Jamaica is called “Blue Mountain”;7 and other locations. These fascinating organisms have been identified as remnants of prehistoric ferns, with early fossils dating back as far as 300 million years.8 Phlegmariurus phlegmaria is a traditional Chinese medicinal plant for the treatment of arthritis, rheumatic pain, sore throat, traumatic injury, urticaria, and edema.9 Previously studies have shown that P. phlegmaria is a rich source of Lycopodium alkaloids (LAs) possessing various structures of unprecedented skeletons.10 In continuing the search for structurally unique and bioactive ingredients
from from the genus Phlegmariurus,11 plegmadine A (1), a Lycopodium alkaloid (LA) with a unique cyclobutane ring and featuring a complex tetracyclo[4.2.2.03,8.03,10]decane moiety, together with biogenetically related compounds 14hydroxyllobscurinol (2), lobscurinol (3)12, and 13 fawcettimine (4) were isolated from P. phlegmaria (Figure 1). 6
H 8 16
OH
5 7
H
4
17
11
12
H 16
N
15
HO
14
3
14
H H
2
13
10
1
5
OH 4 3
12 11
13
9
15
6 7
8
O
O
9
2
1
2 1
10
N
Me 17
OH
H
H HO
O H
N
O N
Me
3
4
Figure 1. Chemical structures of 1-4. Since the discovery of huperizine A, for a long time, the screening of biological activity of LAs always focused on antiacetylcholinesterase activity. However, all of the other LAs identified so far have either not shown any AChE inhibition activity or possessed activity that is significantly lower than that of huperizine A. Thus, it is necessary to expand the screening targets for LAs. In the current study,
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we focused our attention on long-term potentiation (LTP) in the hippocampus. An activity-dependent synaptic plasticity model has been put forward as a cellular mechanism for some types of learning and memory.14 In the present study, we evaluate the protective effects of the compounds 1-4 on LTP impairment caused by intraperitoneal injection of corticosterone (IP-CORT) in mice. Significantly, compound 2 exhibited noticeable protective effects. This paper reports the isolation, structure elucidation, and the protective effects of 1-4 against corticosterone induced long-term potentiation (LTP) impairment in mice as well as the bioinspired semisynthesis of 1. Phlegmadine A (1), colorless needles crystals (EtOH), had the molecular formula of C17H25NO2, as determined by a quasi-molecular ion at m/z 276.1963 ([M+H]+, calcd 276.1958) in its the HRESIMS and its 13C NMR spectroscopic data (Table 1), corresponding to five degrees of unsaturation. Absorption bands at 3432 and 1745 cm-1 in the IR spectrum implied the existence of hydroxy and carbonyl group functionalities. In the 1H NMR spectrum (Table 1), N-methyl (δH 2.27, 3H, s, H-17) and methyl protons (δH 1.23, 3H, s, H-16) were clearly observed. The 13C NMR spectroscopic data and the HSQC spectra (Figure S9) revealed the presence of 17 carbons due to four quaternary carbons (δC 55.7, 64.2, 66.8, and 218.7), four tertiary carbons (δC 42.2, 48.8, 65.6, and 66.9), seven methylenes, and two methyl groups (including one N-methyl (δC 48.8, C17)). Because of the existence of a carbonyl group, the remaining degrees of unsaturation required compound 1 to possess a tetracyclic ring system. 6 c
5
OH
8 7 16
5 7
3 14
N
11
12
4
15
17
13
a 2
1
10
12
9 b
16
9
COSY
compound 1 was determined. Table 1. NMR data of 1 and 2 ( in ppm, J in Hz) 1
2
no.
δH
δC
δH
1a
2.83, t (13.5)
58.0
2.63, t (11.9)
1b
2.42, overlapped
2a
1.84, m
2b
171, m
3
2.96, m
4
δC 57.2
2.50, t (11.9) 28.8
3.25, m
29.5
2.31, m 42.2
5.95, dd (11.7, 6.0)
131.6
64.2
146.5
5
4.09, t (3.0)
66.9
4.36, br s
73.6
6a
2.19, m
46.8
1.69, overlapped
37.2
6b
1.96, dd (13.5, 3.0)
7
2.01, m
48.8
2.79, m
38.2
8a
1.54, dd (12.5, 4.0)
37.4
2.73, dd (19.0, 6.1)
28.8
8b
0.78, d (12.5)
9a
2.68, m
9b
2.42, overlapped
10a
2.05, m
10b
1.71, m
11a
2.30, m
11b
2.07, m
1.69, overlapped
2.20, overlapped 59.7
2.96, t (12.1)
57.3
2.26, m 26.4
1.76, m
25.1
1.60, m 25.4
2.20, overlapped
31.6
1.94, dd (14.5, 8.6)
12
66.8
57.8
13
218.7
195.9
65.6
141.7
14
2.65, d (2.5)
15
55.7
125.8
16
1.23, s
15.8
1.88, s
17.2
17
2.27, s
48.8
2.35, s
48.4
OH
9.27, s
Recorded at 600 (1H) and 150 (13C) MHz in CDCl3.
