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Two new diterpenoids from the roots of Pygmacopremna herbacea
Phytochemistry Letters 12 (2015) 129–132
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Two new diterpenoids from the roots of Pygmacopremna herbacea Kovela Satish a, Galla Srihari a, Gangarajula Sudhakar a, Kura Narsimha b, Tadikamalla Prabhakar Rao b, Madugula Marthanda Murthy a,* a b
Crop Protection Chemicals Division (Organic Chemistry-II), CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India Center for Nuclear Magnetic Resonance, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 January 2015 Received in revised form 21 February 2015 Accepted 6 March 2015 Available online 18 March 2015
Two new diterpenoids, neobharangi-d-lactone (1) and bharangi quinone (2) along with two known compounds neobharangin (3) and bharangin (4) were isolated from the ethyl acetate extract of root nodules of Pygmacopremna herbacea. The structures of the new compounds were established by 1D and 2D NMR spectroscopic data. ß 2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Keywords: Pygmacopremna herbacea Verbenaceae Diterpenoids Neobharangi-d-lactone Bharangi quinone Bharangin Neobharangin
1. Introduction The Verbenaceae family is a rich source of terpenoids and quinonemethides. Pygmacopremna herbacea syn. Premna herbacea belonging to the family of Verbenaceae is a small herb or sometimes an under shrub, arising from a perennial rootstock distributed through the subtropical Himalayas, Assam, West Bengal, Bihar, Orissa and Deccan peninsula (Krishnamurthi, 1969). It is known as Gantubharangi in Telugu, Bharangi in Hindi. P. herbacea roots are used in the ayurvedic system and also a folk medicine against inflammatory and malaria in the Yunnan province of China. The roots of this plant are used in the preparations of ayurvedic medicines either alone or as an ingredient for the treatment of bronchitis, asthma, blood pressure, tumors, inflammation, hiccough, epilepsy, helimenthiasis, etc. (Nayar et al., 1976). Fresh root stocks and roots along with ginger are given in asthma, rheumatism and dropsy. The rootstocks and root bark are used to cure toothache. The leaves are used in fevers and cough, and their poultices are applied to boils (Ambasta, 1996). Previous investigations on P. herbacea by our group have resulted in the
* Corresponding author. Tel.: +91 40 27193933; fax: +91 40 27193382. E-mail address: [email protected] (M. Marthanda Murthy).
isolation and characterization of bharangin (Sankaram et al., 1988a), isobharangin (Sankaram et al., 1989), bharanginin (Sankaram et al., 1988b), pygmacone (Sankaram and Marthandamurthy, 1991), bharangi-d-lactone, bharangi-g-lactone (Srihari et al., 2011), 3-dehydroxy isobharangin and neobharangin (Satish et al., 2011). In order to isolate the minor active constituents, we reinvestigated this plant and obtained two new compounds (1) and (2) from the ethyl acetate extract of the root nodules. In this paper, we described the isolation and structure determination of 1 and 2 using 2D NMR techniques. 2. Results and discussion P. herbacea root powder was extracted successively with nhexane and ethyl acetate in a soxhlet apparatus. The extracts were concentrated under vacuum. The concentrated ethyl acetate extract was subjected to repeated column chromatography over silica gel resulting in the isolation of new compounds (1) and (2) along with known compounds neobharangin (3) (Satish et al., 2011) and bharangin (4) (Sankaram et al., 1988a) (Fig. 1). Compound 1 was obtained as a brown colored semi solid, ½a25 D þ 17:33 (c 0.15, CHCl3). High resolution mass analysis of compound 1 showed the molecular ion [(M+H)+], 403.2112, gave the elemental composition C23H31O6 (calcd. 403.2115) in its
http://dx.doi.org/10.1016/j.phytol.2015.03.010 1874-3900/ß 2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
K. Satish et al. / Phytochemistry Letters 12 (2015) 129–132
130
O
AcO
OCH3
O H
O O 3
2
A
4
19
O
O
HO 18
11 10 9
1 5
B 8
20
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 –OCOCH3 –OCOCH3 –OCH3
O 2
12
HO
O 16 13
C 14
15
18
O
O
10
2 1 A 3 5 4
17
7
6
Position C
H
O 1
Table 2 13 C NMR (125 MHz) data for compounds 1, 2, 3 and 4.
OAc
O
19
3
20
11 9
B 8 6
12
O 13
C 14
16 15 17
7
4
Fig. 1. Structures of isolated compounds from P. herbacea.
