Chemical Constituents From Phalaenopsis Hybrids and Their Bioactivities

Abstract

One new flavone glycoside and 11 known compounds were characterized from the methanolic extracts of Dtps. Tinny Ribbon × Dtps. Plum Rose (Phalaenopsis hybrids) by nuclear magnetic resonance and mass spectrometric analyses. In addition, the major pigment constituents 1 to 3 were examined for their antioxidant and antityrosinase activities. The experimental results indicated that the Phalaenopsis flower extracts were potential for developing new cosmetic products.

Keywords

flavone glycoside anthocyanin orchidaceae antioxidant antityrosinase

Phalaenopsis hybrids, known as moth orchids, are one of the most important orchids with different flower colors, such as white, yellow, red, red-purple, and purple. This orchid is not only suitable for domestic production, but also an important import plant material for the United States, Japan, and many European countries.^1,2 In Taiwan, one of the famous Phalaenopsis Kingdoms, the Phalaenopsis hybrids are most significant ornamental flowers for export worldwide, and has been emphasized by government to be developed as a dedicate agriculture industry. The chemistry of secondary metabolites of the Orchidaceae plants has been intensively investigated and various constituents, such as bibenzyl,^3-6 stilbenoid,^7-10 phenanthrene,^6,11,12 and phenanthropyran derivatives,^13,14 have been reported. In addition, flavonoids and anthocyanins, which are the main pigments in Phalaenopsis hybrid flowers, have also been studied and their structures were elucidated well in the literature.^15,16 Due to the structural diversity, and biological and ecological significance of flavonoids and anthocyanins, researchers continue to study their functions in different plant materials. They affect plant interactions with microsymbionts,¹⁷ insect predators and pollinators,^18,19 and also function in pigmentation and act as protectants against UV irradiation.^20-22 In fact, almost all higher plants produce flavonoids; however, some of them are quite unique, in which many specific compounds are accumulated during plant growth and development. Therefore, plants with different flavonoid accumulation patterns could be used to study the regulation of the flavanone metabolic pathway and to elucidate potential biosynthetic mechanism. Anthocyanins are also important secondary metabolites that constitute a major subgroup of flavonoids. They play one of the major roles as pigments of flowers and the biosynthetic pathway of anthocyanins is one of the most extensively studied pathways of plant secondary products. The common biosynthesis of anthocyanidins starts with general phenylpropanopid metabolism and leads to colorless flavonoids and colored anthocyanidins, successively.²³ Herein this study wishes to report the characterization of the chemical constituents in the flowers of Dtps. Tinny Ribbon × Dtps. Plum Rose (red Phalaenopsis hybrids) as a reference for further study on the biosynthetic pathway for these pigments.

Fresh flowers of Dtps. Tinny Ribbon × Dtps. Plum Rose were extracted with methanolic aqueous solution containing 0.1% trifluoroacetic acid (TFA) at room temperature and concentrated.²⁴ The successive purification by a combination of conventional chromatographic techniques characterized one new compound apigenin 6-C-β-d-ribopyranosyl-7-O-β-d-glucopyranoside (1) (Figure 1). In addition, 11 known compounds including saponarin (2)²⁵ (3',7-di-O-sinapyl-glucosyl)-3-glucosyl cyanidin (3)¹⁵ mixtures of β-sitosteryl acetate (4) and stigmasteryl acetate (5,²⁶ mixtures of β-sitosterol (6) and stigmasterol (7)²⁷ cyclotirucanenol (8)²⁸ vanillic acid (9)²⁷ p-methoxybenzaldehyde (10)²⁹ canthin-6-one (11)²⁷ and methyl 4-β-d-glucopyranosyloxy-trans-cinnamate (12)³⁰ were also identified by spectroscopic data in comparison with the published values reported in the literature.

Figure 1

Structures of compounds 1-3.

