Structural Characterization and Hepatoprotective Activity of Naphthoquinone From Cucumis bisexualis

Abstract

Three new naphthoquinones, namely 5,8-dihydroxy-7′-isopropyl-furan ring-1″-prenyl-naphthoquinone (1), 5,8-dihydroxy-7′-propenyl-furan ring-1″-isopropanol-naphthoquinone (2), and 5,8-dihydroxy-7′-isopropanol-furan ring-1″-(5″-hydroxyphenyl)-naphthoquinone (3), along with 10 known naphthoquinone derivatives (4-13) were isolated from Cucumis bisexualis for the first time. All the compounds were elucidated on the basis of spectroscopic data analysis and chemical evidence. Compounds (1-13) were evaluated for their hepatoprotective activites on human L-O2 cells. Among them, compounds 1, 4, and 8 exhibited significant promotion effects on the proliferation of L-O2 cells.

Cucumis bisexualis belonging to the Cucurbitaceae family is not only used as a favorite wild fruit but also served for an important folk medicine distributed in China. ¹ Up to now, there are only 2 reports on the studies of the chemical components and biological activities of C. bisexualis. In 2017, it was found that its extract could reduce blood glucose in diabetic mice and enhance the antioxidant capacity of type 2 diabetic mice. ² Preliminary studies of our group have found that the coumarin derivatives from C. bisexualis possessed hepatoprotective activities. ³ As part of our continuous phytochemical investigations on the bioactive isolates from C. bisexualis based on the previous research, this prompted us to perform a bioassay-guided investigation of C. bisexualis in order to evaluate its further hepatoprotective activities. We report here on the isolation and structural elucidation of 3 new naphthoquinones (1-3), together with 10 known naphthoquinone derivatives (4-13) that were isolated from the active fraction (EtOAc layer) of C. bisexualis for the first time (Figure 1). Moreover, the hepatoprotective activities of compounds (1-13) were evaluated in cell proliferation inhibition test and hepatoprotective test on CCl ₄-induced injury of human L-O2 cells by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay. It was found that compounds 1, 4, and 8 (μM) exhibited important hepatoprotective activities. These research results may guide the search for new natural products with hepatoprotective attributes.

Figure 1

Structures of compounds 1 to 13.

Compound 1 was obtained as a pale yellow powder. The molecular formula C₂₈H₂₂O₇ was established by the presence of a deprotonated molecular ion [M−H]⁻ at m/z 469.1275 (calcd. for C₂₈H₂₁O₇, 469.1287) in the high resolution-electrospray ionization-mass spectrometry (HR-ESI-MS) data, which requires 18 degrees of unsaturation. The UV spectrum showed absorptions at λ _max 210, 232, 280, and 431 nm, suggesting the presence of the naphthoquinone skeleton in compound 1. ⁴ Its infrared (IR) spectrum showed the presence of hydroxyl (3438 cm⁻¹), carbonyl (1736 cm⁻¹), aromatic ring (1617 cm⁻¹), and methyl (1376 cm⁻¹) functionalities. The ¹H nuclear magnetic resonance (NMR) data of compound 1 showed that a typical prenyl (δ _H 2.23, 2H, d, J = 7.2 Hz, H-2″; δ _H 5.24, 1H, t, J = 7.2, 1.5 Hz, H-3″; δ _H 1.81, 3H, s, 5″-CH₃; δ _H 1.63, 3H, s, 6″-CH₃) was connected to C-1″ by the heteronuclear multiple bond correlation (HMBC) correlations of H-3″ to C-1″, H-1″ to C-3″ (Figure 2). Moreover, an isobutene group (δ _H 6.20, 1H, s, H-9′; δ _H 1.77, 6H, s, 11″/12″-CH₃) was assigned to C-7′ according to the HMBC correlations of H-6′ to C-8′, H-8′ to C-6′ (Figure 2). Two typical broad single peaks (δ _H 7.27, 2H, brs, H-6/7) were found in the low field of ¹H NMR spectrum, which also confirmed that the 1,2-anthraquinone unit was in compound 1.

Figure 2

Key HMBC and ¹H-¹H COSY correlations of compounds 1 to 3.

The other fragments of compound 1 were determined by the HMBC correlations of H-6 to C-10, H-7 to C-9, H-6′ to C-4′/9′ (Figure 2), the ¹H-¹H correlation spectroscopy (COSY) correlations of H-6 to H-7, H-1″ to H-2″, H-2″ to H-3″ (Figure 2), and the nuclear overhauser enhancement spectroscopy (NOESY) correlations of H-6 to H-7, H-6′ to H-8′ (Figure 3). Consequently, compound 1 was elucidated as 5,8-dihydroxy-7′-isopropyl-furan ring-1″-prenyl-naphthoquinone.

