Accepted Manuscript Short communication Seasonal dynamics of total flavonoid contents and antioxidant activity of Dryopteris erythrosora Yinghua Xie, Yunxia Zheng, Xiling Dai, Quanxi Wang, Jianguo Cao, Jianbo Xiao PII: DOI: Reference:
S0308-8146(14)00728-6 http://dx.doi.org/10.1016/j.foodchem.2014.05.024 FOCH 15805
To appear in:
Food Chemistry
Please cite this article as: Xie, Y., Zheng, Y., Dai, X., Wang, Q., Cao, J., Xiao, J., Seasonal dynamics of total flavonoid contents and antioxidant activity of Dryopteris erythrosora, Food Chemistry (2014), doi: http://dx.doi.org/ 10.1016/j.foodchem.2014.05.024
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Short communication for ISPMF2015
Seasonal dynamics of total flavonoid contents and antioxidant activity of Dryopteris erythrosora Yinghua Xie1, Yunxia Zheng1, Xiling Dai1, Quanxi Wang1 , Jianguo Cao1*, Jianbo Xiao1,2* 1
Department of Biology, Shanghai Normal University, 100 Guilin Rd, Shanghai 200234, China.
2
Research Center of Bio-separation Engineering Technology, Anhui Academy of Applied
Technology, Suixi Road 312, Hefei, Anhui 230031, PR China *Correspondence to: Jianguo Cao, Department of Biology, Shanghai Normal University, 100 Guilin Rd, Shanghai 200234, China, Tel(Fax): +86 21 64322526, E-mail:[email protected]; Jianbo Xiao, Department of Biology, Shanghai Normal University, 100 Guilin Rd, Shanghai 200234, China; Tel(Fax): +86 21 64321291, E-mail: [email protected]
Abstract The seasonal dynamics of the total flavonoid contents in various parts of Dryopteris erythrosora, a traditional Chinese medicinal fern, and their antioxidant activity were investigated. The total flavonoids content in various parts of D. erythrosora showed an obvious seasonal dynamic change. The total flavonoid contents in stems (from 4.3% to 12.5%) were much higher than that in leaves with an average content of 2.01%. In spring, the total flavonoid contents in stems were relatively low, but increased rapidly from summer to winter. However, the seasonal dynamics of total flavonoid contents in leaves showed different model. The total flavonoid contents in the stems showed a negative correlation with that in the leaves from January to July. The correlation coefficient of about -0.7 was obtained. The antioxidant activity of the extracts also altered in proportion to the change of total flavonoid contents. In general, the extracts from stems always showed highest antioxidant potentials and it was suggested that the stems can be used as crude medicine.
Key words: Flavonoids; Dryopteris erythrosora; antioxidant activities; habitats
1. Introduction 1
Dryopteris erythrosora, which is rich in bioactive components such as flavonoids, polyphenols, and terpenoids, distributes widely in China (Gao, Li, Wang, & Lu, 2003). The epidemiological and medical evidence have suggested that the dietary flavonoids play an important role in preventing and managing of diseases such as cancer, diabetes, and cardiovascular diseases (Andrae-Marobela, Ghislain, Okatch, & Majinda, 2013; Xiao, Ni, Kai, & Chen, 2013; Delmas & Xiao, 2012; Xiao, 2013; Xiao & Kai, 2012; Xiao, Muzashvili, & Georgiev, 2014; Xiao, Ni, Kai, & Chen, 2015; Martín-Peláez, Covas, Fitó, Kušar, & Pravst, 2013; Panickar, 2013). Some flavonoid compounds have been identified in the genus Dryopteris (Hiraoka, 1978; Harborne, 1966; Imperato, 2007a; Imperato, 2007b). Chang, Hu, Jiang, and Qiang (2005) reported that the total flavonoid contents in the aerial parts of D. erythrosora were about 0.89%. We recently investigated the total flavonoids content and free radical scavenging activity of extract from leaves, rachis, roots and stems of D. erythrosora (Zhang, Cao, Dai, Chen, & Wang, 2012). And 8 flavonoids were identified in D. erythrosora, which were considered to have bioactivities in antioxidant, anticancer and acetylcholinesterase (Cao, Xia, Chen, Xiao, & Wang, 2013). However, the seasonal dynamics of the flavonoid content and their bioactivities in D. erythrosora were still not clear. We collected plant materials monthly from shade (under bamboo forest) and half shade (under broad-leaved forest) habitats. The flavonoid contents and radical scavenging activity were compared. The results provided important information for utilizing this medicinal fern.
2 Materials and methods 2.1 Chemicals and materials Rutin (>98%) was purchased from Sigma Co. (MO, USA). Nitrotetrazolium blue chloride (NBT),
phenazine
metho-sulfate
(PMS),
2,2-diphenyl-1-picrylhydrazyl
(DPPH),
and
2,2’-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) were purchased from Aladdin Reagent Int. (Shanghai, China). Nicotinamide adenine dinucleotide (NADH) was obtained from Sangon Biotech Co. Ltd (Shanghai, China). The materials of D. erythrosora were collected monthly (10th-15th day every month) from Sheshan Mount in Shanghai (China). The identities of the plants were verified by Prof. Dr. Jianguo Cao in College of Life & Environment Science of Shanghai Normal University. Two to three plants of D. erythrosora were collected and the leaves, 2
stems, rachis and roots were separated for flavonoids studies. All other reagents and solvents were of analytical grade and all aqueous solutions were prepared using newly double-distilled water.
2.2 Preparation of plant extract Each time, three plants of D. erythrosora were collected at one site. The whole plant, including the fresh leaves, stems, rachis, and roots were washed and dried in the oven at 65 ºC. The dried sample was powdered and filtered through 40-mesh screen. The dried sample (1.0000 g) was extracted with 50% ethanol (25 mL) for 2h at 50 °C. Then, the ultrasound-assisted extraction was performed on a ultrasound generation system (Kunshan, China) for 20 min. This extraction process was repeated twice. The extracts were filtered with filter paper and collected. The mixture was allowed to cool for 20 min and concentrated to dry by evaporating with a rotary evaporator. The residue was suspended with 50 mL ethanol and filtered through a 0.45 µm membrane (Millipore, USA).
2.3 Determination of total flavonoids The total flavonoid contents in extract were measured by a colorimetric assay (Zhang et al., 2012). The extract (5 mL) was added to a 10 mL flask, and then 5% NaNO2 solution (0.3 mL) was added. After mixed well, the solution was allowed to stand for 6 min at room temperature; and 5% Al(NO3)3 solution (0.3 mL) was added to the flask, mixed well and kept for 6 min at room temperature. At last 4% NaOH solution (4.4 mL) was added, mixed well and kept for 12 min at room temperature. Absorbance was read on a TU-1810 UV-spectrophotometer (Beijing, China) at 510 nm, and the total flavonoid contents (%) were estimated using calibration curves.
