Antiviral activities of compounds isolated from pinus densiflora (Pine tree) against the influenza a virus

Article abstract of DOI:10.3390/biom10050711

Pinus densiflora was screened in an ongoing project to discover anti-influenza candidates from natural products. An extensive phytochemical investigation provided 26 compounds, including two new megastigmane glycosides (1 and 2), 21 diterpenoids (3–23), and three flavonoids (24–26). The chemical structures were elucidated by a series of chemical reactions, including modified Mosher’s analysis and various spectroscopic measurements such as LC/MS and 1D-and 2D-NMR. The anti-influenza A activities of all isolates were screened by cytopathic effect (CPE) inhibition assays and neuraminidase (NA) inhibition assays. Ten candidates were selected, and detailed mechanistic studies were performed by various assays, such as Western blot, immunofluorescence, real-time PCR and flow cytometry. Compound 5 exerted its antiviral activity not by direct neutralizing virion surface proteins, such as HA, but by inhibiting the expression of viral mRNA. In contrast, compound 24 showed NA inhibitory activity in a noncompetitive manner with little effect on viral mRNA expression. Interestingly, both compounds 5 and 24 were shown to inhibit nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in a dose-dependent manner. Taken together, these results provide not only the chemical profiling of P. densiflora but also anti-influenza A candidates.

Full text of DOI:10.3390/biom10050711

biomolecules
Article
Antiviral Activities of Compounds Isolated from
Pinus densiflora (Pine Tree) against the Influenza
A Virus
Thi Kim Quy Ha 1,2,†, Ba Wool Lee 1,† , Ngoc Hieu Nguyen 1, Hyo Moon Cho 1,
Thamizhiniyan Venkatesan 1, Thi Phuong Doan 1, Eunhee Kim 3 and Won Keun Oh 1,
*
1
Korea Bioactive Natural Material Bank, Research Institute of Pharmaceutical Sciences, College of Pharmacy,
Seoul National University, Seoul 08826, Korea; htkquy@ctu.edu.vn (T.K.Q.H.); paul36@snu.ac.kr (B.W.L.);
hieusnu@gmail.com (N.H.N.); chgyand@naver.com (H.M.C.); thamtris@snu.ac.kr (T.V.);
phuongdoan@snu.ac.kr (T.P.D.)
College of Natural Sciences, Cantho University, Campus II, Cantho City 94000, Vietnam
Choong Ang Vaccine Laboratory, 1476-37, Yuseong-daero, Yuseong-gu, Daejeon 34055, Korea;
ehkim@cavac.co.kr
2
3
*
Correspondence: wkoh1@snu.ac.kr; Tel.: +82-2-880-7872
†
These authors contributed equally to this work.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 4 April 2020; Accepted: 29 April 2020; Published: 4 May 2020
Abstract: Pinus densiflora was screened in an ongoing project to discover anti-influenza candidates
from natural products. An extensive phytochemical investigation provided 26 compounds, including
two new megastigmane glycosides (1 and 2), 21 diterpenoids (3–23), and three flavonoids (24–26).
The chemical structures were elucidated by a series of chemical reactions, including modified
Mosher’s analysis and various spectroscopic measurements such as LC/MS and 1D- and 2D-NMR.
The anti-influenza A activities of all isolates were screened by cytopathic effect (CPE) inhibition assays
and neuraminidase (NA) inhibition assays. Ten candidates were selected, and detailed mechanistic
studies were performed by various assays, such as Western blot, immunofluorescence, real-time
PCR and flow cytometry. Compound
5 exerted its antiviral activity not by direct neutralizing
virion surface proteins, such as HA, but by inhibiting the expression of viral mRNA. In contrast,
compound 24 showed NA inhibitory activity in a noncompetitive manner with little effect on viral
mRNA expression. Interestingly, both compounds 5 and 24 were shown to inhibit nitric oxide (NO)
production and inducible nitric oxide synthase (iNOS) expression in a dose-dependent manner. Taken
together, these results provide not only the chemical profiling of P. densiflora but also anti-influenza
A candidates.
Keywords: Pinus densiflora; anti-influenza; neuraminidase; H1N1; cytopathic effect; anti-inflammation
1. Introduction
According to data from the Centers for Disease Control and Prevention (CDC), it is estimated
that 5–10% of the human population is infected by seasonal human influenza virus each year,
and approximately 290,000 to 650,000 people globally die from influenza-related respiratory
diseases [1,2]. The influenza virus is divided into four types (A-D) based on the core protein, and the
influenza A virus (IAV) is considered the main target for drug development, especially since it is the
most devastating in pandemic or epidemic outbreaks [3]. Many efforts have been made to develop
anti-influenza A agents that inhibit each key step of IAV from various resources. Major events that
occur in viral replication of IAV are as follows: (i) attachment of a virion by interaction between the
sialic acid receptor of the host cell and hemagglutinin (HA) on the virion surface; (ii) fusion and
Biomolecules 2020, 10, 711; doi:10.3390/biom10050711
www.mdpi.com/journal/biomolecules

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endocytosis of the virion into the cytoplasm of the host cell; (iii) release of viral ribonucleoprotein (RNP)
into the cytoplasm and nuclear import; (iv) transcription and replication of viral RNA; (v) translation
and protein synthesis; (vi) assembly of virion composition; and (vii) budding and release of a newly
assembled virion by neuraminidase [4].
Pharmaceutical companies have endeavored to develop inhibitors that block any of these
aforementioned steps. Among these targets, blocking viral entry and fusion has been emerging as
a favorable inhibition target because hemagglutinin, especially, HA2 subunit (stem region of HA)
responsible for viral fusion is highly conserved and able to trigger an antibody-mediated immune
response during infection [
even toward various different subtypes of influenza virus [
including cholesterol conjugated short peptide named S-KKWK, chlorogenin 3-O-
derivatives, and fucoidan derivatives have been investigated to date [ 10]. For clinically approved
5
,6
], in which the antibodies induced by HA2 show high cross-reactivity,
]. As a result, several potent HA inhibitors
-chacotrioside
7
β
8–
drugs, umifenovir interrupts viral fusion by targeting viral HA and is being used as an over-the-counter
(OTC) drug in Russia and China [11]. Interestingly, although umifenovir has been used clinically
for more than 30 years in Russia, no reports on emergence of resistant viruses from humans have
been published so far [12]. DAS181, which prevents binding of the influenza virus by cleaving viral
receptors on host epithelical cells, is in phase III clinical trials [13]. Amantadine and rimantadine have
been used to inhibit the release of viral RNP as M2 ion channel inhibitors. However, most influenza
strains have developed resistance to both drugs [14]. Neuraminidase inhibitors, including zanamivir
and oseltamivir, which are most commonly prescribed to treat flu, have been used globally. However,
their use has been facing limitations due to the rapid emergence of resistant influenza strains and the
risk of side effects [15]. Therefore, the discovery of small molecules that inhibit any step of the virus life
cycle and can be used in combination to prevent the rapid occurrence of resistant viruses is still urgent.
Pinus densiflora Siebold and Zucc., commonly known as Korean red pine, is a coniferous evergreen
tree with an irregular or umbrella-shaped crown that can grow up to 100 feet tall. P. densiflora
belongs to the Pinaceae family and is widely distributed in East Asia, including the Russian Far East,
Northern China, Central and Southern Japan, and Korea [16]. Various parts of P. densiflora, such as the
needles, resins and pollen, have been listed in two famous ancient pharmacopoeias, Dong-Eu-Bo-Gam
(Heo, Joon, AD 1713) [17] of Korea and the Compendium of Materia Medica of China (Li, Shizhen,
1578) [18]. Its resin and pollen as medicinal raw materials can still be found in the Korean Herbal
Pharmacopoeia [19]. P. densiflora has been established as having health-promoting properties, including
the treatment of strokes and memory improvement as a nourishing tonic [20
activities such as the improvement of fatigue, depression, anxiety, cancer, and several chronic diseases
have been reported [22 23]. With respect to antiviral properties, potent inhibitory activity of some
,21]. In addition, various
,
fractions from P. densiflora against HSV (herpes simplex virus) has been suggested experimentally [24].
However, to the best of our knowledge, there have been limited studies evaluating the antiviral
properties of P. densiflora, especially against influenza A at the compound level. Regarding its
chemical components, essential oils (0.3–1.3%, antioxidant and antiaging) including α-pinene, β-pinene,
camphene, limonene, borneol (6.8%), bornyl acetate (3.8%), along with cinnamic acid, benzoic acid,
flavonoids, abietane diterpenoid, and stilbenoid (antibacterial and antifungal) have been reported from
its leaves [20,25–27]. It has been reported that the pine cones that contain labdane-type diterpenoids
that have inhibitory activity against acne [28]. Extensive isolation of the compounds from P. densiflora
allowed the purification of 26 compounds with various skeletons, including two previously undescribed
megastigmane glycosides (1 and 2), which contributes to the chemical profiling of constituents in
P. densiflora. Moreover, all isolated compounds were tested by a cytopathic effect (CPE) assay with
H1N1 virus infection, which is an indicator of the potential candidates as being anti-influenza agents.
For selected active candidates, various assays including, Western blot, immunofluorescence assay, flow
cytometry, and NO production assay, were performed to investigate the anti-influenza mode of action.

