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A New Monoterpene from the Flower Buds of Buddleja officinalis
A New Monoterpene from the Flower Buds of Buddleja officinalis
Natural Product Sciences. 2013. Dec, 19(4): 355-359
Copyright © 2013, The Korean Society of Pharmacognosy
  • Received : September 11, 2013
  • Accepted : November 22, 2013
  • Published : December 31, 2013
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About the Authors
Chul Lee
Laboratory of Pharmacognosy, College of Pharmacy, Dankook University, Cheonan 330-714, Korea
Sora Lee
World Class University, Department of Nanobiomedicine, Dankook Univeristy, Cheonan 330-714, Korea
So-Young Park
World Class University, Department of Nanobiomedicine, Dankook Univeristy, Cheonan 330-714, Korea
soypark23@dankook.ac.kr

Abstract
A new monoterpene, crocusatin M ( 1 ), was isolated from the flower buds of Buddleja officinalis , together with four known monoterpenes, (6 R )-hydroxy-1,1,5-trimethylcyclohex-4-enone ( 2 ), (+)-dehydrovomifoliol ( 3 ), 7-epiloliolide ( 4 ), and crocusatin D ( 5 ). Their structures were determined by an extensive analysis of 1D, 2D NMR, HRESI-MS, and CD data as well as by comparison of their spectroscopic data with those of literatures. All isolates were evaluated for inhibitory activities on LPS-induced nitric oxide production in RAW 264.7 cells. Among them, compounds 2 and 3 showed moderate inhibitory effects with IC 50 values of 63.8 and 24.4 μg/ml, respectively.
Keywords
Introduction
The nitric oxide (NO) radical, synthesized by the inducible nitric oxide synthase (iNOS)-catalyzed oxidation of L-argininin, plays a critical role in a number of physiological and pathological processes in mammals. However, excess production of NO by iNOS in macrophages causes various inflammatory diseases (Alderton ., 2001) . Therefore, potent inhibitors of NO production might be regarded as promising anti-inflammatory drugs.
As part of our ongoing discovery for NO inhibitors from medicinal plants, we found the MeOH extract of flower buds of Buddleja officinalis Maxim. (Buddlejaceae) possessed the inhibitory effect on NO production in LPSinduced RAW 264.7 cells. B. officinalis is widely distributed in America, Africa, and Asia, and it has been used traditionally for the treatment of conjunctivitis and ophthalmologic diseases (Piao ., 2003 ; Lee ., 2010) . Previous phytochemical studies on this plant have led to the isolation of triterpenoidal saponins (Guo ., 2004) , phenylethanoids (Tai ., 2009) , flavonoids (Piao ., 2003) , and iridoids (Tai ., 2011) which are well-known for containing anti-oxidative, and antiinflammatory activities (Liao ., 1999 ; Piao ., 2003) . In this work, we have described in the isolation and structure determination of monoterpenoids, including their NO inhibitory effects.
Experimental
General experimental procedures − UV and CD spectra were obtained on JASCO UV-550 and JASCO J-715 spectrometers, respectively. NMR spectra were recorded on a Bruker Avance III 700 MHz NMR spectrometer using CD 3 OD and DMSO- d 6 as solvents with the tetramethylsilane as an internal standard. Chemical shift are presented in ppm. High resolution electrospray-ionization-time-of-flight (HR-ESI-TOF) and low resolution ESI mass spectra were measured on maXis 4G (Bruker) and LCQ Fleet (Thermoscientific), respectively. Semipreparative HPLC was performed using a Shimadzu Prominence UFLC with UV detector. Open column chromatography was performed using a silica gel 60 (Kiesel gel 60, 70 - 230 mesh, Merck) and RP-18 (Part NO. 5982-5752, Agilent). Thin-layer chromatography (TLC) was performed using precoated silica gel 60 F 254 (0.24 mm, Merck) plates.
Plant material − The flower buds of B. officinalis was purchased from a commercial market (Samhong medicinal herb market, Seoul, Korea) in 2013. One of the authors (S.-Y. Park) performed botanical identification, and a voucher specimen has been deposited in Pharmacognosy laboratory of College of Pharmacy, Dankook University, Korea.