spectrum. The UV spectrum showed absorption maxima at 267 nm (log e 3.60). The IR spectrum of 1 showed absorption bands at nmax: 1737, 1687 and 1771 cm1 indicating the presence of d-lactone, saturated carbonyl and carbonyl of acetate groups, respectively. The comparison of 1H NMR spectrum of 1 and 3 (Satish et al., 2011) showed a notable difference in AB doublets present in the B ring at dH 6.44 (1H, d, J = 6.8 Hz) of H-6 and dH 6.61 (1H, d, J = 6.8 Hz) of H7 in the structure of 3 were replaced by methylene groups at C-6, dH 2.74 (1H, dd, J = 12.6, 17.7 Hz) and dH 2.67 (1H, dd, J = 3.9, 17.7 Hz) and saturated carbonyl group at C-7 in the structure of compound 1 (Table 1). The other two AB doublets protons resonated at dH 5.09 (1H, d, J = 13.2 Hz) and dH 4.73 (1H, d, J = 13.7 Hz) of CH2-3 and dH 2.80 (1H, d, J = 13.6 Hz) and dH 2.74 (1H, d, J = 13.6 Hz) of CH2-10 in the structure of 3 were also observed in the structure of 1. The 1H NMR spectrum of 1 also showed one methoxyl group at dH 3.64 and one acetoxyl group dH 2.36. The remaining signals including isopropyl and three methyl groups in 1 were the same in 3. All the 23 carbons corresponding to its molecular formula were observed in the 13C NMR spectrum (Table 2). The 13C NMR spectrum showed signals for three carbonyls dC 196.8 (saturated carbonyl), dC 171.8 (carbonyl of acetate group) and dC 167.7 (d-lactone), six carbon signals in the aromatic region, 13 carbon signals in the aliphatic region and one carbon signal attached to an oxygen atom. The presence of three methine, three methylene and
1 (CDCl3)
3 (CDCl3)
2 (CDCl3)
4 (CDCl3)
dC
dC
dC
dC
46.3 167.7 87.6 36.6 50.6 37.9 196.8 125.8 142.9 45.2 148.2 136.4 142.5 115.2 27.6 23.1 22.9 20.2 25.9 25.3 171.8 20.4 51.4
46.8 173.2 70.4 38.9 159.1 121.0 136.9 127.8 123.0 45.7 146.0 178.4 142.2 133.4 26.9 21.7 21.5 22.2 33.1 31.1 – – –
82.6 171.2 43.8 33.6 57.2 23.1 24.6 147.5 138.7 39.3 179.5 148.3 140.5 185.5 25.6 20.4 20.3 19.1 32.3 25.7 168.1 20.4 –
83.9 169.5 42.2 37.3 159.1 116.9 137.7 133.6 113.7 38.5 147.2 178.7 141.6 137.6 26.7 21.3 21.2 23.4 29.2 28.0 – – –
five methyl signals were differentiated in its DEPT 1358 spectrum. Among the three methine carbons, two were in aliphatic (dC 50.6, 27.6) region and one in aromatic (dC 115.2). Three methylene carbons (dC 87.6, 37.9 and 45.2) and the remaining five methyl carbons (dC 23.1, 22.9, 20.2, 25.9 and 25.3) were in the aliphatic region. Strong correlations between two methylene protons of CH2-3 (dH 4.63 and 4.40), CH2-6 (dH 2.74 and 2.67), CH2-10 (dH 2.35 and 2.25) and methine proton CH-5 (dH 2.55) with CH2-6 (dH 2.74 and 2.67), and methine proton H-15 (dH 3.04) of isopropyl group with two methyl protons of H-16 (dH 1.21) and H-17 (dH 1.20) were observed in its COSY spectrum. The NOESY spectrum showed correlations between H-3 (dH 4.63, 4.40) protons with H-19 (dH 1.14) and H-20 (dH 1.10); H-5 (dH 2.55) protons with H-19 (dH 1.14) and H-20 (dH 1.10); Ha-6 (dH 2.74) proton with Hb-6 (dH 2.67); H-6 (dH 2.74, 2.67) protons with H-19 (dH 1.14) and H-20 (dH 1.10); Ha-10 (dH 2.35) protons with Hb-10 (dH 2.25); H-14 (dH 7.34) protons with H-15 (dH 3.04), H-16 (dH 1.21) and H-17 (dH 1.20) and finally methine proton H-15 (dH 3.04) of isopropyl group correlated with both methyl groups at H-16 (dH 1.21), H-17 (dH 1.20) (Fig. 2).
Table 1 1 H NMR data for Compounds 1 (500 MHz), 2 (500 MHz), 3 (500 MHz), and 4 (500 MHz). Position H 3 5 6 7 10 14 15 16 17 18 19 20 –OAc –OMe
1 (CDCl3) dH (m, J in Hz)
3 (CDCl3) dH (m, J in Hz)
4.63 4.40 2.55 2.67 2.74 –
(d, 8.4) (d, 8.4) (dd, 12.6, 3.9) (dd, 17.7, 3.9) (dd, 12.6, 17.7)
5.09 (d, 13.2) 4.73 (d, 13.7) – 6.44 (d, 6.8)
2.35 2.25 7.34 3.04 1.21 1.20 1.45 1.14 1.10 2.36 3.64
(d, 13.8) (d, 13.8) (s) (sept., 6.8) (d, 6.7) (d, 6.7) (s) (s) (s) (s) (s)
6.61 (d, 6.8) 2.80 2.74 6.88 3.12 1.19 1.18 1.71 1.48 1.43 – –
(d, 13.6) (d, 13.6) (s) (sept., 6.9) (d, 5.5) (d, 6.2) (s) (s) (s)
2 (CDCl3)
dH (m, J in Hz) 2.55 2.46 1.78 1.99 1.25 3.45 2.12 3.54 2.45 – 3.17 1.23 1.21 1.26 1.12 0.99 2.35 –
(d, 16.4) (d, 16.4) (dd, 12.2, 2.6) (m) (m) (dd, 14.8, 6.4) (t, 12.7) (d, 13.7) (d, 13.7) (sept., 7.1) (d, 7.1) (d, 7.1) (s) (s) (s) (s)
4 (CDCl3) dH (m, J in Hz) 2.59 (d, 16.0) 2.45 (d, 16.0) – 6.14 (d, 9.0) 6.50 (d, 9.0) 3.67 2.88 6.80 3.08 1.16 1.13 1.40 1.33 1.27 – –
(d, 15.0) (d, 15.0) (s) (sept., 6.0) (d, 6.0) (d, 6.0) (s) (s) (s)
K. Satish et al. / Phytochemistry Letters 12 (2015) 129–132
O H O H H H
H
H
H O
OAc
131
O H O H
OCH3 H
H
Fig. 2. Selected NOE correlations (
H H
) of 1.