2.4. DPPH free radical scavenging activity The DPPH free radical scavenging activity of flavonoids extract was measured according to our previous report (Cao, Xia, Chen, Xiao, & Wang, 2013). One hundred microliters of samples were added to 900 µ L of DPPH solution (1 mM in 50% ethanol) and incubated in the dark for 30 min. After that, the absorbance at 517 nm was measured. The DPPH free radical scavenging activity was calculated using the following formula: 3
DPPH free radical scavenging activity (%) = (1- A1/A0) × 100 Where A0 is the absorbance of the control, and A1 is the absorbance of samples. Each experiment was repeated three times and found to be reproducible within experimental errors (RSD < 5.0%). 2.5 Superoxide anion radical (O2¯) scavenging activity Superoxide anion (O2¯) scavenging activity of flavonoid exteacts was measured by Robak and Gryglewski with slightly modification (Xiao et al., 2011). The superoxide radical was generated in 1.0 mL of sodium phosphate buffer (100 mM, pH 7.4) containing 300 µL NBT (50 µM) solution, 300 µL NADH (78 µM) solution and different concentrations of flavonoid extract from D. erythrosora. The reaction started by addition of 300 µL PMS (10 µM). The reaction mixture was incubated at room temperature for 8 min, and the absorbance at 560 nm was measured. The capability to scavenge the superoxide radical was calculated using the following equation: O2¯ scavenging activity (%) = (1-A1/A0 ) × 100 Where A0 is the absorbance of the control, and A1 is the absorbance of sample. Each experiment was repeated three times and found to be reproducible within experimental errors (RSD < 5.0%).
2.6 ABTS radical scavenging activity The ABTS assay described by Arts et al. (2002) was used with minor modifications. The ABTS assay assessed the total radical scavenging capacity based on the ability of an antioxidant to ＋
scavenge the stable ABTS radical cation (ABTS• ), which was produced by mixing 1.0 mL ABTS stock solution (7.4 mM) with 1.0 mL potassium persulfate (2.6 mM) and allowing to stand in the ＋
dark at room temperature for 12 h before using. The ABTS• solution was diluted 50 times with ＋
PBS (pH7.4) at 37 ºC. Samples (100 µ L) were added to 900 µ L of ABTS• solution and kept in the dark at room temperature for 60 min. Thus, the absorbance was spectrophotometrically ＋
determined at 734 nm. The ABTS•
scavenging activity was calculated using the following
formula: ＋
ABTS•
scavenging activity (%) = (1- A1/A0) × 100
Where A0 is the absorbance of the control, and A1 is the absorbance of sample. Each experiment was repeated three times and found to be reproducible within experimental errors (RSD < 5.0%).
4
2.7 Statistical analysis In present investigation, some simple statistical analysis, mainly the standard deviation, correlation coefficient, was carried out using the Microsoft Excel.
3 Results and discussion 3.1 Total flavonoids content in D. erythrosora The 50% ethanol was used to extract flavonoids from dried leaves, rachis, roots and stems of D. erythrosora. As shown in Table 1, the total flavonoid contents in D. erythrosora were ranged from 0.78% to 12.53% depending on collecting time within one year and different parts. The annual average contents of total flavonoids in any parts of D. erythrosora in half shade habitat were always slight higher than that in the shade habitat (Table 1). The annual average flavonoid contents of different parts in the shade habitat were determined as: stems (7.65%) > roots (3.83%) > rachis (3.75%) > leaves (1.75%). Meanwhile the annual average flavonoid contents of different parts in the half shade habitat were determined as: stems (8.61%) > roots (4.21%) > rachis (4.17%) > leaves (2.28%). The total flavonoid contents in stems (from 4.3% to 12.5%) were much higher than that in leaves with an average content of 2.01%. It was illustrated that the total flavonoid contents in stems were obviously higher than that in the roots, rachis and leaves (Table 1). In our previous study, the total flavonoid contents in D. erythrosora collected in May ranged from 2.1% to 8.26% (Zhang, Cao, Dai, Chen, & Wang, 2012), which were agree to the present results. Chang, Hu, Jiang, and Qiang (2005) reported that the total flavonoid contents in aerial parts of D. erythrosora was 0.89%, which was similar to that of leaves collected in the summer season in current investigation. In general, the dry weight of stems was slightly higher than that of the leaves, rachis and roots. Therefore, the stems were considered to be the most effective part to be used as crude medicine.
3.2 Seasonal dynamics of total flavonoids content in different parts No matter in the half shade habitat or in the shade habitat, the total flavonoid contents in various parts of D. erythrosora showed a similar seasonal dynamic change (Fig. 1). The seasonal 5
dynamics of the total flavonoid contents in the leaves and stems were negatively related, with a correlation coefficient of about -0.7 (Fig. 1). In January, the total flavonoid contents in the leaves were relatively low (about 1.5%-2.0%). Then, the total flavonoid contents in the leaves increased and reached up to maximum in April (about 5.5%). Thereafter, the total flavonoid contents decreased rapidly and maintained at the low status during the whole summer. In December, the total flavonoid contents in the leaves increased again and then decreased in January (Table 1; Fig. 1). The total flavonoid contents in stems ranged from 5.9 to 6.9% in January, but decreased in spring and got a minimum content of 4.9%. After that, the total flavonoid contents improved rapidly from April and maintained at a high level during the whole summer and autumn (Fig. 1). The changes of the total flavonoid contents in the rachis and roots were relatively low and seemed to have no significant variation (Fig. 1). The typical negative correlation of total flavonoid contents in the stems and the leaves were very interesting. Increasing of the total flavonoid contents in the stems was accompanied with decreasing of the total flavonoids content in the leaves. It is generally considered that the flavonoids were produced in the leaves of plant (Ming, Maia-Almeida, & Conceição, 2012). Polar transportation of flavonoids from the leaves to the stems in the growth season might be reasons of negative correlation. The largest flavonoid production in leaves was observed in Spring. So we can collect fresh leaves at the spring as crude medicine of flavonoids. In other seasons, the flavonoids might transport and accumulate in the stems. As a result, we can use the stems at the late summer and the early autumn as crude medicine of flavonoids.