Extraction and isolation − Dried and pulverized buds (3 kg) were extracted with 100 % MeOH (24 L, 3 times) at room temperature. The MeOH filtrate was evaporated in vacuo to yield the MeOH extract (301 g). The extract was suspended in distilled water, and then partitioned sequentially into n -hexane (55 g), CH 2 Cl 2 (15 g), EtOAc (19 g), and n -BuOH (61 g). The CH 2 Cl 2 soluble fraction (15 g) was chromatographed over a silica gel column chromatography (CC) to yield 8 subfractions (BOC1-BOC8), eluting with a step-gradient solvent system (100% CH 2 Cl 2 to 100% MeOH). The subfraction BOC2 and BOC3 were individually subjected over a RP-18 CC (Biotage MPLC, 10 to 100% MeOH, gradient) to give 6 and 7 subfractions (BOC21-BOC26, and BOC31-BOC37), respectively. The subfraction BOC21 was purified with a RP-18 preparative HPLC (15 to 45% ACN, gradient) to yield compounds 2 (6 mg), 3 (4 mg), and 4 (5 mg). Compounds 1 (6 mg), and 5 (3 mg) were obtained by a RP-18 preparative HPLC (15 to 36% ACN, gradient) from the subfraction BOC31.
Crocusatin M (1) − Colorless oil; [α] D 25 −45.5° ( c 0.08, MeOH); UV (MeOH) λ max 243.0 (3.70) nm; CD ( c 0.06, MeOH): [θ] 323.4 +0.42, [θ] 241.2 −4.45, [θ] 218 +2.96; HRESI-MS m/z 185.1177 [M + H] + (calcd for C 10 H 18 O 3 , 185.1178); 1 H NMR (700 MHz, CD 3 OD), and 13 C NMR (177 MHz, CD 3 OD), see Table 1 .
1H and13C-NMR data of compounds1a
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a) TMS was used as an internal standard; chemical shifts (δ) are expressed in ppm; J values are given in parentheses; 1H (700) and 13C (177) MHz in CD3OD.
(6R)-Hydroxy-1,1,5-trimethylcyclohex-4-enone (2) − Colorless oil; ESI-MS m/z 155 [M + H] + ; 1 H NMR (700 MHz, CD 3 OD) δ H : 5.81 (1H, s, H-4), 3.99 (1H, s, H-6), 2.33 (1H, d, J = 16.1 Hz, H a -2), 2.22 (1H, d, J = 16.1 Hz, H b -2), 2.04 (3H, s, H 3 -10), 1.05 (3H, s, H 3 -9), 0.98 (3H, s, H 3 -8); 13 C NMR (177 MHz, CD 3 OD) δ C : 200.5 (C-3), 164.1 (C-5), 124.9 (C-4), 75.7 (C-6), 48.6 (C-2), 38.1 (C-1), 25.8 (C-9), 20.8 (C-8), 20.2 (C-10).
(+)-Dehydrovomifoliol (3) − Colorless oil; ESI-MS m/z 223 [M + H] + ; 1 H NMR (700 MHz, CD 3 OD) δ H : 7.00 (1H, d, J = 15.4 Hz, H-7), 6.42 (1H, d, J = 15.4 Hz, H-8), 5.93 (1H, s, H-4), 2.60 (1H, d, J = 16.8 Hz, H a -2), 2.30 (3H, s, H 3 -10), 2.26 (1H, d, J = 16.8 Hz, H b -2), 1.89 (3H, s, H 3 -13), 1.05 (3H, s, H 3 -12), 1.01 (3H, s, H 3 -11); 13 C NMR (177 MHz, CD 3 OD) δ C : 199.2 (C-3), 199.0 (C-9), 163.3 (C-5), 146.9 (C-7), 130.3 (C-8), 126.6 (C-4), 78.6 (C-6), 49.2 (C-2), 41.3 (C-1), 26.3 (C-10), 23.4 (C-12), 22.1 (C-11), 17.8 (C-13).