The pseudomolecular formula of compound 1 was assigned as C₂₆H₂₉O₁₄ by high-resolution fast-atom bombardment mass spectrometry (HR-FAB-MS) analysis, which showed an ion adduct peak at m/z 565.1555. In the negative mode electrospray ionization mass spectrometry (ESI-MS)/MS analysis, the base peak at m/z 563 and the fragment ion at m/z 401 suggested the glucosylation of OH-7. The [M-H-162] ion (m/z 401) gave the evidence for an O-linked hexose (glucose) and the fragment [M-H-90-162] ion (m/z 311) indicated the presence of Y₁ fragment (Figure 2) as a result of the elimination of one O-hexose and one C-pentose units.³¹ The infrared (IR) absorption bands at 3370 and 1609 cm⁻¹ displayed the presence of a hydroxyl group and a hydrogen-bonded conjugated carbonyl group. The UV absorption maxima at 227, 271, and 334 nm were characteristic of a flavone skeleton.³² The bathochromic shift in the UV absorption maximum observed after addition of AlCl₃ was in agreement with the presence of the hydrogen-bonded hydroxyl group of flavone at C-5.³³ In the ¹H NMR spectrum of 1, a set of A₂B₂ signals at δ 7.86 (2H, d, J = 8.9 Hz) and 6.90 (2H, d, J = 8.9 Hz) were attributed to H-2', -6' and H-3', -5' of the para-substituted B-ring. A singlet at δ 6.65 was assumed to be H-3 as it showed correlation with carbons at δ 107.0 (C-10), 122.9 (C-1'), 166.7 (C-2), and 184.2 (C-4) in heteronuclear multiple bond correlation (HMBC) spectrum (Table 1). Another singlet proton signal at δ 7.03 was proposed to be H-8 since it exhibited cross peaks with carbon signals at δ 107.0 (C-10), 111.8 (C-6), and 158.8 (C-9) in HMBC spectral analysis. Two anomeric proton signals at δ 4.75 (d, J = 9.9 Hz) and 4.90 (br d, J = 6.1 Hz) suggest the presence of two sugar units in compound 1. Two sets of ox methylene protons [H-6''' at δ 3.70 (dd, J = 12.0, 6.4 Hz), 3.99 (br d, J = 12.0 Hz) and H-5'' at δ 3.65 (d, J = 11.6 Hz), 3.92 (br d, J = 11.6 Hz)] were also observed in the ¹H NMR spectrum. These two sugar moieties were identified as d-ribose (δ 70.1, 71.0, 71.7, 75.2, and 76.4)^34,35 and d-glucose (δ 62.7, 71.6, 74.8, 76.9, 78.6, and 104.0),³⁶ respectively, with the assistance of ¹³C NMR, COSY, and HMBC spectral analysis (see Online Supplemental material). The ribose connected with the aglycone through C-C linkage was indicated with the carbon signal at δ 75.2 in its ¹³C NMR spectrum, which is the characteristic anomeric carbon signal of C-glycosides.³⁶ Furthermore, in the HMBC spectrum, long-range correlations from H-1" (δ 4.75) of ribose to C-5, C-6, and C-7 also confirmed the linkage of ribose to C-6. Another ³ J-HMBC correlation between H-1" and C-5" evidenced the six-membered ring structure of ribose. In addition, cross peaks between the proton signals H-8 and H-1''' of glucose in NOESY spectrum and ³ J-HMBC correlation from H-1''' to C-7 further supported the glucosylation on OH-7 (Figure 3). Consequently, the chemical structure of 1 was established as apigenin 6-C-β-d-ribopyranosyl-7-O-β-d-glucopyranoside as shown.

Figure 2

Chemical structure and mass fragmentation of 1.

Figure 3

Significant heteronuclear multiple bond correlation (—) and nuclear Overhauser effect spectroscopy (---) correlations of 1.