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2. Materials and Methods
2.1. General Experimental Procedures
Optical rotation values were obtained using a JASCO P-2000 polarimeter (JASCO International Co.
Ltd., Tokyo, Japan). IR spectra were acquired using a Nicolet 6700 FT-IR spectrometer (Thermo Electron
Corp., Waltham, MA, USA). NMR spectra were recorded on an Advance 500 MHz spectrometer
(Bruker, Billerica, MA, USA). HRESIMS data were obtained using an Agilent 6530 Q-TOF (Agilent
Technologies, Inc., Santa Clara, CA, USA). Column chromatography (CC) was performed with silica
gel (63–200 µm particle size, Zeochem, Lake Zurich, Switzerland) and RP-C18 (75 µm particle size,
Nacalai Tesque, Kyoto, Japan). For TLC analysis, RP-18 F254S and silica gel 60 F254 plates, from
Merck (Darmstadt, Germany) were used. A Gilson HPLC system equipped with an Optima Pak C18
column (10
×
250 mm, 10 µm particle size; RS Tech, Seoul, Korea) was used for the purification of
compounds, with a flow rate of 2 mL/min and UV detection at 205 and 254 nm. Industrial-grade
solvents (Daejung Chemical, Siheung, Korea) were used for preprocessing, including extraction
and fractionation, and analytical-grade solvents (Daejung Chemical) were used for purification of
the compounds.
2.2. Plant Material
The cortex and leaves of P. densiflora were collected from the medicinal plant garden of the College
of Pharmacy, Seoul National University, Goyang-si, Gyeonggi-do, Korea (37◦710270N, 126◦810880E).
The samples were botanically authenticated by Prof. Won Keun Oh at the College of Pharmacy,
Seoul National University. A voucher specimen (SNUPMHG-001837) was deposited at the College of
Pharmacy, Seoul National University, Seoul, Republic of Korea.
2.3. Extraction and Isolation
The cortex (1.5 kg) and leaves (2.5 kg) of P. densiflora were extracted separately with MeOH with the
assistance of ultrasonic waves. The combined extracts were then evaporated under reduced pressure
to obtain the crude residue. The crude extracts were dispensed in distilled water and successively
partitioned with EtOAc and n-BuOH. The EtOAc and n-BuOH-soluble fractions were then fractionated
and purified by repeated normal-phase and reverse-phase chromatography, as well as by preparative
HPLC, resulting in the isolation of 26 compounds, including two new megastigmane-type compounds
(1 and 2), 21 diterpenoids, and three flavonoids. The isolation scheme and NMR spectra are reported
in the Supplementary Material (Figures S1 and S2). All compounds in this study were confirmed to
have high purity (≥ 95%).
(2S,9S)-2,9-Dihydroxymegastigman-5-ene-2-O-β
-d-glucopyranoside (1): Colorless gum; [α]2D0-14.1
(c = 0.5, MeOH); IR νmax 3360, 2969 cm−1; HRESIMS m/z 419.2282 [M+HCOO]− (calcd for C20H35O9,
419.2287); 1H and 13C NMR see Table 1.
(2R,9S)-2,9-Dihydroxymegastigman-5-ene-2-O-β
-d-glucopyranoside (2): Colorless gum; [α]2D0-4.7
(c = 0.5, MeOH); IR νmax 3358, 2969 cm−1; HRESIMS m/z 419.2292 [M+HCOO]− (calcd for C20H35O9,
419.2287); 1H and 13C NMR see Table 1.
Table 1. NMR Spectroscopic data for compounds 1 and 2.
1 a
2 a
δH (J in Hz)
No.
δH (J in Hz)
δC
δC
1
2
3
41.5
87.9
27.1
40.6
83.0
23.9
3.43 dd (3.0, 11.0)
2.04 m
3.62 dd (3.0, 10.2)
1.86 m
1.79 m
1.74 m
4
2.05 m
31.7
2.11 td (4.8, 16.8)
2.02 m
31.3

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Table 1. Cont.
1 a
2 a
δH (J in Hz)
No.
δH (J in Hz)
δC
δC
5
6
7
127.3
137.3
26.0
127.1
137.5
26.1
2.15 dt (4.5, 13.0)
2.00 m
2.21 dt (4.8, 12.0)
1.98 m
8
9
10
11
12
13
Glu1
Glu2
Glu3
Glu4
Glu5
Glu6
1.51 m
3.72 m
1.18 d (6.0)
1.15 s
1.05 s
1.61 s
4.35 d (8.0)
3.22 t (8.0)
3.36 m
40.7
69.2
23.3
22.5
26.4
19.7
106.6
75.7
78.3
71.7
77.7
62.8
1.53 m
3.73 m
1.19 d (6.6)
1.14 s
40.8
69.2
23.3
22.7
26.7
19.7
101.8
75.1
78.3
71.9
77.8
63.0
1.04 s
1.64 s
4.36 d (7.8)
3.22 dd (7.8, 9.0)
3.39 t (9.0)
3.31 t (9.0)
3.26 m
3.28 m
3.26 m
3.86 dd (2.0, 11.5)
3.68 dd (5.5, 11.5)
a
3.88 dd (1.8, 11.4)
3.69 dd (6.0, 11.4)
Recorded in methanol-d4 at 500 MHz.
2.4. Enzymatic Hydrolysis of 2
A solution of
-glucosidase from almonds (10.1 mg), and the solution was incubated at 37 ◦C for 24 h [29].
The mixture was further incubated after the addition of -glucosidase (5.3 mg) for 28 h. The reaction
2 (4.4 mg) in 20 mM acetate buffer (approximately pH 5.0, 0.5 mL) was treated
with
β
β
mixture was dried under a N2 stream after cooling and subjected to silica gel column chromatography
(16.5 g, Φ = 15 mm, L = 25 cm). The residue was eluted from n-hexane and acetone (3/1) to methanol.
The aglycone 2a (1.2 mg) was obtained in fractions 18–20. Aglycone (2a): Colorless syrup, [α]2D0+15.3
(c = 0.2, CHCl3); 1H-NMR (CH3OD, 400 MHz)
δ
: 3.41 (dd, J = 10.0, 3.6 Hz, H-2), 1.71 (m, H-3), 2.03
(overlapped, H-4), 2.20 (m, H-7a), 1.95 (m, H-7b), 1.50 (m, H-8), 3.71 (m, H-9), 1.17 (d, J = 6.4 Hz, H-10),
0.96 (s, H-11), 1.07 (s, H-12), 1.61 (s, H-13); 13C-NMR (CH3OD, 100 MHz)
: 41.3 (C-1), 76.9 (C-2), 27.9
δ
(C-3), 31.5 (C-4), 127.1 (C-5), 137.3 (C-6), 26.1 (C-7), 40.7 (C-8), 69.2 (C-9), 23.3 (C-10), 21.8 (C-11), 26.4
(C-12), 19.8 (C-13); HRESIMS (positive-ion mode): m/z: 213.1853 [M+H]+ (calcd C13H25O2: 213.1849)
(Figures S13–S16).
2.5. Preparation of 2,9-Di-(S)-MTPA Ester (2b) and 2,9-Di-(R)-MTPA Ester (2c) from 2a
The absolute configuration of C-9 in
After transferring 2a (0.4 mg, 1.9 mol) in dry pyridine (4.8
was added to the mixture for dissolution. Then, R-(–)-MTPA-Cl (15
2
was determined by modified Mosher’s analysis [30].
L, 60 mol), 0.5 mL of anhydrous CH2Cl2
L, 78.3 mol) was added to
µ
µ
µ
µ
µ
the mixture, and the reaction was initiated at ambient temperature for 6 h. The progress of the
reaction was monitored by TLC using n-hexane and acetone (3:1). After the reaction was complete,
the resulting solution was dried under a N2 stream and dissolved in distilled water, followed by
repeated partitioning with CH3Cl. The combined CH3Cl extracts containing 2,9-di-(S)-MTPA ester
(
2b) were dried, and 1H-NMR and 1H-1H COSY were recorded in CDCl3. 2,9-Di-(R)-MTPA ester (2c
)
1
was also prepared in a similar manner as 2b. Compound 2b: H-NMR (600 MHz, CDCl3)
δ: 4.95
(dd, J = 9.6, 3.0 Hz, H-2), 1.83-1.91 (m, H-3), 1.99-2.06 (overlapped, H-4), 2.05 (overlapped, H-7a),
1.93 (overlapped, H-7b), 1.66 (m, H-8a), 1.60 (m, H-8b), 5.10 (m, H-9), 1.28 (d, J = 6.0, H-10), 0.89
(s, H-11), 0.91 (s, H-12), 1.54 (s, H-13); HR-ESI-MS (positive-ion mode): m/z: 662.2899 [M + NH4]+
(calcd C33H42F6NO6: 662.2911) (Figure S17). Compound 2c
(dd, J = 9.0, 3.0 Hz, H-2), 1.77-1.85 (m, H-3), 1.92-1.98 (overlapped, H-4), 1.92 (overlapped, H-7a),
: δ: 4.89
1H-NMR (600 MHz, CDCl3)

