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Mar 17, 2016 - Abstract: Marchantia polymorpha L. is a representative bryophyte used as a traditional Chinese medicinal herb for scald and pneumonia.
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Flavonoids, Antioxidant Potential, and Acetylcholinesterase Inhibition Activity of the Extracts from the Gametophyte and Archegoniophore of Marchantia polymorpha L. Xin Wang 1,2 , Jianguo Cao 3 , Yuhuan Wu 1,4, *, Quanxi Wang 3,5, * and Jianbo Xiao 6 1 2 3 4 5 6

*

Insititute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China; [email protected] University of Chinese Academy of Sciences, 19A Yuquan Road, Beijing 100049, China College of Life and Environmental Sciences, Shanghai Normal University, Shanghai 200234, China; [email protected] College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou 310036, China Shanghai Key Laboratory of Plant Functional Genomics and Resources, Chinese Academy of Sciences, Shanghai Chenshan Botanical Garden, Shanghai 200234, China State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Taipa, Macau; [email protected] Correspondence: [email protected] (Y.W.); [email protected] (Q.W.); Tel.: +86-137-0163-6197 (Y.W.); +86-137-5814-5015 (Q.W.)

Academic Editor: Derek J. McPhee Received: 19 February 2016 ; Accepted: 10 March 2016 ; Published: 17 March 2016

Abstract: Marchantia polymorpha L. is a representative bryophyte used as a traditional Chinese medicinal herb for scald and pneumonia. The phytochemicals in M. polymorpha L. are terpenoids and flavonoids, among which especially the flavonoids show significant human health benefits. Many researches on the gametophyte of M. polymorpha L. have been reported. However, as the reproductive organ of M. polymorpha L., the bioactivity and flavonoids profile of the archegoniophore have not been reported, so in this work the flavonoid profiles, antioxidant and acetylcholinesterase inhibition activities of the extracts from the archegoniophore and gametophyte of M. polymorpha L. were compared by radical scavenging assay methods (DPPH, ABTS, O2´ ), reducing power assay, acetylcholinesterase inhibition assay and LC-MS analysis. The results showed that the total flavonoids content in the archegoniophore was about 10-time higher than that of the gametophyte. Differences between the archegoniophore and gametophyte of M. polymorpha L. were observed by LC-MS analysis. The archegoniophore extracts showed stronger bio-activities than those of the gametophyte. The archegoniophore extract showed a significant acetylcholinesterase inhibition, while the gametophyte extract hardly inhibited it. Keywords: Marchantia polymorpha L.; flavonoids; antioxidant potential; archegoniophore; gametophyte; acetylcholinesterase inhibition

1. Introduction Bryophytes, as the oldest known land plants, are of great significance in phylogenetic evolution. Many bryophyte plants are traditionally used to treating illnesses of the cardiovascular system, bronchitis, and burns and also possess antimicrobial, anticancer, antifungal, antimicrobial activity [1–3]. Approximately, 23,000 bryophyte species exist in the world, among which some 3021 species are found in China and about 50 species are used in medicine [4]. According to the analysis and statistics on the

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on the literature about the flavonoids of bryophytes, the with species with reported flavonoid data only literature about the flavonoids of bryophytes, the species reported flavonoid data only account account about 1.4% of the total of number of bryophytes for aboutfor 1.4% of the total number bryophytes found infound China.in China. Flavonoids, as with over 10,000 known structures [5,6],[5,6], have have vital asplant plantsecondary secondarymetabolites metabolites with over 10,000 known structures functions in plantingrowth development [7]. Evidence on epidemiological and pharmacological vital functions plant and growth and development [7].based Evidence based on epidemiological and data has shown data that has theshown flavonoids play an important in preventing and managing modern pharmacological that the flavonoids play anrole important role in preventing and managing diseases diseases such as cancer diabetes [5,9]; HIV[5,9]; [10];HIV inflammation and obesity At present, modern such as[8]; cancer [8]; diabetes [10]; inflammation and[11]. obesity [11]. Atresearch present, on flavonoids almost allalmost focus on but seldom onseldom bryophytes. research on flavonoids all spermatophytes, focus on spermatophytes, but on bryophytes. Marchantia polymorpha polymorpha is is aarepresentative representativebryophyte bryophyteand andthe thegametophyte gametophyteofof polymorpha M.M. polymorpha L. L. is is traditionally used cure cuts, fractures, poisonous snake bites, burns, scalds, open wounds traditionally used to to cure cuts, fractures, poisonous snake bites, burns, scalds, andand open wounds [4]. [4]. Many studies on gametophyte the gametophyte of polymorpha M. polymorpha L. have reported. extracts of Many studies on the of M. L. have beenbeen reported. TheThe extracts of M. M. polymorpha L. exhibited antifungal activity, antibacterial and antioxidant activities [12–14]. The main polymorpha L. exhibited antifungal activity, antibacterial and antioxidant activities [12–14]. The main bioactive compounds compounds in M. polymorpha polymorpha L. L. are are terpenoids, terpenoids, bis[bibenzyls] bis[bibenzyls] and and polyphenols, polyphenols, especially bioactive in M. especially flavonoids [15,16]. [15,16]. However, However, the thebioactivity bioactivityand andflavonoids flavonoidsprofiles profilesofofdifferent differentparts parts polymorpha flavonoids ofof M.M. polymorpha L. L. have not been reported. Herein, the flavonoid profiles, antioxidant potential, and acetylcholinesterase have not been reported. Herein, the flavonoid profiles, antioxidant potential, and acetylcholinesterase inhibition activity the extracts extracts from from the the gametophyte gametophyte and inhibition activity of of the and archegoniophore archegoniophore of of M. M. polymorpha polymorpha L. L. were compared. were compared. 2. Results Resultsand andDiscussion Discussion 2.1. Total Flavonoid 2.1. Total Flavonoid Contents Contents in in M. M. polymorpha polymorpha L. L. The thethe gametophyte andand archegoniophore of M.ofpolymorpha L. were The total total flavonoids flavonoidscontents contentsinin gametophyte archegoniophore M. polymorpha L. determined as 4.62 ˘ 0.24 mg/g and 47.42 ˘ 0.76 mg/g, respectively (Figure 1), showing that the were determined as 4.62 ± 0.24 mg/g and 47.42 ± 0.76 mg/g, respectively (Figure 1), showing thatotal the flavonoids content of the was ten as haigh as that theofgametophyte. otal flavonoids content ofarchegoniophore the archegoniophore wastimes ten times as haigh as of that the gametophyte.