Table 1

NMR Spectroscopic Data of Compound 1.^a

No.	δ_H	δ_C	HMBC (H→C)	COSY
2		166.7 s
3	6.65 s	104.3 d	C-2, C-4, C-10, C-1'
4		184.2 s
5		161.4 s
6		111.8 s
7		164.8 s
8	7.03 br s	96.3 d	C-6, C-7, C-9, C-10
9		158.8 s
10		107.0 s
1'		122.9 s
2'	7.86 d (8.9)	129.7 d	C-2, C-3', C-4', C-6'	H-3'
3'	6.90 d (8.9)	117.1 d	C-1', C-4', C-5'	H-2'
4'		162.9 s
5'	6.90 d (8.9)	117.1 d	C-1', C-3', C-4'	H-6'
6'	7.86 d (8.9)	129.7 d	C-2, C-2', C-3', C-4'	H-5'
1''	4.75 d (9.9)	75.2 d	C-5, C-6, C-7, C-2'', C-3'', C-5''	H-2'', H-4''
2''	4.59 d (9.5)	70.1 d	C-6, C-3''	H-1'', H-3''
3''	3.56 m	76.4 d	C-2''	H-2'', H-4''
4''	3.92 br d (11.6)	71.0 d
5''	3.65 d (11.6)	71.7 t	C-4''	H-5''
	3.92 br d (11.6)
1'''	4.90 br d (6.1)	104.0 d	C-7, C-2'''
2'''	3.62 m	74.8 d	C-1'''	H-3'''
3'''	3.50 t (9.0)	76.9 d		H-2''', H-4'''
4'''	3.36 br t (9.3)	71.6 d	C-3''', C-5''',C-6'''	H-3''', H-5'''
5'''	3.58 m	78.6 d
6'''	3.70 dd (12.0, 6.4)	62.7 t	C-5'''	H-5''', H-6'''
	3.99 br d (12.0)			H-6'''

NMR, nuclear magnetic resonance; HMBC, heteronuclear multiple bond correlation; COSY, correlation spectroscopy.

^aNMR data were measured in CD₃OD at 500 MHz (¹H) and 125 MHz (¹³C), respectively.

The isolated compounds 1, 2, and 3 were screened for their antioxidant and antityrosinase bioactivities.³⁷ Compounds 1 and 3 exhibited moderate α,α-diphenyl-β-picrylhydrazyl free radical scavenging activity with the half-maximal inhibitory concentration (IC₅₀) values of 27.3 and 307.1 µM, respectively, compared to the reference compound vitamin E (IC₅₀ 12.5 µM). Compound 2 also displayed moderate antityrosinase activity with IC₅₀ value of 41.6 µM, compared to the reference compound kojic acid (IC₅₀ 30.0 µM). Moreover, the total yield of compounds 1 3 in fresh petals is about 0.58%. These experimental data supported the further development of Phalaenopsis flower extracts as ingredients of new cosmetic products.

Experimental

General

Melting points were determined using Yanagimoto MP-S3 micro melting point apparatus without correction. The UV spectra were obtained on a GBC Cintra 101 UV-Vis spectrophotometer. IR spectra were recorded with a Varian Scimitar FTS-2000 FT-IR spectrometer. Optical rotations were measured using a JASCO DIP-370 digital polarimeter. NMR spectra were recorded on Bruker AV-500 and Avance 300 spectrometers. Chemical shifts were reported in δ values (ppm) with reference to the respective residual solvent or deuterated solvent peaks. High-performance liquid chromatography was performed on a Shimadzu LC-10AT ^VP series pumping system equipped with a Shimadzu SPD-6AV UV-Vis spectrophotometric detector at 375 and 530 nm, a Luna C-18 packed column (4.6 × 250 mm, 5 µm) and a Rheodyne injector. MS/MS spectrometry was analyzed on a Perkin-Elmer SCIEX API mass spectrometer equipped with an electrospray ionization interface (ISV = 4700, orifice voltage of 80). The mass spectrometer was operated in the negative-ion mode. High-resolution mass spectra were determined with a JEOL JMS-700 spectrometer with FAB ionization. GR-grade organic solvent: methanol, chloroform, n-butanol, n-hexane, ethyl acetate, acetone, ammonia, and hydrochloric acid (ACS grade) were purchased from Mallinckrodt (St Louis, MO, USA). Deuterated solvent (CD₃OD) was purchased from Sigma-Aldrich (St Louis, MO, USA).

Plant Material

The flowers of Phalaenopsis hybrids, Dtps. Tinny Ribbon × Dtps. Plum Rose, were kindly provided by Ox Orchid Nursery, Tainan, Taiwan and were authenticated by Prof W. H. Chen (Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan). A voucher specimen (TSWu_ PCKuo_20060001) has been deposited in the herbarium of School of Pharmacy, National Cheng Kung University, Tainan, Taiwan.

