A new phenylethanoid glycoside, named rosmacinalis (1), and 6x known compounds {2-phenylethyl O-α-L-rhamnopyranosyl-(1‴→6″)-O-β-D-glucopyranoside (2), clinopodiolide C (3), rosmanol (4), 7α-methoxyrosmanol (5) 7β-methoxyrosmanol (6) and carnosol (7)} were isolated from the leaves of Rosmarinus officinalis. Their structures were determined by extensive analysis of high-resolution electron spray ionization mass spectrum and nuclear magnetic resonance spectral data, as well as by comparison of the spectral data with those reported in the literature. Anti-inflammatory activity of compounds 1‐7 was evaluated by their inhibition of NO production in lipopolysaccharide-stimulated RAW 264.7 cells. At a concentration of 100 µM, compounds 1 and 2 exhibited inhibitory rates of 47.1% ± 2.2% and 44.5% ± 1.3%, respectively, while compounds 3‐7 showed a cytotoxic effect. After dilution to a concentration of 20 µM, except compound 7, compounds 1‐6 did not show a cytotoxic effect. Their NO inhibitory rates ranged from 14.2% ± 1.3% to 31.1% ± 1.9%.
Rosmarinus officinalis Linnaeus (Lamiaceae family) is a perennial herb native to the Mediterranean region, where it is commonly known as rosemary. It can be used either in raw materials (fresh and dry) or in processed products (extract and essential oil) for cosmetic, pharmaceutical, and food applications.1 In traditional medicinal remedies, rosemary leaves are used to treat inflammation-related diseases and pain, enhance circulation, boost the immune system, and promote hair growth.2 Rosemary essential oil mainly contains 1,8-cineole, camphor, pinene, borneol, and limonene, which exhibit antidepressant, antimicrobial, anti-allergic, and smooth muscle relaxant effects.3 In natural cosmetics, rosemary is used not only as an antioxidant agent but also as an important fragrance ingredient. Beside essential oil, recent literature has indicated that the biological activities of rosemary have been attributed to phenolic groups, especially rosmarinic acid and carnosic acid derivatives.2 Many reports suggest that rosmarinic acid has potential antioxidant and anti-inflammatory activity, and hepatoprotective effects that could be developed for pharmaceutical and cosmetic applications.4,5 Carnosic acid and its derivatives are phenolic diterpene compounds. Their structures contain an ortho-dihydroquinone fragment, which easily reacts with free radicals produced in biological systems. Carnosic acid compounds play crucial roles because of their antioxidant, anti-inflammatory, and anticarcinogenic activities.6-8 The finding of phenolic compounds from rosemary therefore attracted medicinal chemists to explore the biomolecules and clarify the therapeutic potential of this medicinal plant.9 With the aim of finding natural anti-inflammatory constituents, we describe herein the isolation and identification of 2 phenylethanoid glycosides (1 and 2) and 5 carnosic acid derivatives (5-7) from rosemary leaves (Figure 1). Compound 1 was determined to be a new compound. The anti-inflammatory activity of compounds (1-7) was evaluated by their ability to inhibit NO production in lipopolysaccharide (LPS)-activated RAW 264.7 cells.
Chemical structure of compounds 1‐7 isolated from the leaves of Rosmarinus officinalis.
Results and Discussion
Compound 1 was obtained as a white amorphous powder. Its molecular formula was deduced to be C21H32O12 based on the cluster of quasi-molecular ion peaks in the high-resolution electron spray ionization mass spectrum (HR-ESI-MS), including m/z 499.1773 [M + Na]+ (calculated for [C21H32O12Na]+, 499.1786), m/z 494.2216 [M+NH4]+ (calculated for [C21H36O12N]+, 494.2232), and m/z 477.1965 [M + H]+ (calculated for [C21H33O12]+, 477.1967) (Supplemental Figure S1). The 1H-nuclear magnetic resonance (NMR) spectrum of 1 exhibited signals corresponding to a 1,3,4-trisubstituted benzene moiety (δH 6.87 1H, d, J = 2.0 Hz], 6.73 [1H, d, J = 8.0 Hz], 6.70 [1H, dd, J = 2.0 and 8.0 Hz]), 2 anomeric protons (δH 4.77 [1H, d, J = 1.5 Hz], 4.31 [1H, d, J = 7.5 Hz]), a methoxy group (δH 3.86 [3H, s]), and a doublet methyl group (δH 1.28 [3H, d, J = 6.5 Hz]) (Supplementarl Figure S2). The 13C-NMR spectrum of 1 (Supplemental Figure S3) indicated the presence of 21 carbon signals, which were then further classified