Figure Total flavonoids contentsofofgametophyte gametophyteand and archegoniophore archegoniophore of . (n(n==3).3). Figure 1. 1. Total flavonoids contents of M. M.polymorpha polymorphaLL. The total flavonoids contents of 132 samples of bryophytes have also been determined, which The total flavonoids contents of 132 samples of bryophytes have also been determined, which ranged from 1.0 mg/g to 5.0 mg/g. It was found that the flavonoids content in the archegoniophore of ranged from 1.0 mg/g to 5.0 mg/g. It was found that the flavonoids content in the archegoniophore of M. polymorpha L. was also the highest in all the bryophyte parts. As secondary metabolites, flavonoids M. polymorpha L. was also the highest in all the bryophyte parts. As secondary metabolites, flavonoids were generally regarded to be associated with growth. Some experts had noticed that quantitative were generally regarded to be associated with growth. Some experts had noticed that quantitative variations in flavonoids when the plant moved into its reproductive phase [17], and then the vital variations in flavonoids when the plant moved into its reproductive phase [17], and then the vital functions of flavonoids on reproductive organs were reported [18–20]. As the female reproductive functions of flavonoids on reproductive organs were reported [18–20]. As the female reproductive organ of M. polymorpha L., the archegoniophore also showed quantitative flavonoid variations, organ of M. polymorpha L., the archegoniophore also showed quantitative flavonoid variations, which which is consistent with the abovementioned view. is consistent with the abovementioned view. 2.2. Antioxidant Activity 2.2. Antioxidant Activity The DPPH free radical scavenging activities of the extracts from the archegoniophore are shown The DPPH free radical scavenging activities of the extracts from the archegoniophore are shown in Figure 2. With increasing doses from 5.0 to 40.0 μL, the DPPH free radical scavenging potential in Figure 2. With increasing doses from 5.0 to 40.0 µL, the DPPH free radical scavenging potential observably increased. Forty μL of the extract from the archegoniophore could scavenge about 68% of observably increased. Forty µL of the extract from the archegoniophore could scavenge about 68% the free radicals. However, the DPPH free radical scavenging potential of the extracts from the of the free radicals. However, the DPPH free radical scavenging potential of the extracts from the gametophyte of M. polymorpha L. was far lower than that of the archegoniophore. The DPPH·radicals scavenging activity IC50 of the archegoniophore extract was determined as 1.3 μg/mL.

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3 of 13 polymorpha L. was far lower than that of the archegoniophore. The DPPH¨ radicals scavenging activity IC50 of the archegoniophore extract was determined as 1.3 µg/mL.

Figure 2. The DPPH free radical scavenging potential of the gametophyte and archegoniophore of M. polymorpha L. (n =free 3). radical scavenging potential of the gametophyte and archegoniophore of Figure 2. The DPPH M. polymorpha L. (n =free 3). radical scavenging potential of the gametophyte and archegoniophore of Figure 2. The DPPH

ABTS·radical scavenging potential of the extracts from the archegoniophore of M. polymorpha M.The polymorpha L. (n = 3). L. increased with increasing volume from 5.0 μLfrom (Figure 3). Furthermore,ofthe gametophyte The ABTS¨ radical scavenging potential of 1.0 the to extracts the archegoniophore M. polymorpha L. extracts of M. polymorpha L. showed a lower scavenging ability than that of the archegoniophore. The The ABTS·radical scavenging potential of the extracts from the archegoniophore of M. polymorpha increased with increasing volume from 1.0 to 5.0 µL (Figure 3). Furthermore, the gametophyte extracts 50 of the archegoniophore extract was 3.01.0 μg/mL. L. increased withL. increasing from toability 5.0 μL (Figure 3).the Furthermore, the gametophyte ofIC M. polymorpha showed avolume lower scavenging than that of archegoniophore. The IC50 of extracts of M. polymorpha L. showed a lower scavenging ability than that of the archegoniophore. The the archegoniophore extract was 3.0 µg/mL. IC50 of the archegoniophore extract was 3.0 μg/mL.

Figure 3. The ABTS radical scavenging potential of flavonoids extract from gametophyte and archegoniophore M. polymorpha L. (n = 3). potential of flavonoids extract from gametophyte and Figure 3. The of ABTS radical scavenging

archegoniophore of M. polymorpha L. (n = 3). The superoxide anion scavenging extractsextract from from the archegoniophore Figure 3. The ABTS radical scavengingactivity potentialofofthe flavonoids gametophyte and and The superoxide anion scavenging activity of the extracts from the archegoniophore gametophyte of M. of polymorpha L. isL.shown from 100 and to (n = 3). in Figure 4. With the increasing volumes archegoniophore M. polymorpha gametophyte of M. polymorpha L. is shown in Figure With the increasing volumes from 100increased to 150 μL, 150 µL, the superoxide anion scavenging potential of 4. the extracts from the archegoniophore theThe superoxide anion scavenging of the extracts from the archegoniophore increased slightly, superoxide anion scavenging activity ofM. the extracts from theshowed archegoniophore and slightly, while the extracts from thepotential gametophyte of polymorpha L. hardly any superoxide while the extracts from the gametophyte of M. polymorpha L. hardly showed any superoxide anion gametophyte of M.activity. polymorpha L. is shown in Figure 4. With the increasing volumes from 100 to 150 μL, anion scavenging scavenging the superoxideactivity. anion scavenging potential of the extracts from the archegoniophore increased slightly, while the extracts from the gametophyte of M. polymorpha L. hardly showed any superoxide anion scavenging activity.

