Nama : Nurafinda

Nim : 1703111046

Judul Artikel : Rimpang dari Bulbophyllum Anggrek Adalah yang Kaya akan Senyawa
Bioaktif Sitotoksik Agen Anti Kanker yang Potensial

Bhinija,K.,Huehne, P.S.,Prawat,H.,dkk. 2021. Rimpang dari Bulbophyllum anggrek adalah

yang kaya akan senyawa bioaktif sitotoksik agen anti kanker yang potensial. Jurnal Botani
Afrika Selatan. Vol. 141. Hal. 367-372.
Laboratorium Bioteknologi, Institut Penelitian Chulabhorn, Bangkok, 10210 Thailand
RINGKASAN ARTIKEL

1. Pendahuluan
Bulpophyllum spesies (Orchidaceae) merupakan sumber obat tradisional yang
digunakan untuk mengobati TBC, radang kronis, patah tulang, menyehatkan paru-paru,
dan menurunkan demam. Dua obat tradisional yang terkenal adalah Bulbophyllum
odoratissimum dan B. Kwangtungense schlecht. Berbagai bagian tanaman anggrek telah
digunakan untuk mengobati berbagai penyakit. Spesies Bulbophyllum mengandung
konsentrasi tinggi senyawa fenolik dan aromatik seperti fenantrena, bibenzil, dan
fenilpropanoid. Beberapa fenantrena alami dan dihidrofenantrena memiliki efek
antikanker dan ini ditemukan terutama dalam famili Orchidaceae termasuk Dendrobium,
Bulbophyllum, Eria, Maxillaria, Bletilla, Coelogyne, Cymbidium, Ephemerantha, dan
Epidendrom spesies. Laporan terbaru membahas bahwa senyawa dihydrodibenzoepin baru
diisolasi dari B. Kwangtungense dikonfirmasi memiliki aktivitas sitotoksik terhadap garis
sel HeLa dan K562, dan fenantukuinon (yaitu densiflorol B) dari B. Odoratissimum
menunjukkan sitotoksitas terhadap garis sel A546, BEL-7402, HL-60,K562 dan SGC-
7901. Namun dihidrofenantrena dari Dendrobium nobile dan D. Venustum, lusianthridin
telah menjadi agen antikanker yang menjanjikan untuk sel induk kanker paru-paru. Oleh
karena itu penelitian ini bertujuan untuk mengetahui bagian tanaman yang paling efektif
dengan sitotoksitas tinggi dan untuk menetapkan profil sitotoksitas senyawa bioaktif yang
diisolasi pada tanaman B. blefarist. Senyawa bioaktif dalam jaringan yang paling
sitotoksik dari B. blefarist mungkin berguna untuk penelitian lebih lanjut terkait dengan
penemuan dan pengembangan obat potensial dari spesies Bulbophyllum.
2. Hasil dan Pembahasan
2.1. Penyelidikan Fitokimia
 Semua jaringan tanaman dibiarkan pada suhu kamar sampai benar-benar kering
sebelum diekstraksi. Pengeringan memakan waktu 3-7 hari tergantung pada jenis jaringan
(daun, pseudobulb, atau rimpang). Ekstrak kasar 3 g dari B. blefarist rimpangnya
dilakukan kromatografi cair vakum untuk menghasilkan 10 fraksi (F1-F10). F1, F3, dan
F4 dipisahkan dengan KLT preparatif, lalu dimurnikan dengan HPLC fase terbalik untuk
menghasilkan senyawa 1 (F3); 2, 3, 4 (F4); dan 8, 9, 10 (F1) yang merupakan campuran
dari turunan alkil akrilat. Senyawa 5 (F6), 6 (F7), dan 7 (F8) dipisahkan dengan KLT
preparatif. Jumlah maksimum senyawa yang diisolasi adalah senyawa 1 (113,5 mg) diikuti
oleh senyawa 4 (39,6 mg), senyawa 2 (37,62 mg), senyawa 5 (8,7 mg), campuran 8, 9, dan
10 (8,7 mg), senyawa 6 (3,6 mg), senyawa 7 (3,2 mg) dan senyawa 3 (1,89 mg). Semua
senyawa yang diisolasi selanjutnya dan dijelaskan dengan kombinasi spektroskopi 1D dan
2D NMR. Senyawa 1 berbentuk getah kemerahan, sesuai dengan 9,10dihydro-3,4,6-
trimethoxy-2,7-phenanthrenediol. Senyawa 2 berbentuk bubuk kekuningan, sesuai dengan
9,10dihydro-3,4,6-trimetoxy-2,7-phenanthrenediol, CID : 442702 (lusianthridin). Senyawa
3 adalah berbentuk getah kemerahan sesuai dengan 3,3-dihidroxy-5-metoxy bibenzil, CID
: 10466989 (batatasin-III). Senyawa 4 berbentuk getah kemerahan sesuai dengan literatur
untuk gigantol, CID : 3085362. Senyawa 5 adalah getah kemerahan sesuai dengan literatur
untuk tristin, CID : 15736297. Senyawa yang dimurnikan 1, 2, 4, dan 5 yang kuantitasnya
memenuhi kriteria uji aktivitas sitotoksik yang selanjutnya dinilai pengaruhnya terhadap
10 garis sel kanker.