3.3 DPPH free radical scavenging activity of different parts of D. erythrosora In present investigation, D. erythrosora collected in March, June, September and December, which represented spring (from Feb to Apr), summer (from May to Jul), autumn (from Aug to Oct) and winter (from Nov to Jan), were chosen to study the DPPH free radical scavenging activities. For different habitats, DPPH free radical scavenging activities of the flavonoids of D. erythrosora showed similar characteristics in the same month (Fig. 2). However, for different seasons, DPPH
6
free radical scavenging activity of the flavonoids extract from D. erythrosora showed different potential (Fig. 2). In spring (March), the DPPH free radical scavenging potential (IC50) of flavonoids extract from the leaves, rachis, roots and stems were determined as 14.8, 18.0, 11.5 and 8.9 µ l, respectively (Fig. 3). The DPPH free radical scavenging activity of flavonoids extract in various parts of D. erythrosora was similar with each other especially for the half shade habitat (Fig. 2A). However, the DPPH free radical scavenging activity of various parts of D. erythrosora began to diverge and showed a great difference in other seasons. The DPPH free radical scavenging abilities (IC50) of flavonoids extract from the leaves, rachis, roots and stems were determined as 36.1, 15.7, 15.9 and 4.7 µ l, respectively (Fig. 3). From the data of the IC50, it illustrated that the DPPH free radical scavenging potential of the extract from stems was much higher than that in other parts. In conclusion, the extract from leaves appears strong DPPH free radical scavenging abilities in spring and decreased in other seasons. The extract of stems shows strong DPPH free radical scavenging potential in all seasons. Thus, we can collect the leaves in spring days and stems in any seasons as crude medicine.
3.4 Superoxide anion scavenging activity of different parts of D. erythrosora Fig. 4 showed the inhibitory effect of flavonoid extracts from D. erythrosora on superoxide radical generation. All the extracts of the parts of the plant showed inhibitory effect on superoxide anion. For the different habitats, D. erythrosora extracts exhibited similar effects of superoxide anion scavenging activity (Fig. 4). In different seasons, the flavonoids extracts from D. erythrosora showed a strong superoxide anion scavenging activity in March, June and September (Fig. 4, A-C, E-G) and much weak superoxide anion scavenging activity in December (Fig. 4, D, H). For various parts, inhibitory effect of the leaves extract on superoxide radical generation was slight stronger in spring (March) (IC50=15.0 µl), but gradually decreased in summer (IC50 =19.6 µl) and autumn days (IC50 =36.2 µ l), afterwards became almost no inhibitory effect on superoxide radical generation in the winter days (December).
7
From spring to autumn (March, June and September), the extracts from stems showed a stronger superoxide anion radical scavenging ability. The superoxide anion radical scavenging potentials (IC50) of flavonoid extracts from the stems of D. erythrosora in March, June and September were determined as 15.7, 9.7 and 8.7 µ l, respectively in the shade habitat. But in December, the superoxide anion radical scavenging ability (IC50) of flavonoids extract in the stems was determined as 47.4 µl in the shade habitat, which indicated that the inhibitory effect of stems extract on superoxide radical generation decrease greatly. In conclusion, the extracts from leaves showed strong superoxide anion radical scavenging potential in spring, which reduced in summer and autumn and diminished in winter. The extracts form stems appear stronger superoxide anion radical scavenging potential than those in other parts. From above data, it was suggested that the leaves in spring and stems in any seasons can be collected as crude medicine.
3.5 ABTS radical scavenging activity of flavonoids extract from D. erythrosora ＋
The ABTS• scavenging activities of flavonoids extract from D. erythrosora were shown in Fig. ＋
5. In the spring, the extracts in all parts of the plant look like to show very weak ABTS•
scavenging activity (Fig. 5, A and E). In other seasons, the extracts from the stems of D. ＋
erythrosora showed relatively high ABTS• ＋
The ABTS•
scavenging activity (Fig. 5, B-D and E-G).
scavenging ability (IC50) of flavonoid extracts in the stems from D. erythrosora
from June to December was determined as 3.7±0.9 µl. The radical scavenging activity of stem extracts (10 µ l) can reach up to 90%. The leaves, rachis and roots of D. erythrosora also showed ＋
relatively weak ABTS•
scavenging activity.
As for the antioxidant activity, the seasonal changes of the DPPH free radical and ABTS radical scavenging potential of flavonoid extracts were seemingly in linear with the seasonal dynamics of the total flavonoid contents. However, the superoxide anion scavenging activity did not show the same trend. Zhang et al. (2012) reported that the flavonoids distribution was significantly different in different parts of D. erythrosora and only dihydromyricetin was detected in all parts of plant.
8
Therefore, we deduced that the extracting solutions contain different kind of flavonoids, and every kind of flavonoids had varies capacity of antioxidant activities. In conclusion, the extracts form stems appeared stronger ABTS scavenging potential than other parts in all seasons. The extracts from stems had weak ABTS scavenging activity in spring while increased in other seasons. From above data, we can collect the stems in any seasons as crude medicine. 4 Conclusion In current research, the seasonal dynamics of the total flavonoid contents in various parts of D. erythrosora and their antioxidant activity were studied in detailed. The total flavonoid contents in D. erythrosora ranged from 0.78% to 12.53%, and showed an obvious seasonal dynamic change. The total flavonoid contents in stems were much higher than that in other parts, which showed a negative correlation with that in the leaves. As for the antioxidant activity, the extracts form stems always-showed stronger scavenging potential than other parts, and it was suggested that the stems can be used as crude medicine.
Acknowledgments The authors are grateful for financial sponsored by the National Natural Science Fund of China (31301442), Natural Science Foundation of Shanghai (30970267), and Leading Academic Discipline Project and Key Project of Shanghai Municipal Education Commission (J50401 and 12ZZ128).