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Figure 4. The superoxide anion scavenging potential of gametophyte and archegoniophore of Figure 4. 4.TheThe superoxide anion scavenging potential of gametophyte and archegoniophore of Figure anion scavenging potential of gametophyte and archegoniophore of L. superoxide (n = 3). M. polymorpha L. (n M. M. polymorpha polymorpha L. =(n3). = 3). Reducing powers of the extracts from the archegoniophore and gametophyte were tested, and Reducing powers of the extracts from the archegoniophore and gametophyte were tested, and the results are shown 5. Within of 20–160 μL of gametophyte extract, the extracts from and the Reducing powersinofFigure the extracts fromthe therange archegoniophore and were tested, the results are shown in Figure 5. Within the range of 20–160 μL of extract, the extracts from the archegoniophore exhibited higher activity than that of gametophyte. the results are shown in Figure 5. Within the range of 20–160 µL of extract, the extracts from the archegoniophore exhibited higher activity than that of gametophyte. archegoniophore exhibited higher activity than that of gametophyte.

Figure 5. The reducing power of archegoniophore and gametophyte of M. polymorpha L. (n = 3).

Figure 5. The reducing power of archegoniophore and gametophyte of M. polymorpha L. (n = 3). Figure 5. The reducing power of archegoniophore and gametophyte of M. polymorpha L. (n = 3). The FRAP assay was used to measure the antioxidant and reductive capacity of the extracts The FRAP assay was used togametophyte measure the antioxidant and reductive capacity ofresults the extracts from from archegoniophore of M. polymorpha L. (Figure 6). The illustrated Thethe FRAP assay was usedand to measure the antioxidant and reductive capacity of the extracts from the archegoniophore and gametophyte of M.and polymorpha L. (Figureof6).M.The results illustrated that the the extracts from archegoniophore the gametophyte L. both that possessed thethat archegoniophore andthe gametophyte of M. polymorpha L. (Figure 6). Thepolymorpha results illustrated the extracts from the archegoniophore and the gametophyte of M. polymorpha L. both possessed antioxidant antioxidant and reductive activity. With the increasing volume, the antioxidant and reductive capacity extracts from the archegoniophore and the gametophyte of M. polymorpha L. both possessed antioxidant and reductive activity. With the increasing volume, the of antioxidant andfrom reductive capacity improved. range 5 to 25 µL, the activity the extracts the archegoniophore was andimproved. reductive Within activity.the With the of increasing volume, the antioxidant and reductive capacity improved. Within the range ofof5the to gametophyte. 25 μL, the activity of the extracts from the archegoniophore was stronger stronger than that Within the range of 5 to 25 μL, the activity of the extracts from the archegoniophore was stronger than that of the gametophyte. than that of the gametophyte.

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Figure 6.6.Antioxidant polymorpha Figure Antioxidantpower powerby byFRAP FRAPassay assayof ofarchegoniophore archegoniophoreand andgametophyte gametophyteof ofM. M.polymorpha 3). 6. Antioxidant power by FRAP assay of archegoniophore and gametophyte of M.polymorpha (n == 3). Figure

(n = 3).

In theextracts extractsfrom fromthe thearchegoniophore archegoniophoreshowed showed more efficient scavenging activity In summary, summary, the more efficient scavenging activity on 2+ on DPPH radical, ABTS radical and anionthose than of the gametophyte. the Fe2+ In radical, summary, the extracts from thesuperoxide archegoniophore showed efficient scavenging activity DPPH ABTS radical and superoxide anion than ofthose themore gametophyte. In the Fe Inreducing reducing power assay, minor differences between the archegoniophore gametophyte were found, on DPPH radical, ABTS radical and superoxide anion than those of and the gametophyte. In the Fe2+ power assay, minor differences between the archegoniophore and gametophyte were found, which which suggested that the levels of archegoniophore and gametophyte constituents capable of reducing reducing assay, minor differences between archegoniophore and gametophyte were found, suggestedpower that the levels of archegoniophore and the gametophyte constituents capable of reducing Fe2+ 2+ Fe almost same. which suggested that the levels of archegoniophore and gametophyte constituents capable of reducing werewere almost same. 2+ The extracts with concentrations of total flavonoids usually showed stronger DPPH radical Fe were The almost extractssame. withhigher higher concentrations of total flavonoids usually showed stronger DPPH scavenging activity [21]. Here, theHere, extracts theflavonoids archegoniophore showed stronger higher total flavonoid Thescavenging extracts with higher concentrations of total showed DPPH radical radical activity [21]. thefrom extracts from the usually archegoniophore showed higher total content and stronger free radical scavenging activity. Although the archegoniophore results support scavenging activity [21]. Here, the extracts from the archegoniophore showed higher total flavonoid flavonoid content and stronger free radical scavenging activity. Although the archegoniophore results the above viewpoint, the radical total flavonoids concentrations and DPPH radical scavenging activity IC50 content and stronger free scavenging activity. concentrations Although the archegoniophore results support support the above viewpoint, the total flavonoids and DPPH radical scavenging values from this workthe were compared with those reported forDPPH other fern species, thefern orders fromIC left the above total flavonoids concentrations radical activity 50 activity ICviewpoint, this work were compared withand those reported forscavenging other species, the 50 values from to right were archegoniophore, Pyrrosia nummulariifolia, Athyrium pachyphyllum, Hicriopteris glauca, values from this work were compared with those reported for other fern species, the orders from left orders from left to right were archegoniophore, Pyrrosia nummulariifolia, Athyrium pachyphyllum, Adiantum capillus-veneris, Pyrrosia petiolosa, nummulariifolia, Araiostegia Selaginella tenera, Selaginella inaequalifolia, to right were archegoniophore, Pyrrosia Athyrium pachyphyllum, Hicriopteris glauca, Hicriopteris glauca, Adiantum capillus-veneris, Pyrrosiaimbricata, petiolosa, Araiostegia imbricata, Selaginella tenera, Dryopteris erythrosora, Dryoathyrium boryanum, Selaginella involvens, Selaginella intermedia (Figure 7) Adiantum capillus-veneris, Pyrrosia petiolosa, Araiostegia imbricata, Selaginella tenera, Selaginella inaequalifolia, Selaginella inaequalifolia, Dryopteris erythrosora, Dryoathyrium boryanum, Selaginella involvens, [22–24]. The higher flavonoids content did not always show a positive correlation with the Dryopteris erythrosora,(Figure Dryoathyrium boryanum, Selaginella involvens, intermedia 7) Selaginella intermedia 7) [22–24]. The higher flavonoids contentSelaginella did not always show(Figure a positive antioxidant activity. [22–24]. Thewith higher flavonoidsactivity. content did not always show a positive correlation with the correlation the antioxidant antioxidant activity.