Extraction and Isolation

Fresh flowers of Dtps. Tinny Ribbon × Dtps. Plum Rose (1.24 kg) were cut into small pieces and were extracted with 0.1% TFA methanolic solution (5 × 2 L) at room temperature for 8 hours and filtered. Evaporation of the solvent under reduced pressure provided dark brown crude extract (70 g). It was partitioned with ethyl acetate (EtOAc) to yield EtOAc (15 g) and H₂O (55 g) soluble residue, respectively. The EtOAc extract was loaded on a silica gel column, and elution was carried out with a stepwise gradient from 5 % to 50 % EtOAc in n-hexane, and finally washed by MeOH to afford five fractions. Fraction 1 was further separated by two successive silica gel open columns with mixtures of EtOAc and n-hexane (5:95) to give mixtures of β-sitosteryl acetate (4) and stigmasteryl acetate (5) (25.0 mg). Fraction 2 was further separated using repeated silica gel column chromatography to yield mixtures of β-sitosterol (6) and stigmasterol (7) (40.0 mg). Fraction 3 was rechromatographed by successive silica gel open columns and thin-layer chromatography (TLC) to afford cyclotirucanenol (8) (5.0 mg). Fraction 4 on column chromatography over silica gel with the mixing eluents of n-hexane and acetone (19:1) and further purification by repeated column chromatography and TLC gave vanillic acid (9) (5.0 mg) and p-methoxybenzaldehyde (10) (3.0 mg). Fraction 5 was further subjected to column chromatography over silica gel using a stepwise gradient of n-hexane/EtOAc (20:1 to 1:1) to afford canthin-6-one (11) (5.0 mg). The H₂O soluble residue was subjected to Sephadex LH-20 column chromatography eluting with increasing concentrations of MeOH in H₂O to give five fractions. Rechromatography of fraction 2 over Sephadex LH-20 eluting with a mixture of H₂O-MeOH and successive purification by preparative TLC on silica gel with mixture of CHCl₃-MeOH (9:1) saturated with water gave methyl 4-β-d-glucopyranosyloxy-trans-cinnamate (12) (15.0 mg). Purification of fraction 3 on Sephadex LH-20 column chromatography eluting with a mixture of H₂O-MeOH led to the isolation of saponarin (2) (5.0 g) and apigenin 6-C-β-d-ribopyranosyl-7-O-β-d-glucopyranoside (1) (2.0 g). Separation of fraction 4 by repeated column chromatography over Sephadex LH-20 eluting with a mixture of H₂O-MeOH yielded (3',7-di-O-sinapylglucosyl)-3-glucosyl cyanidin (3) (160 mg).

Apigenin 6-C-β-Ribopyranosyl-7-O-β-Glucopyranoside (1)

Colorless powder.

MP: 205-207°C.

[α]_D ²⁵: + 48 (c 0.2, MeOH).

UV (MeOH) λ _max (log ε): 334 (3.20), 271 (3.40), 227 (4.10, sh), 208 (4.30) nm.

IR (KBr) ν _max: 3370, 2917, 1649, 1609, 1482, 1453, 1350, 1185 cm^–1.

¹H and ¹³C NMR: Table 1.

ESI-MS m/z (relative intensity %): 563 [M−H]⁻ (100), 473 (7), 401 (31), 341 (9), 311 (16), 297 (12).

HR-FAB-MS m/z 565.1555 [M+H]⁺ (calcd for C₂₆H₂₉O₁₄, 565.1557).

Antioxidant and Antityrosinase Activities

See supporting information.

Footnotes

Acknowledgments

Authors are thankful to the Ox Orchid Nursery, Tainan, Taiwan for their kind support of the plant materials.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: financial support from the Council of Agriculture, Executive Yuan, Taiwan, and Ministry of Science and Technology (MOST), Taiwan.

Supplemental Material

References

Globaltrade atlas database . 2017. https://www.gtis.com/gta/

Tsai

. The importance of local culture, innovation, path dependence, and locality in the Taiwanese Phalaenopsis industry. STUST J Humanit Soc Sci. 2018;18:109-144.

Majumder

PL.

Basak

. Two bibenzyl derivatives from the orchid Cirrhopetalum andersonii . Phytochemistry. 1991;30(1):321-324.doi:10.1016/0031-9422(91)84146-J

Majumder

PL.