H
H
H O
OAc
H
OCH 3 H
Fig. 3. Selected HMBC correlations (H ! C) of 1.
The 1JCH coupled carbons were assigned from the HSQC spectrum (Table 3). The carbonyl group was placed at C-7 position since a long correlation with C-14 and C-6 protons were observed in the HMBC spectrum (Fig. 3). According to the HMBC spectrum, the acetoxyl group and methoxyl groups were placed at C-11 and C-12, respectively (Fig. 3). The HMBC spectrum showed correlations between H-14 (dH 7.34): C-7 (dC 196.8), C-13 (dC 142.5), C-11 (dC 148.2), C-12 (dC 136.4); H-15 (dH 3.04): C-14 (dC 115.2), C-12 (dC 136.4), C-13 (dC 142.5); H-10 (dH 2.35, 2.25): C-5 (dC 50.6) and C-11 (OAc, dC 171.8); H-6 (dH 2.74, 2.67): C-7 (dC 196.8), C-8 (dC 125.8), C-1 (dC 46.3), C-4 (dC 36.6); H-5 (dH 2.55): C-3 (dC 87.6), C-4 (dC 36.6), C-19 (dC 25.9) and C-20 (dC 25.3); H-3 (dH 4.63, 4.40): C-5 (dC 50.6) which was confirmed the non-protonated carbons (Fig. 3). As there was no correlation observed between H-5 and C-1 methyl in the NOESY spectrum of 1, the proposed relation between H-5 and C-1 methyl may be anti and the relative configuration is as shown in Fig. 1. Thus from the foregoing spectral studies the structure of 1 was elucidated as neobharangi-d-lactone (IUPAC name: (4aS,11aR)-8-isopropyl-9-methoxy-4,4,11a-trimethyl-1,6dioxo-1,3,4,4a,5,6,11,11a-octahydrobenzo[5,6]cyclohepta[1,2c]pyran-10-yl acetate). Compound 2 was obtained as a brown colored semi-solid, ½a27 D þ 40:0 (c 0.3, CHCl3). High resolution mass analysis of compound 2 showed the molecular ion [M+NH4]+, 406.2119 and gave the elemental composition C22H32O6N (calcd. 406.2229) in its spectrum. The UV spectrum showed intense absorption peak at lmax (methanol) 274 nm (log e 4.08). The IR spectrum displayed absorption bands at nmax: 1773 cm1 (O–CO–CH3), 1737 cm1 (dlactone >C5 5O), 1658 cm1 (quinone carbonyls both >C5 5O). Comparison of the 1H NMR of 2 with 4 (Sankaram et al., 1988a) showed the presence of d-lactone (1737 cm1 as observed in its IR spectrum) and further confirmed from its 13C NMR spectrum (dC 171.2) (Tables 1 and 2). The methylene protons at C-3 and C-10 present in the structure of 4 were also remained in the 1H NMR spectrum of 2 at C-3: dH 2.55 (1H, d, J = 16.4 Hz) and dH 2.46 (1H, d, J = 16.4 Hz), and C-10: dH 3.54 (1H, d, J = 13.7 Hz) and dH 2.45 (1H, d, J = 13.7 Hz), respectively. A notable difference in the 1H NMR spectra of 4 and 2 was the absence of the aromatic proton at C-14 position in the structure of 2. A sharp singlet signal at dH 2.35
integrating for three protons was assigned to acetoxyl group in the H NMR spectrum of 2. The 13C NMR spectrum of 2 showed the presence of quinone carbonyls at dC 179.5 and 185.5. All methyl groups present in the structure of 4 were the same in 2 which confirmed the same skeleton for them. The methine, methylene and methyl carbons were differentiated in its DEPT 1358 spectrum. Two methine carbons were in aliphatic region (dC 57.2 and 25.6) and four methylene carbons were also in the aliphatic region (dC 43.8, 39.3, 24.6 and 23.1). The remaining five methyl carbons (dC 32.3, 25.7, 20.4, 20.3 and 19.1) were in the aliphatic region. Strong correlations between two methylene protons of CH2-3 (dH 2.55 and 2.46), CH2-6 (dH 1.99 and 1.25), CH2-7 (dH 3.45 and 2.12), CH2-10 (dH 3.54 and 2.45); CH-5 (dH 1.78) and CH2-6 (dH 1.99 and 1.25), and methine proton H-15 (dH 3.17) of isopropyl group with two methyl protons of H-16 (dH 1.23) and H-17 (dH 1.21) were observed in its COSY spectrum. The 1JCH coupled carbons were assigned from its HSQC spectrum (Table 3). The NOESY spectrum showed correlations between Ha-3 (dH 2.55) with Hb-3 (dH 2.46); Ha-6 (dH 1.99) with Hb-6 (dH 1.25); Ha-7 (dH 3.45) with Hb-7 (dH 2.12); Ha-10 (dH 3.54) with Hb-10 (dH 2.45); and finally methine proton H15 (dH 3.17) of isopropyl group correlated with both methyl groups H-16 (dH 1.23) and H-17 (dH 1.21) (Fig. 4). The assignment