from the heteronuclear single-quantum coherence spectrum (Supplemental Figure S4) as 6 aromatic carbons (δC 148.9, 145.9, 131.6, 122.4, 116.1, 113.8), 2 anomeric carbons (δC 104.5, 102.3), 8 oxygenated methines (δC 78.1, 76.9, 75.1, 74.0, 72.4, 72.1, 71.7, 69.8), 2 oxygenated methylenes (δC 72.1, 68.2), 1 aliphatic methylene (δC 36.8), 1 methoxy group (δC 56.5), and 1 methyl group (δC 18.0). In the heteronuclear multiple bond correlation (HMBC) spectra, correlations between H-2 (δH 2.87) and C-1′ (δC 131.6)/ C-2′ (δC 113.8)/ C-6′ (δC 122.4), H-2′ (δH 6.87)/ H-6′ (δH 6.70) and C-2 (δC 36.8) were observed establishing the connection between C-2 and C-1′ (Figure 2). The deshielded carbon signal of C-4′ (δC 145.9) together with HMBC correlations between H-2′ (δH 6.87)/H-6′ (δH 6.70) and C-4′ suggested the presence of a hydroxy group at C-4′. Additionally, HMBC correlations between H-5′ (δH 6.73) and C-3′ (δC 148.9), methoxy protons (δH 3.86), and C-3″ clearly confirmed a methoxy group at C-3′ (Supplemental Figure S5). Besides the 1,3,4-trisubstituted benzene moiety, the correlation spectroscopy (COSY) spectrum of 1 (Supplemental Figure S6) exhibited 3 additional spin systems (Figure 2) of an ethylene group (H-1 [δH 4.02, 3.75]/ H-2 [δH 2.87]) and 2 hexose sugar units (H-1″ [δH 4.31]/ H-2″ [δH 3.20]/ H-3″ [δH 3.36]/ H-4″ [δH 3.31]/ H-5″ [δH 3.43]/ H-6″ [δH 3.99, 3.63] and H-1‴ [δH 4.77]/ H-2‴ [δH 3.84]/ H-3‴ [δH 3.68]/ H-4‴ [δH 3.38]/ H-5‴ [δH 3.69]/ H-6‴ [δH 1.28]). Vicinal coupling J values between protons H-1″ and H-2″ (J = 7.5 Hz), H-2″ and H-3″ (J = 9.0 Hz), H-3″ and H-4″ (J = 9.0 Hz), and H-4″ and H-5″ (J = 9.0 Hz) indicated the presence of a β-glucopyranose moiety, while vicinal coupling J values between protons H-1‴ and H-2‴ (J = 1.5 Hz), H-2‴ and H-3‴ (J = 3.0 Hz), H-3‴ and H-4‴ (J = 9.0 Hz), and H-4‴ and H-5‴ (J = 9.0 Hz) indicated the presence of an α-rhamnopyranose moiety. Furthermore, HMBC correlations of Glc H-1″ (δH 4.31)/ C-1 (δC 72.1) and Rha H-1‴ (δH 4.77)/Glc C-6″ (δC 68.2) revealed O-glycosidic linkages of the glucose moiety to C-1 and the rhamnose moiety to Glc C-6″, respectively. Finally, the presence of D-glucose and L-rhamnose in the acid hydrolysis products of compound 1 were confirmed by gas chromatography (GC) analysis of their trimethylsilyl derivatives, as previously described.10 Consequently, compound 1 was determined to be 2-(4′-hydroxy-3′-methoxyphenyl)-ethyl O-α-L-rhamnopyranosyl-(1‴→6″)-O-β-D-glucopyranoside, a new phenylethanoid glycoside and named as rosmacinalis.
Important HMBC and COSY correlations of compound 1.COSY, correlation spectroscopy; HMBC, heteronuclear multiple bond correlation.
Compound 2 was determined to be an additional phenylethanoid glycoside as 2-phenylethyl O-α-L-rhamnopyranosyl-(1‴→6″)-O-β-D-glucopyranoside, while compounds 3-7 were the abietane diterpenoids clinopodiolide C (3), rosmanol (4), 7α-methoxyrosmanol (5), 7β-methoxyrosmanol (7), and carnosol (7). The NMR spectral data of compounds 2-7 were consistent with those previously reported in the literature (Supplemental Figure S7-S18).11-15 Except for compounds 1-3, phenolic diterpenes 4-7 were previously reported from R. officinalis. Anti-inflammatory activity of compounds 1-7 was evaluated by their ability to inhibit NO production in LPS-stimulated RAW 264.7 cells. First, the cytotoxic effects of the compounds on RAW 264.7 cells were examined to ensure that the NO inhibitory activity was not affected by cytotoxic activity. Each compound was assessed at 2o concentrations, 100 and 20 µM. At a concentration of 100 µM, compounds 1 and 2 did not show a cytotoxic effect, and they exhibited NO inhibitory rates of 47.1% ± 2.2% and 44.5% ± 1.3%, respectively. However, at 100 µM, the effects on NO production of compounds 3-7 were not significant due to their cytotoxic effect (cell viability below 80%, Table 1). At a diluted concentration of 20 µM, compound 7 was cytotoxic, but compounds 1-6 did not show a cytotoxic effect. The NO inhibitory activities of compounds 1-6 were then obtained with inhibitory rates ranging from 14.2% ± 1.3% to 31.1% ± 1.9%. L-NMMA (NG-monomethyl-L-arginine) was used as a positive control. Its NO inhibitory values were 92.5% ± 1.1% and 82.2% ± 2.5% at concentrations of 100 and 20 µM, respectively.