Figure 7. Figure 7. The The comparison comparison of of total totalflavonoids flavonoidscontent content(TFC) (TFC)and andIC IC5050.. Figure 7. The comparison of total flavonoids content (TFC) and IC50. AChE Inhibition Inhibition Activity 2.3. AChE

2.3. AChE Inhibition Activity AChE inhibitors are chemicals chemicals that that inhibit inhibit AChE AChE for— for—Alzheimer’s disease (AD) AChE Alzheimer’s disease (AD) [25]. [25]. AChE AChE inhibitors are inhibitors were used first to treat glaucoma, but nowadays AChE has proven to be the most viable inhibitors used first to treat glaucoma, butAChE nowadays has proven to be(AD) the most viable Alzheimer’s disease [25]. AChE AChEwere inhibitors are chemicals that inhibit for—AChE therapeutic target forfirst symptomatic improvement in AD AD [26]. [26].AChE As shown shown in Figure Figure 8, the extracts from therapeutic target for symptomatic improvement in As in from inhibitors were used to treat glaucoma, but nowadays has proven to 8, bethe theextracts most viable therapeutic target for symptomatic improvement in AD [26]. As shown in Figure 8, the extracts from

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exhibited a dose-dependent inhibition against AChE (IC50 = 0.1256 However, the gametophyte extracts hardly inhibited the AChEagainst activity. the archegoniophore exhibited a dose-dependent inhibition AChE (IC50 = 0.1256 mg/mL). the archegoniophore exhibited aa dose-dependent dose-dependent inhibition inhibition against against AChE AChE (IC (IC5050 == 0.1256 0.1256 mg/mL). mg/mL). However, the gametophyte extracts hardly inhibited the AChE activity. However, the gametophyte gametophyte extracts extracts hardly hardly inhibited inhibited the the AChE AChE activity. activity.

Figure 8. AChE inhibitory activity of archegoniophore of M. polymorpha L. (n = 3). Figure 8. AChE inhibitory activity of archegoniophore of M. polymorpha L. (n = 3). Figure 8. AChE AChE inhibitory inhibitory activity of archegoniophore archegoniophore ofand M.polymorpha polymorphaL. L.(n (n= =3). 3).polymorpha L. Figure 8. activity of M. 2.4. Identification of Flavonoids in Extracts from the Archegoniophoreof Gametophyte of M. 2.4. Identification of Flavonoids in Extracts from the Archegoniophore and The Gametophyte M. polymorpha L. LC-DAD-ESI/MS data were used to identify the flavonoids. retentionof (tR), UVλmax 2.4. Identification of Flavonoids Flavonoids in Extracts Extracts from the Archegoniophore Archegoniophore and Gametophyte Gametophyte oftime M. polymorpha polymorpha L. 2.4. Identification of in from the and of M. L. value, the molecular ions and structures of the flavonoids are listed in Table 1. TIC chromatograms LC-DAD-ESI/MS data were used to identify the flavonoids. The retention time (tR), UVλmax LC-DAD-ESI/MS data were used to identify identify the the flavonoids. flavonoids. The retention retention time(t(tR),),UV UVλmax LC-DAD-ESI/MS data used to time and DAD (254 nm) chromatograms of the from the and archegoniophore of λmax R value, the molecular ions andwere structures of extracts the flavonoids aregametophyte listedThe in Table 1. TIC chromatograms value, the molecular ions and structures of the flavonoids are listed in Table 1. TIC chromatograms value, the (254 molecular ions and structures of extracts the flavonoids aregametophyte listed in Table 1. TIC chromatograms M. polymorpha L.nm) are chromatograms shown in Figures respectively. and DAD of9–12, the from the and archegoniophore of and DAD DAD (254 (254 nm) nm) chromatograms chromatograms of of the the extracts extracts from from the the gametophyte gametophyte and and archegoniophore archegoniophore of of and M. polymorpha L. are shown in Figures 9–12, respectively. M. polymorpha L. are shown in Figures 9–12, respectively. M. polymorpha L. are shown in Figures 9–12 respectively.

Figure 9. ESI-MS chromatograms of flavonoid extract of M. polymorpha L. gametophyte. Figure 9. ESI-MS chromatograms of flavonoid extract of M. polymorpha L. gametophyte. Figure 9. ESI-MS chromatograms of flavonoid extract of M. polymorpha L. gametophyte. Figure 9. ESI-MS chromatograms of flavonoid extract of M. polymorpha L. gametophyte.

Figure 10. DAD (254 nm) chromatograms of flavonoid extract from M. polymorpha L. gametophyte. Figure 10. DAD (254 nm) chromatograms of flavonoid extract from M. polymorpha L. gametophyte.