7-Epiloliolide (4) − Colorless oil; ESI-MS m/z 197 [M + H] + ; 1 H NMR (700 MHz, CD 3 OD) δ H : 5.74 (1H, s, H-3), 4.21 (1H, m, H-7), 2.42 (1H, dt, J = 14.7, 2.8 Hz, H a -8),1.98 (1H, dt, J = 14.0, 2.8 Hz, H b -8), 1.76 (3H, s, H 3 -12), 1.74 (1H, dd, J = 14.7, 3.5 Hz, H a -6), 1.53 (1H, dd, J = 14.7, 3.5 Hz, H b -6), 1.46 (3H, s, H 3 -11), 1.27 (3H, s, H 3 -10); 13 C NMR (177 MHz, CD 3 OD) δ C : 184.3 (C-4), 173.1 (C-2), 111.9 (C-3), 87.6 (C-9), 65.8 (C-7), 46.6 (C-6), 45.0 (C-8), 35.8 (C-5), 29.6 (C-11), 26.1 (C-10), 25.6 (C-12).
Crocusatin D (5) − Colorless oil; ESI-MS m/z 185 [M + H] + ; 1 H NMR (700 MHz, CD 3 OD) δ H : 4.33 (1H, dd, J = 14.0, 5.6 Hz, H-3), 4.27 (1H, d, J = 11.9 Hz, H a -7), 4.22 (1H, d, J = 11.9 Hz, H b -7), 2.02 (1H, dd, J = 12.6, 5.6 Hz, H a -2), 1.97 (3H, s, H 3 -10), 1.76 (1H, t, J = 12.6 Hz, H b -2), 1.26 (3H, s, H 3 -9), 1.22 (3H, s, H 3 -8); 13 C NMR (177 MHz, CD 3 OD) δ C : 200.7 (C-4), 160.8 (C-6), 130.7 (C-5), 69.1 (C-3), 57.8 (C-7), 45.8 (C-1), 35.4 (C-2), 28.2 (C-9), 24.3 (C-8), 10.4 (C-10).
Cell cultures − RAW 264.7 mouse macrophage cells were purchased from Korean Cell Line Bank. The cells were routinely cultured in RPMI medium supplemented with 10% fetal bovine serum (Equitech-Bio, USA) at 37 ℃ under 5% CO 2 . These cells were utilized for experiments during the exponential growth phase.
Cytotoxicity test − The cytotoxic effects of extracts, fractions or isolated pure compounds were tested using MTT assay. Briefly, RAW 264.7 cells were plated on a 96 well plate and incubated overnight. Following morning, cells were treated with samples for 24 h. Then, cells were incubated with MTT (5 mg/ml) for 3 h and followed by lysis buffer for overnight. Absorbance was measured at 540 nm using a microplate reader (Molecular Device E -max). All determinations were carried out in triplicates and repeated at least three times.
Determination of NO production − The NO production was determined using Griess reagents (Sigma Aldrich). The cells pre-treated with samples for 1 h were incubated with LPS for additional 24 h. Then, the supernatants were transferred to Eppendorf tubes and centrifuged at 12,000 rpm for 15 min in order to remove the cell debris. The same amount of Griess reagent and supernatant was mixed and incubated at 37 ℃ for 20 min in the dark. The absorbance was determined at 540 nm using a microplate reader.
Results and Discussion
Repeated column chromatographic separation of the CH 2 Cl 2 -soluble extract of the flower buds of B. officinalis led to isolation of a new monoterpene, crocusatin M ( 1 ), along with four known monoterpenes ( Fig. 1 ). The known compounds identified as (6 R )-hydroxy-1,1,5-trimethylcyclohex-4-enone ( 2 ) (Li and Wu, 2002) , (+)-dehydrovomifoliol ( 3 ) (Kim ., 2004) , 7-epiloliolide ( 4 ) (Borkosky ., 1996) , and crocusatin D ( 5 ) (Li and Wu, 2002) , by comparison of their spectroscopic data with those of literatures.