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1.88 (overlapped, H-7b), 1.61 (m, H-8a), 1.56 (m, H-8b), 5.13 (m, H-9), 1.35 (d, J = 6.6, H-10), 0.87
(s, H-11), 0.92 (s, H-12), 1.46 (s, H-13); HR-ESI-MS (positive-ion mode): m/z: 662.2911 [M + NH4]+
(calcd C33H42F6NO6: 662.2911) (Figure S18).
2.6. Sugar Analysis of Compounds 1 and 2
The absolute configuration of the sugars was determined by means of HPLC analysis after
derivatization, which was proposed by Tanaka [31]. A solution of
1 (0.6 mg) in 2 M hydrochloric acid
was incubated at 100 ◦C for 4.5 h. The hydrolysates were neutralized with Na2CO3 and concentrated
under a N2 stream. l-Cysteine methyl ester (0.6 mg) in pyridine was added to the dried residue
and subjected to incubation at 60 ◦C for 1 h. Then, 0.6 mg of phenyl isothiocyanate was added to
the resulting mixture and incubated under the same conditions for 1 h. After cooling and filtration,
10
µL of the solution was directly analyzed by reverse-phase HPLC [HPLC: Thermo Fisher Ultimate
3000 system (Germering, Germany); YMC Triart C18 column (4.6
Co., Kyoto, Japan); conditions were as follows: MeCN: H2O = 25: 75 (v:v); a diode array detector at
250 nm; and a flow rate of 0.8 mL/min]. The UV pattern and retention time of the derivatized sugar in
×
250 mm, 5 m particle size; YMC
µ
compound
of the sugar in compound
d-glucose is 19.753 min, whereas that of l-glucose is 17.663 min. As derivatives of compounds
1
were compared to those of derivatized authentic d,l-glucose. The absolute configuration
was determined in a similar manner. The retention time of derivatives of
and
were proven
2
1
2
were eluted at 19.767 min and 19.800 min, respectively, the sugar moieties of both
1 and 2
to be in the d-configuration.
2.7. Cell Culture and Virus Stock
Madin–Darby Canine Kidney (MDCK) cells and RAW264.7 cells were provided by American
Type Culture Collection (ATCC, Manassas, VA 20108, USA). The cells were maintained in Dulbecco’s
modified Eagle’s medium (DMEM) (HyClone, Logan, UT) supplemented with 10% fetal bovine serum
(FBS) (HyClone, Logan, UT), 100 U/mL penicillin and 100 µg/mL streptomycin (GIBCO-BRL, Grand
Island, NY, USA). Influenza viruses (H1N1 A/PR/8/34 virus, H9N2 A/chicken/Korea/01210/2001 virus)
were obtained from Choong Ang Vaccine Laboratory, Korea and stored at −80 ◦C.
2.8. Cytopathic Effect (CPE) Inhibition Assay
Cytopathic effect and cytotoxicity assays were performed as previously described [32]. Briefly,
MDCK cells were grown in 96-well plates for 24 h. The cells were inoculated with influenza
viruses (H1N1 A/PR/8/34 virus or H9N2 A/chicken/Korea/01210/2001 virus) at 0.01 MOI using DMEM
containing 0.15 µg/mL trypsin and 5 µg/mL bovine serum albumin (BSA) (Sigma, St Louis, MO, USA).
After 2 h of incubation, the cells were washed with phosphate-buffered saline (PBS) (TaKaRa, Japan)
and the medium was replaced with new medium containing the test compounds. The cells were
continually incubated for 3 days at 37 ◦C under a 5% CO2 atmosphere. Then, the medium was replaced
with fresh DMEM, and 20
bromide (MTT) solution (Sigma, St Louis, MO, USA) was added to each well. After 4 h of incubation,
the supernatant was removed and formazan crystals were dissolved in 100 L of DMSO. The absorbance
µL of 2 mg/mL 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
µ
was measured at a wavelength of 550 nm. Similarly, the cytotoxicity assay was carried out using
96-well plates. After 24 h of incubation, MDCK cells were washed with PBS and treated with the test
compounds. The culture was treated with 20 µL of 2 mg/mL MTT solution in each well after 2 days of
incubation. The final steps followed those described above.
2.9. Quantitative Real-Time PCR
MDCK cells were maintained in 6-well plates and infected with the H1N1 A/PR/8/34 virus for
2 h. The cells were washed twice with PBS and the medium was replaced with new media containing
the test compounds. The TRIzol method was used to isolate total RNA from the cells after 24 h of
incubation [33]. Total RNA was reverse transcribed using random primers (Invitrogen, USA) according

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to the manufacturer’s instructions. Real-time PCR was performed using selective primers for H1N1
(Table S5) (Enotech, Korea) using the Maxima SYBR Green qPCR master mix 2X kit (Thermo Sci.,
Rockford, IL, USA). The StepOnePlusTM real-time PCR system was used with cycling conditions as
follows: 95 ◦C for 10 min, followed by 40 cycles of 95 ◦C for 15 sec and 60 ◦C for 1 min. The data were
calculated using StepOne software v2.3 (Applied Biosystems).
2.10. Western Blotting Analysis
The cultures were prepared similarly to the cultures prepared for the quantitative real-time PCR
assay. However, after 24 h of incubation, the cells were washed with cold PBS and stored at
80 ◦C
−
until use. The cells were then lysed with lysis buffer [50 mM Tris-HCl (pH 7.6), 120 mM NaCl, 1 mM
EDTA, 0.5% NP-40, and 50 mM NaF]. The protein concentrations of the lysates were determined using
a protein assay kit (Bio-Rad Laboratories Inc., USA) and electrophoresed on 12% SDS-polyacrylamide
gels [34]. The gels were electrotransferred to polyvinylidene fluoride membranes (PVDF 0.45 µm,
Immobilon-P, USA). The membranes were blocked with a 5% skim milk solution (Becton Dickinson,
USA) and then incubated with primary antibodies: neuraminidase (NA; Gene Tex, San Antonio, TX,
USA), hemagglutinin (HA; Sigma, St Louis, MO, USA) or mouse monoclonal actin (Abcam, Cambridge,
UK). After overnight incubation, the membranes were further incubated with secondary antibodies
(Goat anti-Rabbit IgG (H + L) HRP or Goat anti-Mouse IgG (H + L) HRP; Thermo Sci., Rockford,
IL, USA) for 2 h and detected by a chemiluminescence Western blotting detection kit (Thermo Sci.,
Rockford, IL, USA) using Image Reader LAS-4000 software.
2.11. Immunofluorescence Assay
MDCK cells were grown on sterilized glass coverslips for 24 h. Then, the cells were infected
with the H1N1 A/PR/8/34 virus for 2 h, washed twice with PBS and the medium was replaced with
new media containing the test compounds. The cultures were continually incubated at 37 ◦C under
a 5% CO2 atmosphere for 24 h. After washing with PBS (pH 7.4), the cells were fixed with a 4%
paraformaldehyde solution for 30 min at room temperature and blocked with a 1% BSA solution for
1 h. The cells were incubated overnight with a rabbit monoclonal antibody against NA (GeneTex,
San Antonio, TX, USA) diluted 1:50 in PBS (pH 7.4). After being washed with PBS, the cells were
incubated with a secondary FITC-conjugated Goat anti-Rabbit IgG antibody (Abcam, Cambridge,
UK). After washing with PBS (pH 7.4) [34], the cells were stained with 500 nM DAPI solution for
10 min at room temperature and washed again with PBS (pH 8.0). Slides were mounted and imaged
by fluorescence microscopy (Olympus ix70 Fluorescence Microscope, USA).
2.12. Flow Cytometric Analysis of the Cell Cycle
MDCK cells were maintained in 36-mm culture dishes and infected with the H1N1 A/PR/8/34
virus for 2 h. After washing with PBS and replacing the media with new media containing the test
compounds, the cultures were incubated for 3 days at 37 ◦C under a 5% CO2 atmosphere. The cell
suspensions were collected from adherent cells and detached cells. After that, the cells were washed
with cold PBS and fixed with precooled 70% ethanol solution. After overnight incubation at 4 ◦C,
the cells were stained with 20 µg/mL PI (propidium iodide) in PBS solution for 30 min at room
temperature [35]. The cellular DNA contents were analyzed using a flow cytometer (BD, FACSCalibur,
San Jose, CA, USA). Data were collected for at least 10,000 cells per sample. In each phase of the
viral-infected cell cycle, the distribution of cells is displayed using histograms.
2.13. Neuraminidase Inhibition and Kinetic Assays
NA inhibition and kinetic assays were performed as previously described [32
activity was measured using 20-(4-methylumbelliferyl)-
-D-N-acetylneuraminic acid (4-MU-NANA)
(Sigma-Aldrich Co., USA) as the fluorescent substrate in 96-well plates. Briefly, 10 L of test compounds
diluted in enzyme buffer (32.5 mM 2-(N-morpholino) ethanesulfonic acid, 4 mM CaCl2, pH 6.5)
,36]. In general, NA
α
µ