of quinone carbonyls determined from its HMBC spectrum. According to the HMBC spectrum, the acetoxyl group was placed at C-12 (Fig. 5). The major HMBC correlations are H-15 (dH 3.17): C-14 (dC 185.5), C-12 (dC 148.3), C-13 (dC 140.5); H-10 (dH 3.54, 2.45): C-11 (dC 179.5), C-8 (dC 147.5), C-9 (dC 138.7); H-7 (dH 3.45, 2.12): C-14 (dC 185.5), C-8 (dC 147.5), C-9 (dC 138.7); H-6 (dH 1.99, 1.25): C-8 (dC 147.5); H-5 (dH 1.78): C-1 (dC 82.6); H-3 (dH 2.55, 2.46): C-2 (dC 171.2), C-4 (dC 33.6), C-19 (dC 32.3) (Fig. 5). The compound 2 may also have anti relation between H-5 and C-1 methyl since there was no correlation observed between H-5 and C-1 methyl in the NOESY spectrum, and the relative configuration is shown in Fig. 1. Thus from the foregoing spectral studies the structure of 2 was elucidated as bharangi quinone (IUPAC name of 2: (4aS,11aR)-8-isopropyl-4,4,11a-trimethyl2,7,10-trioxo-2,3,4,4a,5,6,7,10,11,11a-decahydrobenzo[5,6]cyclohepta[1,2-b]pyran-9-yl acetate). 1
3. Experimental Table 3 1 H–13C COSY (HSQC) 1JCH data of 1 and 2. C/H
3 5 6 7 10 14 15 16 17 18 19 20
1
3.1. General experimental procedures 2
dH
dC
dH
4.63, 4.40 2.55 2.74, 2.67 – 2.35, 2.25 7.34 3.04 1.21 1.20 1.45 1.14 1.10
87.6 50.6 37.9 – 45.2 115.2 27.6 23.1 22.9 20.2 25.9 25.3
2.55, 1.78 1.99, 3.45, 3.54, – 3.17 1.23 1.21 1.26 1.12 0.99
dC 2.46 1.25 2.12 2.45
43.8 57.2 23.1 24.6 39.3 – 25.6 20.4 20.3 19.1 32.3 25.7
Optical rotations were measured on a Horiba high sensitive polarimeter. Spectra were recorded with the following instruments:
Fig. 4. Selected NOE correlations (
) of 2.
K. Satish et al. / Phytochemistry Letters 12 (2015) 129–132
132
O H H
H H
O
(7.0 mg) whereas elution with 15% ethyl acetate in hexane gave compound 2 (7.5 mg).
OAc
O
H H O H H H H
Fig. 5. Selected HMBC correlations (H ! C) of 2.
IR, Perkin Elmer spectrophotometer; UV, Agilent technologies, carry series UV-VIS-NIR spectrophotometer; NMR, Avance 500 MHz; 1H NMR and 13C NMR were recorded in CDCl3 solvent on 500 MHz and 125 MHz spectrometer, respectively, at ambient temperature. Chemical shifts are reported as d values relative to internal CHCl3 d 7.26 or TMS d 0.0 for 1H NMR and CHCl3 d 77.0 for 13C NMR. Mass spectra were recorded for ESI and are given in mass units (m/z). High resolution mass spectra (HRMS) [ESI+] were obtained using either a TOF or a double focusing spectrometer. Column chromatography was carried out using silica gel (60–120 or 100–200 or 230–400 mesh) and the column was eluted with ethyl acetate-hexane and TLC with silica gel GF254. Visualization of the spots on TLC plates was achieved either by UV light or by staining the plates in 5% sulfuric acid in methanol or in methanol–phosphomolybdic acid–sulphuric acid solution and charring on hot plate. 3.2. Plant material
3.3.1. Acetylation of IM To a stirred solution of IM (14.0 mg) in dichloromethane (2 mL) at 0 8C, Et3N (0.02 mL), Ac2O (0.01 mL) and DMAP (catalytic amount) were added and the mixture was stirred for 30 min. After the completion of the reaction, water was added and stirred for 10 min. The residue was extracted with ethyl acetate (1 30 mL) and washed with water and brine. Organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 1 (7.0 mg) and compound 2 (7.5 mg) as brown colored semi-solids. 3.4. Neobharangi-d-lactone (1) Brown colored semi-solid; yield: 7.0 mg; ½a25 D þ 17:33 (c 0.15, CHCl3); lmax (methanol): 267 nm (log e 3.60); IR (KBr): nmax 1737, 1687 and 1771 cm1; 1H NMR (500 MHz, CDCl3): see Table 1; 13C NMR (125 MHz, CDCl3): see Table 2; HRMS (ESI): calcd. for C23H31O6 [M+H] + 403.2115, found 403.2113. 3.5. Bharangi quinone (2) Brown colored semi-solid; yield: 7.5 mg; ½a27 D þ 40:0 (c 0.3, CHCl3); lmax (methanol): 274 nm (log e 4.08); IR (KBr): nmax 1773, 1737 and 1658 cm1; 1H NMR (500 MHz, CDCl3): see Table 1; 13C NMR (125 MHz, CDCl3): see Table 2; HRMS (ESI): calcd. for C22H32O6N [M+NH4]+ 406.2229, found 406.2119.