3 14
O
1H-1H
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HMBC
ROESY
Figure 2. Selected 2D-NMR correlations of 1.
In the ROESY spectra, the correlations of H-3/H-5, H-5/H16, and H-14/H-16 suggested that H-3, H-5, and H-14 (Figure 2) were in the same face, whereas H-7 and H-9a were the same orientation. 5
The planar structure of compound 1 was determined by the 2D NMR data, including 1H-1H COSY and HMBC spectra. Its 1H-1H COSY spectrum revealed the presence of spin systems a (C-1 to C-3/C-14), b (C-9 to C-11), and c (C-5 to C8) by the correlations of H-1/H-2/H-3/H-14, H-9/H-10/H11, and H-5/H-6/H-7/H-8 (Figure 2). The HMBC correlations were observed from H-1a (δH 2.83) to C-9, H-9a (δH 2.68) to C-1, and H3-17 (δH 2.27) to C-1 (δC 58.0) and C9 (δC 59.7) indicated the connection of C-1, C-9, and C-17 by a nitrogen atom. And the HMBC cross-peaks of H-11a (δH 2.30) to C-13, C-12, C-7, and C-4 (δC 64.2), as well as H-7 (δH 2.01) to C-12, C-11, and C-4 were clearly apparent. Therefore, it could be deduced that C-7 and C-11 were connected to C-12. In addition, the HMBC correlations from H-3 (δH 3.76) to C-15, C-14, C-13, C-12, C-5, and C-4, and H5 (δH 4.09) to C-4, suggested that C-3 and C-5 were connected by C-4. Furthermore, the HMBC correlations from H-14 to C-16, C-15, C-12, and C-13, and cross-peaks from H-16 to C-15, C-14, and C-8 were seen. Meanwhile, its 1H-1H COSY spectrum revealed the OH was located at C-5 by the cross-peaks of H-5 (δH 4.09) and hydroxyl proton (δH 9.27). These data, finally, led to the planar structure of
4
6 7
3
8
10
2 1
9 Tetracyclo[4.2.2.03,8 .03,10]decane
Figure 3. X-ray ORTEP drawing of compound 1 and its
nomenclatures of the unique bridged systems. The absolute configuration of compound 1 was deduced by the single-crystal X-ray analysis. Fortuitously, good quality colorless crystals of 1 were grown from ethanol by recrystallization, which makes the single-crystal X-ray diffraction experiment successful performance by employing graphite monochromated Cu Kα radiation (λ =1.54178 A). The single-crystal X-ray diffraction investigate not only revealed a unique cyclobutane ring and featuring a complex tetracclo[4.2.2.03,8.03,10]decane moiety in 1 as deduced by NMR analysis but also established its absolute configurations of C-3, C-4, C-5, C-7, C-12, C-14, and C-15 as (3R, 4S, 5S, 7S, 12R, 14S, 15S) (Figure 3). Thus, the structure
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of 1 was elucidated as phlegmadine A. 14-Hydroxyllobscurinol (2), colorless crystals obtained from ethanol, exhibited an [M+H]+ ion peak at m/z 292.1907 in its positive HRESIMS, which was corresponding with the molecular formula C17H26NO3. Absorption bands at 3422, 1671, and 1634 cm-1 in the IR spectrum implied the existence of the hydroxy and the α,β-unsaturated ketonic functionalities. The 13C NMR spectroscopic data of 2 (Table 1) displayed the existence of 17 carbon signals. Among them, the four sp2 carbon signals at δC 125.8, 131.6, 141.7, and 146.5, and one carbonyl signal at δC 195.9, revealing 2 as lycophlegmarine is a tricyclic LA.15 Comparison of NMR data of 2 with those of 3 implied that the structure of 2 was analogue to that of 3.12 The main difference reflected in the chemical shifts of C-14 (δC 141.7 (s) in 2; 124.8 (d) in 3) and C-15 (δC 125.8 (s) in 2; 157.1 (s) in 3). In addition, according to the molecular formula of 2, one more oxygen atom was needed. Based on the above analysis, suggested that H-14 of compound 3 was substituted by a hydroxyl group in compound 2. Therefore, the planar structure of 2 was determined.