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and 10
A/chicken/Korea/01210/2001 virus, wild-type (wt) pandemic 2009 H1N1 virus (A/California/07/2009),
oseltamivir-resistant virus (H274Y mutation) were coincubated for 30 min at 37 ◦C. Then, 30
L of
µL of virus suspension containing NAs from four subtypes (H1N1 A/PR/8/34 virus, H9N2
µ
42
µ
M 4-MU-NANA su◦bstrate in enzyme buffer was added to each well. The enzymatic reactions
were maintained at 37 C for 2 h and quenched with 150 µL of stop solution (25% EtOH, 0.1 M
glycine, pH 10.7). The fluorescence was measured with an excitation wavelength of 360 nm and
an emission wavelength of 440 nm using a SpectraMax GEMINI XPS microplate reader (Molecular
Devices, Sunnyvale, CA, USA). The enzymatic kinetic assay was performed in a similar manner as that
of the NA inhibition assay. The product 4-methylumbelliferone was measured immediately without
adding stop solution. The inhibition type was analyzed using SigmaPlot 11.0 (SPCC Inc., Chicago,
IL, USA).
2.14. Cell Protection Assay for H1N1 Infection via Coincubation
In order to evaluate the effect of coincubation of compounds
5
and 24 with the host cells, MDCK
cells were preincubated with the two test compounds (20
µ
M) for 4 h at 37 ◦C, followed by H1N1
A/PR/8/34 virus infection [37]. After 1 h incubation at 37 ◦C under a 5% CO2 atmosphere, the cells
were washed twice with PBS. Then, the medium was replaced with fresh medium and the cells were
incubated for 3 days. The antiviral activity was evaluated by a cytotoxicity assay.
2.15. Virus Particles Assay for H1N1 Infection via Coincubation
In order to evaluate the effect of coincubation of compounds
infectivity, H1N1 A/PR/8/34 virus diluted with DMEM containing 0.15
BSA was incubated with or without the test compounds (20
5
and 24 with virus regarding its
g/mL trypsin and 5 g/mL
M) for 1 h at 4 ◦C [37]. Then, MDCK cells
µ
µ
µ
were incubated with the viral medium for 1 h at 37 ◦C under a 5% CO2 atmosphere. The monolayers
of cells were washed twice with PBS and incubated for 3 days in new medium. The antiviral activity
was evaluated by a cytotoxicity assay.
2.16. Nitric Oxide (NO) Production Assay
DMEM without phenol red (WelGene, Korea) was used for the NO production assay. Briefly,
RAW 264.7 cells were seeded into 12-well plates and incubated for 1 day at 37 ◦C under a 5% CO2
atmosphere. The cells were treated with the test compounds for 10 h before infection with the H1N1
A/PR/8/34 virus at high MOI (1.0) and low MOI (0.01) for 1 h [38]. The medium was replaced with
new medium and the cells were further incubated for 12 h. Nitric oxide production was evaluated by
using the Griess reagent method, and the absorbance was measured at 540 nm [39]. For the NO assay
stimulated by lipopolysaccharide (LPS), RAW 264.7 cells were pretreated with the test compounds for
10 h, and 100 ng/mL LPS was added to the cells. After 12 h of incubation, NO production was measured
as described above. Inducible nitric oxide synthase (iNOS) expression was measured by Western blot.
Briefly, RAW 264.7 cells were maintained in 6-well plates for 24 h. The cells were preincubated with test
compounds for 10 h, and then 100 ng/mL LPS was added to the culture. The cells were collected after
12 h of incubation. The Western blot method was performed as described above with a monoclonal
iNOS antibody (Thermo Sci., Rockford, IL, USA).
2.17. Simulation of Binding Affinity via Molecular Docking
In order to evaluate the binding affinity between the ligands and influenza viral polymerase or
NA in silico, the docking study using Discovery Studio 4.0/CDOCKER (Accelrys, San Diego, CA) was
performed [40]. The crystal structure of the influenza A virus nucleoprotein (NP; PDB ID: 3RO5),
the PA-PB1 complex from influenza virus RNA polymerase (PDB ID: 2ZNL), the 2009 pandemic
H1N1 NA complex (PDB ID: 3TI4), and the N1 NA H274Y (PDB ID: 3CL0) were supplied from
the Protein Data Bank (http://www.pdb.org) [41
–44]. Ligand interaction affinity was calculated by
CDOCKER interaction energy and interacting bonds such as van der Waals, salt bridge, attractive