Figure 10. DAD (254 nm) chromatograms of flavonoid extract from M. polymorpha L. gametophyte. Figure 10. DAD (254 nm) chromatograms of flavonoid extract from M. polymorpha L. gametophyte.

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Figure 11. ESI-MS chromatograms of flavonoid extract of M. polymorpha L. archegoniophore. Figure 11. ESI-MS chromatograms of flavonoid extract of M. polymorpha L. archegoniophore. Figure 11. ESI-MS chromatograms of flavonoid extract of M. polymorpha L. archegoniophore.

Figure 12. DAD (254 nm) chromatograms of flavonoid extract from M. polymorpha L. archegoniophore. Figure 12. DAD (254 nm) chromatograms of flavonoid extract from M. polymorpha L. archegoniophore.

Figure 12. DAD (254 nm) chromatograms of flavonoid extract from M. polymorpha L. archegoniophore.

From the reported data, it was From the comparison comparison with with chromatograms, chromatograms, mass mass spectral spectral and and UV UVλmax λmax reported data, it was tentatively identified identified that that there there were were 10 in archegoniophore and gametophyte extracts tentatively 10 flavonoids flavonoids mass in the the spectral archegoniophore andreported gametophyte From the comparison with chromatograms, and UVλmax data,extracts it was (Table 1). (Table 1). identified that there were 10 flavonoids in the archegoniophore and gametophyte extracts tentatively With UV UVλmax 292, and 344 nm, and molecular ions at m/z H]+ , 593.2 [M−, ´ H]´1, λmaxatat With 292, and 344 nm, and molecular ions at m/z 595.1595.1 [M +[M H]++, 593.2 [M − H] peak (Table 1). peak 1 was tentatively identified as kaempferol-3-O-rutinoside. PeakUV 2 with at UV at 268,−336 300 nm, and λmax was tentatively as 344 kaempferol-3-O-rutinoside. with 268, 300 With UVλmaxidentified at 292, and nm, and molecular+ions Peak at m/z2 595.1 [M +λmax H]+, 593.2 [M −and H] , peak 1 ´ , was tentatively identified as 336 nm, and molecular ions at m/z 609.2 [M + H] , 607.1 [M ´ H] + − , 607.1 [M − H] , was tentatively identified as chrysoeriol and molecular ions at m/z 609.2 [M + H] was tentatively identified as kaempferol-3-O-rutinoside. Peak 2 with UVλmax at 268, 300 and 336 nm, chrysoeriol 7-O-neohesperidoside [27]. For peak 3, the positive ESI-MS gave aatmolecular 7-O-neohesperidoside peak ESI-MS gave spectrum a molecular m/z 433.1 607.1 [M − H]−,spectrum was tentatively identifiedion as chrysoeriol and molecular ions at[27]. m/z+ For 609.2 [M 3,+ the H]+,positive ´ , and the ion at m/z 433.1 [M + H] , whereas the negative ESI-MS spectrum at m/z 431 [M ´ H] + − [M + H] , whereas the [27]. negative ESI-MS spectrum m/z 431 [M − H]gave , anda the UV spectrum showed 7-O-neohesperidoside For peak 3, the positiveat ESI-MS spectrum molecular ion at m/z 433.1 UV spectrum showedabsorptions characteristic flavone absorptions atso268, 288 tentatively and 340 nm, so it wasas tentatively + − characteristic flavone at 268, 288 and 340 nm, it was identified apigenin [M + H] , whereas the negative ESI-MS spectrum at m/z 431 [M − H] , and the UV spectrum showed identified as apigenin 7-O-glucoside Peak 4 nm, hadlike UVλmax at 258 and 330 nm, like baicalein 7-O-glucoside [28]. Peak 4 had UVλmax at[28]. 258 6,7-di-O-β-gluco-pyranuronoside characteristic flavone absorptions at 268, 288and and330 340 nm, sobaicalein it was tentatively identified as apigenin ´ 6,7-di-O-β-gluco-pyranuronoside and molecular ions at m/z 623.1 [M + H]+λmax , 621.1 [Mand ´ H] + − and molecular[28]. ionsPeak at m/z 623.1 H]258 , 621.1 [M −nm, H]like [29]. Peak 5 6,7-di-O-β-gluco-pyranuronoside had UV at 254 348 [29]. nm, 7-O-glucoside 4 had UV[M λmax+at and 330 baicalein Peak 5 was had similar UVλmaxtoatkaempferol, 254 and 348and nm,molecular which was similar to287 kaempferol, and[M molecular ions at−m/z +, 285 −, 571 + − which ions at m/z [M + H] − H] [2M H]− and molecular ions at m/z 623.1 [M + H] , 621.1 [M − H] [29]. Peak 5 had UVλmax at 254 and 348 nm, + , 285 [M ´ H]´ , 571 [2M ´ H]´ [30]. For peak 6, the positive ESI-MS spectrum gave a 287 [M + H] + +, 285 −, 571 [30]. For peak 6, thetopositive ESI-MS spectrum gave a molecular m/z 447.1 , whereas the− which was similar kaempferol, and molecular ions at m/z 287 ion [M at + H] [M[M − +H]H] [2M − H] molecularESI-MS ion at m/z 447.1 [M peak + H]+ ,at whereas the negative ESI-MS peak at m/zwhich 445 [Mshowed ´ H]´ , −, and spectrum + negative spectrum m/z 445 [M − H] the UV spectrum [30]. For peak 6, the positive ESI-MS spectrum gave a molecular ion at m/z 447.1 [M + H] , whereas the and the UV spectrum which showed characteristic absorptions atto268 336 nm,identified allowed characteristic flavone absorptions and445 336 [M nm,flavone thisthe product beand tentatively negative ESI-MS spectrum peakatat268m/z −allowed H]−, and UV spectrum which showed this product to be tentatively identified as apigenin 7-O-glucuronide [31]. The molecular ions of + peak as apigenin 7-O-glucuronide [31].atThe molecular ions of peak were attom/z 271 [M + H] in the7 characteristic flavone absorptions 268 and 336 nm, allowed this7product be tentatively identified + in the positive ESI-MS spectrum and 269 [M ´ H]´ in the negative ESI-MS were at m/z 271 [M + H] − positive ESI-MS spectrum and[31]. 269 The [M −molecular H] in theions negative ESI-MS spectrum. This, as apigenin 7-O-glucuronide of peak 7 were at m/z 271 [Mtogether + H]+ inwith the spectrum. This, together with the UV at 268 and 338 nm tentatively identified it as apigenin [31]. λmax − the UV λmax at 268 and 338 nm tentatively identified it as apigenin [31]. For peak 8, the UV λmax atwith 266 positive ESI-MS spectrum and 269 [M − H] in the negative ESI-MS spectrum.+This, together ´ For UV peak 8, the UVmolecular at 266 and 340 nm,301.1 and molecular at m/z 301.1 [M + H] 8, , and 299 ´ H] −, tentatively λmax338 and 340λmax nm, ions at m/z [M + H] , ions and 299 [M[31]. − H] as, the at and 268 and nm tentatively identified it +as apigenin For peak theidentified UV[M λmax atit 266 tentatively identified it9asand chrysoeriol [31]. Peak had times (tR ), but +, and10 chrysoeriol [31]. peakat10m/z had similar times (tsimilar R), but−,retention the UVλmax of peak 9 were and 340 nm, andPeak molecular ions 301.1 [M9 and +retention H]peak 299 [M − H] tentatively identified itthe as + , 461 [M ´ H]´ , and the UV UV of peak 9 were at 254 and 348 nm with m/z at 463 [M + H] of + − λmax λmax at 254 and 348 nm with9 m/z 463 10 [Mhad + H]similar , 461 [M − H] , and the(tUV of peak 10 were at 266 and chrysoeriol [31]. Peak andat peak retention times R), λmax but the UV λmax of peak 9 were + , 637.1 [M ´ H]´ , so they were tentatively peak 10 were at 266 and 366 nm with m/z at 639.1 [M + H] + − 366254 nm with 639.1 H][M , 637.1 H] −, H] so −they tentatively identified luteolin at and 348m/z nmat with m/z[M at +463 + H]+,[M 461− [M , andwere the UV λmax of peak 10 were as at 266 and 1 -O-β-d-glucuronide [30], and tricin-7-O-rutinoside [32], respectively. identified as luteolin 3 + − 3′-O-βD -glucuronide [30], and tricin-7-O-rutinoside [32], respectively. 366 nm with m/z at 639.1 [M + H] , 637.1 [M − H] , so they were tentatively identified as luteolin Apigenin-7-O-β-D-glucuronide andapigenin apigeninhad had been previously reported reported in in the the gametophytes Apigenin-7-O-β-glucuronide and been previously gametophytes 3′-O-βD-glucuronideD[30], and tricin-7-O-rutinoside [32], respectively. of M. polymorpha L. [33], and now apigenin 7-O-glucuronide and apigenin were also found in in the of M.Apigenin-7-O-βpolymorpha L. [33], and now apigenin 7-O-glucuronide and apigenin were also found the D-glucuronide and apigenin had been previously reported in the gametophytes archegoniophore. It was thus clear that there are no consistent differences in the flavonoid patterns of archegoniophore. It was thus clear that there are no consistent differences in the flavonoid patterns of M. polymorpha L. [33], and now apigenin 7-O-glucuronide and apigenin were also found in the gametophytes and archegoniophore of M. polymorpha L. of gametophytes and archegoniophore M. polymorpha L. archegoniophore. It was thus clear thatof there are no consistent differences in the flavonoid patterns