2.2 In Vitro Sitotoksitas Ekstrak Kasar

Viabilitas sel dari empat garis sel kanker yang berbeda, A549, HepG2, HuCCA-1
dan MOLT-3, dievaluasi setelah paparan selama 48 jam terhadap ekstrak etanol daun,
pseudobulb, dan rimpang B. blefarist. Hasil uji MTT dan XTT mengungkapkan bahwa
ekstrak rimpang memiliki aktivitas sitotoksik paling banyak dan sangat efisien dalam
menginduksi aktivitas anti-proliferasi dan kematian garis sel kanker aktif MOLT-3, A549,
dan HepG2. Ekstrak pseudobulb menunjukkan aktivitas sitotoksik yang signifikan
terhadap garis sel MOLT-3 saja, sedangkan ekstrak daun memiliki aktivitas yang rendah
terhadap keempat garis sel yang digunakan dalam penelitian ini. Untuk membuat profil
anti kanker dan sitotoksik dari ekstrak Bubophyllum, pelarut yang sangat efisien dan
ekonomis, etanol, telah dilakukan untuk mendapatkan polar dan beberapa non metabolit
polar. Oleh karena itu, kami menggunakan EtOH sebagai pelarut ekstraksi untuk studi
isolasi senyawa kimia dari rimpang Bulbophyllum.

2.3 Profil Sitotoksitas In Vitro Dari Senyawa Yang Diisolasi

Uji sitotoksitas in vitro mengungkapkan tiga dari empat senyawa 9,10-dihydro-3,4,6-
trimetoxy-2,7-fenantrenadiol (1), lusianthridin (2), dan tristin (5) menunjukkan berbagai
efek sitotoksik pada H69AR, HeLa, HepG2, HL-60, MOLT-3, dan garis sel HepG2.
Lusianthridin (2) memiliki sitokin berbasis luas terhadap banyak garis sel : HL-60,
MOLT-3, HepG2, dan H69AR dengan IC 50 level 26.9, 30.3, 42.3, 142.6, dan 142.7 M.
Sebaliknya, senyawa 1 dan 5 menunjukkan sitokin terbesar toksisitas terhadap garis sel
MOLT-3 dengan level IC 50 25,9 dan 21,6 M, masing-masing. Efek anti-proliferatif
moderat dari lusianthridin ( 2 ) diamati terhadap pertumbuhan H69ARdan HepG2 pada
hari ke-2 pengobatan. Hasil yang diperoleh untuk lusianthridin ( 2 ) konsisten dengan
lusianthridin yang diisolasi dari Dendrobium nobile yang ditemukan memiliki efek
sitotoksik terhadap beberapa garis sel (karsinoma paru-paru manusia A549, SK-OV-3
huadenokarsinoma ovarium pria, dan HL-60). lusianthridin diisolasi dari B. blepharistes ,
huadenokarsinoma ovarium pria, dan HL-60). Lusianthridin diisolasi dari B. blepharistes ,
dalam penelitian ini, memiliki sitotoksisitas terhadap HL-60 pada tingkat IC 50 26,9 M
tetapi sitotoksik terhadap A549 jauh lebih rendah daripada D. nobile . Dalam general,
turunan fenantrena, lusianthridin ( 2 ), tampaknya lebih efektif daripada senyawa 1 (9,10-
dihydro-3,4,6-trimethoxy-2,7-fenantrenadiol). Derivatif termetilasi tidak menunjukkan
aktivitas apapun, menunjukkan bahwa gugus hidroksi fenolik bebas diestimasi penting
untuk aktivitas penghambatan. Ini adalah laporan pertama dari lusianthridin menampilkan
aktivitas antikanker terhadap paru-paru yang resistan terhadap banyak obat garis sel
kanker (H69AR) pada tingkat IC 50 142,6 M.

Dua bibenzil, gigantol ( 4 ) dan tristin ( 5 ), memiliki efek terhadap pertumbuhan

HeLa, HL-60 dan MOLT-3, dengan tristin ( 5 ) menunjukkan sitotoksisitas yang lebih
tinggi terhadap MOLT-3, dengan level IC 50 dari 21,6 M. Gigantol ( 4 ) dari B.
blepharistes dan dari B. odoratissinum menunjukkan aktivitas sitotoksik terhadap
serangkaian kanker yang serupa garis sel.

Penelitian ini berhasil menemukan bahwa ekstrak rimpang dari Bulbophyllum

blepharistes Rchb.f memiliki sitotoksisitas paling tinggi terhadap sel A549, HepG2, dan
MOLT-3 dibanding dengan ekstrak daun dan pseudobulb. The B. blepharistes ‘s rimpang
mengandung konsentrasi tinggi fenantrena dan bibenzil. Lusianthridin ( 2 ), rimpang
senyawa bioaktif pertama yang diisolasi dari B. blepharistes menunjukkan aktivitas
sitotoksik tertinggi terhadap bermacam-macam garis sel kanker diikuti oleh 9,10-dihydro-
3,4,6-trimethoxy-2,7-fenantrenadiol ( 1 ) dan tristin ( 5).
 South African Journal of Botany 141 (2021) 367–372

Contents lists available at ScienceDirect

South African Journal of Botany

journal homepage: www.elsevier.com/locate/sajb

The rhizome of Bulbophylllum orchid is the rich source of cytotoxic

bioactive compounds, the potential anticancer agents
Kisana Bhinija a, Pattana Srifah Huehne a,∗, Hunsa Prawat b, Somsak Ruchirawat c,
Busakorn Saimanee d, Skorn Mongkolsuk a, Jutamaad Satayavivad e
a
Laboratory of Biotechnology, Chulabhorn Research Institute, Bangkok, 10210 Thailand
b
Laboratory of Natural Products, Chulabhorn Research Institute, Bangkok, 10210 Thailand
c
Laboratory of Medicinal Chemistry, Chulabhorn Research Institute, Bangkok, 10210 Thailand
d
Office of Research, Chulabhorn Research Institute, Bangkok, 10210 Thailand
e
Laboratory of Pharmacology, Chulabhorn Research Institute, Bangkok, 10210 Thailand

