# Pogonatherumol, a Novel Highly Oxygenated Norsesquiterpene with Flavone C-Glycosides fromPogonatherum crinitum

Authors: Lin Ni; Wei Huang; He-shan Wang; Hui-you Xu
Journal: Journal of Chemistry
 (2018)

---

## Abstract

A novel highly oxygenated norsesquiterpene, pogonatherumol (1), with two known flavone C-glycosides (2-3), was isolated from Pogonatherum crinitum. The structure of the new compound was illuminated based on its spectroscopic data and X-ray analysis. Compounds 1 and 3 inhibited NO production in the mouse peritoneal macrophage (64.5 ± 7.2% and 61.6 ± 5.8%, respectively, at a concentration of 50 μM). The three compounds were inactive when tested against two human cancer cell lines (IC50 values > 50 μM).


---

## Body

## 1. Introduction

Pogonatherum crinitum, is one of the species in the genus Pogonatherum of Gramineae family, which has been used as a natural medicine in Chinese folk [1], for treatment of diabetes, urinary tract infection, jaundice hepatitis, and nephritis [2–5]. Previous phytochemical studies on this plant have led to a series of flavones and lignins, some of which showed anti-inflammatory and antifungal activities [6, 7]. In order to discover structurally and biologically interesting compounds from P. crinitum, a novel highly oxygenated norsesquiterpene, pogonatherumol (1) with two known flavone C-glycosides was investigated (Figure 1). Herein, the isolation and structural characterization of the compounds and their biological activities are reported in this paper.Figure 1
The structure of compounds1–3. ∗New compound.


## 2. Experimental

### 2.1. General Experimental Procedures

X-ray dif-segment intensity data of1 were collected on an MAC DIP-2030K diffractometer with graphite-monochromated Mo Kα radiation and were corrected by Lorentz and polarization. An XT-5B micromelting point apparatus was used to determine the melting point. JEOL ECZ-400S, JASCO V-650, and Nicolet iS5N spectrophotometers manufactured by Thermo Scientific were used to record NMR, UV, and IR spectra, respectively. Thermo Scientific LTQ Orbitrap XL was used to collect HRESIMS spectra. Column chromatography (CC) was performed with silica gel (300–400 mesh), polyamide (60–100 mesh), Sephadex LH-20, D101 macroporous, and ODS (50 μm). Preparative HPLC made up for an LC-20AP instrument, an SPD-20A detector, and an ODS-A column (YMC, 250 × 20 mm, 5 μm) was used to purify the compounds.


### 2.2. Plant Material

Pogonatherum crinitum was collected in Jianyang, Fujian, China, in September 2015. Professor Xiaoxing Zou, from the Fujian Agriculture and Forestry University (FAFU), People’s Republic of China (PRC), identified the plant. The plant specimen was deposited at the lab of the material basis and utilization of TCM, College of Plant Protection, FAFU, PRC.


### 2.3. Extraction and Isolation

Air-dried and powdered herba ofPogonatherum crinitum (10 kg) was heated under reflux two times with 75% aqueous EtOH (80 L × 2 h × 2) at 80°C. The extract was evaporated under reduced pressure, and the residue was suspended in H2O and then extracted with EtOAc (8 L × 3). The ethyl acetate segment (113.2 g) was dissolved in water and subjected to passage over polyamide eluting with H2O and 50% EtOH-H2O (v/v), successively (segments A1 and A2). The 50% EtOH-water extract (segment A2, 43.7 g) was passed over CC on silica gel eluting with CH2Cl2-MeOH (15 : 1–1 : 1) to afford 8 segments (B1–B8). Segment B3 (1.032 g) was subjected to passage over Sephadex LH-20 eluting with MeOH and finally purified by preparative HPLC to give 1 (23 mg), using chromatographic condition as MeOH-H2O (30 : 70, v/v), and detected at 210 nm, 8 mL/min. Segment B5 (5.273 g) was subjected to passage over an RP-18 column with MeOH-H2O (15%, 30%, and 45%, v/v, successively) to afford 3 segments (C1–C3). Finally, segment C2 (1.518 g) was purified by preparative HPLC to give 2 (10 mg) and 3 (8 mg), under chromatographic condition as MeCN-H2O (15 : 85, v/v), and detected at 210 nm, 8 mL/min.