Biomolecules 2020, 10, 711
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charge, conventional hydrogen bond, carbon hydrogen bond, pi-cation, pi-anion, pi-alkyl, and pi-pi
T-shaped bonds.
2.18. Statistical Analysis
The results were obtained from three independent experiments. Statistical calculations were
conducted with Sigma Plot Statistical 11.0, SPSS Statistics 23, StepOne software v2.3, and a LAS 4000
luminescent image analyzer. The results were calculated as the means
experiments (* p < 0.05, ** p < 0.01, and *** p < 0.001 using one-way ANOVA statistics).
±
SD of two to three independent
3. Results and Discussion
3.1. Isolation and Structural Elucidation of Compounds Isolated from P. densiflora
The cortex and leaves of P. densiflora were extracted with MeOH. Following ioassay-guided
fractionation, the EtOAc and n-BuOH soluble fractions were then fractionated and purified by column
chromatography and preparative HPLC, resulting in the isolation of 26 compounds, including two
new megastigmane-type compounds (1 and 2), 21 diterpenoids (3–23), and three flavonoids (24–26).
Compound
1 was isolated as colorless gum with the molecular formula C19H34O7, which was
established from the negatively charged ion [M + HCOO]− at m/z 419.2282 (calcd. for C20H35O9,
419.2287) in the HRESIMS spectrum (Figure S3), corresponding to three double-bond equivalents.
The 1H NMR data showed the presence of three singlet methyl groups [(δH 1.15 (s, H-11), δH 1.05
(s, H-12) and δH 1.61 (s, H-13)], one doublet methyl group at δH 1.18 (d, J = 6.0 Hz, H-10), two
oxygenated methine protons [δH 1.61 (dd, J = 3.0, 11.0 Hz, H-2) and δH 3.72 (m, H-9)], and sugar ring
protons at δH 3.22 - 3.86 (H-2’ - H-5’) along with one anomeric doublet signal at δH 4.35 (d, J = 8.0 Hz)
(Figure S4). The 13C NMR spectrum showed 13 resonances except for the sugar moiety, including
two sp2 carbons at δC 127.3 and 137.3, two oxygenated sp3 carbons at δC 87.9 and 69.2, and nine
sp3 carbons in the δC 20-50 ppm range (Figure S5). The characteristic deshielded chemical shifts
at δH 3.72 (m)/δC 69.2 of H-9/C-9, the number of carbons of the core structure, and the existence of
1
a long chain (H7-H8-H9-H10) and a fragment H2-H3-H4 in the structure supported by the H-1H
COSY correlations demonstrated that compound
1 is an ionone derivative of the megastigmane
class (Figure S7). As oxygenated C13-norisoprenoids, megastigmanes are classified into two series,
the damascene series (C-7) and the ionone series (C-9), according to the position of oxygenation.
The linkage of the sugar molecule with the aglycone was determined by the HMBC correlation from
H-1’ (δH 4.35) to C-2 (δC 87.9) (Figure S6). The planar structure of
1 was also supported by comparison
of NMR data with those of the previously reported platanionoside J after hydrolysis, except for the
difference in chemical shift of C-2 (δC 87.9 vs. 77.0) where glycosylation occurred in 1 [45].
Compound
2 was also isolated in the same fraction as 1 by preparative HPLC. Its molecular
formula was determined to be C19H34O7 by a negative peak [M + HCOO]− at m/z 419.2294 (calcd
for C20H35O9, 419.2287) in the HRESIMS spectrum (Figure S8). Interestingly, the 1H and 13C NMR
data of
2 were found to be similar to those of 1 (Table 1), indicating that those two compounds might
share the same skeleton but differentiate from each other in absolute configuration. Compound
2
was also determined to be of megastigmane type by four methyl signals at δH 1.14 (s, H-11), 1.04
(s, H-12), 1.64 (s, H-13) and 1.19 (d, J = 6.6 Hz, H-10) and 13 carbons constituting the aglycone and
sugar moiety, which was observed by an anomeric proton signal resonating at δH 4.36 (d, J = 7.8 Hz)
and an anomeric carbon at δC 101.8 (Figures S9–S12). By spectral comparison with
the absolute configuration of compound lies at the C-2 position. The spectral differences originating
from C-2 between and were observed in the C-1, 2, 3, and C-1’ positions. Compound exhibited its
carbon resonances of C-1, 2, 3 and 1’ at δC 41.5, 87.9, 27.1 and 106.6, respectively, whereas compound
showed its corresponding resonances at δC 40.6, 83.0, 23.9, and 101.8, which indicated the possibility
of opposite stereochemistry of the C-2 position (Table 1). As the sugar moieties of and were
determined to be -d-glucose (Table 1) [46] and
-d-glucose by 3J1’,2’, which is distinct from that of
1, the difference in
2
1
2
1
2
1
2
β
α

Biomolecules 2020, 10, 711
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the derivatizing method for HPLC analysis (Figure S21) [31], the absolute configuration of C-2 in
1
and
This can be applied to
and
The C-1’ (the first carbon of the sugar moiety) resonance of glucosides of S-alcohols is deshielded,
appearing at δC 106.4 0.5 ppm, whereas this carbon resonance in an R-alcohol appears in a relatively
upfield region, δC 101.6 0.9 ppm. Moreover, the signal of the chiral -carbon where hydroxylation
occurs in alcohols is generally displaced by +7.0 0.6 ppm upon glycosylation, whereas a more
remarkable deshielding of the -carbon signal is observed (by approximately +10 - 11 ppm) in the
case of -d-glucosides of secondary S-alcohols containing at least one equatorial alkyl substituent
on one of its -positions. Since the carbon resonances of C-1’ in compounds and appeared at δC
106.6 and 101.8 ppm, respectively, and the chemical shift difference of the C-2, -carbons, between
δC 83.0) and the aglycone of δC 76.9), the absolute configurations of C-2 in and were readily
determined to be S and R, respectively. Compounds and possess the opposite configuration
2
could be determined by the application of
β-d-glucopyranosylation-induced chemical shifts [47].
β
-d-glucosides of secondary alcohols, especially isoprenoid-
β-d-glucopyranosides
β
-d-mannopyranoside, having at least one equatorial alkyl substituent on one of its β-carbons.
±
±
α
±
α
β
β
1
2
β
2
(
2
(
1
2
1
2
at C-2 and their NMR spectra were not identical, suggesting that they are in a diastereomeric
relationship, and they will have the same stereochemistry at C-9. Therefore, elucidating the absolute
configuration of C-9 of either compound will enable the confirmation of C-9 of the other compound.
As compound
to enzymatic hydrolysis and modified Mosher’s analysis [29
and S20), the stereochemistry at C-9 was determined to be 9S and that of C-2 was also confirmed
to be 2R, initially derived from glycosylation-induced chemical shifts. Hence, compounds and
were determined as (2S,9S)-2,9-dihydroxymegastigman-5-ene-2-O- -d-glucopyranoside ( ) and
(2R,9S)-2,9-dihydroxymegastigman-5-ene-2-O-β-d-glucopyranoside (2).
2
was isolated with a relatively higher yield compared to
1, compound 2 was subjected
,
30]. As a result (Figure 1, Figures S19
1
2
β
1
Figure 1.
(A) Key HMBC (arrows) and COSY (bold) correlations of compounds 1 and 2. (B) Data from
the modified Mosher’s method for 2a.
The structures of all known compounds isolated from the cortex and leaves of P. densiflora
were elucidated by extensive analyses of spectroscopic data including NMR and MS
spectra and by comparison with previously reported data as follows:
acid [48]; ( ) 12-hydroxydehydroabietic acid [49]; ( ) 7 -methoxydehydroabietic acid [50];
15-hydroxydehydroabietic acid 51]; 16-nor-15-oxodehydroabietic acid 52 53];
abieta-8,11,13,15-tetraen-18-oic acid 53]; -hydroxyabieta-8,11,13,15-tetraen-18-oic
54]; 10 karamatsuic acid 11 palustric acid 56];
12) 12-methoxy-7,13-abietadien-18-oic acid [57]; (13) 7-oxo-13 -hydroxyabiet-8(14)-en-18-oic acid [58];
14) 9,13 -epidioxy-8(14)-abieten-18-oic acid [59]; (15) 14 ,15-epoxyabiet-9(11)-en-12-oxo-18-oic
52]; (17) 8(14)-podocarpen-7,13-dion-18-oic
(3) dehydroabietic
4
5
α
(
(
6
8
)
)
[
(
7
)
[ ,
[
(9) 7α
acid
[
(
)
[
55];
(
)
[
(
(
β
β
α
acid [60]; (16) 8(14)-podocarpen-13-on-18-oic acid [51
acid 51 52]; 18 4-epi-trans-communol 61];
20 4-epi-trans-communic acid 63]; 21
,
[
,
(
)
[
(
(
19
)
4-epi-trans-communal
[
[
62];
64];
(
)
[
)
3β-hydroxy-12,13(E)-biformen