a r t i c l e i n f o a b s t r a c t

Article history: Bulbophyllum orchid is used as a folk medicine in Asia. However, the concentrations of bioactive con-
Received 15 December 2020 stituents in the plant may vary in different tissues. Thus, this study determined the most cytotoxic tissue
Revised 9 April 2021
extract in Bulbophyllum blepharistes Rchb.f. and identified its cytotoxic bioactive compounds. The ethanol
Accepted 8 May 2021
extract from rhizomes of B. blepharistes exhibits more cytotoxicity than extracts from the leaf and pseu-
dobulb. Ten compounds isolated from the rhizome are phenanthrenes (1, 2), bibenzyls (3, 4, 5), phenyl-
Edited by P Bhattacharyya propanoids (6), methyl benzoate (7), along with a mixture of three alkyl acrylates (8, 9, 10). Compound 2,
lusianthridin (9,10-dihydro-7-methoxy-2,5-phenanthrenediol), which was reported here for the first time
Keywords:
Bulbophyllum in Bulbophyllum, showed cytotoxicity to H69AR, HeLa, HL-60, HepG2, and MOLT-3 cells. Compound 1 and
Anticancer agent 5 (tristin) significantly inhibited the growth of MOLT-3. The rhizome of B. blepharistes appears to be a
Lusianthridin good source for the potential anticancer agents (i.e. lusianthridin).
Orchid
© 2021 SAAB. Published by Elsevier B.V. All rights reserved.
Phenanthrene

1. Introduction

Abbreviations: H69AR, multidrug resistance lung cancer; HeLa, human cervi-
cal carcinoma; HL-60, acute promyelocytic leukemia; HepG2, liver hepatocellu-
lar carcinoma; MOLT-3, acute lymphoblastic leukemia; EtOH, ethanol; EtOAc, ethyl
acetate; HPLC, High Performance Liquid Chromatography; λ, wavelength; MeOH,
methanol; TLC, preparative Thin Layer Chromatography; CH2 Cl2 , dichloromethane;
NMR, Nuclear Magnetic Resonance spectroscopy; CDCl3 , deuterochloroform; DMSO-
d6 , hexadeuterodimethyl sulfoxide; Me4 Si, tetramethylsilane; δ , chemical shift;
J, coupling constant; DEPT, Distortionless Enhancement by Polarization Trans-
fer; HMQC, Heteronuclear Multiple Quantum Correlation; HMBC, Heteronuclear
Multiple-Bond Correlation; COSY, Correlation Spectroscopy; NOESY, Nuclear Over-
hauser Effect Spectroscopy; A549, lung cancer,; HuCCA-1, human cholangiocar-
cinoma; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; T47-
D, hormone-dependent breast cancer; MDA-MB-231, hormone-independent breast
cancer; S102, human liver cancer; XTT, 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-
2H-tetrazolium-5-carboxyanilide salt; IC50 , Half-maximal inhibitory concentration;
RPMI-1640, Roswell Park Memorial Institute Medium; HRESI-MS, High-Resolution
Electrospray Ionization Mass Spectrometry; MHz, MegaHertZ; m/z, a value of mass
divided by charge; Calcd, calculated; CID, Compound ID number; Hz, Hertz; δ C ,
chemical shift of carbon; δ H , chemical shift of proton; mult., multiplicity; S, sin-
glet; d, doublet; m, multiplet; t, triplet; dd, doublet of doublet; SK-OV-3, human
ovary adenocarcinoma; BEL-7402, human hepacarcinoma; SGC-7901, gastric cancer
cell lines.
∗
Corresponding author.
E-mail addresses: kisana@cri.or.th (K. Bhinija), pattana@cri.or.th (P.S. Huehne),
hunsa@cri.or.th (H. Prawat), somsak@cri.or.th (S. Ruchirawat), busakorn@cri.or.th (B.
Saimanee), skorn@cri.or.th (S. Mongkolsuk), jutamaad@cri.or.th (J. Satayavivad).
Bulbophyllum species (Orchidaceae) are a folk medicine source
used to treat tuberculosis, chronic inflammation, and frac-
tures, nourish the lungs, and reduce fever (Yi et al., 2005;
Gutierrez, 2010; Hossain, 2011). Two well-known folk medicines
are Bulbophyllum odoratissimum (Sm.) Lindl. (Chen et al., 2007) and
B. kwangtungense Schlecht (Shi dou-lan) (Wu et al., 2006). Differ-
ent parts of the orchid plants have been used to treat various dis-
eases. These parts include the entire plant (B. leopardinum (Wall.)
Lindl., B. odoratissimum (Sm.) Lindl., and B. retusiusculum Rchb.f.),
the pseudobulb (B. careyanum (Hook.) Sprengel and B. cariniflorum
Rchb.f.), the tuber (B. kwangtungensis Schltr), and the roots (B. neil-
gherrense Whight) (Gutierrez, 2010; Hossain, 2011).
The Bulbophyllum species contain high concentrations of phe-
nolic and aromatic compounds, such as phenanthrenes, bibenzyls,
and phenylpropanoids (Leong and Harrison, 20 04; Yi et al., 20 05).
These natural plant-derived phytochemical compounds may play
important roles in cancer prevention (Chen et al., 2008; Dai and
Mumper, 2010). Some natural phenanthrenes and dihydrophenan-
threnes have an anticancer effect, and these are found mainly in
the Orchidaceae family including Dendrobium, Bulbophyllum, Eria,
Maxillaria, Bletilla, Coelogyne, Cymbidium, Ephemerantha, and Epi-