#### 2.3.1. Pogonatherumol (1)

Colorless needles; mp 153°C-154°C; UV (MeOH)λmax (log ε) 232 (3.53) nm; IR νmax 3313, 2960, 2867, 1672, 1566, 1510, 1294, 1182, 922, 866 cm−1; 1H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz) (Table 1); HRESIMS m/z 233.11728 [M + H]+ (calcd. for C14H17O3, 233.12722).Table 1
1H and 13C NMR data of compound 1.
Number
δ
c
a
δ
H
b (mult. J/Hz)
1
191.5
—
2
138.7
—
3
26.1
3.43,s
4
139.1
—
5
151.0
—
6
112.1
6.76,s
7
142.1
—
8
126.7
—
9
137.7
—
10
129.5
6.56,d (7.1)
11
14.8
1.89,d (7.1)
12
26.2
4.06,m
13
23.2
1.12,d (6.8, overlap)
14
23.2
1.12,d (6.8, overlap)
aIn DMSO-d6 (100 MHz); bin DMSO-d6 (400 MHz).


#### 2.3.2. Crystallographic Data of1

Bond precision: C-C = 0.0059; wavelength = 0.71073; cell:a = 8.214 (4), b = 6.783 (3), c = 22.236 (11); alpha = 90; beta = 90; gamma = 90; temperature: 296 K; C14H16O3, Mr 232.27; space group Pnma; hall group-p2ac2n; Dx = 1.245 g·cm−3; 1.092 g·cm−3, F(000) = 496; Mu = 0.087 mm−1; nref = 1607; Tmin = 0.468, Tmax = 0.746; data completeness = 0.982; theta(max) = 28.149; R(reflections) = 0.0888(946); WR2(reflections) = 0.2143(1607); s = 1.130; npar = 114.The crystallographic data of the structure of pogonatherumol (deposition number: CCDC 1842928) were deposited at the Cambridge Crystallographic Data Centre.


### 2.4. Biological Activities

Two cell lines, human ovarian neoplasm cell (A2780) and human colon cancer cell line (HCT-116), were used to test the tumor cytotoxic activities, and Taxol was used as a positive control. The method was according to the previously described [8]. LPS-induced RAW264.7 macrophage was used to evaluate the NO production inhibition activity of the three compounds. Dexamethasone was used as a positive control, and the test method was according to the literature [9, 10].


## 2.1. General Experimental Procedures

X-ray dif-segment intensity data of1 were collected on an MAC DIP-2030K diffractometer with graphite-monochromated Mo Kα radiation and were corrected by Lorentz and polarization. An XT-5B micromelting point apparatus was used to determine the melting point. JEOL ECZ-400S, JASCO V-650, and Nicolet iS5N spectrophotometers manufactured by Thermo Scientific were used to record NMR, UV, and IR spectra, respectively. Thermo Scientific LTQ Orbitrap XL was used to collect HRESIMS spectra. Column chromatography (CC) was performed with silica gel (300–400 mesh), polyamide (60–100 mesh), Sephadex LH-20, D101 macroporous, and ODS (50 μm). Preparative HPLC made up for an LC-20AP instrument, an SPD-20A detector, and an ODS-A column (YMC, 250 × 20 mm, 5 μm) was used to purify the compounds.


## 2.2. Plant Material

Pogonatherum crinitum was collected in Jianyang, Fujian, China, in September 2015. Professor Xiaoxing Zou, from the Fujian Agriculture and Forestry University (FAFU), People’s Republic of China (PRC), identified the plant. The plant specimen was deposited at the lab of the material basis and utilization of TCM, College of Plant Protection, FAFU, PRC.