Pal (née Ray)

Pal

. Cumulatin and tristin, two bibenzyl derivatives from the orchids Dendrobium cumulatum and Bulbophyllum triste . Phytochemistry. 1993;32(6):1561-1565.doi:10.1016/0031-9422(93)85180-Y

Majumder

PL.

Roychowdhury (Née Basak)

Chakraborty

. Bibenzyl derivatives from the orchid Bulbophyllum protractum . Phytochemistry. 1997;44(1):167-172.doi:10.1016/S0031-9422(96)00402-5

Harrison

LJ.

Powell

AD.

Leong

YW.

Kang

. Phenanthrenes, dihydrophenanthrenes and bibenzyls from the orchid Bulbophyllum vaginatum . Phytochemistry. 1997;44(1):157-165.doi:10.1016/S0031-9422(96)00387-1

Majumder

PL.

Basak

. Two stilbenoids from the orchid Cirrhopetalum andersonii . Phytochemistry. 1991;30(10):3429-3432.doi:10.1016/0031-9422(91)83222-7

Majumder

PL.

Ghosal

. Two stilbenoids from the orchid Arundina bambusifolia . Phytochemistry. 1993;32(2):439-444.doi:10.1016/S0031-9422(00)95011-8

Majumder

PL.

Ghosal (Née Chatterjee)

. Two stilbenoids from the orchid Arundina bambusifolia . Phytochemistry. 1993;35(1):205-208.doi:10.1016/S0031-9422(00)90535-1

10.

Majumder

PL.

Lahiri

Mukhoti

. Four stilbenoids from the orchid Agrostophyllum khasiyanum . Phytochemistry. 1996;42(4):1157-1161.doi:10.1016/0031-9422(96)00022-2

11.

Majumder

PL.

Sen

. Pendulin, a polyoxygenated phenanthrene derivative from the orchid Cymbidium pendulum . Phytochemistry. 1991;30(7):2432-2434.doi:10.1016/0031-9422(91)83675-B

12.

Majumder

PL.

Maiti

. Flaccidinin and oxoflaccidin, two phenanthrene derivatives of the orchid Coelogyne flaccida . Phytochemistry. 1989;28(3):887-890.doi:10.1016/0031-9422(89)80137-2

13.

Majumder

PL.

Sabzabadi

. Agrostophyllin, a naturally occurring phenanthropyran derivative from Agrostophyllum khasiyanum . Phytochemistry. 1988;27(6):1899-1901.doi:10.1016/0031-9422(88)80477-1

14.

Manako

Wake

Tanaka

Shimomura

Ishimaru

. Phenanthropyran derivatives from Phalaenopsis equestris . Phytochemistry. 2001;58(4):603-605.doi:10.1016/S0031-9422(01)00249-7

15.

Griesbach

. Flavonoid copigments and anthocyanin of Phalaenopsis schilleriana . Lindleyana. 1990;5:231-234.

16.

Tatsuzawa

Saito

Seki

Hara

Yokoi

Honda

. Acylated cyanidin glycosides in the red-purple flowers of Phalaenopsis . Phytochemistry. 1997;45(1):173-177.doi:10.1016/S0031-9422(96)00802-3

17.

Romeo

Downum

Verpoorte

. Recent Advances in Phytochemistry. New York: Plenum Publishing; 1998:32.

18.

Harborne

Cody

Middleton Jr

Harborne

JB.

Alan

RL.

, eds.. Plant Flavonoids in Biology and Medicine: Biochemical, Pharmacological, and Structure-Activity Relationships. New York: John Wiley & Sons Inc; 1986:p. 15.

19.

Stafford

. Roles of flavonoids in symbiotic and defense functions in legume roots. Bot Rev. 1997;63(1):27-39.doi:10.1007/BF02857916

20.

Ylstra

Touraev

Moreno

et al . Flavonols stimulate development, germination, and tube growth of tobacco pollen. Plant Physiol. 1992;100(2):902-907.doi:10.1104/pp.100.2.902

21.

Brouillard

. The Flavonoids – Advances in Research Since 1980. 525. New York, NY: Chapman and Hall; 1988.

22.