https://doi.org/10.1016/j.sajb.2021.05.013
0254-6299/© 2021 SAAB. Published by Elsevier B.V. All rights reserved.
K. Bhinija, P.S. Huehne, H. Prawat et al.
Fig. 1. Bulbophyllum blepharistes Rchb.f. orchid plant.
dendrum species (Kovac et al., 2008; Gong et al., 2004). Recent re-
ports discussed that new dihydrodibenzoepin compounds isolated
from B. kwangtungense were confirmed to possess cytotoxic activ-
ity against the HeLa and K562 cell lines (Wu et al., 2006), and
a phenanthoquinone (i.e. densiflorol B) from B. odoratissimum ex-
hibited cytotoxicity against the A546, BEL-7402, HL-60, K562, and
SGC-7901 cell lines (Chen et al., 2007). However, a dihydrophenan-
threne from Dendrobium nobile (Lee et al., 1995) and D. venustum,
lusianthridin, has become a promising anticancer agent for lung
cancer stem cells (Bhummaphan et al., 2019).
In Thailand, the Bulbophyllum orchid has 154 known species,
making it the country’s second most prevalent orchid genus af-
ter the Dendrobium orchid (Boonkorkaew, 2010). Bulbophyllum ble-
pharistes Rchb.f. is the one of Thai native Bulbophyllum orchid that
grows widely spread in the North and Northeast due to its toler-
ance to a drought environment. The ethanol extract of B. blephar-
istes demonstrated higher DPPH radical scavenging activity than
the extract of B. vaginatum (Lindl.) Rchb.f. (Malaysian herb) (un-
published data). Therefore, this study aimed to determine the most
effective plant part with high cytotoxicity and to establish the cy-
totoxicity profile of the isolated bioactive compounds in B. blephar-
istes. The bioactive compounds in the most cytotoxic tissue of B.
blepharistes may be useful for further research related to potential
drug discovery and development from Bulbophyllum species.
2. Materials and methods
2.1. Plant materials
Samples of three-year-old Bulbophyllum blepharistes Rchb.f. were
used in this study (Fig. 1). The plants were derived from seedlings
grown in a greenhouse at Saman orchid, Ratchaburi. Voucher spec-
imens were deposited in the Bangkok Herbarium, Plant Varieties
Protection Office, Department of Agriculture, Ministry of Agricul-
ture and Cooperatives (No: BK071398).
2.2. Crude extraction for preliminary cytotoxicity test
The leaf, pseudobulb, and rhizome explants (200 g, each part)
were cleaned, left to air-dry, and blended into fine powder. Then,
the sample was extracted three times with EtOH (200 mL each) at
room temperature for 24 h. The ethanol extracts were combined
and concentrated under evaporation and vacuum to produce a final
around 3 g of crude extract. The explant that showed the most
cytotoxic activity will be selected for further investigation.
368
South African Journal of Botany 141 (2021) 367–372
2.3. Chemical compound extraction and isolation
The 3 g ethanol extract from B. blepharistes’s rhizome was sub-
jected to vacuum liquid chromatography (3 × 5 cm, silica gel,
Merck Art 7729) using a stepped gradient elution from 100% n-
hexane to 100% EtOAc giving 10 fractions (F1-F10). Fraction F3
(135.5 mg) was further purified by preparative HPLC using isocratic
elution 50% MeOH-H2 O for 50 min (column hichrom C18 , 250 × 21
mm, detected with λ 225 and 254 nm, flow rate 8 mL/min) to yield
compound 1 (113.5 mg). Fraction F4 (264.7 mg) was separated
using preparative thin layer chromatography (TLC) with CH2 Cl2 -
MeOH (98:2) to give 6 bands (I-VI). Bands I-III were further puri-
fied by preparative HPLC using isocratic elution 50% MeOH-H2 O for
50 min (column hichrom C18 , 250 × 21 mm, detected with λ 225
and 254 nm, flow rate 8 mL/min) to produce compounds 2 (37.62
mg), 3 (1.89 mg), and 4 (39.6 mg), respectively. Fraction F6 (90
mg) was purified using preparative TLC with CH2 Cl2 -MeOH (95:5)
to produce compound 5 (8.7 mg). Fraction F7 (59 mg) was sep-
arated using preparative TLC with CH2 Cl2 -MeOH as eluent (95:5)
to yield compound 6 (3.6 mg). Fraction F8 (37 mg) was separated
using preparative TLC with CH2 Cl2 -MeOH as eluent (95:5) to yield
compound 7 (3.2 mg). Fraction F1 (45 mg) was separated using
preparative TLC with hexane-EtOAc (90:10) to give 3 bands (I-III).
Band I was further purified by preparative HPLC using 100% MeOH
(column hichrom C18 , 250 × 21 mm, detected with λ 225 and 254
nm, flow rate 8 mL/min) to produce a mixture (8.7 mg) of 3 com-
pounds 8, 9 and 10 (Fig. 2).
2.4. General experimental procedures
High resolution mass spectrometry (HR-MS) was performed on
a Bruker (Micro ToF) spectrometer. 1 H and 13 C NMR spectra were
recorded in CDCl3 or CDCl3 + DMSO-d6 solution containing Me4 Si
as an internal standard on a Bruker AVANCE300 III and AVANCE
400 III HD spectrometer. Chemical shifts (δ ) were given in parts
per million downfield from tetramethylsilane and coupling con-
stants (J) were measured in Hertz. DEPT, HMQC, HMBC, COSY, and
NOSEY experiments were analyzed using standard Bruker software.
HPLC was carried out on a Waters 600 system equipped with a
Waters Delta 600 pump, a Waters 600 Controller, and a Waters
2998 photodiode array detector, using the Waters Empower 2 soft-
ware. The reversed phase column hichrom C18 , 250 × 4.6 mm was
used for analytical applications with 20 μl of injection volume,
the solvent flow rate was 1.0 mL/min, the reversed phase column