Figure 3

Key NOESY correlations of compounds 1 to 3.

Compound 2 was isolated as a pale yellow powder. Its molecular formula was determined as C₂₅H₁₈O₈, evidenced from the HR-ESI-MS data (m/z 445.0952 [M−H]⁻, calcd. for C₂₅H₁₇O₈, 445.0923) with 17 degrees of unsaturation. Its UV and IR spectral data were similar to those of compound 1, which showed the absorptions at λ _max 210, 233, 280, and 430 nm and indicated the existence of hydroxyl (3432 cm⁻¹), carbonyl (1736 cm⁻¹), aromatic ring (1616 cm⁻¹), and methyl (1374 cm⁻¹) functional groups, separately. It was concluded that compound 2 was an analog of compound 1. The ¹H and ¹³C NMR spectral data of compound 2 (Table 1) were similar to those of compound 1 except for 1″-isopropanol (δ _H 1.27, 6H, s, 3″/4″-CH₃) that was connected to C-1″ by the HMBC correlations of H-1″ to C-4/4′ and 7′-propenyl (δ _H 3.16, 2H, d, J = 6.9 Hz, H-9′; δ _H 5.91, 1H, dd, J = 17.2, 10.3 Hz, H-10′; δ _H 5.06, 1H, d, J = 17.2 Hz, H-11′a; δ _H 5.03, 1H, d, J = 10.3 Hz, H-11′b) ⁵ that was assigned to C-7′ by the HMBC correlations of H-6′ to C-9′, H-10′ to C-7′. The other fragments of compound 2 were determined by the HMBC correlations of H-6 to C-10, H-7 to C-9 (Figure 2), the ¹H-¹H COSY correlations of H-6 to H-7, H-9′ to H-10′, H-10′ to H-11′ (Figure 2), and the NOESY correlations of H-6 to H-7, H-6′ to H-9′ (Figure 3). Consequently, compound 2 was elucidated as 5,8-dihydroxy-7′-propenyl-furan-ring-1″-isopropanol-naphthoquinone.

Table 1

¹H NMR (400 MHz, Acetone-d ₆), ¹³C NMR (100 MHz, Acetone-d ₆), and Key HMBC Correlations of Compounds 1 to 3.

No.	1		2		3
	δ H	δ C	δ H	δ C	δ H	δ C
1	-	186.6	-	186.5	-	186.6
2	-	133.2	-	133.5	-	133.3
3	-	148.5	-	148.3	-	143.7
4	-	183.7	-	183.3	-	183.6
5	-	159.1	-	159.0	-	159.4
6	7.27(brs)	130.5	7.28(brs)	130.7	7.26(brs)	130.6
7	7.27(brs)	130.5	7.28(brs)	130.7	7.26(brs)	130.6
8	-	154.4	-	154.6	-	154.7
9	-	113.9	-	114.0	-	114.2
10	-	111.6	-	111.7	-	111.5
1′	-	176.1	-	175.8	-	175.9
2′	-	133.2	-	133.0	-	133.1
3′	-	148.5	-	148.8	-	148.6
4′	-	177.8	-	177.3	-	177.5
5′	-	143.3	-	142.7	-	142.9
6′	7.21(s)	106.4	6.78(s)	106.1	7.04(s)	106.0
7′	-	158.5	-	162.8	-	172.1
8′	-	163.2	-	160.7	-	160.1
9′	6.20(s)	115.2	3.16(d,6.9)	32.3	-	70.1
10′	-	143.6	5.91(dd,17.2,10.3)	137.2	1.31(s)	28.5
11′	1.79(s)	22.4	5.06(d,17.2), 5.03(d,10.3)	116.2	1.31(s)	28.5
12′	1.71(s)	16.8	-	-	-	-
1″	2.88(t,6.0,1.5)	30.3	2.72(s)	44.8	4.20(s)	-
2″	2.23(d,7.2)	28.9	-	68.2	-	32.7
3″	5.24(t,7.2,2.5)	120.5	1.27(s)	26.9	6.95(d,8.0)	129.8
4″	-	133.2	1.27 (s)	26.9	6.62(d,8.0)	116.2
5″	1.81(s)	24.9	-	-	-	-
6″	1.63(s)	18.8	-	-	6.62(d,8.0)	116.2
7″	-	-	-	-	6.95(d,8.0)	129.8