of gametophytes and archegoniophore of M. polymorpha L.

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Table 1. Flavonoids identified tentatively in the archegoniophore and the gametophyte of M. polymorpha L. Peak No.

TR (min)

MW

m/z

UVλmax/nm

Identification

Archegoniophore

Gametophyte

H]+

1

28.25

594

595.1 [M + 593.2 [M ´ H]´

292 344

Kaempferol-3-O-rutinoside

none

exist

2

49.78

608

609.2 [M + H]+ 607.1 [M ´ H]´

268 300 336

Chrysoeriol 7-O-neohesperidoside

none

exist

3

52.19

432

433.1 [M + H]+ 431 [M ´ H]´

268 288 340

Apigenin-7-O-β-D-glucoside

none

exist

4

55.82

622

623.1 [M + H]+ 621.1 [M ´ H]´

258 330

none

exist

254 348

Kaempferol

exist

none

Baicalein 6,7-di-O-β-D-glucopyranuronoside

5

55.87

286

287 [M + H]+ 285 [M ´ H]´ 571 [2M ´ H]

6

58.19

446

447.1 [M + H]+ 445 [M ´ H]´

268 336

Apigenin-7-O-β-D-glucuronide

none

exist

7

63.03

270

271 [M + H]+ 269 [M ´ H]´

268 338

Apigenin

none

exist

8

64.5

300

301.1 [M + H]+ 299 [M ´ H]´

266 340

Chrysoeriol

exist

none

9

64.98

462

463 [M + H]+ 461 [M ´ H]´

254 348

Luteolin 31 -O-β-D-glucuronide

exist

none

10

68.50

638

639.1 [M + H]+ 637.1 [M ´ H]´

210 266 336

Tricin-7-O-rutinoside

exist

none

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3. Materials Materialsand andMethods Methods 3.1. Plant Materials Materials 3.1. Plant Gametophytes and archegoniophores of M. M. polymorpha polymorpha L. L. (Figure (Figure 13) 13) were Gametophytes and archegoniophores of were collected collected from from Mountain Tianmu National Natural Reserve, Zhejiang Province, China. The plant was identified by Mountain Tianmu National Natural Reserve, Zhejiang Province, China. The plant was identified by Prof. Yuhuan Wu. Voucher specimens (Gametophyte-2013070447; Archegoniephore-2013042116) are Prof. Yuhuan Wu. Voucher specimens (Gametophyte-2013070447; Archegoniephore-2013042116) are kept kept in in the the HTC HTC of of the the College College of of Life Life & & Environmental Environmental Science, Science, Hangzhou Hangzhou Normal Normal University. University.