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acetylcholinesterase inhibition activities. Food and Chemical Toxicology, 51, 242-250. Cao, H., Chen, X. Q., & Yamamoto, K. (2012). Bovine Serum Albumin Significantly Improves the DPPH Free Radical Scavenging Potential of Dietary Polyphenols and Gallic Acids. Anti-cancer Agents in Medicinal Chemistry, 12, 940-948. Chang, Y., Hu J. L., Jiang, S. J., & Qiang, S. (2005). Study on the distribution and the total flavonoids content of medicial pteridophytes in Nanjing Zijin Mountain. Journal of Northeast Agricultural University, 36, 320-323. Delmas, D., & Xiao, J. B. (2012). Natural polyphenols properties: Chemopreventive and chemosensitizing activities. Anti-Cancer Agents in Medicinal Chemistry, 12, 835. Gao, Z. P., Li, R. F., Wang, B. H., & Lu, Y. R. (2003). Progress in chemical constituents of genus Dryopteris. Chinese Journal of Experimental Traditional Medical Formulae, 9(3), 50-55. Harborne, J. B. (1966). Comparative biochemistry of flavonoids-II: 3-Desoxyanthocyanins and their systematic distribution in ferns and gesnerads. Phytochemistry, 5, 589-600. Hiraoka, A. (1978). Flavonoid patterns in Athyriaceae and Dryopteridaceae. Biochemical Systematics and Ecology, 6, 171-175. Imperato, F. (2007a). A new flavonoid, quercetin 3-O-(X"-acetyl-X"-cinnomoyl -glucoside) and a new fern consituent quescetin 3-O-(glucosylrhamnoside) from Dryopteris villarii. American Fern Journal, 97, 124-126. Imperato, F. (2007b). Three new flavonoid glycosides, kaempferol 3-O-(caffeoylrhamnoside), apigenin 4'-O- (caffeoylglucoside) and 4'-O-(feruloylglucoside) from Dryopteris villarii. American Fern Journal, 97, 233-236. Martín-Peláez, S., Covas, M. I., Fitó, M., Kušar, A., & Pravst, I. (2013). Health effects of olive oil polyphenols: Recent advances and possibilities for the use of health claims. Molecular Nutrition & Food Research, 57, 760-771. Ming, L. C., Maia-Almeida, C. I., & Conceição, D. M. (2012). Phytomass and flavonoid production in different organs and phenological stages of Passiflora alata Dryander. Journal of Medicinal Plants Research. 6(45), 5695-5700. Panickar, K. S. (2013). Effects of dietary polyphenols on neuroregulatory factors and pathways that mediate food intake and energy regulation in obesity. Molecular Nutrition & Food 10
Research, 57, 34-47. Xiao, J. B., Huo, J. L., Jiang, H. X., & Yang, F. (2011). Chemical compositions and bioactivities of crude polysaccharides from tea leaves beyond their useful date. International Journal of Biological Macromolecules 49, 1143-1151. Xiao, J. B., & Kai, G. Y. (2012). A review of dietary polyphenol-plasma protein interactions: Characterization, influence on the bioactivity, and structure-affinity relationship. Critical Reviews in Food Science and Nutrition, 52, 85-101. Xiao, J. B., Ni, X. L., Kai, G. Y., & Chen, X. Q. (2013). A review on structure-activity relationship of dietary polyphenols inhibiting α-amylase. Critical Reviews in Food Science and Nutrition, 53, 497-506. Xiao, J. B. (2013). Polyphenol-plasma proteins interaction: its nature, analytical techniques, and influence on bioactivities of polyphenols. Current Drug Metabolism, 14, 367-368. Xiao, J. B., Muzashvili, T. S., Georgiev, M. I. (2014). Advance on biotechnology for glycosylation of high-value flavonoids. Biotechnology Advances, Doi:10.1016/j.biotechadv.2014.04.006. (in press) Xiao, J. B., Ni, X. L., Kai, G. Y., Chen X. Q. (2015). Advance in dietary polyphenols as aldose reductases inhibitors: Structure-activity relationship aspect. Critical Reviews in Food Science and Nutrition, Doi: 10.1080/10408398.2010.548108. (in press) Zhang, M., Cao, J. G., Dai, X. L., Chen, X. F., & Wang, Q. X. (2012). Flavonoids contents and free radical scavenging activity of extracts from leaves, stems, rachis and roots of Dryopteris erythrosora. Iranian Journal of Pharmaceutical Research, 11, 991-997.
Table 1 The total flavonoid contents in roots, stems, leaves and rachis of D. erythrosora under shade 11
and half shade habitats. Shade habitat (%)
Half-shade habitat (%)
Date Leaves
Rachis
Roots
Stems
Leaves
Rachis
Roots
Stems
Jan.
1.498
4.641
5.060
6.990
2.008
5.181
4.083
5.870
Feb.
1.651
4.224
4.066
6.272
2.814
4.783
4.359
7.162
Mar.
2.258
3.495
4.460
4.275
4.670
5.486
3.536
5.470
Apr.
3.937
4.306
2.932
4.930
5.503
4.233
3.387
4.908
May
1.735
2.357
5.495
8.810
1.004
2.607
6.925
8.945
Jun.
1.301
3.544
3.072
7.616
1.523
3.984
5.301
12.525
Jul.
0.786
3.793
2.731
8.330
1.004
2.102
3.082
7.655
Aug.
0.884
3.771
1.129
7.782
0.933
4.154
3.041
11.959
Sep.
0.846
3.972
3.313
9.066
1.843
3.850
4.846
9.792
Oct.
2.040
2.685
2.455
9.985
1.535
4.512
4.443
9.802
Nov.
0.890
3.288
6.717
7.657
0.957
4.605
4.379
9.287
Dec.
3.205
4.908
4.522
10.105
3.535
4.554
3.153
9.933
1.75
3.75
3.83
7.65
2.28
4.17
4.21
8.61
0.99
0.75
1.53
1.82
1.54
0.97
1.13
2.44
Average content Standard deviation
12
14.0 Leaves
10.0
Total flavonoid content %
Total flavonoid content %
12.0
Rachis Roots
8.0
Stems
6.0 4.0 2.0
Leaves 12.0
Rachis Roots
10.0
Stems 8.0 6.0 4.0 2.0 0.0
0.0 Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Jan
Dec
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
monthes
monthes
A
B
Fig. 1. The total flavonoid contents in leaves, stems, rachis and roots (A: shade habitat; B: half shade habitat).
13
Fig. 2. The DPPH scavenging activity of flavonoid extracts from D. erythrosora. A-D, half shade habitat (A, March; B, June; C, September; D, December) E-H, shade habitat (E, March; F, June; G, September; H, December)
14
50 45
value of IC50 (ul)
40
Spring other seasons
35 30 25 20 15 10 5 0 leaves
rachis
roots
stems
Fig. 3. The DPPH scavenging abilities (IC50 value) of flavonoid extracts from the leaves, rachis, roots and stems.
15
Fig. 4. The superoxide anion scavenging activity of flavonoid extracts from D. erythrosora. A-D, half shade habitat (A, March; B, June; C, September; D, December) E-H, shade habitat (E, March; F, June; G, September; H, December)
16
Fig. 5. The ABTS scavenging activity of flavonoid extracts from D. erythrosora. A-D, half shade habitat (A, March; B, June; C, September; D, December) E-H, shade habitat (E, March; F, June; G, September; H, December)
17
Highlights
•
The total flavonoids content in various parts of D. erythrosora obviously appear an obvious seasonal dynamic change.
•
The total flavonoids content in the stems show a negative correlation with that in the leaves.
•
The total flavonoids content in stems is much higher than that in the roots, rachis and leaves.
•
The antioxidant activities of the extracts also altered in proportion to the change of total flavonoid contents.
•
The extracts from stems always showed highest antioxidant potentials.