hichrom C18 , 250 × 21 mm was used for preparative applications,
the injection volume was 200 μl and the flow rate was 8.0 mL/min.
All commercial grade solvents were distilled prior to use and spec-
tral grade solvents were used for spectroscopic measurements.
2.5. In vitro cytotoxicity assay of crude extract
Crude ethanol extracts of leaf, rhizome, and pseudobulb were
re-suspended with dimethyl sulfoxide, diluted with cell culture
medium to the desired concentration (50, 10, 2, 0.4, and 0.08
mg/mL) and then the cytotoxic activity was evaluated in four
cancer cell lines (A549 lung cancer, HepG2 liver hepatoblas-
toma carcinoma, HuCCA-1 human cholangiocarcinoma, and MOLT-
3 acute lymphoblastic leukemia), by using MTT assay (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (Roche Di-
agnostic, USA) (Scudiero et al., 1988). Cytotoxicity in the MOLT-
3 acute lymphoblastic leukemia line (Table S1) was done us-
ing the XTT assay (2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-
tetrazolium-5-carboxyanilide salt) (Dolye and Griffiths, 1997). In
the XTT assay, the cell suspensions were plated and incubated for
4 h after the addition of 50 μL of a mixture of the tetrazolium
K. Bhinija, P.S. Huehne, H. Prawat et al.
Fig. 2. Isolation chart of the chemical constituents of the rhizome ethanol extract of Bulbophyllum blepharistes Rchb.f. VLC: vacuum liquid chromatography; EtOAc: acetyl
acetate; prep TLC: preparative thin layer chromatography; prep-HPLC: preparative high-performance liquid chromatography; CH2 Cl2 : dichloromethane; MeOH: methanol.
salt of XTT (1 mg/mL) in XTT reagent (5 mL) and N-methyl diben-
zopyrazine methyl sulfate (100 μL of a 0.383 mg/mL solution).
The absorbance of the orange formazan compounds formed was
measured at the wavelengths 450 nm and 690 nm. The IC50 lev-
els were determined for each drug concentration at 50% inhibi-
tion of cell growth. Cells were grown on RPMI-1640 medium con-
taining 100 U/mL penicillin-streptomycin, sodium pyruvate (Sigma-
Aldrich, USA), 4.5 g/L glucose, 2 mML-glutamine and 10% fetal
bovine serum. The reference substances used were etoposide and
doxorubicin (Sigma-Aldrich, USA).
2.6. In vitro cytotoxicity assay of purified compounds
Four isolated compounds including: 9,10-dihydro-3,4,6-
trimethoxy-2,7-phenanthrenediol (1); lusianthridin (2); gigantol
(4) and tristin (5) that were present in high concentration in B.
blepharistes were examined for their cytotoxicity to six adhesive
cell lines (A549, H69AR multidrug resistance lung cancer, HeLa hu-
man cervical carcinoma, HepG2, T47-D hormone-dependent breast
cancer, HuCCA-1, MDA-MB-231 hormone-independent breast can-
cer and S102 human liver cancer) and two non-adhesive cell lines
(HL-60 acute promyelocytic leukemia and MOLT-3) (Table S1). The
inhibition capacity of adhesive and non-adhesive cell lines was
assessed using MTT assay (Scudiero et al., 1988) and XTT assay
(Dolye and Griffiths, 1997), respectively.
3. Results and discussion
3.1. Phytochemical investigation
All plant tissues were left at room temperature until they were
completely dried before extraction. Air-drying took 3-7 days de-
pending on the types of tissues (leaf, pseudobulb, or rhizome). The
explants were exposed to air at ambient temperature and did not
369
South African Journal of Botany 141 (2021) 367–372
force dry with high temperature, therefore the heat-labile com-
pounds are preserved (Azwanida, 2015). The 3 g crude extract from
B. blepharistes’s rhizome was subjected to vacuum liquid chro-
matography to give 10 fractions (F1-F10). The F1, F3, and F4 were
separated by preparative TLC, further purified by reversed phase
HPLC to yield compound 1 (F3); 2, 3, 4 (F4); and 8, 9, 10 (F1)
which are a mixture of three alkyl acrylate derivative. The com-
pound 5 (F6), 6 (F7), and 7 (F8) were separated by preparative TLC
(Figs. 2 and 3). The maximum amount of the isolated compound
was compound 1 (113.5 mg), followed by compound 4 (39.6 mg),
compound 2 (37.62 mg), compound 5 (8.7mg), mixed of compound
8, 9, and 10 (8.7 mg), compound 6 (3.6 mg), compound 7 (3.2 mg)
and compound 3 (1.89 mg). All isolated compounds were subse-
quently and elucidated by a combination of 1D and 2D NMR spec-
troscopic and HRESI-MS data to confirm their structures (Table 1
and S2, Fig. S1−S33).
Compound 1 was a reddish gum, in agreement with 9,10-
dihydro-3,4,6-trimethoxy-2,7-phenanthrenediol (Majumder et al.,
1999); for 13 C (75 MHz) and 1 H (300 MHz) NMR data (CDCl3 )
(Table 1); HRESI-MS (negative); m/z 301.1086 [M−H]− (calcd. for
C17 H17 O5 , 301.1082). It was CID: 181637. The structure of com-
pound 1 was further supported by COSY, HMQC, HMBC, and
NOESY.
Compound 2 was a yellowish powder, in agreement with
9,10-dihydro-7-methoxy-2,5-phenanthrenediol, CID: 442702 (lu-
sianthridin) (Majumder and Lahiri, 1990); for 13 C (100 MHz) and
1 H (400 MHz) NMR data (CDCl + DMSO-d ) (Table 1); HRESI-MS
3 6
(negative); m/z 241.0870 [M−H]− (calcd for C15 H13 O3 , 241.0870).
Compound 3 was a reddish gum, in agreement with 3,3 -
dihydroxy-5-methoxy bibenzyl, CID: 10466989 (batatasin-III)
(Majumder et al., 1997); for 13 C (100 MHz) and 1 H (400 MHz)
NMR data (CDCl3 + DMSO-d6 ) (Table 1), HRESI-MS (positive); m/z