Figure 13. Gametophyte (A) and archegoniophore (B) of M. polymorpha L. Figure 13. Gametophyte (A) and archegoniophore (B) of M. polymorpha L.

3.2. Chemicals and Reagents 3.2. Chemicals and Reagents Rutin (purity > 99.0%), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzothiazolineRutin (purity > 99.0%), 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,21 -azinobis-(3-ethylbenzothiazoline6-sulfonic acid) (ABTS), 2,4,6-tri-2-pyridyl-s-triazine (TPTZ), acetylthiocholine (ATCh), and AChE 6-sulfonic acid) (ABTS), 2,4,6-tri-2-pyridyl-s-triazine (TPTZ), acetylthiocholine (ATCh), and AChE were purchased from Sigma Co. (Shanghai, China). Nitrotetrazolium blue chloride (NBT), phenazine were purchased from Sigma Co. (Shanghai, China). Nitrotetrazolium blue chloride (NBT), phenazine methosulfate (PMS), nicotinamide adenine dinucleotide (NADH) and 5, 5′-dithiobis-(2-nitrobenzoic methosulfate (PMS), nicotinamide adenine dinucleotide (NADH) and 5, 51 -dithiobis-(2-nitrobenzoic acid) (DTNB) were purchased from Aladdin Reagent Int. (Shanghai, China). Other reagents were of acid) (DTNB) were purchased from Aladdin Reagent Int. (Shanghai, China). Other reagents were of analytical grade, except for acetonitrile, which was HPLC grade and purchased from Thermo Fisher analytical grade, except for acetonitrile, which was HPLC grade and purchased from Thermo Fisher Scientific (Shanghai, China). Scientific (Shanghai, China). 3.3. Preparation of Plant Plant Extracts Extracts 3.3. Preparation of Fresh plant plant materials, materials, after after being being cleaned cleaned and and dried dried under under shady shady conditions, conditions, were were dried dried at at 75 75 ˝°C Fresh C for 48 h, and then powdered and filtered through a 40-mesh screen. The dried samples (1.00 g) were for 48 h, and then powdered and filtered through a 40-mesh screen. The dried samples (1.00 g) were separately extracted extracted with with 60% 60% ethanol ethanol (25 (25 mL) mL) for for 22 h h at at 50 50 ˝°C. Then, ultrasound-assisted ultrasound-assisted extraction extraction separately C. Then, was performed performed for for 20 20 min, min, the the extraction extraction processes processes were were repeated repeated twice. twice. Finally, the mixture mixture was was was Finally, the filtered via a vacuum suction filter pump, and the extract solutions, which would be used to measure filtered via a vacuum suction filter pump, and the extract solutions, which would be used to measure the total total flavonoids flavonoids content, content, were were collected. collected. Eight extract solution solution was was extracted extracted twice twice with with the Eight mL mL of of extract petroleum ether ether for for removing removing the the chlorophyll, chlorophyll, and and the the residue residue solution solution was was concentrated concentrated to to dryness dryness petroleum by evaporation on a rotary evaporator, and then dissolved with ethanol. Before testing the solutions by evaporation on a rotary evaporator, and then dissolved with ethanol. Before testing the solutions were filtered filteredthrough through a 0.45 membrane (Millipore, Billerica, MA, Samples USA). Samples foranalysis HPLC were a 0.45 µmμm membrane (Millipore, Billerica, MA, USA). for HPLC analysis then prepared. were thenwere prepared. 3.4. Determination Flavonoids Content Content 3.4. Determination of of Total Total Flavonoids The method flavonoids content waswas a colorimetric assay. To rutin samples, with The methodof ofdetermination determinationofof flavonoids content a colorimetric assay. To rutin samples, the same volume and different concentration were successively added 5% NaNO 2 (0.3 mL, 6 min), 5% with the same volume and different concentration were successively added 5% NaNO2 (0.3 mL, 6 min), Al(NO 3)3 (0.3 mL, 6 min), 4% NaOH (4.4 mL, 12 min). According to the optical density (OD) at 510 nm, 5% Al(NO 3 )3 (0.3 mL, 6 min), 4% NaOH (4.4 mL, 12 min). According to the optical density (OD) at calibration curves rutin drawn with the software Origin 7.5. This would givegive A, BA,and R2. 510 nm, calibration for curves forwas rutin was drawn with the software Origin 7.5. This would B and 2 . Total Total flavonoids content waswas determined thethe same way as as rutin. The formula used was as as follows: R flavonoids content determined same way rutin. The formula used was follows: Total flavonoids content (%) = [(OD1 + OD2 + OD3)/3 − A]/B × 10/2 × Total flavonoids content p%q “ rpOD1 ` OD2 ` OD3 q{3 ´ As{B ˆ 10{2 ˆ volume{1000 ˆ 100% volume/1000 × 100%