18
S0308-8146(14)00728-6 http://dx.doi.org/10.1016/j.foodchem.2014.05.024 FOCH 15805
To appear in:
Food Chemistry
Please cite this article as: Xie, Y., Zheng, Y., Dai, X., Wang, Q., Cao, J., Xiao, J., Seasonal dynamics of total flavonoid contents and antioxidant activity of Dryopteris erythrosora, Food Chemistry (2014), doi: http://dx.doi.org/ 10.1016/j.foodchem.2014.05.024
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Short communication for ISPMF2015
Seasonal dynamics of total flavonoid contents and antioxidant activity of Dryopteris erythrosora Yinghua Xie1, Yunxia Zheng1, Xiling Dai1, Quanxi Wang1 , Jianguo Cao1*, Jianbo Xiao1,2* 1
Department of Biology, Shanghai Normal University, 100 Guilin Rd, Shanghai 200234, China.
2
Research Center of Bio-separation Engineering Technology, Anhui Academy of Applied
Technology, Suixi Road 312, Hefei, Anhui 230031, PR China *Correspondence to: Jianguo Cao, Department of Biology, Shanghai Normal University, 100 Guilin Rd, Shanghai 200234, China, Tel(Fax): +86 21 64322526, E-mail:[email protected]; Jianbo Xiao, Department of Biology, Shanghai Normal University, 100 Guilin Rd, Shanghai 200234, China; Tel(Fax): +86 21 64321291, E-mail: [email protected]
Abstract The seasonal dynamics of the total flavonoid contents in various parts of Dryopteris erythrosora, a traditional Chinese medicinal fern, and their antioxidant activity were investigated. The total flavonoids content in various parts of D. erythrosora showed an obvious seasonal dynamic change. The total flavonoid contents in stems (from 4.3% to 12.5%) were much higher than that in leaves with an average content of 2.01%. In spring, the total flavonoid contents in stems were relatively low, but increased rapidly from summer to winter. However, the seasonal dynamics of total flavonoid contents in leaves showed different model. The total flavonoid contents in the stems showed a negative correlation with that in the leaves from January to July. The correlation coefficient of about -0.7 was obtained. The antioxidant activity of the extracts also altered in proportion to the change of total flavonoid contents. In general, the extracts from stems always showed highest antioxidant potentials and it was suggested that the stems can be used as crude medicine.
Key words: Flavonoids; Dryopteris erythrosora; antioxidant activities; habitats
1. Introduction 1
Dryopteris erythrosora, which is rich in bioactive components such as flavonoids, polyphenols, and terpenoids, distributes widely in China (Gao, Li, Wang, & Lu, 2003). The epidemiological and medical evidence have suggested that the dietary flavonoids play an important role in preventing and managing of diseases such as cancer, diabetes, and cardiovascular diseases (Andrae-Marobela, Ghislain, Okatch, & Majinda, 2013; Xiao, Ni, Kai, & Chen, 2013; Delmas & Xiao, 2012; Xiao, 2013; Xiao & Kai, 2012; Xiao, Muzashvili, & Georgiev, 2014; Xiao, Ni, Kai, & Chen, 2015; Martín-Peláez, Covas, Fitó, Kušar, & Pravst, 2013; Panickar, 2013). Some flavonoid compounds have been identified in the genus Dryopteris (Hiraoka, 1978; Harborne, 1966; Imperato, 2007a; Imperato, 2007b). Chang, Hu, Jiang, and Qiang (2005) reported that the total flavonoid contents in the aerial parts of D. erythrosora were about 0.89%. We recently investigated the total flavonoids content and free radical scavenging activity of extract from leaves, rachis, roots and stems of D. erythrosora (Zhang, Cao, Dai, Chen, & Wang, 2012). And 8 flavonoids were identified in D. erythrosora, which were considered to have bioactivities in antioxidant, anticancer and acetylcholinesterase (Cao, Xia, Chen, Xiao, & Wang, 2013). However, the seasonal dynamics of the flavonoid content and their bioactivities in D. erythrosora were still not clear. We collected plant materials monthly from shade (under bamboo forest) and half shade (under broad-leaved forest) habitats. The flavonoid contents and radical scavenging activity were compared. The results provided important information for utilizing this medicinal fern.
2 Materials and methods 2.1 Chemicals and materials Rutin (>98%) was purchased from Sigma Co. (MO, USA). Nitrotetrazolium blue chloride (NBT),
phenazine
metho-sulfate
(PMS),
2,2-diphenyl-1-picrylhydrazyl
(DPPH),
and
2,2’-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS) were purchased from Aladdin Reagent Int. (Shanghai, China). Nicotinamide adenine dinucleotide (NADH) was obtained from Sangon Biotech Co. Ltd (Shanghai, China). The materials of D. erythrosora were collected monthly (10th-15th day every month) from Sheshan Mount in Shanghai (China). The identities of the plants were verified by Prof. Dr. Jianguo Cao in College of Life & Environment Science of Shanghai Normal University. Two to three plants of D. erythrosora were collected and the leaves, 2
stems, rachis and roots were separated for flavonoids studies. All other reagents and solvents were of analytical grade and all aqueous solutions were prepared using newly double-distilled water.
2.2 Preparation of plant extract Each time, three plants of D. erythrosora were collected at one site. The whole plant, including the fresh leaves, stems, rachis, and roots were washed and dried in the oven at 65 ºC. The dried sample was powdered and filtered through 40-mesh screen. The dried sample (1.0000 g) was extracted with 50% ethanol (25 mL) for 2h at 50 °C. Then, the ultrasound-assisted extraction was performed on a ultrasound generation system (Kunshan, China) for 20 min. This extraction process was repeated twice. The extracts were filtered with filter paper and collected. The mixture was allowed to cool for 20 min and concentrated to dry by evaporating with a rotary evaporator. The residue was suspended with 50 mL ethanol and filtered through a 0.45 µm membrane (Millipore, USA).
2.3 Determination of total flavonoids The total flavonoid contents in extract were measured by a colorimetric assay (Zhang et al., 2012). The extract (5 mL) was added to a 10 mL flask, and then 5% NaNO2 solution (0.3 mL) was added. After mixed well, the solution was allowed to stand for 6 min at room temperature; and 5% Al(NO3)3 solution (0.3 mL) was added to the flask, mixed well and kept for 6 min at room temperature. At last 4% NaOH solution (4.4 mL) was added, mixed well and kept for 12 min at room temperature. Absorbance was read on a TU-1810 UV-spectrophotometer (Beijing, China) at 510 nm, and the total flavonoid contents (%) were estimated using calibration curves.
