Antimicrobial Terpenoids from Seed of Chamaecyparis obtusa (Siebold & Zucc.) Endl.

Abstract

Chamaecyparis obtusa (Siebold & Zucc.) Endl. is an evergreen tree of the family Cupressaceae well known for its unique scents. The seed extract of this cypress tree was phytochemically investigated to isolate a novel abietane-diterpene compound (1) along with fifteen known terpenoids (2-16). All of the isolated compounds were subjected to the screening of antimicrobial activities against Cutibacterium acnes and Staphylococcus epidermidis including erythromycin resistant strains. Among the isolates, 1α-hydroxy-hinokione (1), hinokione (3), 1,2-dehydrohinokione (4) and ferruginol (9) showed significant antibacterial activities against both acne-causing strains. This study demonstrated that abietane-type diterpenoids are the main antibacterial components in C. obtusa seed extract, and some isolated compounds can be further developed as potential acne-treatment agents.

INTRODUCTION

Acne is a chronic inflammatory disease occurring in the sebaceous glands of the skin.¹ The Gram-positive bacteria such as Cutibacterium acnes and Staphylococcus epidermidis are directly involved in the process of causing acne. In fact, C. acnes secretes lipolytic enzymes to break down the triglycerides of sebum to form free fatty acids, which stimulate the skin to cause inflammation in the cells around the pores.² Meanwhile, S. epidermidis normally distributed in the skin is not a major cause of inflammation, but is known to play a role in exacerbating acne by further expanding the inflammatory areas.³

Among the ways to treat acne, antibiotics such as erythromycin and clindamycin are locally applied to the skin to suppress the proliferation of strains.⁴ However, this type of treatment requires a long period of time and then causes antibiotic-resistant bacteria along with several side effects.⁵ In practice, the occurrence of resistant bacteria is recognized as a limitation in antibiotic treatment in acne.⁶

Natural products attract attention as a way to develop treatment with fewer side effects in the field of skin diseases.⁷ Naturally derived microbial compounds are especially attractive in solving problems related to resistant bacteria.⁸

Chamaecyparis obtusa (Siebold & Zucc.) Endl. is an evergreen tree of the family Cupressaceae growing up to 40 meters in height. This cypress tree is native to Japan, but has been planted long ago to build a forest in southern area of Korea including Jeju Island.⁹ These trees are widely used as indoor decoration wood and household goods due to their own unique fragrance. As the main cause of these scents, terpene compounds have been identified in the leaves and branches as well as in seeds. In addition to research on terpene ingredients, studies on the antibacterial effect of C. obtusa have also been revealed.^10-14 However, there are no reports on the verification of the antibacterial effect on the strain C. acnes related to acne treatment.

In this study, we tried to find natural products possessing activities against C. acnes and S. epidermidis including erythromycin resistant strains from cypress tree seeds. As a result of phytochemical studies, identified were one new diterpene compound (1) as well as 15 known terpenoids (2-16), and some of which exhibit potent antimicrobial activities.

EXPERIMENTAL

General Experimental Procedures

All solvents of analytical grade were used without further purification. Optical rotation was measured with Model 343 polarimeter (λ 589 nm (D), 20 ℃, PerkinElmer Inc., USA). HR-ESI-MS data was performed on the SYNAPT G2 mass spectrometer (Waters Co., USA).1D and 2D NMR spectra were performed on JNM-EXC 400 (FT-NMR system, 400 MHz, JEOL Co., Japan) using CDCl₃, CD₃OD, acetone-d₆ and pyridine-d₅ as a solvent for measurement. The chemical shift values are reported in ppm relative to the solvent used. Sephadex^TM LH-20 (GE healthcare Co., Sweden) and silica gel 60 (Merck Co., Germany) were used for column chromatography, and precoated silica gel 60 F-254 plates (Merck Co., Germany) were used for thin-layer chromatography (TLC). The spots on TLC were detected by spraying with 3% KMnO₄ aqueous solution and anisaldehyde solution with 5% H₂SO₄ and then heating on a hot plate. Bacto^TM tryptic soy agar (TSA), and Bacto^TM tryptic soy broth (TSB) were purchased from Difco (USA). Gifu Anaerobic Medium (GAM) broth was purchased from MB cell (Korea).

Plant Material

The seeds of C. obtusa were collected in Gwangpyeong-ri, Seogwipo city of Korea, in December 2019. Voucher specimen (No. 497) was deposited at Natural Product Chemistry Laboratory, Department of Chemistry and Cosmetics, Jeju National University.