267.0998 [M+Na]+ (calcd for C15 H16 O3 Na, 267.0992).
K. Bhinija, P.S. Huehne, H. Prawat et al.
Fig. 3. Structures of isolated compounds 1−10 from Bulbophyllum blepharistes Rchb.f.
Compound 4 was a reddish gum, in agreement with the liter-
ature for gigantol, CID: 3085362 (Chen et al., 20 08); for 13 C (10 0
MHz) and 1 H (400 MHz) NMR data (CDCl3 ) (Table 1); HRESI-MS
(negative); m/z 273.1140 [M−H]− (calcd for C16 H17 O4 , 273.1132).
Compound 5 was a reddish gum, in agreement with the litera-
ture for tristin, CID: 15736297 (Majumder and Pal, 1993); 13 C (100
MHz) and 1 H (400 MHz) NMR data (CDCl3 ) (Table 1); HRESI-MS
(negative); m/z 259.0964 [M−H]− (calcd for C15 H15 O4 , 259.0976).
Compound 6 was a white power, in agreement with the lit-
erature for 3-(4-hydroxyphenyl)propanoic acid, CID: 10394 (Fang
et al., 2018); 1 H NMR (300 MHz, CDCl3 ) δ 7.10 (2H, d, J = 8.0 Hz,
H-2 and H-6 ), 6.78 (2H, d, J = 8.0 Hz, H-3 and H-5 ), 2.92 (2H, t,
J = 7.2 Hz, H-2), 2.67 (2H, t, J = 7.2 Hz, H-3). HRESI-MS (negative);
m/z 165.0564 [M−H]− (calcd for C9 H9 O3 , 165.0557).
Compound 7 was a colorless powder, in agreement with
the literature for methyl-3,4-dihydroxybenzoate, CID: 287064
(Azizuddini et al., 2010); 1 H NMR (300 MHz, CDCl3 ) δ 7.73 (1H,
dd, J = 8.3, 1.9 Hz, H-6), 7.61 (1H, d, J = 1.9 Hz, H-2), 6.99 (1H,
d, J = 8.3 Hz, H-5), 3.98 (3H, s, OCH3 ). HRESI-MS (negative); m/z
167.0353 [M−H]− (calcd for C8 H7 O4 , 167.0350).
Finally, a mixture was obtained as a colorless powder of com-
pound 8 as (E)-docosyl 3-(4-hydroxy-3-methoxyphenyl)acrylate,
CID: 14238616 (Chang et al., 2001); compound 9 was (E)-tetracosyl
3-(4-hydroxy-3-methoxyphenyl)acrylate (Addae-Mensah et al.,
1992; Lee et al., 2005), compound 10 was (E)-hexacosyl 3-(4-
hydroxy-3-methoxyphenyl)acrylate, (Addae-Mensah et al., 1992;
Maurice et al., 1994). 1 H NMR (400 MHz, CDCl3 ) of the mixture
δ 7.61 (1H, d, J = 15.9 Hz, H-3), 7.07 (1H, dd, J = 8.2, 1.8 Hz,
H-6 ), 7.04 (1H, d, J = 1.8 Hz, H-2 ), 6.92 (1H, d, J = 8.2 Hz, H-5 ),
6.30 (1H, d, J = 15.9 Hz, H-2), 4.19 (2H, t, J = 6.7 Hz, H-1 ),
3.93 (3H, s, OCH3 ), 1.70 (2H, m, H-2 ), 0.88 [3H, t, J = 7.0 Hz,
(OCH2 (CH2 )n CH3 )]; 13 C NMR (100 MHz, CDCl3 ) of the mixture δ
167.4 (C-1), 147.9 (C-4 ), 146.7 (C-3 ), 144.6 (CH-3), 127.0 (C-1 ),
123.0 (CH-6 ), 115.6 (CH-2), 114.6 (CH-5 ), 109.2 (CH-2 ), 64.6 (CH2 -
1 ), 55.9 (3 -OCH3 ), 14.1 (OCH2 (CH2 )n CH3 ); HRESI-MS (positive)
of the mixture; m/z 503.4096 [M+H]+ (calcd for compound 8
C32 H55 O4 , 503.4096); m/z 531.4426 [M+H]+ (calcd for compound
9 C34 H59 O4 , 531.4408); m/z 559.4750 [M+H]+ (calcd for compound
10 C36 H63 O4 , 559.4721).
370
South African Journal of Botany 141 (2021) 367–372
The purified compound 1, 2, 4, and 5 that the quantity meets
the criteria for cytotoxic activity test were further assessed the im-
pact of their effects against 10 cancer cell lines.
3.2. In vitro cytotoxicity of crude extract
Cell viabilities of four different cancer cell lines, A549, HepG2,
HuCCA-1 and MOLT-3, were evaluated after exposure for 48 h to
leaf, pseudobulb, and rhizome ethanol extracts of B. blepharistes.
The MTT and XTT assay results revealed that rhizome extracts had
the most cytotoxic activity and were highly efficient at inducing
anti-proliferative activity and death of the active cancer cell lines
MOLT-3, A549, and HepG2. The pseudobulb extract showed signifi-
cant cytotoxic activity against the MOLT-3 cell line only, while leaf
extract had low activity against all four cell lines used in this study
(Table 2).
Liver and lung cancer has become the top 10 of the most life-
threatening diseases in Thailand (International Agency for Research
on Cancer, 2020). Therefore searching for high potential Bulbo-
phyllum of native species in Thailand possessing anticancer activ-
ity would be useful for medicinal use in human health related
researches. To create the anticancer and cytotoxic profiles of the
Bulbophyllum extracts, a highly efficient and economical solvent,
ethanol, has been performed to obtain the polar and some non-
polar metabolites. It is easy to evaporate the eluents and can be
purified quickly by practical chromatography methods. In the cur-
rent study, we have successfully identified that the ethanol crude
extracts of the B. blepharistes rhizome exhibited significant cyto-
toxicity against the proliferation of A549, HepG2, and MOLT-3. Un-
like, the alcoholic extract of B. vaginatum (Malaysian herb used for
ear drop to treat earache) showed no anticancer activity with the
tested cell lines (unpublished data), although the purified com-
pounds from hexane extract of B. vaginatum contained most of
the common phenanthrenes, dihydrophenanthrenes, bibenzyls, and
phenanthro(4,3-b)furan (Leong and Harrison, 2004). In addition,
the ethanol extracts of B. kaitense Rechib. presented high con-
tent phenolic compounds, coumarin, quinine, and carbohydrate,
but these compounds completely absent in petroleum ether and
chloroform extracts (Kalaiyarasan et al., 2012). The petroleum ether
extract of pseudobulb and root tissues of B. sterile (Lam.) Suresh
(an Indian folk medicine) were used to treat rheumatism and re-
K. Bhinija, P.S. Huehne, H. Prawat et al. South African Journal of Botany 141 (2021) 367–372