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Chemical structures of isolated compounds 1 - 5.
Compound 1 was isolated as colorless oil. The molecular formula was determined as C 10 H 17 O 3 from quasi-molecular ion peak at m/z 185.1177 [M + H] + (calcd for C 10 H 18 O 3 , 185.1178). This formula indicated 3 degrees of unsaturation, and the UV spectrum at 243.0 nm implied a characteristic absorption band for a conjugated carbonyl group in the molecule (Li and Wu, 2002) . The 1 H NMR spectrum of 1 exhibited characteristic proton signals due to a gem -dimethyl group at δ H 1.11 (3H, s) and 1.01 (3H, s), a vinyl methyl proton at δ H 2.02 (3H, d, J = 1.4 Hz), two oxygenated methylene protons at δ H 3.76 (1H, d, J = 11.2 Hz), and 3.64 (1H, d, J = 11.2 Hz), two methylene protons 2.92 (1H, d, J = 17.5 Hz), and 2.08 (1H, d, J = 17.5 Hz), and a vinyl proton at δ H 5.92 (1H, s). The 13 C and DEPT spectra of 1 showed 10 distinct carbon signals including three methyl carbons at δ C 23.8, 22.7, and 18.6, two methylene carbons at δ C 64.5, and 49.6, a methine carbon at δ C 126.9, and four quaternary carbons at δ C 200.5, 166.3, 77.5, and 39.9. These spectral data suggested that 1 was closely comparable to those of crocusatin C except for the additional hydroxyl group at C-6 instead of the proton signal of crocusatin C (H-6), which is a monocyclic monoterpene isolated from Crocus sativus (Li and Wu, 2002) . The large coupling constant of H-7 ( J = 11.2 Hz), the quaternary carbon signal at δ C 77.5, and the observed HMBC correlation between C-6 (δ C 77.5) and H-7 (δ H 3.76 and 3.64) clearly supported this structure ( Fig. 2 ). The relative stereochemistry of 1 was deduced on the basis of NOE experiment ( Fig. 3 ). The key NOE correlations between H 2 -7/H-9, H 2 -7/H-2α, H-2α/H-9, and H-2β/H-8 were observed, corresponding to a α-quasiaxial orientation of H 2 -7 (Machida ., 1998) . Furthermore, the CD spectrum of 1 showed the Cotton effects at 323.4 (Δε +0.42), 241.2 (Δε -4.45), and 218.0 (Δε +2.96) nm (Straubinger ., 1997 ; Kim ., 2004) , indicating the chirality of C-6 was R configuration ( Fig. 4 ). Therefore, compound 1 was elucidated as (6 R )-6-hydroxy-6-hydroxymethyl-1,1,5-trimethylcyclohex-4-enone, named as crocusatin M.
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The HMBC correlations of compound 1.
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The key NOE correlation of compound 1.
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CD spectrum of compound 1.
Inhibitory effects of compounds1 - 5against LPSinduced NO production
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a) Concentration required to achieve 50% inhibition of NO production b) Cell cytotoxicity was found c) A positive control.
All isolates ( 1 - 5 ) were evaluated for their inhibitory effects on NO production in LPS-stimulated RAW 264.7 cells ( Table 2 ). Compounds 2 and 3 exhibited the inhibitory effects on NO production with IC 50 values of 63.8 and 24.4 μg/ml, respectively. The inhibitory effect of compound 3 is comparable to that of caffeic acid, the positive control used for this assay (IC 50 27.6 μg/ml). However, compounds 1 and 5 had no significant effect (IC 50 > 100 μg/ml) and compound 4 was cytotoxic to RAW 264.7 cells with the tested concentration.
In summary, five monoterpenes including one new monoterpene, crocusatin M, were isolated from the flower buds of B. officinalis , and their structures were elucidated based on the comparison of their spectroscopic data with those of literatures. Their inhibitory effects on NO production were also evaluated in LPS-stimulated RAW 264.7 cells. Compounds 2 and 3 showed the moderate inhibitory effect on NO production. Taken together, our findings suggest that compounds 2 and 3 are nitric oxide inhibitors.