Extraction and Isolation

The seeds of C. obtusa (0.5 kg) were extracted two times with 50% aqueous ethanol (4.0 L) under stirring for 24 h at room temperature. The extracted solution was filtered, and the filtrate was concentrated under reduced pressure and freeze dried to afford a tan powder (25.5 g). A portion of the extract (20.7 g) was suspended in water (2.0 L) and fractionated into n-hexane (Hex, 6.3 g), ethyl acetate (EtOAc, 6.9 g), n-butanol (BuOH, 2.8 g) and water (H₂O, 4.0 g) portions.

The n-Hex soluble fraction (4.8 g) was subjected to vacuum liquid chromatography (VLC) over silica gel using step gradient solvents (n-Hex-EtOAc-methanol) to give 21 subfractions (VH1 to VH21). The fraction VH14 (222.2 mg) was further purified using Sephadex LH-20 column chromatography (CC) with chloroform-methanol (30:1) eluents to afford compound 1 (49.7 mg). The fraction VH6 (645.4 mg) was chromatographed by silica gel with chloroformmethanol (80:1) eluents to afford compounds 12 (32.2 mg), 15 (29.6 mg) and 14 (17.9 mg). A Fr. VH9 (820.3 mg) was further purified using Sephadex LH-20 CC with chloroform-methanol (60:1) eluents to yield compounds 3 (29.7 mg), 4 (45.2 mg) and 16 (84.8 mg). A Fr. VH10~11 (560.2 mg) was also purified using Sephadex LH-20 CC with chloroform-methanol (50:1) eluents to give compounds 2 (58.3 mg) and 7 (49.8 mg). The compound 8 (50.4 mg) was obtained from fraction VH7 (240.8 mg) by recrystallization with methanol. A fraction VH12~13 (777.0 mg) was further purified using Sephadex LH-20 CC with chloroform-methanol (40:1) eluents to afford compound 6 (45.8 mg). The compounds 9 (68.7 mg) and 10 (110.8 mg) were obtained from the fraction VH4 (226.5 mg) by silica gel CC using chloroform eluent.

The EtOAc soluble fraction (5.0 g) was also subjected to VLC over silica gel using step gradient solvents (n-hexane-EtOAc-methanol) to give 32 subfractions (VE1 to VE32). A fraction VE4~5 (310.5 mg) was further purified using silica gel CC with n-Hex-EtOAc (10:1) eluents to give compound 13 (9.8 mg). Silica gel CC of the fraction VE6 (230.4 mg) with chloroform-methanol (60:1) led to isolation of the compound 11 (7.1 mg). A fraction EV18~19 (350.3 mg) was purified using Sephadex LH-20 CC with chloroformmethanol (15:1) eluents to afford compound 5 (16.9 mg).

Antimicrobial Activities against C. acnes and S. epidermidis

The strains of C. acnes (CCARM 0081, 9009, 9010, 9089) and S. epidermidis (CCARM 3709, 3710, 3711) were purchased from the Culture Collection of Antimicrobial Resistant Microbes (CCARM, Korea). C. acnes were cultured in GAM in anaerobic condition using Becton Dickinson Gas Pak^TM EZ gas generating container system. Subculture of C. acnes was conducted every 48 h in a 37 ℃ incubator. S. epidermidis were cultured in TSB and TSA. Subculture of S. epidermidis was conducted every 24 h under aerobic conditions in a 37 ℃ incubator.

The experimental protocol accompanied a previous report with modifications.¹⁵ Ethanol extract, n-Hex, EtOAc, n-BuOH and H₂O fractions as well as isolated compounds 1-16 of C. obtuse were evaluated against C. acnes by determining the MIC values through the broth-dilution method. A freshly grown culture was diluted with GAM and prepared bacteria (2×10⁵ CFU/mL). Erythromycin was tried as a positive control. Samples were diluted in sterile broth and subsequently mixed with broth incubated with C. acnes. Dilutions were proceeded by 2-fold until to the desired final concentration. The test concentrations of ethanol extract, n-Hex, EtOAc, n-BuOH, and H₂O fractions were the range of 4,000-3.91 μg/mL. The test concentrations of isolated compounds were the range of 4,000–3.91 µM. Test samples were incubated under anaerobic conditions at 37 ℃ for 48 h until visible growth of the test microorganisms was observed in the control. The O.D. value was measured using a microplate reader at 600 nm. Also, S. epidermidis (2×10⁵ CFU/mL) was prepared, and the test concentrations of 50% EtOH extract, n-Hex, EtOAc, n-BuOH, and H₂O fractions were 10,000-9.76 μg/mL. Test samples were incubated at 37 ℃ for 24 h until visible growth of the test microorganisms was observed in the control.