Table 2
The cytotoxicity of crude extracts from Bulbophyllum blepharistes Rcbh.f. against
four cancer cell lines.

Cytotoxicity Activities (IC50 μg/mL)

A549 HepG2 HuCCA-1 MOLT-3

6.67, dd (8.0, 1.8)

δ H , mult (J in Hz)

Leaf I I 65.22±4.81 I
49.99±3.98 57.69±5.02 13.96±2.29
6.61, d (1.8)

6.23, d (2.1)

6.23, d (2.1)
6.82, d (8.0) Rhizome I

6.19, t (2.1)
Pseudobulb I I I 42.67±2.11
2.81, m

2.79, m
Etoposide ND ND ND 0.024±0.004

3.84, s
Doxorubicin 0.19±0.01 0.27±0.01 0.79±0.08 0.008±0.001

I = inactive; ND = Not determined

111.1, CH

114.1, CH
120.9, CH

108.1, CH

100.4, CH

108,1, CH
37.1, CH2

38.0, CH2
55.8, CH3
133.5, C

146.2, C
143.7, C

144.8, C

156.6, C

156.6, C
δ C , type

duce swelling (Shanavaskhan et al., 2012). Therefore, we used EtOH

5a

-
as an extraction solvent for the further study of chemical com-
pound isolation from Bulbophyllum rhizome.
6.68, dd (8.1, 1.8)
δ H , mult (J in Hz)

6.62, d (1.8)

6.83, d (8.1)

t (1.8)

3.3. In vitro cytotoxicity profiling of isolated compounds

2.81, m

6.25, m

6.25, m

m
s
s
6.32,
2.80,
3.84,
3.75,

As shown in Table 3, in vitro cytotoxicity assays revealed that

three out of the four compounds 9,10-dihydro-3,4,6-trimethoxy-
2,7-phenanthrenediol (1); lusianthridin (2); and tristin (5) showed
111.1, CH

114.2, CH
120.9, CH

106.8, CH
108.0, CH
37.2, CH2

32.2, CH2
55.8, CH3
55.2, CH3
99.0, CH
133.6, C

146.2, C
143.7, C

144.5, C

156.5, C

160.8, C
δ C , type

varying cytotoxic effects on H69AR, HeLa, HepG2, HL-60, MOLT-

3, and HepG2 cell lines. Lusianthridin (2) had broad based cyto-
4a

toxicity against numerous cell lines: HL-60, HeLa, MOLT-3, HepG2,

and H69AR with IC50 levels of 26.9, 30.3, 42.3, 142.6, and 142.7
δ H , mult(J in Hz)

μM. By contrast, compound 1 and 5 showed the greatest cyto-

6.27, t (2.2)
t (7.8)

toxicity against the MOLT-3 cell line with IC50 levels of 25.9 and
m

6.33, m

6.25, m
2.81, m
6.70, m

21.6 μM, respectively. A moderate anti-proliferative effect of lu-

3.74, s
6.67,
7.10,
6.68,
2.80,

sianthridin (2) was observed against H69ARand HepG2 growth

on day 2 of treatment. The results obtained for lusianthridin
(2) were consistent with the lusianthridin isolated from Dendro-
115.5, CH

113.0, CH
129.2, CH
119.5, CH

108.2, CH

105.4, CH
37.4, CH2

37.8, CH2

55.1, CH3
99.1, CH
143.4, C

144.2, C

158.1, C
157.0, C

160.6, C
δ C , type

bium nobile which was found to exert cytotoxic effects against

multiple cell lines (A549 human lung carcinoma, SK-OV-3 hu-
3b

man ovary adenocarcinoma, and HL-60) (Lee et al., 1995). The lu-
sianthridin isolated from B. blepharistes, in this study, had cytotox-
C (100 MHz) and 1 H (400 MHz) NMR spectroscopic data for compounds 1, 2, 3, 4, and 5.

5 -OMe
3-OMe

icity against HL-60 at an IC50 level of 26.9 μM but the cytotoxi-

No.

1
2
3
4
5
6
7
1
2
3
4
5
6
7

city against A549 was much lower than that of D. nobile. In gen-
eral, the phenanthrene derivative, lusianthridin (2), appeared to be
6.72, dd (8.5, 1.5)
δ H , mult (J in Hz)

more effective than compound 1 (9,10-dihydro-3,4,6-trimethoxy-

Assignments for 1–5 were based on DEPT, COSY, HMQC, HMBC experiments.