2.4. DPPH free radical scavenging activity The DPPH free radical scavenging activity of flavonoids extract was measured according to our previous report (Cao, Xia, Chen, Xiao, & Wang, 2013). One hundred microliters of samples were added to 900 µ L of DPPH solution (1 mM in 50% ethanol) and incubated in the dark for 30 min. After that, the absorbance at 517 nm was measured. The DPPH free radical scavenging activity was calculated using the following formula: 3
DPPH free radical scavenging activity (%) = (1- A1/A0) × 100 Where A0 is the absorbance of the control, and A1 is the absorbance of samples. Each experiment was repeated three times and found to be reproducible within experimental errors (RSD < 5.0%). 2.5 Superoxide anion radical (O2¯) scavenging activity Superoxide anion (O2¯) scavenging activity of flavonoid exteacts was measured by Robak and Gryglewski with slightly modification (Xiao et al., 2011). The superoxide radical was generated in 1.0 mL of sodium phosphate buffer (100 mM, pH 7.4) containing 300 µL NBT (50 µM) solution, 300 µL NADH (78 µM) solution and different concentrations of flavonoid extract from D. erythrosora. The reaction started by addition of 300 µL PMS (10 µM). The reaction mixture was incubated at room temperature for 8 min, and the absorbance at 560 nm was measured. The capability to scavenge the superoxide radical was calculated using the following equation: O2¯ scavenging activity (%) = (1-A1/A0 ) × 100 Where A0 is the absorbance of the control, and A1 is the absorbance of sample. Each experiment was repeated three times and found to be reproducible within experimental errors (RSD < 5.0%).
2.6 ABTS radical scavenging activity The ABTS assay described by Arts et al. (2002) was used with minor modifications. The ABTS assay assessed the total radical scavenging capacity based on the ability of an antioxidant to ＋
scavenge the stable ABTS radical cation (ABTS• ), which was produced by mixing 1.0 mL ABTS stock solution (7.4 mM) with 1.0 mL potassium persulfate (2.6 mM) and allowing to stand in the ＋
dark at room temperature for 12 h before using. The ABTS• solution was diluted 50 times with ＋
PBS (pH7.4) at 37 ºC. Samples (100 µ L) were added to 900 µ L of ABTS• solution and kept in the dark at room temperature for 60 min. Thus, the absorbance was spectrophotometrically ＋
determined at 734 nm. The ABTS•
scavenging activity was calculated using the following
formula: ＋
ABTS•
scavenging activity (%) = (1- A1/A0) × 100
Where A0 is the absorbance of the control, and A1 is the absorbance of sample. Each experiment was repeated three times and found to be reproducible within experimental errors (RSD < 5.0%).
4
2.7 Statistical analysis In present investigation, some simple statistical analysis, mainly the standard deviation, correlation coefficient, was carried out using the Microsoft Excel.
3 Results and discussion 3.1 Total flavonoids content in D. erythrosora The 50% ethanol was used to extract flavonoids from dried leaves, rachis, roots and stems of D. erythrosora. As shown in Table 1, the total flavonoid contents in D. erythrosora were ranged from 0.78% to 12.53% depending on collecting time within one year and different parts. The annual average contents of total flavonoids in any parts of D. erythrosora in half shade habitat were always slight higher than that in the shade habitat (Table 1). The annual average flavonoid contents of different parts in the shade habitat were determined as: stems (7.65%) > roots (3.83%) > rachis (3.75%) > leaves (1.75%). Meanwhile the annual average flavonoid contents of different parts in the half shade habitat were determined as: stems (8.61%) > roots (4.21%) > rachis (4.17%) > leaves (2.28%). The total flavonoid contents in stems (from 4.3% to 12.5%) were much higher than that in leaves with an average content of 2.01%. It was illustrated that the total flavonoid contents in stems were obviously higher than that in the roots, rachis and leaves (Table 1). In our previous study, the total flavonoid contents in D. erythrosora collected in May ranged from 2.1% to 8.26% (Zhang, Cao, Dai, Chen, & Wang, 2012), which were agree to the present results. Chang, Hu, Jiang, and Qiang (2005) reported that the total flavonoid contents in aerial parts of D. erythrosora was 0.89%, which was similar to that of leaves collected in the summer season in current investigation. In general, the dry weight of stems was slightly higher than that of the leaves, rachis and roots. Therefore, the stems were considered to be the most effective part to be used as crude medicine.
3.2 Seasonal dynamics of total flavonoids content in different parts No matter in the half shade habitat or in the shade habitat, the total flavonoid contents in various parts of D. erythrosora showed a similar seasonal dynamic change (Fig. 1). The seasonal 5
dynamics of the total flavonoid contents in the leaves and stems were negatively related, with a correlation coefficient of about -0.7 (Fig. 1). In January, the total flavonoid contents in the leaves were relatively low (about 1.5%-2.0%). Then, the total flavonoid contents in the leaves increased and reached up to maximum in April (about 5.5%). Thereafter, the total flavonoid contents decreased rapidly and maintained at the low status during the whole summer. In December, the total flavonoid contents in the leaves increased again and then decreased in January (Table 1; Fig. 1). The total flavonoid contents in stems ranged from 5.9 to 6.9% in January, but decreased in spring and got a minimum content of 4.9%. After that, the total flavonoid contents improved rapidly from April and maintained at a high level during the whole summer and autumn (Fig. 1). The changes of the total flavonoid contents in the rachis and roots were relatively low and seemed to have no significant variation (Fig. 1). The typical negative correlation of total flavonoid contents in the stems and the leaves were very interesting. Increasing of the total flavonoid contents in the stems was accompanied with decreasing of the total flavonoids content in the leaves. It is generally considered that the flavonoids were produced in the leaves of plant (Ming, Maia-Almeida, & Conceição, 2012). Polar transportation of flavonoids from the leaves to the stems in the growth season might be reasons of negative correlation. The largest flavonoid production in leaves was observed in Spring. So we can collect fresh leaves at the spring as crude medicine of flavonoids. In other seasons, the flavonoids might transport and accumulate in the stems. As a result, we can use the stems at the late summer and the early autumn as crude medicine of flavonoids.
3.3 DPPH free radical scavenging activity of different parts of D. erythrosora In present investigation, D. erythrosora collected in March, June, September and December, which represented spring (from Feb to Apr), summer (from May to Jul), autumn (from Aug to Oct) and winter (from Nov to Jan), were chosen to study the DPPH free radical scavenging activities. For different habitats, DPPH free radical scavenging activities of the flavonoids of D. erythrosora showed similar characteristics in the same month (Fig. 2). However, for different seasons, DPPH
6
free radical scavenging activity of the flavonoids extract from D. erythrosora showed different potential (Fig. 2). In spring (March), the DPPH free radical scavenging potential (IC50) of flavonoids extract from the leaves, rachis, roots and stems were determined as 14.8, 18.0, 11.5 and 8.9 µ l, respectively (Fig. 3). The DPPH free radical scavenging activity of flavonoids extract in various parts of D. erythrosora was similar with each other especially for the half shade habitat (Fig. 2A). However, the DPPH free radical scavenging activity of various parts of D. erythrosora began to diverge and showed a great difference in other seasons. The DPPH free radical scavenging abilities (IC50) of flavonoids extract from the leaves, rachis, roots and stems were determined as 36.1, 15.7, 15.9 and 4.7 µ l, respectively (Fig. 3). From the data of the IC50, it illustrated that the DPPH free radical scavenging potential of the extract from stems was much higher than that in other parts. In conclusion, the extract from leaves appears strong DPPH free radical scavenging abilities in spring and decreased in other seasons. The extract of stems shows strong DPPH free radical scavenging potential in all seasons. Thus, we can collect the leaves in spring days and stems in any seasons as crude medicine.