RESULTS AND DISCUSSION

Isolation and Structure Elucidation of Compounds

The n-Hex and EtOAc fractions of the 50% aqueous ethanol extract from C. obtusa seed were subjected to repeated column chromatography over silica gel and Sephadex LH-20 eluting with various solvent systems to afford one novel abietane-diterpene (1) along with fifteen known terpenoids (2-16, Fig. 2). The NMR spectra for the isolates (1-16) can be found in supplementary materials.

Compound 1 was obtained as a white needle crystal. Its molecular formula was determined as C₂₀H₂₈O₃ according to the [M+Na]⁺ peak at m/z 339.1937 (Calcd. 339.1936) in the positive HR-ESI-MS, indicating 7 degrees of unsaturation. The specific optical rotation was [α]²⁰_D = + 45.2 (c 0.2, CHCl₃).

Inspection of ¹³C-NMR and DEPT-135° spectra revealed the presence of total 20 carbons including 3 methylene carbons (δ_C 42.1, 30.5, 20.2), 7 quaternary carbons (δ_C 215.8, 151.8, 140.5, 133.4, 129.9, 47.3, 43.6) and 10 methyl and methine carbons (Table 1). Among them, the peak of δ_C 215.8 is expected to be from a carbonyl group, and the remaining 6 sp² carbons indicate the presence of an aromatic ring with a phenolic hydroxyl group presumed based on the deshielded signal (δ_C 151.8). The signal at δ_C 74.3 is a characteristic of the aliphatic carbon directly attached to the oxygen atom. ¹H-NMR spectral analysis of compound 1 shows that the signals at δ_H 6.89 (1H, s) and 6.64 (1H, s) are from aromatic protons and the signals at δ_H 1.11-4.05 are from aliphatic carbon-linked protons, where the signal at δ_H 4.50 (1H, dd, J = 5.0, 3.2 Hz) predicts the presence of oxygen-bound aliphatic carbon. The deshielded septet splitting of the peak δ_H 3.10 (1H, J = 6.9 Hz) is a characteristic of isopropyl proton attached to the sp² carbon. Five methyl proton signals were identified by the integrated value at δ_H 1.22 (3H, d, J = 6.9 Hz), 1.21 (3H, d, J = 6.9 Hz), 1.24 (3H, s), 1.16 (3H, s) and 1.11 (3H, s). Judging by the information of 20 carbon atoms, the compound 1 was presumed to have the abietane-type diterpene framework. The suggested structure was further conformed by COSY and HMBC spectrum (Fig. 1a). The HMBC connections of the carbon at carbonyl group (δ_C 215.8) were established with the aliphatic carbons at δ_C 42.1, 26.7 and 21.4. Also, oxygenbearing δ_C 74.3 was connected to the aliphatic carbons δ_C 42.1 and 22.8, which confirmed that hydroxy group is in the C-1 position and the carbonyl is in the C-3 position in the diterpene skeleton. The relative configuration of hydroxy group at C-1 was established on the basis of the NOESY spectrum (Fig. 1b). The proton at the C-1 shows NOESY cross-peaks with the aromatic ring proton at the C-11, the methyl proton at C-20 position and the aliphatic proton of C-2 position, thus confirming the configuration of hydroxy group in axial (α) position. Based on these results, compound 1 was named 1α-hydroxy-hinokione, which was first isolated in nature.

Figure 1. ¹H-¹H COSY and key HMBC correlation (a) and key NOESY correlation (b) in the compound 1.

Table 1. 1D and HMBC (H → C) data for compound 1 in CDCl₃ (400 MHz)

Further phytochemical studies of the fractions led to the isolation of fifteen known terpene compounds (Fig. 2); 12-methoxy-8,11,13-abietatriene-7β,11-diol-3-one (2),¹² hinokione (3),¹⁶ 1,2-dehydrohinokione (4),¹⁷ 1α-hydroxyhinokiol (5),¹⁸ hinokiol (6),¹⁹ isohinokiol (7),²⁰ sugiol (8),²¹ ferruginol (9),²² cryptojaponol (10),²³ 7α,11-dihydroxy-12-methoxy-8,11,13-abietatriene(11),²⁴ 7β-hydroxydeoxocryptojaponol (12),²⁵ 6,7-dehydrodeoxocryptojaponol (13),²⁶ trans-communic acid (14),²⁷ α-eudesmol (15)²⁸ and hinokiic acid (16).²⁹ Besides abietane-type diterpenes (1-13), labdane-type diterpene 14 and sesquiterpens (15, 16) were identified. All of these compounds were elucidated based on the NMR studies as well as comparison of the spectral data to the literature values.

Figure 2. Isolated compounds 1-16 from the seeds of C. obtuse.