The plant material of P. herbacea (Kingdom: Plantae; Phylum: Magnoliophyta; Class: Magnoliate; Order: Lamiales; Family: Lamiaceae; Sub-Family: Verbenaceae; Genus: Premna or (Pygmacopremna); Species: herbacea) was collected from Chintapalli forest area, Visakhapatnam (District), A.P., India in June 2009 and identity was confirmed by Dr. P. Padma Rao, Centre for Homeopathic standardization unit, Hyderabad. The specimen voucher No. 1596 is kept in the herbarium of the Pharmacognosy Division of Andhra University, Visakhapatnam, Andhra Pradesh.
We thank Dr. Lakshmi kantam, Director, CSIR-IICT, Hyderabad and Dr. V.J. Rao, Head, Crop Protection Chemicals Division (Organic-II) for constant encouragement and financial support. The author (K.S.) thanks CSIR, New Delhi, India for the award of senior research fellowship.
3.3. Extraction and isolation
Appendix A. Supplementary data
P. herbacea air died root nodules (8.0 kg) were powdered and then successively extracted with n-hexane (90 L) and ethyl acetate (90 L) in a Soxhlet apparatus. The extracts were concentrated under reduced pressure to afford n-hexane extract (50 g) and ethyl acetate extract (130 g). The ethyl acetate extract (130 g) was chromatographed on silica gel eluted with hexane followed by increasing polarity of eluents resulted 44 fractions of 250 mL each. The eluates were combined based on TLC gave eight fractions (EA1–EA8). Fraction EA4 (20 g) was subjected to silica gel column chromatography [60–120 mesh, 1800 g, column was prepared using hexane] eluted with hexane followed by increasing polarity with ethyl acetate yielded five fractions (EA 4-1–EA 4-5). Fraction EA4-3 (400 mg) on further purification by column chromatography over silica gel (100–200 mesh) elution with 10% ethyl acetate resulted the isolation of known compounds neobharangin (3) (12 mg) and elution with 15% ethyl acetate bharangin (4) (200 mg). Fraction EA4-4 (250 mg) on further purification by column chromatography over silica gel (230–400 mesh) elution with 12% ethyl acetate resulted the isolation of known compound (4) (90 mg) whereas elution with 15% ethyl acetate gave inseparable mixture designated as IM (14.0 mg). Successful acetylation of IM gave the mixture of two spots exclusively on TLC which were separated by column chromatography over silica gel [100–200 mesh] elution with 12% ethyl acetate in hexane gave compound 1
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2015.03.010.
Acknowledgments
References Ambasta, S.P., 1996. The Useful Plants of India. Publications and Information Directorate, CSIR, New Delhi, India, pp. 489. Krishnamurthi, A., 1969. The Wealth of India. Publication and Information Directorate, CSIR, New Delhi VIII, 239. Nayar, R.C., Yognarsimha, S.N., Subramanyam, K., 1976. Pharmacognosy of a local market sample of bharangin: P. herbacea. Indian J. Pharm. 38, 39–44. Sankaram, A.V.B., Marthandamurthy, M., 1991. Pygmacone, a phenalenone derivative of diterpenoid origin from P. herbacea. Phytochemistry 30, 359–360. Sankaram, A.V.B., Marthandamurthy, M., Bhaskraiah, K., Narasimharao, G.L., Subrahmanyam, M., Shoolery, J.N., 1988a. Bharangin, a novel diterpenoid quinonemethide from P. herbacea (Roxb.) Moldenke. Tetrahedron Lett. 29, 245–248. Sankaram, A.V.B., Bhaskraiah, K., Marthandamurthy, M., Subrahmanyam, M., 1988b. Bharanginin, a novel heterocyclic ortho-quinone from P. herbacea (Roxb.) Moldenke. Tetrahedron Lett. 29, 4881–4884. Sankaram, A.V.B., Bhaskraiah, K., Marthandamurthy, M., Subrahmanyam, M., 1989. Isobharangin, a new biogenetically significant diterpenoid quinonemethide from P. herbacea (Roxb.) Moldenke. Tetrahedron Lett. 30, 867–868. Satish, K., Vishnuvardhan, M.V.P.S., Lakshma Nayak, V., Srihari, G., Subramanyam, M., Prabhakar Rao, T., Ramakrishna, S., Ravikumar, K., Sridhar, B., Marthanda Murthy, M., 2011. Cytotoxic diterpenoid quinonemethides from the roots of P. herbacea. Bioorg. Med. Chem. Lett. 21, 4581–4584. Srihari, G., Vishuvardhan, M.V.P.S., Satish, K., Subramanyam, M., Raju, T.V., Prabhakar, T., Marthandamurthy, M., 2011. Two new diterpenoids from the root nodules of P. herbacea. Phytochem. Lett. 4, 109–112.