Compound 3 was obtained as a pale yellow powder. Its molecular formula was determined as C₂₈H₁₈O₉, evidenced from the HR-ESI-MS data (m/z 497.0861 [M−H]⁻, calcd. for C₂₈H₁₇O₉, 497.0873) with 20 degrees of unsaturation. The UV absorptions (λ _max 210, 231, 280, and 432 nm) were attributed to naphthoquinone skeleton, ⁴ and the IR absorptions implied the presence of hydroxyl (3436 cm⁻¹), carbonyl (1736 cm⁻¹), aromatic ring (1619 cm⁻¹), and methyl (1377 cm⁻¹) functionalities and exhibited compound 3 that was an analog of compound 2. In the ¹H NMR spectrum of compound 3, a typical AA′BB′ system at δ _H 6.95 (2H, d, J = 8.0 Hz, H-3″/7″) and 6.62 (2H, d, J = 8.0 Hz, H-4″/6″) (Table 2) was connected to C-1″ by the correlations of H-1″ to C-3″/7″ in the HMBC spectrum (Figure 2). The 7′-isopropanol group (δ _H 1.31, 6H, s, 10′/11′-CH₃) was linked with C-7′ by the HMBC correlation of H-6′ to C-9′ (Figure 2). The other fragments of compound 3 were determined by the HMBC correlations of H-6 to C-10, H-7 to C-9, H-6′ to C-4′, H-4″/6″ to C-2″ (Figure 2), the ¹H-¹H COSY correlations of H-6 to H-7, H-3″ to H-4″, H-6″ to H-7″ (Figure 2), and the NOESY correlations of H-6 to H-7, H-3″ to H-4″, H-6″ to H-7″ (Figure 3). Consequently, compound 3 was elucidated as 5,8-dihydroxy-7′-isopropanol-furan ring-1″-(5″-hydroxyphenyl)-naphthoquinone.

Table 2

Results of Hepatoprotection of 1, 4, and 8 on CCl₄ -Induced Injury of L-O2.

Group(μM/mL)	A a , Survival rate (%)
	1	4	8
125	0.1512 ± 0.020 b , 36	0.1536± 0.020 b , 38	0.1502 ± 0.020 b , 35
250	0.1928 ± 0.020 b , 42	0.1951 ± 0.074 b , 41	0.1973 ± 0.031 b , 44
500	0.2130 ± 0.0521 b , 55	0.2146 ± 0.0637 b , 53	0.2174 ± 0.0548 b , 57
Bifendatatum	0.2143 ± 0.0527 b , 45	0.2185 ± 0.0532 b , 45	0.2153 ± 0.0601 b , 45
CCl4 injury	0.1102 ± 0.020 b , 25	0.1056 ± 0.074 b ,25	0.1118 ± 0.031 b ,25
Blank control	0.4821 ± 0.1143 b , 100	0.4882 ± 0.1052 b , 100	0.4823 ± 0.1201 b , 100

^aAbsorbance of living cells number.

^bCompared with blank control group and CCl₄ injury group, P < 0.05.

In addition, 10 known naphthoquinones derivatives (4-13) were identified by comparison of their spectroscopic data and references. Their structures were determined as shikometabolin G (4), ⁶ naphthofuranin A (5), ⁶ (2R)-6-hydroxy-7-methoxy-dehydroiso-α-lapachone (6), ⁷ 2-(1,4-dioxo-1,4-dihydronaphthalen-2-yloxy)-3-hydroxynaphthalene-1,4-dione (7), ⁸ zeylanone (8),¹⁰ 1-methyl-2,4-dimethoxy-3-hydroxyanthraquinone (9), ⁹ javanicin (10), ¹⁰ anhydrofusarubin (11), ¹⁰ shikonin (12), ¹¹ and lapachol (13). ¹¹ The known isolates (4-13) were identified as derivatives of naphthoquinones from this plant for the first time.

Compounds (1-13) were evaluated for their hepatoprotective effects on human L-O2 cells with Bifendatatum as the positive control. The results showed that compounds 1, 4, and 8 had significantly promoted proliferative effects on L-O2 cells at concentrations of 25, 50, 100, 200, 400, and 600 µg/mL, but had weak inhibitory effects with the respective inhibitory rates of 14%, 19%, and 17% at the concentration of 800 µg/mL, and cytotoxicity of compounds 1, 4, and 8 was very weak with IC₅₀ above 800 µg/mL. In the results of hepatoprotective test by CCl₄-injury (Table 2), the survival rates of compounds 1, 4, and 8 at 3 levels were significantly higher than that of CCl₄-injury group (P < 0.05), and were also higher than that of Pifendatatum (P < 0.05) at high concentrations. Consequently, compounds 1, 4, and 8 had the significant hepatoprotective activities. In contrast, compounds 2, 3, 5 to 7, and 9 to 13 showed no hepatoprotective activities.