8
7
11
5
amplitudes. High-frequency stimulation (HFS) was given at the end of 30 min baseline recording of PS, subsequently, PS was recorded in the next 60 min. The mean PS amplitudes were normalized as 100% during the control period (0–30 min) and the relative PS amplitudes were normalized to this value at every point. (B) The average relative PS amplitudes in the 60 min post-HFS (31–90 min). Among the five groups (mifepristone, 1, 2, 3, and 4), mifepristone (Mif) and 2 showed significantly protective effects. Data are displayed as the means ± SEM, n = 6-7. Analysis was conducted by using one-way ANOVA adhere to the Student-NewmanKeuls test. ∗∗∗p < 0.001 compared to the control group; ###p < 0.001 compared to the corticosterone (Cort) group. All compounds were assayed their effects on corticosterone induced LTP impairment in mice. The results (Figure 5) suggest that compound 2 exhibited noticeable protective effects. Phlegmadine A (1) represents an unreported type of LAs with a rare cyclobutane ring. In spite of “[2+2]-ase”has not yet been identified, it is commonly believed that such cyclobutane derivatives originate from the direct coupling of the two olefinic carbon bonds.1 So, we think that 3 might be a precursor of 1 through an intramolecular [2+2] photocycloaddition reaction (Scheme 1). Scheme 1. Hypothetical Biosynthetic Pathway for 1-2 keto-amine
carbinolamine
10
H
O
HO
ROESY
H
H
H O
H
N
O H
H N Fawcettimine (4)
Figure 4. Key ROESY correlations and X-ray ORTEP drawing of 2. Base on ROESY experiment, the relative configuration of compound 2 was deduced. The ROESY correlations of H7/H-10a and H-7/H-11b were observed, which suggested that H-7 was α-oriented. In addition, the correlations of H5/H-8a suggested that H-5 was β-oriented. The structure and absolute configuration (5S, 7S, 12S) of 2 were further confirmed by single-crystal X-ray diffraction (Figure 4).
300
###
#
200
*** 100
0
Control
-
Mif
1
2
H 16
15
8
HO
14
6
5
7
OH 4
6
OH
H
H 8
3 12 11
13
O
16 2
10 9
oxidation
1
N
2
O
17
Me
hv 2+2
3
N
Me
5 7
15 14
H H
OH H
4
17
11
12
N 9
3 13
2
1
10
O 1
Inspired by the complex cyclobutane core of plegmadine A (1), we attempt to construct the cyclobutane ring. At first, we confirmed by experiments that the compound 1 was not produced during isolation. Compound 3 (2mg) was dissolved in solvent (CHCl3/MeOH, 1:1) and added into silica gel (10mg). 48 hours later, we found that compound 3 is stable and did not produce compound 1. And compound 3 (1mg) was exposed to the sunshine, after 8 hours we found that compound 3 is also stable. Moreover, we detected compound 1 in the alkaloidal extract by LC-MS analysis (Figures S1−S3). Table 2. Optimization of solvents for the intramolecular [2+2] photocycloaddition of 3 to 1. entrya λ [nm] solvent tb [h] yieldc [%] 1 200-400 CH2Cl2 9 62 2 200-400 MeCN 7 46 3 200-400 t-BuOH 12 41 aAll reactions were carried out at a substrate concentration of 36 μM at λ = 200-400 nm and ambient temperature. bIrradiation time when the reaction no longer occurs. cYield of isolated product.
B Average PS after HFS (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
The Journal of Organic Chemistry
3
4
Cort
Figure 5. Effects of compounds 1-4 on the LTP recording. (A) The time course of average relative population spike (PS)
The strategy of obtaining cyclobutane ring units in organic synthesis is generally through intramolecular [2+2] photocycloaddition. Compound 3 was direct UV irradiation (λ = 200-400 nm, light source: 500 W mercury lamp, glasses
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vessel), an efficient [2+2] photocycloaddition reaction was observed to give the desired tetracyclic product 1. The best results were succeeded by using dichloromethane as the solvent at room temperature (Table 2, entry 1), by this means, compound 1 could be obtained in yields of 62%. Which was superior to acetonitrile (entry 2), or tert-butanol (entry 3). In the latter solvents the reaction remained incomplete after an irradiation time of 12 hours, while severe product decomposition was found to occur in tertbutanol. In conclusion, we have determined a one-step bioinspired semisynthesis for the formation of 6/5/4/9 tetracyclic of skeleton from fawcettimine-type skeleton. Xray diffraction analysis proved that 1 is a natural LA featuring a complex tetracclo[4.2.2.03,8.03,10]decane moiety. In addition, compound 2 exhibited noticeable protective effects on LTP impairment caused by intraperitoneal injection of corticosterone (IP-CORT) in mice. The present results suggest that 2 could improve the storage of certain types of memory through enhancing the strength of synaptic plasticity. It may be a potential therapeutic candidate for neurodegenerative diseases such as Alzheimer's disease (AD).