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Two new diterpenoids from the roots of Pygmacopremna herbacea Kovela Satish a, Galla Srihari a, Gangarajula Sudhakar a, Kura Narsimha b, Tadikamalla Prabhakar Rao b, Madugula Marthanda Murthy a,* a b
Crop Protection Chemicals Division (Organic Chemistry-II), CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India Center for Nuclear Magnetic Resonance, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 January 2015 Received in revised form 21 February 2015 Accepted 6 March 2015 Available online 18 March 2015
Two new diterpenoids, neobharangi-d-lactone (1) and bharangi quinone (2) along with two known compounds neobharangin (3) and bharangin (4) were isolated from the ethyl acetate extract of root nodules of Pygmacopremna herbacea. The structures of the new compounds were established by 1D and 2D NMR spectroscopic data. ß 2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
Keywords: Pygmacopremna herbacea Verbenaceae Diterpenoids Neobharangi-d-lactone Bharangi quinone Bharangin Neobharangin
1. Introduction The Verbenaceae family is a rich source of terpenoids and quinonemethides. Pygmacopremna herbacea syn. Premna herbacea belonging to the family of Verbenaceae is a small herb or sometimes an under shrub, arising from a perennial rootstock distributed through the subtropical Himalayas, Assam, West Bengal, Bihar, Orissa and Deccan peninsula (Krishnamurthi, 1969). It is known as Gantubharangi in Telugu, Bharangi in Hindi. P. herbacea roots are used in the ayurvedic system and also a folk medicine against inflammatory and malaria in the Yunnan province of China. The roots of this plant are used in the preparations of ayurvedic medicines either alone or as an ingredient for the treatment of bronchitis, asthma, blood pressure, tumors, inflammation, hiccough, epilepsy, helimenthiasis, etc. (Nayar et al., 1976). Fresh root stocks and roots along with ginger are given in asthma, rheumatism and dropsy. The rootstocks and root bark are used to cure toothache. The leaves are used in fevers and cough, and their poultices are applied to boils (Ambasta, 1996). Previous investigations on P. herbacea by our group have resulted in the
* Corresponding author. Tel.: +91 40 27193933; fax: +91 40 27193382. E-mail address: [email protected] (M. Marthanda Murthy).
isolation and characterization of bharangin (Sankaram et al., 1988a), isobharangin (Sankaram et al., 1989), bharanginin (Sankaram et al., 1988b), pygmacone (Sankaram and Marthandamurthy, 1991), bharangi-d-lactone, bharangi-g-lactone (Srihari et al., 2011), 3-dehydroxy isobharangin and neobharangin (Satish et al., 2011). In order to isolate the minor active constituents, we reinvestigated this plant and obtained two new compounds (1) and (2) from the ethyl acetate extract of the root nodules. In this paper, we described the isolation and structure determination of 1 and 2 using 2D NMR techniques. 2. Results and discussion P. herbacea root powder was extracted successively with nhexane and ethyl acetate in a soxhlet apparatus. The extracts were concentrated under vacuum. The concentrated ethyl acetate extract was subjected to repeated column chromatography over silica gel resulting in the isolation of new compounds (1) and (2) along with known compounds neobharangin (3) (Satish et al., 2011) and bharangin (4) (Sankaram et al., 1988a) (Fig. 1). Compound 1 was obtained as a brown colored semi solid, ½a25 D þ 17:33 (c 0.15, CHCl3). High resolution mass analysis of compound 1 showed the molecular ion [(M+H)+], 403.2112, gave the elemental composition C23H31O6 (calcd. 403.2115) in its
http://dx.doi.org/10.1016/j.phytol.2015.03.010 1874-3900/ß 2015 Published by Elsevier B.V. on behalf of Phytochemical Society of Europe.
K. Satish et al. / Phytochemistry Letters 12 (2015) 129–132
130
O
AcO
OCH3
O H
O O 3
2
A
4
19
O
O
HO 18
11 10 9
1 5
B 8
20
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 –OCOCH3 –OCOCH3 –OCH3
O 2
12
HO
O 16 13
C 14
15
18
O
O
10
2 1 A 3 5 4
17
7
6
Position C
H
O 1
Table 2 13 C NMR (125 MHz) data for compounds 1, 2, 3 and 4.