Effect of Compounds 1‐7 on NO Production in LPS-Stimulated RAW 264.7 cells
Optical rotation was measured on a Jasco P-2000 polarimeter. HR-ESI-MS was acquired on an Agilent 6530 Accurate Mass Q-TOF system, and NMR spectra on a Bruker Avance III 500 MHz spectrometer. Flash column chromatography was performed using either silica gel or reversed phase (RP-18) resins as adsorbent. Thin-layer chromatography was carried out on precoated silica gel 60 F254 and/or RP-18 F254S plates. Traces of compounds were visualized under ultraviolet irradiation (254 and 365 nm) and by spraying with H2SO4 solution (5%) followed by heating with a heat gun.
Plant Material
Rosmarinus officinalis L. samples, identified by Dr Nguyen The Cuong at the Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology (VAST), were collected in Ha Giang province in January 2019. A voucher specimen (number: RO.19.01) is kept at the Lab of Pharmaceutical Chemistry, VNU University of Science.
Extraction and Isolation
Dried and powdered R. officinalis leaves (2 kg) were ultrasonically extracted with methanol, 3 times (each 5 L for 30 minutes at room temperature). After filtration, the filtrate was evaporated in vacuo to give the methanol extract (156 g). This was suspended in water and successively separated with n-hexane, ethyl acetate, and n-butanol to give n-hexane, ethyl acetate-, and n-butanol-soluble fractions.
The ethyl acetate extract (ROE, 85 g) was loaded on a silica gel column (Ф = 7 cm) and eluted with n-hexane and ethyl acetate (1 L step wise, 40/0, 20/1, 10/1, 5/1, 3/1, 1/1, v/v) to give 6 fractions ROE1-ROE6. Fraction ROE2 was chromatographed on a reverse-phase C-18 column, eluting with acetone/water (5/2, v/v) to give 2 fractions ROE2A and ROE2B. Fraction ROE2A was purified on a reverse-phase C-18 column, eluting with acetone/water (3/1, v/v) to give compound 3 (3.3 mg). Fraction ROE3 was first separated on a reverse-phase C-18 column eluting with acetone/water (2/1, v/v) and then further purified on another reverse-phase C-18 column eluting with methanol/water (3/1, v/v) to give compounds 5 (12.6 mg) and 7 (7.3 mg). Fraction ROE5 was separated on a reverse-phase C-18 column, eluting with acetone/water (5/2, v/v) to give 3e fractions ROE3A-ROE3C. Fractions ROE3A and ROE3B were purified on a reverse-phase C-18 column, eluting with methanol/water (3/1, v/v) to give compounds 4 (45.6 mg) and 5 (29 mg), respectively. The n-butanol extract (ROB, 23 g) was separated by silica gel column chromatography using a solvent system of dichloromethane/ methanol/water (4/1/0.1, v/v/v) to give 4 fractions ROB1-ROB4. Fraction ROB1 was purified on a silica gel column, eluting with acetone/dichloromethane/water (3/1/0.2, v/v/v) to give compound 2 (26 mg). Fraction ROB3 was purified on a reverse-phase C-18 column, eluting with methanol/water (1/1, v/v) to give compound 1 (15 mg).
Rosmacinalis (1)
White amorphous powder, − 53.8° (c 0.1, MeOH); HR-ESI-MS m/z 499.1773 [M + Na]+ (calculated for [C21H32O12Na]+, 499.1786), m/z 494.2216 [M+NH4]+ (calculated for [C21H36O12N]+, 494.2232), and m/z 477.1965 [M + H]+ (calculated for [C21H33O12]+, 477.1967);1H-NMR (CD3OD, 500 MHz) and 13C-NMR (CD3OD, 125 MHz) data are given in Table 2.
1H-NMR and 13C-NMR Spectroscopic Data for Compound 1 in Deuterated Methanol.