2,7-phenanthrenediol). The methylated derivatives did not exhibit

6.31, d (1.9)

6.43, d (1.9)

8.19, d (8.5)

6.69, d (1.5)

any activity, suggesting that a free phenolic hydroxy group is es-

2.69, m
2.70, m

3.76, s

sential for inhibitory activity. This is the first report of lusianthridin

displaying anticancer activity against a multidrug-resistant lung
cancer cell line (H69AR) at an IC50 level of 142.6 μM.
104.9, CH

128.6, CH
112.9, CH

114.3, CH
100.7, CH

29.9, CH2
30.7, CH2

55.0, CH3

The two bibenzyls, gigantol (4) and tristin (5), had a moderate
157.9, C

154.8, C
115.0, C
125.0, C

154.9, C

138.9, C

140.0, C
δ C , type

effect against the growth of HeLa, HL-60 and MOLT-3, with tristin
(5) showing higher cytotoxicity against MOLT-3, with an IC50 level
2b

-
-
-

C (75 MHz) and 1 H (300 MHz) NMR data.

of 21.6 μM. The gigantols (4) from B. blepharistes and from B. odor-
δ H , mult (J in Hz)

atissinum showed cytotoxic activity against a similar set of cancer

cell lines. Although the basis for cytotoxic activity is not fully un-
derstood, gigantol extracted from the entire B. odoratissinum plant
2.65, m
2.66, m
6.64, s

7.91, s

6.79, s

3.98, s
3.75, s
3.92, s

was reported to be a potent inhibitor of tumor necrosis factor-

alpha, interleukin-1beta and interleukin-6 (Won et al., 2006).
Tristin (5) from B. blepharistes displayed cytotoxicity effects
in CDCl3 + DMSO-d6
110.3, CH

110.3, CH

113.7, CH

against more cancer cell lines than the tristin from B. odoratiss-
61.2, CH3

56.2, CH3
29.0, CH2
30.2, CH2

60.1, CH3
147.5, C
139.0, C
150.3, C
120.4, C
124.5, C

144.8, C
143.9, C

131.4, C

135.1, C
δ C , type

inum (Chen et al., 2008). Three dihydrodibenzoxepins and the

1a , c

known compound, densiflorol A, from B. kwangtungense displayed

-

in CDCl3

anticancer activity against the tumor cell lines HeLa and K562
2-OMe
3-OMe
4-OMe
6-OMe

(Wu et al., 2006). Two natural dihydrostilbenes isolated from B.

Table 1

10a

c 13
No.

odoratissimum and the corresponding synthetic compounds exhib-

4b

10
4a

8a
1
2
3
4

5
6
7
8

b
a

ited significant cytotoxicity against the human SGC-7901, KB, and

13

HT-1080 cell lines (Zhang et al., 2007), while densiflorol B was

the most active compound against the K562, HL-60, A549, BEL-
7402, and SGC-7901 cell lines (Chen et al., 2008). Furthermore,
two dimericphenanthrenes, bulbophythrins A and bulbophythrins

371
K. Bhinija, P.S. Huehne, H. Prawat et al. South African Journal of Botany 141 (2021) 367–372

Table 3
The cytotoxicity of isolated compounds from the rhizome of Bulbophyllum blepharistes Rchb.f. against cancer cell lines.

Cytotoxicity activities (IC50 = μM)

Compound
A549 H69AR HeLa HepG2 HL-60 HuCCA-1 MDA-MB-231 MOLT-3 S102 T47-D

1 I ND 135.8±3.6 143.8±5.2 69.4±4.4 I I 25.9±1.5 ND I

2 I 142.6±0.8 30.3±1.4 142.7±1.1 26.9±1.5 I I 42.3±1.0 I I
4 I I 169.5±5.5 I 93.7±7.0 I I 77.9±1.9 I I
5 I ND 176.7±0.3 I 113.1±5.7 I I 21.6±0.6 ND I
Etoposide ND ND ND 26.8±4.3 0.73±0.03 ND ND 0.03±.005 ND ND
Doxorubicin 0.31±.56 38.3±.35 0.26±.02 0.38±.04 ND 0.81±.12 1.96±.10 ND 2.05±.15 14.6±.09

I = Inactive at IC50 > 200 μM (IC50 > 50 μg/ml); ND = Not determined; Etoposide and Doxorubicin were used for positive control.

B, exhibited selective cytotoxicity; the former against HL-60 and
BEL-7402 cells and the latter against A549 cells (Xu et al., 2009).
In contrast to flavone C-glycoside and bibenzyl, the bulbotetusine
isolated from B. retusiusculum (Yang et al., 2016) and the phenan-
threne, bobulretin A, and dihydrophenanthrene, bobulretin B iso-
lated from B. retusiusculum showed no obvious activity against five
human tumor cell lines (Sun et al., 2018).
4. Conclusions
Our study was successfully discovered that the rhizome ex-
tract of Bulbophyllum blepharistes Rchb.f. had the most cytotoxi-
city against A549, HepG2, and MOLT-3 cells compared with the
leaf and pseudobulb extracts. The B. blepharistes’s rhizome con-
tained high concentration of phenanthrenes and bibenzyls. The lu-
sianthridin (2), first isolated bioactive compound from B. blephar-
istes’s rhizome showed the highest cytotoxic activity against var-
ious cancer cell lines followed by 9,10-dihydro-3,4,6-trimethoxy-
2,7-phenanthrenediol (1) and tristin (5).
Declaration of Competing Interest
The authors declare that they have no conflicts of interest.
Acknowledgments
This work was supported by the Chulabhorn Research Institute
[grant number; PH2020-03].
Author Statement
KB conducted the experiments. PSH conceived and designed the
experiment, and wrote the manuscript. HP analyzed chemical com-
pound structures. SR provided facility for chemical analysis. BS per-
formed cytotoxic screening. SM corrected the manuscript and data.
JS provided cell lines and corrected the manuscript. All authors
read and approved the final manuscript.
Supplementary materials
Supplementary material associated with this article can be
found, in the online version, at doi:10.1016/j.sajb.2021.05.013.
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