3.4 Superoxide anion scavenging activity of different parts of D. erythrosora Fig. 4 showed the inhibitory effect of flavonoid extracts from D. erythrosora on superoxide radical generation. All the extracts of the parts of the plant showed inhibitory effect on superoxide anion. For the different habitats, D. erythrosora extracts exhibited similar effects of superoxide anion scavenging activity (Fig. 4). In different seasons, the flavonoids extracts from D. erythrosora showed a strong superoxide anion scavenging activity in March, June and September (Fig. 4, A-C, E-G) and much weak superoxide anion scavenging activity in December (Fig. 4, D, H). For various parts, inhibitory effect of the leaves extract on superoxide radical generation was slight stronger in spring (March) (IC50=15.0 µl), but gradually decreased in summer (IC50 =19.6 µl) and autumn days (IC50 =36.2 µ l), afterwards became almost no inhibitory effect on superoxide radical generation in the winter days (December).
7
From spring to autumn (March, June and September), the extracts from stems showed a stronger superoxide anion radical scavenging ability. The superoxide anion radical scavenging potentials (IC50) of flavonoid extracts from the stems of D. erythrosora in March, June and September were determined as 15.7, 9.7 and 8.7 µ l, respectively in the shade habitat. But in December, the superoxide anion radical scavenging ability (IC50) of flavonoids extract in the stems was determined as 47.4 µl in the shade habitat, which indicated that the inhibitory effect of stems extract on superoxide radical generation decrease greatly. In conclusion, the extracts from leaves showed strong superoxide anion radical scavenging potential in spring, which reduced in summer and autumn and diminished in winter. The extracts form stems appear stronger superoxide anion radical scavenging potential than those in other parts. From above data, it was suggested that the leaves in spring and stems in any seasons can be collected as crude medicine.
3.5 ABTS radical scavenging activity of flavonoids extract from D. erythrosora ＋
The ABTS• scavenging activities of flavonoids extract from D. erythrosora were shown in Fig. ＋
5. In the spring, the extracts in all parts of the plant look like to show very weak ABTS•
scavenging activity (Fig. 5, A and E). In other seasons, the extracts from the stems of D. ＋
erythrosora showed relatively high ABTS• ＋
The ABTS•
scavenging activity (Fig. 5, B-D and E-G).
scavenging ability (IC50) of flavonoid extracts in the stems from D. erythrosora
from June to December was determined as 3.7±0.9 µl. The radical scavenging activity of stem extracts (10 µ l) can reach up to 90%. The leaves, rachis and roots of D. erythrosora also showed ＋
relatively weak ABTS•
scavenging activity.
As for the antioxidant activity, the seasonal changes of the DPPH free radical and ABTS radical scavenging potential of flavonoid extracts were seemingly in linear with the seasonal dynamics of the total flavonoid contents. However, the superoxide anion scavenging activity did not show the same trend. Zhang et al. (2012) reported that the flavonoids distribution was significantly different in different parts of D. erythrosora and only dihydromyricetin was detected in all parts of plant.
8
Therefore, we deduced that the extracting solutions contain different kind of flavonoids, and every kind of flavonoids had varies capacity of antioxidant activities. In conclusion, the extracts form stems appeared stronger ABTS scavenging potential than other parts in all seasons. The extracts from stems had weak ABTS scavenging activity in spring while increased in other seasons. From above data, we can collect the stems in any seasons as crude medicine. 4 Conclusion In current research, the seasonal dynamics of the total flavonoid contents in various parts of D. erythrosora and their antioxidant activity were studied in detailed. The total flavonoid contents in D. erythrosora ranged from 0.78% to 12.53%, and showed an obvious seasonal dynamic change. The total flavonoid contents in stems were much higher than that in other parts, which showed a negative correlation with that in the leaves. As for the antioxidant activity, the extracts form stems always-showed stronger scavenging potential than other parts, and it was suggested that the stems can be used as crude medicine.
Acknowledgments The authors are grateful for financial sponsored by the National Natural Science Fund of China (31301442), Natural Science Foundation of Shanghai (30970267), and Leading Academic Discipline Project and Key Project of Shanghai Municipal Education Commission (J50401 and 12ZZ128).