## 2.3. Extraction and Isolation

Air-dried and powdered herba ofPogonatherum crinitum (10 kg) was heated under reflux two times with 75% aqueous EtOH (80 L × 2 h × 2) at 80°C. The extract was evaporated under reduced pressure, and the residue was suspended in H2O and then extracted with EtOAc (8 L × 3). The ethyl acetate segment (113.2 g) was dissolved in water and subjected to passage over polyamide eluting with H2O and 50% EtOH-H2O (v/v), successively (segments A1 and A2). The 50% EtOH-water extract (segment A2, 43.7 g) was passed over CC on silica gel eluting with CH2Cl2-MeOH (15 : 1–1 : 1) to afford 8 segments (B1–B8). Segment B3 (1.032 g) was subjected to passage over Sephadex LH-20 eluting with MeOH and finally purified by preparative HPLC to give 1 (23 mg), using chromatographic condition as MeOH-H2O (30 : 70, v/v), and detected at 210 nm, 8 mL/min. Segment B5 (5.273 g) was subjected to passage over an RP-18 column with MeOH-H2O (15%, 30%, and 45%, v/v, successively) to afford 3 segments (C1–C3). Finally, segment C2 (1.518 g) was purified by preparative HPLC to give 2 (10 mg) and 3 (8 mg), under chromatographic condition as MeCN-H2O (15 : 85, v/v), and detected at 210 nm, 8 mL/min.


### 2.3.1. Pogonatherumol (1)

Colorless needles; mp 153°C-154°C; UV (MeOH)λmax (log ε) 232 (3.53) nm; IR νmax 3313, 2960, 2867, 1672, 1566, 1510, 1294, 1182, 922, 866 cm−1; 1H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz) (Table 1); HRESIMS m/z 233.11728 [M + H]+ (calcd. for C14H17O3, 233.12722).Table 1
1H and 13C NMR data of compound 1.
Number
δ
c
a
δ
H
b (mult. J/Hz)
1
191.5
—
2
138.7
—
3
26.1
3.43,s
4
139.1
—
5
151.0
—
6
112.1
6.76,s
7
142.1
—
8
126.7
—
9
137.7
—
10
129.5
6.56,d (7.1)
11
14.8
1.89,d (7.1)
12
26.2
4.06,m
13
23.2
1.12,d (6.8, overlap)
14
23.2
1.12,d (6.8, overlap)
aIn DMSO-d6 (100 MHz); bin DMSO-d6 (400 MHz).


### 2.3.2. Crystallographic Data of1

Bond precision: C-C = 0.0059; wavelength = 0.71073; cell:a = 8.214 (4), b = 6.783 (3), c = 22.236 (11); alpha = 90; beta = 90; gamma = 90; temperature: 296 K; C14H16O3, Mr 232.27; space group Pnma; hall group-p2ac2n; Dx = 1.245 g·cm−3; 1.092 g·cm−3, F(000) = 496; Mu = 0.087 mm−1; nref = 1607; Tmin = 0.468, Tmax = 0.746; data completeness = 0.982; theta(max) = 28.149; R(reflections) = 0.0888(946); WR2(reflections) = 0.2143(1607); s = 1.130; npar = 114.The crystallographic data of the structure of pogonatherumol (deposition number: CCDC 1842928) were deposited at the Cambridge Crystallographic Data Centre.


## 2.3.1. Pogonatherumol (1)

Colorless needles; mp 153°C-154°C; UV (MeOH)λmax (log ε) 232 (3.53) nm; IR νmax 3313, 2960, 2867, 1672, 1566, 1510, 1294, 1182, 922, 866 cm−1; 1H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz) (Table 1); HRESIMS m/z 233.11728 [M + H]+ (calcd. for C14H17O3, 233.12722).Table 1
1H and 13C NMR data of compound 1.
Number
δ
c
a
δ
H
b (mult. J/Hz)
1
191.5
—
2
138.7
—
3
26.1
3.43,s
4
139.1
—
5
151.0
—
6
112.1
6.76,s
7
142.1
—
8
126.7
—
9
137.7
—
10
129.5
6.56,d (7.1)
11
14.8
1.89,d (7.1)
12
26.2
4.06,m
13
23.2
1.12,d (6.8, overlap)
14
23.2
1.12,d (6.8, overlap)
aIn DMSO-d6 (100 MHz); bin DMSO-d6 (400 MHz).


## 2.3.2. Crystallographic Data of1

Bond precision: C-C = 0.0059; wavelength = 0.71073; cell:a = 8.214 (4), b = 6.783 (3), c = 22.236 (11); alpha = 90; beta = 90; gamma = 90; temperature: 296 K; C14H16O3, Mr 232.27; space group Pnma; hall group-p2ac2n; Dx = 1.245 g·cm−3; 1.092 g·cm−3, F(000) = 496; Mu = 0.087 mm−1; nref = 1607; Tmin = 0.468, Tmax = 0.746; data completeness = 0.982; theta(max) = 28.149; R(reflections) = 0.0888(946); WR2(reflections) = 0.2143(1607); s = 1.130; npar = 114.The crystallographic data of the structure of pogonatherumol (deposition number: CCDC 1842928) were deposited at the Cambridge Crystallographic Data Centre.