EXPERIMENTAL SECTION General experimental procedures. Melting points were measured on X-4 micro melting point apparatus. Optical rotations were obtained on Horiba SEPA-300 polarimeter. UV spectra were recorded with the Shimadzu UV2401PC spectrometer. IR spectra were measured on Bruker Tensor27 spectrometer with KBr pellets. Crystallographic data were collected using graphite-monochromatized Cu Kα radiation on Bruker APEX DUO diffractometer. The NMR were recorded with Bruker AV 600 spectrometer. ESIMS and HRESIMS were collected on an Agilent 1290 UPLC/6540 Q-TOF. Medium pressure liquid chromatography (MPLC) was run on a Lisui EZ Purify III System (Shanghai Lisui Chemical Engineering Company, Shanghai, China). Sephadex LH-20 (Amersham Pharmacia Biotech, Sweden). Column chromatography (CC) was run on silica gel (200–300 mesh; Qingdao Marine Chemical Factory, Qingdao, China). Compounds were visualized by spraying the dried plates with 10% H2SO4 in EtOH. followed by heating until dryness or by spraying Dragendorff's reagent. TLC was performed on silica gel GF254 (SiO2; Qingdao Haiyang Chemical Factory, Qingdao, China). Petroleum ether, ethyl acetate, chloroform, acetone, and methanol were purchased from Tianjing Chemical Reagents Co. (Tianjing, People’s Republic of China). Male BALB/c mice (weight 18-20 g) were obtain from the animal center of the Academy of Military Medical Sciences (AMMS). Plant material. The whole plants of P. phlegmaria were collected in Yunnan Province, PR China, in August 2016. The sample was identified by Dr. Xiao Cheng. A voucher specimen (2016-8-16) has been deposited in the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, PR China. Extraction and Isolation. Dried whole plants of P. phlegmaria (20 kg) were powdered and extracted with 60% EtOH/H2O (40L, 24 h × 3) at room temperature, the extract was dissolved in water and adjusted to pH 3 with 1.0 % HCl/H2O. Subsequently, the acidic suspension was extracted with EtOAc. Then, water-soluble materials, which
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were adjusted to pH 10 with saturated Na2CO3 aq, were extracted with CHCl3 to obtain an alkaloidal extract (52 g). The alkaloidal crude extract was run on MPLC (RP-18 gel) and eluted with MeOH/ H2O (1:9-1:0) to afford five fractions A-D. Fraction A (6.0 g) was subjected repeatedly to silica gel columns chromatography (EtOAc/MeOH) and Sephadex LH-20 (MeOH) to obtain four subfractions A01-04. Fraction A03 (2.5 g) was further purified by a silica gel column chromatography (CHCl3/MeOH, 20:1) to yield compound 1 (6 mg). Fraction C (18 g) was subjected to silica gel column chromatography (CHCl3/MeOH, 20:1) and Sephadex LH-20 to afford three subfractions C01-03. Fraction C01 (12.5 g) was separated repeatedly using a silica gel columns chromatography and eluted with EtOAc/MeOH (30:1) to yield fawcettimine (4) (106 mg).Further purification of fraction C02 (4.5 g) by silica gel column chromatography (EtOAc/MeOH, 20:1) to obtain lobscurinol (3) (52 mg). Compound 2 (8 mg) was isolated from fraction D (10 g) by silica gel column chromatography (EtOAc/MeOH, 30:1). Phlegmadine A (1): colorless crystals (EtOH); mp 131.5132.5 ℃; [α]18.9 D+43.88 (c 0.10, CH3OH); UV (CH3OH) λmax (log ε): 202 (3.32) nm; IR (KBr) νmax: 3432 ， 2948, 2867, 1745, 1632, 1459 ， 1055 cm-1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 276.1963 ([M+H]+, calcd for C17H26NO2, 276.1958). 