Biomolecules 2020, 10, 711
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(
22
)
18
α
,3
α
-dihydroxy-12,13(E)-biformen
[
65];
(
23
)
(13S)-15-hydroxylabd-8(17)-en-18-oic
acid [66]; (24) ampelopsin [67]; (25) 5,7,40-trihydroxy-3-methoxy-6-C-methylflavone [68]; and
(
26) 5,40-dihydroxy-3,6,7-trimethoxy-8-C-methylflavone [69] (see Supplementary Material) (Figure 2).
Figure 2. Chemical structures of compounds isolated from Pinus densiflora.
3.2. Influenza Viral Inhibition and Structure-Activity Relationships (SARs) of Isolated Compounds from
P. densiflora
The 26 compounds isolated from P. densiflora (
antiviral activities by using CPE inhibition and NA inhibition assays. As shown in Figure 3A and Figure
S22, compounds 10 13 16 17 20 23, and 24 showed significant inhibition of the cytopathic
effect induced by the influenza H1N1 virus at 10 M with ribavirin as a positive control. Especially,
the inhibition activity of compound was comparable to that of ribavirin at the same concentration.
Interestingly, nine compounds out of the ten candidates are diterpenoids, and our results are very
consistent with the antiviral activity results of some diterpenoids in previously reported papers [70 72].
1–26) (Figure 1, Figure 2) were screened for their
5,
6,
8
,
,
,
,
,
,
µ
5
–
On the other hand, compounds 24–26 were selected as strong candidates from the NA inhibition
assay (Figure 3B). The flavonoid substances from P. densiflora inhibited NA, and these results can be
effectively explained by previously reported studies about the structure–activity relationships (SARs) of
compounds in the flavonoid class [73]. Compounds 5 and 24, which showed the strongest activity from
the CPE inhibition assay and NA inhibition assay, respectively, were shown to have CPE inhibitory
activity in a dose-dependent manner against the H1N1 and H9N2 subtypes (Figure 3C and Figure S23).
From the assessment of effects on cell protection and viral particle neutralization, neither compound
protected cells after they were preincubated in the cell protection assay, and they did not neutralize the
virulent particles by direct binding (Figure S24). The effect of compound 5 on the cellular apoptosis
in infected MDCK cells was also evaluated using flow cytometric analysis. As shown in Figure S27,
the percent of apoptotic cells (Sub-G1) significantly decreased from 72.22 to 51.36 and 12.91% after
treatment with compound 5 (5 and 20 µM, respectively). These results also suggest that compound 5
has antiviral activity, in part because it inhibits virus-induced cell apoptosis.
To discuss the structure–activity relationships (SARs) of diterpenoids isolated from the P. densiflora
cortex and leaves, 21 compounds (
tricyclic phenolic ( 10), tricyclic (11
data of group 1 ( 10) (Figure 3A and Figure S22) indicated that the methoxylation at C-7 (R2) could
improve the antiviral properties and reduce the cytotoxicity (when comparing compound with
and ). Compounds 10, which replaced the isopropyl group with other groups, still maintained an
3–
23) were divided into three groups based on abietane-type
3–
–
17), and labdane-type bicyclic diterpenoids (18 23). Biological
–
3–
5
3
4
6–

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antiviral effect. However, the presence of the hydroxy moiety at C-7 slightly increased the cytotoxicity
(comparing compound with ). The antiviral profile of group 2 (11 17) indicated that the existence
of one or two ketone groups at C-7 or C-13 significantly contributed to the anti-influenza activity of
the abietane type (comparing compounds 13 16, and 17 with 11 12 14, and 15). The final group
18 23) demonstrated that substitution of the carboxyl group at C-4 clearly increased H1N1 inhibition.
However, replacement of the carboxyl moiety with other groups significantly reduced the antiviral
8
9
–
,
,
,
(
–
properties, while the cytotoxicity significantly increased (comparing compounds 20 and 23 with 18
, 19
and 21).
Figure 3. Antiviral activities of isolated compounds against the H1N1 A/PR/8/34 virus in the cytopathic
effect (CPE) inhibition and neuraminidase (NA) inhibition assays. (
was evaluated by the effects of all isolated compounds ( 26) and ribavirin (Rib), a positive control,
at 10 M with the use of the CPE inhibition assay. ( ) The inhibitory effects of compounds 26 at
50 M and oseltamivir (Oselt; 100 nM), a positive control, on NA from H1N1. ( ) The influence of
compounds and 24 (5, 10, and 20 M) on cell viability was investigated using the CPE inhibition
assay. Values are expressed as the mean SD of three independent experiments, * p < 0.05, ** p < 0.01,
A) The percent cell survival
1–
µ
B
3–
µ
C
5
µ
±
and *** p < 0.001 compared to the virus control group (one-way analysis of variance followed by a
two-tailed Student’s t-test).

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3.3. The Effects of Diterpenoids on mRNA and Protein Expression during H1N1 Replication
During the viral life cycle, there are several proteins (NA, HA, matrix, NP) that play key roles
for replication of the influenza virus. Therefore, inhibition of the synthesis of these proteins could be
potentially promising targets for influenza antiviral drug discovery. Based on the CPE inhibition data
in Figure 3A, some candidates (compounds 5, 8, 10, 20, 23 and 24) were obtained and screened for NA
and HA expression by Western blot analysis. After 2 h of infection with the H1N1 virus, the cells
were treated with the test compounds at various concentrations with ribavirin as a positive control.
The changes in NA and HA expression were analyzed after 24 h of incubation. As shown in Figure 4A,
compounds
compound 24 did not show clear inhibition. Compound
production of NA and HA in a dose-dependent manner at 5, 10 and 20
5
,
8
,
10
,
20 and 23 significantly decreased the protein expression of NA and HA, while
was also found to significantly decrease the
M. These aforementioned
5
µ
results were supported by an immunofluorescence assay (Figure 4B). The green fluorescence of tagged
NA was significantly detected in the cell cytoplasm in the viral-infected cells, whereas the fluorescence
was not detected in the noninfected cells. Interestingly, when H1N1-infected cells were treated with
compound
5
at different concentrations (5, 10, and 20
µ
M), the green fluorescence similarly decreased
M). It was further confirmed by
5 clearly reduced the mRNA expression of NA and HA at the early stage
in the cell cytoplasm compared to the ribavirin-treated group (10
real-time PCR that compound
of the viral cycle (Figure 4C).
µ
Figure 4. Relative mRNA and protein viral expression levels decreased after treatment with several
compounds from P. densiflora. (A) Madin–Darby Canine Kidney (MDCK) cells were infected with the
H1N1 virus for 2 h and then treated with compounds
After 24 h of incubation, viral NA and HA were evaluated by Western blot analysis. Data are presented
as the mean SD (n = 2–4), * p < 0.05, ** p < 0.01, and # p < 0.05, ## p < 0.01 compared to the NA and
HA virus control groups, respectively. ( ) Compound reduced NA in the cytoplasm of viral-infected
cells as measured by the immunofluorescence method. After infection with the H1N1 virus for 2 h,
MDCK cells were incubated with compound or ribavirin as a positive control for 24 h. The cells were
then fixed, stained, and visualized using a fluorescence microscope. ( ) Compound decreased the
mRNA levels of NA and HA induced by virus replication in MDCK cells as assessed by real-time PCR.
Values are expressed as the mean SD (n = 3), *** p < 0.001, and # p < 0.05, ## p < 0.01 compared to the
NA and HA virus control groups, respectively.
5, 8, 10, 20, 23, and 24 at various concentrations.
±
B
5
5
C
5
±

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3.4. Detailed Neuraminidase Inhibition Assay of Compound 24 against Various Subtypes of Influenza A Virus
Based on the screening data of the isolated compounds in Figure 3B, compound 24 was examined for
detailed inhibitory NA activity [(including H1N1 A/PR/8/34 virus, H9N2 A/chicken/Korea/01210/2001
virus, wild-type (wt) pandemic 2009 H1N1 virus (A/California/07/2009), oseltamivir-resistant virus
(H274Y mutation)]. As shown in Table 2, compound 24 exhibited moderate activity on the percent NA
activity, ranging from 35.34
±
0.97 to 87.33
±
1.67% in a dose-dependent manner (5, 10, and 20.0 µM).
In brief, upon comparison of efficacy of compound 24 with that of oseltamivir, NA activity of H1N1
was inhibited by about 50% by 20 µM of compound 24, whereas oseltamivir inhibited around 50% at
100 nM. In case of NA of H9N2, NA activity was inhibited around 65% upon treatment of compound
24 at 10
compound 24 showed around 40% enzyme inhibitory activity at 20
activity was reduced by 100 nM of oseltamivir. Based on our previously reported paper [32], the IC50
of oseltamivir against NA from H274Y was 7.42 1.35 M, which is consistent with our present data,
but compound 24 inhibited around 53% of inhibitory activity at 20 M. Notably, the inhibitory activity
µM, whereas oseltamivir showed around 90% of inhibitory activity even at 100 nM. Similarly,
µM, whereas 60% of enzymatic
±
µ
µ
of oseltamivir (100 nM) against the oseltamivir-resistant novel H1N1 (H275Y mutant) decreased
greatly compared to the activity against H1N1 A/PR/8/34 and novel H1N1 (wt) (from 49.90 ± 3.74 and
38.57 ± 4.32 to 91.10
±
4.07%, respectively), whereas compound 24 retained its inhibitory activity even
against the oseltamivir-resistant novel H1N1 (from 48.40
±
1.35 and 58.76
±
1.85 to 57.22
±
1.11%,
respectively). Compound 24 was further assessed for its inhibitory mode against NA from H1N1 and
H9N2 using a double-reciprocal Lineweaver–Burk plot. The result in Figure 5 shows that compound
24 was found to be a noncompetitive inhibitor in both enzyme subtypes (H1N1 and H9N2), because
raising the concentration of substrate resulted in the convergence of lines that intersected at a nonzero
point on the negative x-axis. Thus, compound 24 may allosterically interrupt NA activity that cleaves
the binding between the sialic acid residues of the receptor of cells and a newly formed virion [74].
Table 2. Percent influenza NA activity (compared with DMSO as a control).
Comp. Name
H1N1
H9N2
H1N1 (wt)
H274Y
DMSO
100 ± 2.69
49.90 ± 3.74
75.93 ± 1.17
62.55 ± 0.94
48.40 ± 1.35
100 ± 0.62
10.79 ± 0.18
64.19 ± 1.92
49.79 ± 1.74
35.34 ± 0.97
100 ± 1.04
38.57 ± 4.32
87.33 ± 1.67
76.23 ± 2.67
58.76 ± 1.85
100 ± 6.51
91.10 ± 4.07
76.73 ± 1.59
72.38 ± 6.96
57.22 ± 1.11
Oseltamivir (100 nM)
Comp. 24 (5 µM)
Comp. 24 (10 µM)
Comp. 24 (20 µM)
Figure 5.
A/Chicken/Korea/O1310/2001 enzymes. Lineweaver–Burk plots for the inhibition of compound
24 ) at three concentrations (C1 = 10 M, C2 = 20 M, and C3 = 40 M) on NA from the H1N1 virus
Inhibition mode of compound 24 on the NA H1N1/PR/8/34 and H9N2
(A
µ
µ
µ