Position
δCa
δHb (mult., J in Hz)
Position
δCa
δHb (mult., J in Hz)
1
72.1
4.02 (m) 3.75 (m)
3″
78.1
3.36 (t, 9.0)
2
36.8
2.87 (m)
4″
71.7
3.31 (t, 9.0)
1′
131.6
-
5″
76.9
3.43 (m)
2′
113.8
6.87 (d, 2.0)
6″
68.2
3.99 (dd, 2.0, 12.0) 3.63 (dd, 5.5, 12.0)
3′
148.9
-
6″-O-Rha
4′
145.9
-
1‴
102.3
4.77 (d, 1.5)
5′
116.1
6.73 (d, 8.0)
2‴
72.1
3.84 (dd, 1.5, 3.0)
6′
122.4
6.70 (dd, 2.0, 8.0)
3‴
72.4
3.68 (dd, 3.0, 9.0)
3′-OCH3
56.5
3.86 (s)
4‴
74.0
3.38 (t, 9.0)
1-O-Glc
5‴
69.8
3.69 (m)
1″
104.5
4.31 (d, 7.5)
6‴
18.0
1.28 (d, 6.5)
2″
75.1
3.20 (dd, 7.5, 9.0)
Abbreviation: NMR, nuclear magnetic resonance.
Measured at (a)125 MHz, (b)500 MHz.
Acid hydrolysis and confirmation of monosaccharide
The RAW264.7 cells were received from Perugia University, Italy and were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 10 mM HEPES, and 1 mM sodium pyruvate. The cells were dispensed into 96-well plate (2 × 105 cells/well) and incubated at 37 °C in a humidified atmosphere (5% CO2 and 95% air). After 24 hours of incubation, the culture medium was replaced with DMEM without FBS and continuously incubated for 3 hours. The cells were treated with either compounds or vehicle solution and then stimulated with LPS (1 µg/mL) in the next 2 hours. After an additional 24 hours incubation, the cell culture medium (100 µL) was mixed with an equal volume of Griess reagent (Promega, Fitchburg, WI, USA) for 10 minutes and the absorbance was read at 540 nm. The amount of nitrite, an indicator of NO production in the medium, was obtained from a standard curve, which was constructed by NaNO2 serial dilution. L-NMMA was used as a positive control.
Cell viability was determined by adding 10 µL MTT solution (5 mg/mL) and incubating for 4 hours. Formazan crystals were dissolved in 50 µL of DMSO. Absorbance was read at 540 nm and compared with the vehicle group. Data were expressed as mean ± SD of triplicate experiments. Statistical analysis was performed by GraphPad Prism software.
Conclusions
Two phenylethanoid glycosides, 2-(4′-hydroxy-3′-methoxyphenyl)-ethyl O-α-L-rhamnopyranosyl-(1‴→6″-O-β-D-glucopyranoside (rosmacinalis, 1) and 2-phenylethyl O-α-L-rhamnopyranosyl-(1‴→6″-O-β-D-glucopyranoside (2) along with 5 carnosic acid derivatives, clinopodiolide C (3), rosmanol (4), 7α-methoxyrosmanol (5), 7β-methoxyrosmanol (6) and carnosol (7) were isolated from the leaves of R. officinalis. Of these, 1 is a new compound. Compounds (1-7) were evaluated for their inhibition of NO production in LPS-activated RAW 264.7 cells. At a concentration of 100 µM, compounds 1 and 2 exhibited inhibitory rates of 47.1% ± 2.2% and 44.5% ± 1.3%, respectively, while compounds 3-7 showed cytotoxic effects. After dilution to a concentration of 20 µM, except compound 7, compounds 1-6 did not show a cytotoxic effect. Their NO inhibitory rates ranged from 14.2% ± 1.3% to 31.1 %± 1.9%.
Supplemental Material
Supplementary Material 1 - Supplemental material for A New Phenylethanoid Glycoside From the Leaves of Rosmarinus officinalis With Nitric Oxide Inhibitory Activity
Supplemental material, Supplementary Material 1, for A New Phenylethanoid Glycoside From the Leaves of Rosmarinus officinalis With Nitric Oxide Inhibitory Activity by Le Thi Huyen, Le Thi Oanh, Nguyen Thi Son, Nguyen Thi Minh Thu, Nguyen Huy Hoang, Pham Hai Yen, Nguyen Xuan Nhiem, Bui Huu Tai and Phan Van Kiem in Natural Product Communications
Footnotes
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research is funded by the Vietnam National University, Hanoi (VNU) under project number QG.19.11.
ORCID iD
Phan Van Kiem
Supplemental Material
Supplemental material for this article is available online.
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