## 2.4. Biological Activities

Two cell lines, human ovarian neoplasm cell (A2780) and human colon cancer cell line (HCT-116), were used to test the tumor cytotoxic activities, and Taxol was used as a positive control. The method was according to the previously described [8]. LPS-induced RAW264.7 macrophage was used to evaluate the NO production inhibition activity of the three compounds. Dexamethasone was used as a positive control, and the test method was according to the literature [9, 10].


## 3. Results and Discussion

Pogonatherumol (1) was obtained as colorless needles (MeOH). The IR spectrum of 1 showed 3313, 1672, and 1566 cm−1 absorption bands, which were assigned to hydroxyl, carbonyl, and olefin functionalities, respectively. The molecular formula was determined to be C14H16O3 on the basis of HRESIMS (an [M + H]+, m/z 233.11728), which corresponded to the molecular formula, indicating 7 degrees of unsaturation. Analysis of the 1H NMR spectroscopic data of 1 (Table 1) revealed three methyls (δH 1.12, 1.12 (3H each, both d, J = 6.8 Hz, H3-13, 14), and 1.89 (3H, d, J = 7.1 Hz, H3-11)), a methylene (δH 3.43 (2H, s, H-3)), an aromatic proton δH 6.76 (1H, s, H-6), an olefin proton δH 6.56 (1H, m, H-10), and two hydroxyls (10.06 (1H, s) and 8.74(1H, s)). And the 13C NMR (Table 1) and DEPT spectra showed fourteen carbons, including a carbonyl, a methene, three methines, three methyls, and six aromatic carbons, two of them bearing oxygen atoms (C-4 and C-5). HMBC correlations of H-6/C-4, C-5, and C-8; H-3/C-1, C-2, C-8, and C-9; 4-OH/C-5 and C-9; and 5-OH/C-4, C-5, and C-6 confirmed the presence of the indanone skeleton (Figure 2). An isopropyl (δH 1.12, 1.12 (3H each, both d, J = 6.8 Hz, H3-13, 14) and 4.06 (1H, m, H-12)) was attached to C-7, which could be confirmed by the HMBC correlation from H-13 and H-14 to C-12 and C-7 and from H-12 to C-6, C-7, and C-8. Further HMBC correlations of H-11/C-10 and C-2 and of H-10/C-1 indicated that an ethylidene was fixed in C-2, and NOESY correlation from H3-11 to H-3 suggested the trans-configuration of double bonds between C-2 and C-10 (Figure 2). Finally, the proposed structure of 1, (E)-2-ethylidene-4,5-dihydroxy-7-isopropyl-2,3-dihydro-1H-inden-1-one, named “pogonatherumol” was confirmed by an X-ray crystallography analysis. The ORTEP drawing, with the atom-number scheme indicated, is shown in Figure 3.Figure 2
Key1H, 1H COSY, HMBC, and NOESY correlations of 1.Figure 3
ORTEP drawing of compound1.In addition to pogonatherumol, two known C-glycosides, schaftoside (2) [11] and isoschaftoside (3) [12], were identified by comparing their ESIMS and 1H and 13C NMR data with those reported in the literature.Pogonatherumol (1) with a fused indanone skeleton has not been previously reported as a natural product. Notably, it seems to be a novel rearrangement of norsesquiterpene.Meanwhile, the inhibited NO production and the tumor cytotoxic activities of three compounds were evaluated. Compounds1 and 3 inhibited NO production in the mouse peritoneal macrophage (64.5 ± 7.2% and 61.6 ± 5.8%, respectively, at a concentration of 50 μM), while the positive control dexamethasone gave an inhibitory ratio of 59.4.1 ± 6.2% at a concentration of 10 μM. All compounds were also evaluated in vitro for cytotoxicity against A2780 and HCT-116 cell lines employing an MTT assay with Taxol as the positive control. The three compounds were inactive when tested against the two human tumor cell lines (IC50 values ≥ 50 μM).



---

*Source: 5029610-2018-08-23.xml*