OAc
O
19
3
20
11 9
B 8 6
12
O 13
C 14
16 15 17
7
4
Fig. 1. Structures of isolated compounds from P. herbacea.
spectrum. The UV spectrum showed absorption maxima at 267 nm (log e 3.60). The IR spectrum of 1 showed absorption bands at nmax: 1737, 1687 and 1771 cm1 indicating the presence of d-lactone, saturated carbonyl and carbonyl of acetate groups, respectively. The comparison of 1H NMR spectrum of 1 and 3 (Satish et al., 2011) showed a notable difference in AB doublets present in the B ring at dH 6.44 (1H, d, J = 6.8 Hz) of H-6 and dH 6.61 (1H, d, J = 6.8 Hz) of H7 in the structure of 3 were replaced by methylene groups at C-6, dH 2.74 (1H, dd, J = 12.6, 17.7 Hz) and dH 2.67 (1H, dd, J = 3.9, 17.7 Hz) and saturated carbonyl group at C-7 in the structure of compound 1 (Table 1). The other two AB doublets protons resonated at dH 5.09 (1H, d, J = 13.2 Hz) and dH 4.73 (1H, d, J = 13.7 Hz) of CH2-3 and dH 2.80 (1H, d, J = 13.6 Hz) and dH 2.74 (1H, d, J = 13.6 Hz) of CH2-10 in the structure of 3 were also observed in the structure of 1. The 1H NMR spectrum of 1 also showed one methoxyl group at dH 3.64 and one acetoxyl group dH 2.36. The remaining signals including isopropyl and three methyl groups in 1 were the same in 3. All the 23 carbons corresponding to its molecular formula were observed in the 13C NMR spectrum (Table 2). The 13C NMR spectrum showed signals for three carbonyls dC 196.8 (saturated carbonyl), dC 171.8 (carbonyl of acetate group) and dC 167.7 (d-lactone), six carbon signals in the aromatic region, 13 carbon signals in the aliphatic region and one carbon signal attached to an oxygen atom. The presence of three methine, three methylene and
1 (CDCl3)
3 (CDCl3)
2 (CDCl3)
4 (CDCl3)
dC
dC
dC
dC
46.3 167.7 87.6 36.6 50.6 37.9 196.8 125.8 142.9 45.2 148.2 136.4 142.5 115.2 27.6 23.1 22.9 20.2 25.9 25.3 171.8 20.4 51.4
46.8 173.2 70.4 38.9 159.1 121.0 136.9 127.8 123.0 45.7 146.0 178.4 142.2 133.4 26.9 21.7 21.5 22.2 33.1 31.1 – – –
82.6 171.2 43.8 33.6 57.2 23.1 24.6 147.5 138.7 39.3 179.5 148.3 140.5 185.5 25.6 20.4 20.3 19.1 32.3 25.7 168.1 20.4 –
83.9 169.5 42.2 37.3 159.1 116.9 137.7 133.6 113.7 38.5 147.2 178.7 141.6 137.6 26.7 21.3 21.2 23.4 29.2 28.0 – – –
five methyl signals were differentiated in its DEPT 1358 spectrum. Among the three methine carbons, two were in aliphatic (dC 50.6, 27.6) region and one in aromatic (dC 115.2). Three methylene carbons (dC 87.6, 37.9 and 45.2) and the remaining five methyl carbons (dC 23.1, 22.9, 20.2, 25.9 and 25.3) were in the aliphatic region. Strong correlations between two methylene protons of CH2-3 (dH 4.63 and 4.40), CH2-6 (dH 2.74 and 2.67), CH2-10 (dH 2.35 and 2.25) and methine proton CH-5 (dH 2.55) with CH2-6 (dH 2.74 and 2.67), and methine proton H-15 (dH 3.04) of isopropyl group with two methyl protons of H-16 (dH 1.21) and H-17 (dH 1.20) were observed in its COSY spectrum. The NOESY spectrum showed correlations between H-3 (dH 4.63, 4.40) protons with H-19 (dH 1.14) and H-20 (dH 1.10); H-5 (dH 2.55) protons with H-19 (dH 1.14) and H-20 (dH 1.10); Ha-6 (dH 2.74) proton with Hb-6 (dH 2.67); H-6 (dH 2.74, 2.67) protons with H-19 (dH 1.14) and H-20 (dH 1.10); Ha-10 (dH 2.35) protons with Hb-10 (dH 2.25); H-14 (dH 7.34) protons with H-15 (dH 3.04), H-16 (dH 1.21) and H-17 (dH 1.20) and finally methine proton H-15 (dH 3.04) of isopropyl group correlated with both methyl groups at H-16 (dH 1.21), H-17 (dH 1.20) (Fig. 2).