Experimental

General Procedures

The UV spectra were measured by an Australia GBC UV-916 spectrophotometer (GBC Scientific Equipment Pty. Ltd., Braeside, Australia) and the IR spectral data were recorded on a Nicolet 5700 fourier transformation infrared (FT-IR) spectrometer with KBr pellets (Shanghai Xiangrun Industry Co. Ltd., Shanghai, China). The HR-ESI-MS spectral data were measured by an Agilent 1100 series LC/MSD ion trap mass spectrometer (Agilent Technologies, California, United States), and the ESI-MS data were measured by a Q-Trap liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS) spectrometer (Turbo Ionspray Source). The NMR spectral data were run on a Bruker-400 with tetramethyl silane (TMS) as internal standard. The reversed-phase high-performance liquid chromatography (HPLC) was performed on Agilent 1200 series with a DIKMA (4.6 × 250 mm) analytical column packed with C18 (5 µm) (Agilent Technologies, California, United States). The prep-HPLC separation was performed on a Shimadzu LC-6AD instrument with a SPD-20A detector and an YMC-Pack ODS-A column (250 × 20 mm, 5 µm) (Shimadzu Corporation, Japan). The column chromatography was performed on Sephadex LH-20, Toyopearl HW-40C, silica gel (100, 200, or 200-300 mesh) (Qingdao Marine Chemical Inc., Qingdao, China). The thin layer chromatography was performed on precoated silica gel GF254 plates and the spots were visualized under UV light (254 or 356 nm) or by spraying with 10% H₂SO4 in 95% EtOH followed by heating. ³

Plant Material

The fruits of C. bisexualis were harvested from Nanyang city of Henan Province of China in September 2016 and were authenticated by Dr Rongrui Wei of Jiangxi University of Traditional Chinese Medicine. A voucher specimen of C. bisexualis (XMP-201609) has been deposited in Jiangxi University of Traditional Chinese Medicine, Nanchang, China.

Extraction and Isolation

The air-dried fruits of C. bisexualis (15.5 kg) were extracted with 90% EtOH (16 L × 3) under reflux 3 times at room temperature. The filtrates were combined and concentrated under reduced pressure until the elimination of ethanol to obtain a black residue (1.6 kg). The extract was suspended in distilled water (15.0 L) and partitioned with petroleum ether, EtOAc, and n-BuOH, consecutively, yielding petroleum ether layer (98.6 g), EtOAc layer (235.5 g), and n-BuOH layer (305.8 g), respectively. The EtOAc layer was confirmed hepatoprotective site according to the screening results of bioactivity-guided investigation. ³ The EtOAc layer was performed on silica gel (100-200 mesh) column and eluted with a gradient elution (n-hexane/EtOAc = 12:1 to 3:1, v/v) to yield 4 fractions: A, B, C, and D. The fraction A (29.6 g) was subjected to silica gel (200-300 mesh) column and eluted with a gradient elution (petroleum ether/EtOAc = 20:1-5:1) to afford 3 subfractions: A₁ (7.56 g), A₂ (10.85 g), and A₃ (8.63 g). The subfraction A₂ was fractionated on Toyopearl HW-40C (95% MeOH) and Sephadex LH-20 (CH₂Cl₂:MeOH = 1:1), then separated by prep-HPLC (detection at 210 nm, 3 mL/min), successively, yielding 1 (6.58 mg, purity >98.2% by HPLC), 2 (7.25 mg, purity >96.7% by HPLC), 3 (9.30 mg, purity >98.0% by HPLC), 4 (8.20 mg, purity >97.6% by HPLC), and 8 (11.35 mg, purity >96.5% by HPLC). Meanwhile, the subfraction A₃ was separated over silica gel, Sephadex LH-20, and Toyopearl HW-40C, prep-HPLC, repeatedly, yielding 5 (12.05 mg, purity >96.8% by HPLC), 6 (10.22 mg, purity >98.6% by HPLC), 7 (8.62 mg, purity >98.8% by HPLC), 9 (11.20 mg, purity >97.0% by HPLC), 10 (14.54 mg, purity >96.3% by HPLC), 11 (10.21 mg, purity >96.7% by HPLC), 12 (11.55 mg, purity >96.1% by HPLC), and 13 (9.84 mg, purity >98.5% by HPLC). The structures of compounds (1-13) are shown in Figure 1.