References Andrae-Marobela, K., Ghislain, F. W., Okatch, H., & Majinda, R. R. T. (2013). Polyphenols: A diverse class of multi-target anti-HIV-1 agents. Current Drug Metabolism, 14, 392-413. Arts, M. J. T. J., Haenen, G. R. M. M., Wilms, L. C., Beetstra, S. A. J. N., Heijnen, C. G. M., & Voss, H. P. (2002). Interactions between flavonoids and proteins: Effect on the total antioxidant capacity. Journal of Agricultural and Food Chemistry, 50, 1184-1187. Cao, J. G., Xia, X., Chen, X. F., Xiao, J. B., & Wang, Q. X. (2013). Characterization of flavonoids from Dryopteris erythrosora and evaluation of their antioxidant, anticancer and 9
acetylcholinesterase inhibition activities. Food and Chemical Toxicology, 51, 242-250. Cao, H., Chen, X. Q., & Yamamoto, K. (2012). Bovine Serum Albumin Significantly Improves the DPPH Free Radical Scavenging Potential of Dietary Polyphenols and Gallic Acids. Anti-cancer Agents in Medicinal Chemistry, 12, 940-948. Chang, Y., Hu J. L., Jiang, S. J., & Qiang, S. (2005). Study on the distribution and the total flavonoids content of medicial pteridophytes in Nanjing Zijin Mountain. Journal of Northeast Agricultural University, 36, 320-323. Delmas, D., & Xiao, J. B. (2012). Natural polyphenols properties: Chemopreventive and chemosensitizing activities. Anti-Cancer Agents in Medicinal Chemistry, 12, 835. Gao, Z. P., Li, R. F., Wang, B. H., & Lu, Y. R. (2003). Progress in chemical constituents of genus Dryopteris. Chinese Journal of Experimental Traditional Medical Formulae, 9(3), 50-55. Harborne, J. B. (1966). Comparative biochemistry of flavonoids-II: 3-Desoxyanthocyanins and their systematic distribution in ferns and gesnerads. Phytochemistry, 5, 589-600. Hiraoka, A. (1978). Flavonoid patterns in Athyriaceae and Dryopteridaceae. Biochemical Systematics and Ecology, 6, 171-175. Imperato, F. (2007a). A new flavonoid, quercetin 3-O-(X"-acetyl-X"-cinnomoyl -glucoside) and a new fern consituent quescetin 3-O-(glucosylrhamnoside) from Dryopteris villarii. American Fern Journal, 97, 124-126. Imperato, F. (2007b). Three new flavonoid glycosides, kaempferol 3-O-(caffeoylrhamnoside), apigenin 4'-O- (caffeoylglucoside) and 4'-O-(feruloylglucoside) from Dryopteris villarii. American Fern Journal, 97, 233-236. Martín-Peláez, S., Covas, M. I., Fitó, M., Kušar, A., & Pravst, I. (2013). Health effects of olive oil polyphenols: Recent advances and possibilities for the use of health claims. Molecular Nutrition & Food Research, 57, 760-771. Ming, L. C., Maia-Almeida, C. I., & Conceição, D. M. (2012). Phytomass and flavonoid production in different organs and phenological stages of Passiflora alata Dryander. Journal of Medicinal Plants Research. 6(45), 5695-5700. Panickar, K. S. (2013). Effects of dietary polyphenols on neuroregulatory factors and pathways that mediate food intake and energy regulation in obesity. Molecular Nutrition & Food 10
Research, 57, 34-47. Xiao, J. B., Huo, J. L., Jiang, H. X., & Yang, F. (2011). Chemical compositions and bioactivities of crude polysaccharides from tea leaves beyond their useful date. International Journal of Biological Macromolecules 49, 1143-1151. Xiao, J. B., & Kai, G. Y. (2012). A review of dietary polyphenol-plasma protein interactions: Characterization, influence on the bioactivity, and structure-affinity relationship. Critical Reviews in Food Science and Nutrition, 52, 85-101. Xiao, J. B., Ni, X. L., Kai, G. Y., & Chen, X. Q. (2013). A review on structure-activity relationship of dietary polyphenols inhibiting α-amylase. Critical Reviews in Food Science and Nutrition, 53, 497-506. Xiao, J. B. (2013). Polyphenol-plasma proteins interaction: its nature, analytical techniques, and influence on bioactivities of polyphenols. Current Drug Metabolism, 14, 367-368. Xiao, J. B., Muzashvili, T. S., Georgiev, M. I. (2014). Advance on biotechnology for glycosylation of high-value flavonoids. Biotechnology Advances, Doi:10.1016/j.biotechadv.2014.04.006. (in press) Xiao, J. B., Ni, X. L., Kai, G. Y., Chen X. Q. (2015). Advance in dietary polyphenols as aldose reductases inhibitors: Structure-activity relationship aspect. Critical Reviews in Food Science and Nutrition, Doi: 10.1080/10408398.2010.548108. (in press) Zhang, M., Cao, J. G., Dai, X. L., Chen, X. F., & Wang, Q. X. (2012). Flavonoids contents and free radical scavenging activity of extracts from leaves, stems, rachis and roots of Dryopteris erythrosora. Iranian Journal of Pharmaceutical Research, 11, 991-997.
Table 1 The total flavonoid contents in roots, stems, leaves and rachis of D. erythrosora under shade 11
and half shade habitats. Shade habitat (%)
Half-shade habitat (%)
Date Leaves
Rachis
Roots
Stems
Leaves
Rachis
Roots
Stems
Jan.
1.498
4.641
5.060
6.990
2.008
5.181
4.083
5.870
Feb.
1.651
4.224
4.066
6.272
2.814
4.783
4.359
7.162
Mar.
2.258
3.495
4.460
4.275
4.670
5.486
3.536
5.470
Apr.
3.937
4.306
2.932
4.930
5.503
4.233
3.387
4.908
May
1.735
2.357
5.495
8.810
1.004
2.607
6.925
8.945
Jun.
1.301
3.544
3.072
7.616
1.523
3.984
5.301
12.525
Jul.
0.786
3.793
2.731
8.330
1.004
2.102
3.082
7.655
Aug.
0.884
3.771
1.129
7.782
0.933
4.154
3.041
11.959
Sep.
0.846
3.972
3.313
9.066
1.843
3.850
4.846
9.792
Oct.
2.040
2.685
2.455
9.985
1.535
4.512
4.443
9.802
Nov.
0.890
3.288
6.717
7.657
0.957
4.605
4.379
9.287
Dec.
3.205
4.908
4.522
10.105
3.535
4.554
3.153
9.933
1.75
3.75
3.83
7.65
2.28
4.17
4.21
8.61
0.99
0.75
1.53
1.82
1.54
0.97
1.13
2.44
Average content Standard deviation
12
14.0 Leaves
10.0
Total flavonoid content %
Total flavonoid content %
12.0
Rachis Roots
8.0
Stems
6.0 4.0 2.0
Leaves 12.0
Rachis Roots
10.0
Stems 8.0 6.0 4.0 2.0 0.0
0.0 Jan
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Jan
Dec
Feb
Mar
Apr
May
Jun
Jul
Aug
Sep
Oct
Nov
Dec
monthes
monthes
A
B
Fig. 1. The total flavonoid contents in leaves, stems, rachis and roots (A: shade habitat; B: half shade habitat).
13
Fig. 2. The DPPH scavenging activity of flavonoid extracts from D. erythrosora. A-D, half shade habitat (A, March; B, June; C, September; D, December) E-H, shade habitat (E, March; F, June; G, September; H, December)
14
50 45
value of IC50 (ul)
40
Spring other seasons
35 30 25 20 15 10 5 0 leaves
rachis
roots
stems
Fig. 3. The DPPH scavenging abilities (IC50 value) of flavonoid extracts from the leaves, rachis, roots and stems.
15
Fig. 4. The superoxide anion scavenging activity of flavonoid extracts from D. erythrosora. A-D, half shade habitat (A, March; B, June; C, September; D, December) E-H, shade habitat (E, March; F, June; G, September; H, December)
16
Fig. 5. The ABTS scavenging activity of flavonoid extracts from D. erythrosora. A-D, half shade habitat (A, March; B, June; C, September; D, December) E-H, shade habitat (E, March; F, June; G, September; H, December)
17
Highlights
•
The total flavonoids content in various parts of D. erythrosora obviously appear an obvious seasonal dynamic change.
•
The total flavonoids content in the stems show a negative correlation with that in the leaves.
•
The total flavonoids content in stems is much higher than that in the roots, rachis and leaves.
•
The antioxidant activities of the extracts also altered in proportion to the change of total flavonoid contents.
•
The extracts from stems always showed highest antioxidant potentials.
18