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3.5. Antioxidant Activity 3.5.1. DPPH Assay The DPPH free radical scavenging activity of the extracts was measured according to our previous report [21]. Briefly, a solution of DPPH (0.1 mM) in methanol was prepared and 1 mL added to different concentrations of extract sample (1.0 mL). The mixture was shaken vigorously and incubated for 30 min in the dark. The absorbance value was measured at 517 nm. In the control, methanol was substituted for sample. The inhibitory ratio (%) was calculated by the following equation: DPPH scavenging percentage p%q “ p1 ´ Asample517 {Acontrol517 q ˆ 100 All the determinations were performed in triplicate and found to be reproducible within the experimental error. 3.5.2. ABTS Assay The ABTS assay of the extracts was performed according to our previous report [21]. ABTS and potassium persulfate were dissolved in ultrapure water to a final concentration of 7 mM and 2.45 mM, respectively. The mixture was allowed to remain in the dark for 12 h before use. Then, 500 µL extract samples of different concentrations were added to appropriately diluted ABTS solutions; the absorbance at 734 nm was read after 6 min. In the control, methanol was substituted for sample. The inhibitory ratio (%) was calculated by the following equation: ABTS scavenging percentage p%q “ p1 ´ Asample734 {Acontrol734 q ˆ 100 All the determinations were performed in triplicate and found to be reproducible within the experimental error. 3.5.3. Reducing Power Assay The reducing power of the extracts was quantified by the method described by Xiao et al. [34]. Extract samples of various concentrations (1 mL) were mixed with 2.5 mL phosphate buffer (PH = 6.0) and 2.5 mL of potassium ferricyanide (1%, w/v) at 50 ˝ C for 20 min. 10% TCA was used to terminate the reaction. The mixture was centrifuged at 3000 rpm for 10 min, then 2.5 mL of supernatant, together with 2.5 mL of distilled water and 0.5 mL of 0.1% ferric chloride were mixed, and the absorbance was read at 700 nm. The mixture of 2.5 mL of supernatant and 3 mlL of distilled water was taken as blank. Every experiment was done in triplicate (n = 3) and found to be reproducible within the experimental error (RSD < 5.0%). 3.5.4. Superoxide Anion (O2´ ) Scavenging Activity The measure of superoxide anion (O2´ ) scavenging activity was carried out as described previously by Xiao et al. [34]. Superoxide anion (O2´ ) was generated from 3.0 mL of sodium phosphate buffer (100 mM, PH = 7.4), which contained 1.0 mL of extract of various concentrations, 1.0 mL of NBT (150 µM) and 1.0 mL of NADH (468 µM). With the addition of 1.0 mL of PMS (60 µM), the reaction started and the mixture was incubated at 25 ˝ C for 5 min. The absorbance at 560 nm was recorded. Capability to scavenging superoxide anion (O2´ ) was calculated using the formula: Superoxide anion scavenging activity p%q “ p1 ´ A1 {A0 q ˆ 100 where A0 is the absorbance of the control sample, and A1 is the absorbance of samples. Each sample was tested three times (n = 3). The absorbance was found to be reproducible within the experimental error.

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3.5.5. FRAP Assay The FRAP assay of the extracts was carried out according to our previous report [35]. The FRAP reagent contained 10 mM TPTZ in 40 mM HCl solution and 20 mM FeCl3 in 0.25 L acetate buffer (pH 3.6). It was freshly prepared and warmed to 37 ˝ C. Briefly, 1.5 mL of FRAP reagent was added to 50 µL of extract of variable concentration. The absorbance at 593 nm was read after 4 min. The FRAP assay was expressed by using FeSO4 calibration curves. All the determinations were performed in triplicate and found to be reproducible within the experimental error. 3.6. AChE Inhibitory Activity AChE inhibition activity of the flavonoid extracts was measured by the method adopted by Xiao et al. [36]. AChE was added to a mixture which contained 140 µL of sodium phosphate buffer (pH = 8.0), 20 µL of DTNB and 20 µL of tested extract and then incubated at 25 ˝ C for 15 min. When this time is up, acetylthiochline (10 µL) was added to the mixture to activate the reaction. Finally, AChE inhibitory activity was evaluated by the percentage of AChE activity rate reduction from 100%. Each sample was tested three times (n = 3, RSD < 5.0%). 3.7. LC/DAD/ESI–MS Analysis The LC-DAD-ESI/MS instrument consisted of an Agilent 1100 HPLC equipped with a diode array detector and an Agilent mass spectrometer (LC/MSD SL) (Agilent Technologies, Santa Clara, CA, USA). A Symmetry column (C18, 250 ˆ 4.6 mm, 5 µm) (Waters, Milford, MA, USA) was used at a flow rate of 1.0 mL/min. The column oven temperature was set at 25 ˝ C. The mobile phase consisted of 0.2% formic acid (A) and acetonitrile (B) with the following gradient program: 0–10 min, 5% B; 10–15 min, 5%–15% B; 15–25 min, 15% B; 25–35 min, 15%–25% B; 35–40 min, 25% B; 40–50 min, 25%–35% B; 50–60 min, 35% B; 60–70 min, 35%–50% B; 70–80 min, 50%–5% B. The flow-rate was kept at 0.30 mL/min. The DAD was set at 254 nm to provide real time chromatograms and the UV/Vis spectra from 190 to 650 nm were recorded for plant component identification. Mass spectra were simultaneously acquired using electro-spray ionization in the positive (PI) and negative ionization (NI) modes, at low (70 V) and high fragmentation voltages (250 V) for both ionization modes. For brevity, the high and low fragmentation voltages of the PI and NI modes will be identified as PI250, PI70, NI250, and NI70 in the text. The mass spectra were recorded for the range of m/z 100–1000, a drying gas temperature of 350 ˝ C, a nebulizer pressure of 50 psi, and capillary voltages of 4000 V for PI and 3500 V for NI, were used. The LC system was directly connected with MSD without stream splitting [37]. Acknowledgments: The work was sponsored by Shanghai Gaofeng & Gaoyuan Project for University Academic Program Development, and supported by the National Natural Science Foundation of China (No. 41571049, 41461010). Author Contributions: Conceived and designed the experiments: Quanxi Wang and Jianbo Xiao. Performed the experiments in key laboratory of cryptogam in Shanghai Normal University : Xin Wang. Analyzed the data: Xin Wang, Quanxi Wang, Jianbo Xiao. Revising manuscript: Quanxi Wang, Jianbo Xiao, Yuhuan Wu, Jianguo Cao. Wrote the paper: Xin Wang. Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Not available. © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons by Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).