5,8-Dihydroxy-7′-Isopropyl-Furan Ring-1″-Prenyl-Naphthoquinone (1)

Pale yellow powder; HR-ESI-MS: m /z 469.1275 [M−H]⁻ (calcd. for C₂₈H₂₁O₇, 469.1287); UV (MeOH) λ _max: 210, 232, 280, 431 nm; IR ν _max (KBr): 3438, 1736, 1617, 1376 cm^{−1 ; 1}H NMR (Acetone-d ₆, 400 MHz) and ¹³C NMR (Acetone-d ₆, 100MHz) data; see Table 1.

5,8-Dihydroxy-7′-Propenyl-Furan-Ring-1″-Isopropanol-Naphthoquinone (2)

Pale yellow powder; HR-ESI-MS: m/z 445.0952 [M−H]⁻ (calcd. for C₂₅H₁₇O₈, 445.0923); UV (MeOH) λ _max: 210, 233, 280, 430 nm; IR ν _max (KBr): 3432, 1736, 1616, 1374 cm^{−1 ; 1}H NMR (Acetone-d ₆, 400 MHz) and ¹³C NMR (Acetone-d ₆, 100MHz) data; see Table 1.

5,8-Dihydroxy-7′-Isopropanol-Furan Ring -1″-(5″-Hydroxyphenyl)-Naphthoquinone (3)

Pale yellow powder; HR-ESI-MS: m/z 497.0861 [M−H]⁻ (calcd. for C₂₈H₁₇O₉, 497.0873); UV (MeOH) λ _max: 210, 231, 280, 432 nm; IR ν _max (KBr): 3436, 1736, 1619, 1377 cm^{−1 ; 1}H NMR (Acetone-d ₆, 400 MHz) and ¹³C NMR (Acetone-d ₆, 100MHz) data; see Table 1.

Hepatoprotective Assay

Compounds (1-13) were evaluated for their hepatoprotective activities on CCl₄-induced injury of human L-O2 cells in vitro with Bifendatatum (Absin Bioscience Inc.; purity >98% by HPLC) as the positive control. The human L-O2 cells at logarithmic growth phase were digested by trypsin (Absin Bioscience Inc.; purity >97% by HPLC) and then were made into a 1 × 10⁴ mL⁻¹ cell suspension in RPMI1640 containing bovine serum (100 mL/L). The L-O2 cells were inoculated in a 96-well plate and the suspension (100 µL) was added to each well and there were 6 duplicate wells in each group. The L-O2 cells were incubated in an atmosphere containing 50 mL of CO₂ and 950 mL of air at 37°C. The medium was aspirated and replaced with fresh medium containing 0 (blank control), 12.5, 25, 50, 100, 200, 400, 600, and 800 µg/mL compounds (1-13) after 24 hours. The L-O2 cells were supplemented with 20 µL MTT (Fluka, 5 g/L) and incubated for another 4 hours. Then, the supernatant was discarded and the L-O2 cells preparation was shaken with DMSO (100 µL) until the crystal gets fully dissolved. The value of A ₅₇₀ (nm) for each well was measured by a microplate reader. Meanwhile, the L-O2 cells were inoculated in a 96-well plate and the suspension (100 µL) was added to each well and divided into CCl₄ injury group, experimental group, positive control, and blank control group and there were 12 duplicate wells in each group. The L-O2 cells were incubated in an atmosphere containing 50 mL of CO₂ and 950 mL of air at 37°C. The medium was aspirated and replaced with fresh medium (100 µL) containing blank control group, injury group (10 mM CCl₄), positive control (250 mg/mL Bifendatatum and 10 mM CCl₄), experimental group (125, 250, and 500 µg/mL compounds 1, 4, 8 and 10 mM CCl₄) after 12 hours. After 8 hours, the L-O2 cells were supplemented with 20 µL of MTT (Fluka, 5 g/L) and incubated for another 4 hours. Then, the supernatant was discarded and the L-O2 cells preparation was shaken with DMSO (100 µL) for 20 minutes. A microplate reader was used to measure the value of A ₅₇₀ (nm) for each well. ¹²

Conclusion

Three new naphthoquinones (1-3) and 10 known naphthoquinone derivatives (4-13) were isolated from C. bisexualis for the first time. All the compounds were elucidated on the basis of spectral data and references. Compounds (1-13) were evaluated for their hepatoprotective activites on human L-O2 cells. Among them, compounds 1, 4, and 8 exhibited significant promotion effects on the proliferation of L-O2 cells.