14-Hydroxyllobscurinol (2): colorless crystals (EtOH); mp 89.1-90.1 ℃; [α]23.6 D+297.88 (c 0.11, CH3OH); UV (CH3OH) λmax (log ε): 279.5 (3.84), 204 (3.79) nm; IR (KBr) νmax: 3422, 2920, 2852, 1671, 1634, 1389, 1151,1030 cm-1; 1H and 13C NMR data, see Table 1; HRESIMS m/z 292.1915 ([M+H]+, calcd for C17H26NO3, 292.1907). X-ray crystal structure analysis. Colorless crystals of compounds 1 and 2 were obtained in ethanol. Crystallographic data for 1 and 2 were collected using graphite-monochromatized Cu Kα radiation (100 K) on Bruker APEX DUO diffractometer. Data reduction and cell refinement were carried out by a Bruker SAINT. The structures were solved by direct methods using SHELXS-97 and refined using SHELXL-97. Hydrogen atoms were placed in the calculated positions and refined using a riding model. Molecular graphics were generated with PLATON. The crystallographic data for 1 and 2 has been deposited in the Cambridge Crystallographic Data Centre (nos. 1908834 and 1908838, respectively). And copies of the thermal ellipsoid plot for crystal structures are included in the SI (Figures S4 and S5). Crystallographic data for compound 1. C17H25NO2, M = 275.38, a = 7.4961(2) Å, b = 7.4595(2) Å, c = 13.2851(3) Å, α = 90°, β = 104.7710(10)°, γ = 90°, V = 718.32(3) Å3, T = 100(2) K, space group P21, Z = 2, μ(CuKα) = 0.648 mm-1, 6749 reflections measured, 2475 independent reflections (Rint = 0.0293). The final R1 values were 0.0326 (I > 2σ(I)). The final wR(F2) values were 0.0854 (I > 2σ(I)). The final R1 values were 0.0326 (all data). The final wR(F2) values were 0.0855 (all data). The goodness of fit on F2 was 1.004. Flack parameter = 0.12(6). Crystallographic data for compound 2. C17H25NO3•H2O, M = 309.39, a = 13.2222(3) Å, b = 14.2829(4) Å, c = 17.1194(4) Å, α = 90°, β = 90°, γ = 90°, V = 3233.02(14) Å3, T = 100(2) K, space group P212121, Z = 8, μ(CuKα) = 0.726 mm-1, 20954 reflections measured, 5923 independent reflections (Rint = 0.0328). The final R1 values were 0.0285 (I > 2σ(I)). The final wR(F2) values were 0.0754 (I > 2σ(I)). The final R1 values were 0.0286 (all data). The final wR(F2)
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The Journal of Organic Chemistry values were 0.0755 (all data). The goodness of fit on F2 was 1.096. Flack parameter = 0.10(3). Biomimetic synthesis of 1. In a vial with a stir bar, compound 3 (10.0 mg, 0.0363 mmol) was dissolved in dichloromethane, acetonitrile, and tert-butanol (2 ml), respectively. The reaction was capped and stirred, placed under direct irradiation (λ = 200-400 nm, light source: 500 W mercury lamp, glasses vessel) at room temperature for 9, 7, and 12 hours, respectively. The reaction mixture was concentrated directly and further purified by silica gel chromatography (30:1 CHCl3/MeOH, 30:1) to afford 1 (6.2 mg, 62% yield; 4.6 mg, 46% yield; 4.1 mg, 41% yield; respectively). Protective effects against corticosterone induced LTP impairment. The animals were housed in plastic cages on a 12 h light/12 h dark cycle (room temperature of 22 ± 1°C and humidity of 50-60%). Animals have 3 days to adapt to the new environment before the experiments. Meanwhile, they had free access to a standard diet and water. Animal use, care, and the experimental protocol were approved by the Institute Animal Care and Use Committee (IACUC) of the National Beijing Center for Drug Safety Evaluation and Research (NBCDSER). Corticosterone was dissolved in distilled water with 5% ethanol, and was injected subcutaneously (50 mg/kg) 60 min before high-frequency stimulation (HFS). Corresponding control animals were subcutaneously injected with distilled water with 5% ethanol. Compounds 1-4 and mifepristone were administrated via intracerebroventricular injection 30 min before corticosterone 2 microgram per mouse. The method of LTP recording have been described previously.16 The experimental results are expressed as the mean ± S.E.M., the SAS software packages and Origin 8.0 were used to analyze and plot the data. The student’s t-test was used when comparing two groups of data. A one-way repeatedmeasures analysis of variance (ANOVA) was used, followed by a Student-Newman-Keuls test. Values of P