Harborne

. The Flavonoids – Advances in Research Since 1986. 500. New York, NY: Chapman and Hall; 1994.

23.

Hsu

B-D

. Cloning and expression of a putative cytochrome P450 gene that influences the colour of Phalaenopsis flowers. Biotechnol Lett. 2003;25(22):1933-1939.doi:10.1023/B:BILE.0000003989.19657.53

24.

Kouakou

TH.

Konkon

NG.

Ayolié

Obouayeba

AP.

Abeda

ZH.

Koné

. Anthocyanin production in calyx and callus of Roselle (Hibiscus sabdariffa L.) and its impact on antioxidant activity. J Pharmacogn Phytochem. 2015;4:9-15.

25.

Ohkawa

Kinjo

Hagiwara

et al . Three new anti-oxidative saponarin analogs from young green barley leaves. Chem Pharm Bull. 1998;46(12):1887-1890.doi:10.1248/cpb.46.1887

26.

Kuo

P-C.

Damu

AG.

Lee

K-H.

T-S

. Cytotoxic and antimalarial constituents from the roots of Eurycoma longifolia . Bioorg Med Chem. 2004;12(3):537-544.doi:10.1016/j.bmc.2003.11.017

27.

Khan

AQ.

Ahmed

Kazmi

N-ul-H.

Malik

Afza

. The structure and absolute configuration of cyclotirucanenol, a new triterpene from Euphorbia tirucalli Linn. Z Naturforsch. 1988;43(8):1059-1062.doi:10.1515/znb-1988-0826

28.

Chang

F-R.

Lee

Y-H.

Yang

Y-L

et al . Secoiridoid Glycoside and Alkaloid Constituents of Hydrangea c hinensis . J Nat Prod. 2003;66(9):1245-1248.doi:10.1021/np0302394

29.

Yamauchi

Abe

Taki

. Protoplumericin, an iridoid bis-glucoside in Allamanda neriifolia . Chem Pharm Bull. 1981;29(10):3051-3055.doi:10.1248/cpb.29.3051

30.

Abou-Zaid

MM.

Lombardo

DA.

Kite

GC.

Grayer

RJ.

Veitch

. Acylated flavone C-glycosides from Cucumis sativus . Phytochemistry. 2001;58(1):167-172.doi:10.1016/S0031-9422(01)00156-X

31.

Mabry

TJ.

Markham

KR.

Thomas

. The Systematic Identification of Flavonoids. 45. New York, NY: Springer-Verlag; 1970.

32.

T-S.

Chern

H-J.

Damu

et al . Flavonoids and ent-labdane diterpenoids from Andrographis paniculata and their antiplatelet aggregatory and vasorelaxing effects. J Asian Nat Prod Res. 2008;10(1-2):17-24.doi:10.1080/10286020701273627

33.

Araki

Shiraishi

. Mode of interaction of Cu(II) and Mn(II) with D-ribose and D-arabinoseas studied by ¹³C-NMR spectromety. Carbohydr Res. 1986;148(1):121-126.doi:10.1016/0008-6215(86)80043-X

34.

Ahmed

Shimonishi

Bhuiyan

SH.

Utamura

Takada

Izumori

. Biochemical preparation of L-ribose and L-arabinose from ribitol: a new approach. J Biosci Bioeng. 1999;88(4):444-448.doi:10.1016/S1389-1723(99)80225-4

35.

Nørbæk

Brandt

Kondo

. Identification of flavone C-glycosides including a new flavonoid chromophore from barley leaves (Hordeum vulgare L.) by improved NMR techniques. J Agric Food Chem.2000;48(5):1703-1707.doi:10.1021/jf9910640

36.

Kuo

PC.

Chen

GF.

Yang

ML.

. High-performance liquid chromatography profiling of pigments from Phalaenopsis hybrids and their contribution to antioxidant and antityrosinase activities. Acta Hortic.2010;878(878):89-95.doi:10.17660/ActaHortic.2010.878.9

37.

Zhang

Geoffroy

Miesch

et al . Gram-scale chromatographic purification of β-sitosterol. Synthesis and characterization of β-sitosterol oxides. Steroids. 2005;70(13):886-895.doi:10.1016/j.steroids.2005.06.003

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