Acknowledgements
We thank the Korea Basic Science Institute for providing CD experiment and prof. J. S. Oh, Dr. S. S. Hong, and W. S. Jeong at Gyeonggi Natural Products Research Institute for their help for NMR experiments.
References
Alderton W.K. , Cooper C.E. , Knowles R.G. (2001) Nitric oxide synthases: structure, function, and inhibition. Biochem. J. 357 593 - 615    DOI : 10.1042/0264-6021:3570593
Borkosky S. , Valdes D.A. , Bardon A. , Diaz J.G. , Herz W. (1996) Sesquiterpene lactones and other consitituents of Eirmocephala megaphylla and Cyrtocymura cincta. Phytochemistry. 42 1637 - 1639    DOI : 10.1016/0031-9422(96)82943-8
Guo H. , Koike K. , Li W. , Satou T. , Guo D. , Nikaido T. (2004) Saponins from the flower buds of Buddleja officinalis. J. Nat. Prod. 67 10 - 13    DOI : 10.1021/np0300131
Kim I. , Chin Y.-W. , Lim S.W. , Kim Y.C. , Kim J. (2004) Norisoprenoids and hepatoprotective flavone glycosides from the aerial parts of Beta vulgaris var. cicla. Arch. Pharm. Res. 27 600 - 603    DOI : 10.1007/BF02980156
Lee Y.J. , Moon M.K. , Hwang S.M. , Yoon J.J. , Lee S.M. , Seo K.S. , Kim J.S. , Kang D.G. , Lee H.S. (2010) Anti-inflammatory effect of Buddleja officinalis on vascular inflammation in human umbilical vein endothelial cells. Am. J. Chin. Med. 38 585 - 598    DOI : 10.1142/S0192415X1000807X
Li C.-Y. , Wu T.-S. (2002) Constituents of the pollen of Crocus sativus L. and their tyrosinase inhibitory activity. Chem. Pharm. Bull. 50 1305 - 1309    DOI : 10.1248/cpb.50.1305
Liao Y.-H. , Houghton P.J. , Hoult J.R.S. (1999) Novel and known constituents from Buddleja species and their activity against leukocyte eicosanoid generation. J. Nat. Prod. 62 1241 - 1245    DOI : 10.1021/np990092+
Machida K. , Onodera R. , Furuta K. , Kikuchi M. (1998) Studies of the constituents of Gardenia species. I. monoterpenoids from Gardieniae Fructus. Chem. Pharm. Bull. 46 1295 - 1300    DOI : 10.1248/cpb.46.1295
Piao M.S. , Kim M.R. , Lee D.G. , Park Y. , Hahm K.S. , Moon Y.H. , Woo E.R. (2003) Antioxidative constituents from Buddleja officinalis. Arch. Pharm. Res. 26 453 - 457    DOI : 10.1007/BF02976861
Straubinger M. , Jezussek M. , Waibel R. , Winterhalter P. (1997) Novel glycosidic constituents from Saffron. J. Agric. Food Chem. 45 1678 - 1681    DOI : 10.1021/jf960861k
Tai B.H. , Jung B.Y. , Cuong N.M. , Linh P.T. , Tung N.H. , Nhiem N.X. , Huong T.T. , Anh N.T. , Kim J.A. , Kim S.K. , Kim Y.H. (2009) Total peroxynitrite scavenging capacity of phenylethanoid and flavonoid glycosides from the flowers of Buddleja officinalis. Biol. Pharm. Bull. 32 1952 - 1956    DOI : 10.1248/bpb.32.1952
Tai B.H. , Nhien N.X. , Quang T.H. , Ngan N.T.T. , Tung N.H. , Kim Y. , Lee J.-J. , Myung C.-S. , Cuong N.M. , Kim Y.H. (2011) A new iridoid and effect on the rat aortic vascular smooth muscle cell proliferation of isolated compounds from Buddleja officinalis. Bioorg. Med. Chem. Lett. 21 3462 - 3466    DOI : 10.1016/j.bmcl.2011.03.078