Biomolecules 2020, 10, 711
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and (B) at three other concentrations (C1 = 5 µM, C2 = 10 µM, and C3 = 20 µM) on NA from the
H9N2 virus for hydrolysis of the substrate. The values were analyzed in three replicates at each
substrate concentration.
3.5. Molecular Docking Simulation of Compounds 5 and 24 with Expected Target Proteins
From the results in Figure 4, it is evident that compound 5 inhibits the synthesis of viral membrane
proteins, HA and NA, but it is not obvious how this inhibition occurs. Therefore, molecular docking
(MD) analysis was performed to predict which steps are disturbed for the inhibition of synthesis
of HA and NA through binding affinity calculation and this MD analysis will serve as reference
data for the future mechanistic in vitro studies. Since there are numerous target points in cascade
reaction for the synthesis of two proteins and there become more relevant factors as the cascades
proceeds further, it is advantageous to try first MD simulation with proteins in the upstream of the
cascade, such as polymerase subunits and NP which are responsible for viral protein synthesis.
Polymerase proteins including polymerase acidic protein (PA), polymerase basic protein 1 and 2
(PB1 and PB2), and NP, structural proteins of RNP particles play important roles in the life cycle
of the influenza virus, such as during replication and transcription. NP is especially involved in
RNP trafficking between the nucleus and cytoplasm and in the replication of RNPs and directly
binds with M1, PB1, and PB2 [75,76]. Therefore, without NP, synthesis of all the viral proteins
including HA and NA are ultimately prohibited via inhibition of mRNA transcription and vRNA
replication. Moreover, previous studies have reported that the maximum amino acid difference
of NP among influenza A strains was less than 11%, which makes NP as a potential therapeutic
target for the treatment of all influenza virus strains [77]. In addition to NP, after polymerase
subunits (PA, PB1, and PB2) are newly synthesized via initial mRNA expression and imported
back into nucleus, each subunit plays its own distinct roles in mRNA/vRNA transcription. PB1
subunit serves as a place where complementary RNA (cRNA) is elongated [78]. Also, in the cap
snatching step, which is a crucial for mRNA transcription, PB2 and PA are used for binding 5’ cap of
a host mRNA and cleaving nucleotides of the 5’ cap, respectively [79
in Figure 4 and importance of NA and polymerase subunits in terms of synthesis of HA and NA,
we investigated whether compound can be docked to polymerase proteins and NP of influenza virus.
,80]. Considering the results
5
According to the data in the Protein Data Bank (PDB), the binding pocket between the active ligand
4-(2-chloro-4-nitrophenyl)piperazin-1-yl][3-(2-methoxyphenyl)-5-methyl-1,2-oxazol-4-yl]methanone
(LGH) and NP (PDB ID: 3RO5) showed stable interaction energy at residues Arg305, Ser376, Tyr52,
Tyr289, and Arg99 [41] (Figure 6). In our study, compound
5 showed several interactions with Arg305,
Ser376, Tyr52, Trp104, Arg99, Tyr289, Typ313, and Glu294 in active site AC1 with a low CDOCKER
energy and CDOCKER interaction energy (
and S2).
−
17.2458 and
−
40.9050 kcal/mol, respectively; Tables S1
In addition to the NP complex, docking analysis of
5
with the structures of the C-terminal region
of PA (PAC) complexed with a peptide from PB1 was carried out. A previous report suggested that
residues Met1, Val3, Asn4, Pro5, Leu7, Leu8, Phe9, and Leu10 on the PB1 peptide are important key
residues for interactions with PA and show the lowest binding free energy [42]. In order to determine
the mechanism of binding of
polymerase complex (PDB ID: 2ZNL) were identified at residues Pro5, Phe9, Lys643, Gln591, Phe658,
Phe707, Leu666, and Arg663 with a low CDOCKER energy and CDOCKER interaction energy ( 7.5123
and 32.6849 kcal/mol, respectively; Figure 6 and Tables S1 and S2). When comparing CDOCKER
5 with PA-PB1 polymerase, the interactions of compound 5 and the
−
−
energy values of two docking enzymes of compound
more stable manner than with PA-PB1.
5, it is likely that compound 5 binds with NP in a

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Figure 6. Illustration of the molecular docking simulation and 2D diagram of the interactions between
) NP and compound , ( ) PA-PB1 and compound , ( ) NA and compound 24, and ( ) NA mutant
(A
5
B
5
C
D
and compound 24 using Discovery Studio Client v19.1.0.18287/CDOCKER software. The structures of
the proteins were obtained from the Protein Data Bank (http://www.pdb.org) (PDB ID code: 3RO5,
2ZNL, 3TI4, and 3CL0 for A-D, respectively).
Based on the data in Figure 3B and Table 2, in which compound 24 showed strong NA inhibitory
activity against four types of IVA, docking analysis of 24 with NA (PDB ID: 3TI4) [43] and its mutant
(PDB ID: 3CL0) [44] was implemented. Since results in Figure 5 clearly indicated that compound
24 inhibits neuraminidase in a noncompetitive manner, in other words, it binds to allosteric sites,
docking sites were obtained by defining binding sites from receptor cavities of whole structures of
the enzyme to perform blind docking experiments. As shown in Figure 6, Figure S28 and Tables
S1–S3, compound 24 showed stable docking with nine binding sites out of 21 sites. Among the nine
binding sites, compound 24 bound to site 1 with the lowest CDOCKER energy ( 38.5234 kcal/mol),
−
which means its binding is fairly stable compared to the value of docking of laninamivir octanoate
(positive control). Although interaction amino acid residues from both cases cannot be compared
with each other since laninamivir octanoate inhibits the NA in a competitive manner, only CDOCKER
energy and CDOCKER interaction energy could be compared to see how the docking of our compound
24 is stable. It binds to Glu174, Asn208, Leu127, and Cys129 via conventional hydrogen bonds and
with Glu128 via pi-sigma interaction (Figure 6). On the other hand, compound 24 was stably docked
to four binding sites of NA mutant out of seven sites (Figure 6 and Tables S1, S2, and S4). Among
them, the compound was successfully docked to site 6 with the lowest energy of CDOCKER energy
(
−
24.4057 kcal/mol) compared to that of docking of oseltamivir (Figure S28). It interacts with His46
and His329 via a conventional hydrogen bond and has pi-alkyl interaction with Ala74 residue of the
enzyme (Figure 6). Taken together, the MD analysis of compound with NP and PA-PB1 suggests
5
that it may have inhibitory influences on NP rather than on PA-PB1 and serves as reference for the
future in vitro assay to verify. Also, the result of MD analysis of compound 24 supports its strong
inhibitory activity toward NA and NA mutant and suggests the possible allosteric interaction residues
of enzymes.
3.6. Anti-Inflammatory Effects of the Isolated Compounds
Combination chemotherapy between NA inhibitors and anti-inflammatory agents may become
a new strategy against drug resistance in patients with influenza viral infections. Previous studies
have reported that during influenza viral infections, proinflammatory cytokines and nitric oxide (NO)
production could be excessive in macrophages. In this study, we evaluated the ability of isolated