Antimicrobial Activities of Solvent Fractions and Isolated Compounds (1-16)

As the bacterial strains on the skin are closely involved in the occurrence of acne, examination of the antimicrobial activities for the plant extracts was conducted as the first step in order to develop natural acne treatment. In this experiment, the extract and solvent fractions (n-hexane, ethyl acetate, n-butanol and water fr.) of C. obtusa seed were subjected to screening of antimicrobial activities against C. acnes (CCARM 0081, 9009, 9010, 9089) and S. epidermidis (CCARM 3709, 3710, 3711) by determining the inhibition ratio using the broth dilution method. Among the strains, CCARM 9009, 9010 and 3710 possess a resistance to erythromycin, an antibiotics used clinically to treat acne. The results showed that the seed extracts and their non-polar fractions (n-Hex and EtOAc fr.) have significant antimicrobial activities against C. acnes (MIC 31.2 - 62.5 μg/mL) and S. epidermidis (MIC 156.25 - 2500 μg/mL) including resistant strains (the data were provided in supplementary material). On the other hand, the polar fractions (n-BuOH and H₂O fr.) showed very low activities with MIC values over 1000 μg/mL.

As previously mentioned, separation and purification was carried out using the active fractions (n-Hex and EtOAc fr.) leading to identify sixteen terpenoids (1-16). In the subsequent studies, the antimicrobial activities for each isolates (1-16) were screened against C. acnes and S. epidermidis (Table 2) respectively. In an activity experiment with C. acnes, all of the isolated compounds except hinokiol (6) showed good to moderate antibacterial activities. As a whole, the diterpenes (1-14) exhibited better antibacterial activities compared to the sesquiterpenes (15, 16), indicating that the abietane-type diterpenes are the main antimicrobial components of C. obtusa seeds extract. The ferruginol (9) showed the most potent activities against resistant strains with MIC value of 7.81 μM. Diterpene compounds 1,2-dehydrohinokione (4), 7β-hydroxydeoxocryptojaponol (12), and trans-communic acid (14) also exhibited excellent activities with MIC of 15.6 - 31.2 μM. On the other hand, in an experiment with S. epidermis, the terpenoids showed relatively very lower antimicrobial activities compared to the test with C. acnes. The best activity here was also observed in the ferruginol (9) with MIC of 15.6 μM. Only three compounds such as 1α-hydroxy-hinokione (1), hinokione (3), and 1,2-dehydrohinokione (4) other than 9 were observed as active substances with minimum inhibitory concentration (MIC) lower than 1000 μM. Analyzing the relationship between chemical structure and activity here revealed that the phenol at the C-12 position is the key factor for better activity against S. epidermidis, as observed in the previous report.³⁰ As shown results in compounds 1, 3 and 4, substitution of carbonyl instead of hydroxyl at the C-3 seems to be also important in exhibiting better activities.

Table 2. MIC values of isolated compounds 1-16 from C. obtusa seed on C. acnes and S. epidermidis

^[1]Erythromycin-resistant strains

^[2]Positive control

CONCLUSION

Chamaecyparis obtusa is an evergreen tree of the family Cupressaceae widely distributed on Jeju Island. In this study, the extracts and solvent fractions from C. obtusa seeds were investigated on antibacterial activities against C. acnes and S. epidermidis including resistant strains. As the results of the experiment, good to moderate activities were observed in n-Hex and EtOAc fractions. Phytochemical studies were conducted for the two fractions leading to identification of an abietane diterpene 1α-hydroxy-hinokione (1) as a new compound. In addition, fifteen known compounds were isolated; 12-methoxy-8,11,13-abietatriene-7β,11-diol-3-one (2) hinokione (3), 1,2-dehydrohinokione (4), 1α-hydroxy-hinokiol (5), hinokiol (6), isohinokiol (7), sugiol (8), ferruginol (9), cryptojaponol (10), 7α,11-dihydroxy-12-methoxy-8,11,13-abietatriene (11), 7β-hydroxydeoxocryptojaponol (12), 6,7-dehydrodeoxocryptojaponol (13), trans-communic acid (14), α-eudesmol (15), hinokiic acid (16). Among the isolated compounds (1-16), a phenolic diterpene ferruginol (9) was found to have uniquely potent antibacterial activities against both C. acnes and S. epidermidis at the same time. Its activities were excellent regardless of bacterial resistance strains. Subsequently, as shown in Table 2, diterpenes 1, 3 and 4 exhibited good activities in both strains C. acnes and S. epidermidis. Compounds 12 and 14 showed strong activities in C. acnes, whereas they showed little activities in S. epidermidis. In conclusion, C. obtusa seed extracts containing terpenoids 1α-hydroxyhinokione (1), hinokione (3), 1,2-dehydrohinoki-one (4) and ferruginol (9) can be applied as anti-acne ingredients in pharmaceutical and/or cosmetic preparations.