Biomolecules 2020, 10, 711
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compounds from P. densiflora to suppress the production of NO and inducible nitric oxide synthase
(iNOS) expression in RAW cells. As shown in Figure 7A and Figure S25, compounds and 24 did
not show cytotoxicity at 30 M and inhibited NO production in a dose-dependent manner at 5, 10,
and 20 M in RAW cells stimulated with 100 ng/mL LPS. This tendency was reproduced in RAW
5
µ
µ
cells upon viral infection. In our preliminary experiments with low MOI 0.01 infections of the H1N1
A/PR/8/34 virus, NO production was low and inconsistent (Figure S26). Therefore, infections with a
high MOI 1.0 were implemented. As shown in Figure 7B, with a high MOI 1 infection, NO production
was 11-fold higher than that in the low MOI 0.01 infections. Moreover, NO production was clearly
reduced in the presence of compounds
decreased when stimulated RAW cells were treated with these compounds at the concentration of
20 M (Figure 7C). Based on the above data, we gained insight into the effects of the two compounds
5 and 24 (20 µM). Moreover, the expression of iNOS significantly
µ
on the inhibition of NO production. Considering the high mortality induced by complications such
as secondary bacterial pneumonia and otitis media following influenza infection, prevention of
excessive inflammatory responses is also important. It has been demonstrated that severe IAV infection
can give rise to pulmonary infiltration and hypoxemia that ultimately leads to acute respiratory
distress syndrome (ARDS), the major contributor to severe mortality [81]. Pathophysiologically, it has
been shown that inflammation upon viral infection occurs to adapt to the body by increasing body
temperature and recruiting monocytes/macrophages in order to resist and fight against invasion as a
normal defensive mechanism. However, it is possible that the immune system of our body releases
excessive levels of proinflammatory cytokines and chemokines. A strong Th1-type immune response
stimulated by proinflammatory cytokines, such as TNF-
Although detailed experiments, such as the effects on the aforementioned cytokines in lung or spleen
tissue, are needed, our results indicated that compounds and 24 from P. densiflora can effectively
inhibit not only viral growth in MDCK cells but also NO production by multitargeting.
α and IL-1β, causes tissue damage [82,83].
5
Figure 7. Effects of compounds
Treatment of the test compounds at various concentrations to inhibit NO production in RAW 264.7 cells
by either ( ) 100 ng/mL LPS treatment or ( ) H1N1 A/PR/8/34 viral infection. ( ) Western blot analysis
of iNOS expression in activated RAW 264.7 cells decreased by compounds and 24 at 20 M. Data are
presented as the mean ± SD (n = 3), * p < 0.05, ** p < 0.01, compared to the positive control group.
5 and 24 on NO production and iNOS expression in RAW 264.7 cells.
A
B
C
5
µ
4. Conclusions
Taken together, 26 compounds, including two new megastigmane glycosides, 23 diterpenoids,
and three flavonoids, were isolated from the cortex and leaves of P. densiflora. Whole compounds were
evaluated for their CPE and NA inhibitory properties, which in turn allowed us to anticipate SAR
information. Some candidates that showed potent activity were selected and subjected to various
detailed mechanistic studies to identify the mode of action, such as effects on mRNA expression,
cellular apoptosis, neutralization of viral particles, cell protection against viral infection and inhibition

Biomolecules 2020, 10, 711
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of inflammation. While flavonoids exerted their anti-influenza activity via direct NA inhibition,
the diterpenes isolated here are likely to affect the gene expression of various proteins that are essential
for viral propagation rather than direct neutralization of surface proteins. Moreover, considering that
compound
5 exhibited anti-inflammatory activity, other isolated diterpenes may also have that activity.
To date, this study demonstrates that P. densiflora can be considered an abundant natural anti-influenza
viral source to find new bioactive compounds against influenza virus outbreaks in the future.
Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/10/5/711/s1.
Figure S1: Isolation scheme of the compounds from the leaves of Pinus densiflora. Figure S2: Isolation scheme of
the compounds from the cotex of Pinus densiflora. Figure S3: HRESIMS data of compound
spectrum (methanol-d4, 500 MHz) of compound
. Figure S5: 13C NMR spectrum (methanol-d4, 125 MHz) of
compound . Figure S6: HMBC spectrum (methanol-d4, 500 MHz) of compound . Figure S7: COSY spectrum
(methanol-d4, 500 MHz) of compound . Figure S8: HRESIMS data of compound
. Figure S9: 1H NMR
spectrum (methanol-d4, 500 MHz) of compound
. Figure S10: 13C NMR spectrum (methanol-d4, 125 MHz) of
compound . Figure S11: HMBC spectrum (methanol-d4, 500 MHz) of compound . Figure S12: COSY spectrum
(methanol-d4, 500 MHz) of compound
1
. Figure S4: 1H NMR
1
1
1
1
2
2
2
2
2
. Figure S13: HRESIMS data of compound 2a. Figure S14: 1H NMR
spectrum (methanol-d4, 400 MHz) of compound 2a. Figure S15: 13C NMR spectrum (methanol-d4, 100 MHz) of
compound 2a. Figure S16: HSQC NMR spectrum (methanol-d4, 400 MHz) of compound 2a. Figure S17: HRESIMS
data of compound 2b. Figure S18: HRESIMS data of compound 2c. Figure S19: 1H NMR spectrum (CDCl3,
600 MHz) of compound 2b and 2c. Figure S20: COSY spectrum (CDCl3, 600 MHz) of compound 2b and 2c
.
Figure S21: Comparison of chromatograms after sugar analysis for the determination of absolute configuration of
sugar moiety in compound and . Figure S22: Percent cell viability evaluated by the effects of all compounds
26) at 10 M using cytotoxicity assay. Figure S23: Inhibition of cytopathic effect of compounds and 24 against
H9N2 virus. Figure S24: The effects of compounds and 24 on the cell protection from viral infection and the
H1N1 particles. Figure S25: Percent cell viability of RAW 264.7 cells evaluated by the effects of compounds
and 24 at 30 M using cytotoxicity assay. Figure S26: Effects of compounds and 24 on the NO production in
infected-RAW 264.7 cells with low MOI. Figure S27: The effects of compound on the cellular DNA contents
after H1N1 virus infection. Figure S28: Images of global representation of NA surface with selected docking
sites for compound 24. Table S1: CDOCKER and CDOCKER interaction energies of compounds and 24 with
1
2
(
1–
µ
5
5
5
µ
5
5
5
nucleoprotein, PA-PB1 polymerase, neuraminidase, and neuraminidase mutant. Table S2: Molecular docking and
interaction images of positive control with nucleoprotein, neuraminidase, and neuraminidase mutant. Table S3:
CDOCKER and CDOCKER interaction energies of compounds 24 with neuraminidase. Table S4: CDOCKER
and CDOCKER interaction energies of compounds 24 with neuraminidase mutant. Table S5: Primers used for
real-time PCR. Supporting NMR data of isolated compounds.
Author Contributions: T.K.Q.H. and T.V. designed and performed the biological experiments. B.W.L. determined
the absolute configurations of the two new compounds by performing the modified Mosher’s analysis and sugar
analysis and also wrote the manuscript. N.H.N. performed the isolation of the whole compounds and determined
their structures. H.M.C. and T.P.D. aided in fractionation, isolation and characterization of the compounds from
P. densiflora. E.K. purified and provided virus stocks of two subtypes of the influenza virus. W.K.O. designed
the research and is also the corresponding author. All authors have read and agreed to the published version of
the manuscript.
Funding: This work was supported financially by grants from the Korea Bioactive Natural Material Bank
(NRF-2017M3A9B8069409) through the National Research Foundation of Korea funded by the Ministry of Science
and ICT and from the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry
(IPET) through the Animal Disease Management Technology Development Program funded by the Ministry of
Agriculture, Food and Rural Affairs (MAFRA) (318031031SB010) from the Korean government.
Conflicts of Interest: The authors declare no conflict of interest.
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