Table 1 1 H NMR data for Compounds 1 (500 MHz), 2 (500 MHz), 3 (500 MHz), and 4 (500 MHz). Position H 3 5 6 7 10 14 15 16 17 18 19 20 –OAc –OMe
1 (CDCl3) dH (m, J in Hz)
3 (CDCl3) dH (m, J in Hz)
4.63 4.40 2.55 2.67 2.74 –
(d, 8.4) (d, 8.4) (dd, 12.6, 3.9) (dd, 17.7, 3.9) (dd, 12.6, 17.7)
5.09 (d, 13.2) 4.73 (d, 13.7) – 6.44 (d, 6.8)
2.35 2.25 7.34 3.04 1.21 1.20 1.45 1.14 1.10 2.36 3.64
(d, 13.8) (d, 13.8) (s) (sept., 6.8) (d, 6.7) (d, 6.7) (s) (s) (s) (s) (s)
6.61 (d, 6.8) 2.80 2.74 6.88 3.12 1.19 1.18 1.71 1.48 1.43 – –
(d, 13.6) (d, 13.6) (s) (sept., 6.9) (d, 5.5) (d, 6.2) (s) (s) (s)
2 (CDCl3)
dH (m, J in Hz) 2.55 2.46 1.78 1.99 1.25 3.45 2.12 3.54 2.45 – 3.17 1.23 1.21 1.26 1.12 0.99 2.35 –
(d, 16.4) (d, 16.4) (dd, 12.2, 2.6) (m) (m) (dd, 14.8, 6.4) (t, 12.7) (d, 13.7) (d, 13.7) (sept., 7.1) (d, 7.1) (d, 7.1) (s) (s) (s) (s)
4 (CDCl3) dH (m, J in Hz) 2.59 (d, 16.0) 2.45 (d, 16.0) – 6.14 (d, 9.0) 6.50 (d, 9.0) 3.67 2.88 6.80 3.08 1.16 1.13 1.40 1.33 1.27 – –
(d, 15.0) (d, 15.0) (s) (sept., 6.0) (d, 6.0) (d, 6.0) (s) (s) (s)
K. Satish et al. / Phytochemistry Letters 12 (2015) 129–132
O H O H H H
H
H
H O
OAc
131
O H O H
OCH3 H
H
Fig. 2. Selected NOE correlations (
H H
) of 1.
H
H
H O
OAc
H
OCH 3 H
Fig. 3. Selected HMBC correlations (H ! C) of 1.
The 1JCH coupled carbons were assigned from the HSQC spectrum (Table 3). The carbonyl group was placed at C-7 position since a long correlation with C-14 and C-6 protons were observed in the HMBC spectrum (Fig. 3). According to the HMBC spectrum, the acetoxyl group and methoxyl groups were placed at C-11 and C-12, respectively (Fig. 3). The HMBC spectrum showed correlations between H-14 (dH 7.34): C-7 (dC 196.8), C-13 (dC 142.5), C-11 (dC 148.2), C-12 (dC 136.4); H-15 (dH 3.04): C-14 (dC 115.2), C-12 (dC 136.4), C-13 (dC 142.5); H-10 (dH 2.35, 2.25): C-5 (dC 50.6) and C-11 (OAc, dC 171.8); H-6 (dH 2.74, 2.67): C-7 (dC 196.8), C-8 (dC 125.8), C-1 (dC 46.3), C-4 (dC 36.6); H-5 (dH 2.55): C-3 (dC 87.6), C-4 (dC 36.6), C-19 (dC 25.9) and C-20 (dC 25.3); H-3 (dH 4.63, 4.40): C-5 (dC 50.6) which was confirmed the non-protonated carbons (Fig. 3). As there was no correlation observed between H-5 and C-1 methyl in the NOESY spectrum of 1, the proposed relation between H-5 and C-1 methyl may be anti and the relative configuration is as shown in Fig. 1. Thus from the foregoing spectral studies the structure of 1 was elucidated as neobharangi-d-lactone (IUPAC name: (4aS,11aR)-8-isopropyl-9-methoxy-4,4,11a-trimethyl-1,6dioxo-1,3,4,4a,5,6,11,11a-octahydrobenzo[5,6]cyclohepta[1,2c]pyran-10-yl acetate). Compound 2 was obtained as a brown colored semi-solid, ½a27 D þ 40:0 (c 0.3, CHCl3). High resolution mass analysis of compound 2 showed the molecular ion [M+NH4]+, 406.2119 and gave the elemental composition C22H32O6N (calcd. 406.2229) in its spectrum. The UV spectrum showed intense absorption peak at lmax (methanol) 274 nm (log e 4.08). The IR spectrum displayed absorption bands at nmax: 1773 cm1 (O–CO–CH3), 1737 cm1 (dlactone >C5 5O), 1658 cm1 (quinone carbonyls both >C5 5O). Comparison of the 1H NMR of 2 with 4 (Sankaram et al., 1988a) showed the presence of d-lactone (1737 cm1 as observed in its IR spectrum) and further confirmed from its 13C NMR spectrum (dC 171.2) (Tables 1 and 2). The methylene protons at C-3 and C-10 present in the structure of 4 were also remained in the 1H NMR spectrum of 2 at C-3: dH 2.55 (1H, d, J = 16.4 Hz) and dH 2.46 (1H, d, J = 16.4 Hz), and C-10: dH 3.54 (1H, d, J = 13.7 Hz) and dH 2.45 (1H, d, J = 13.7 Hz), respectively. A notable difference in the 1H NMR spectra of 4 and 2 was the absence of the aromatic proton at C-14 position in the structure of 2. A sharp singlet signal at dH 2.35
integrating for three protons was assigned to acetoxyl group in the H NMR spectrum of 2. The 13C NMR spectrum of 2 showed the presence of quinone carbonyls at dC 179.5 and 185.5. All methyl groups present in the structure of 4 were the same in 2 which confirmed the same skeleton for them. The methine, methylene and methyl carbons were differentiated in its DEPT 1358 spectrum. Two methine carbons were in aliphatic region (dC 57.2 and 25.6) and four methylene carbons were also in the aliphatic region (dC 43.8, 39.3, 24.6 and 23.1). The remaining five methyl carbons (dC 32.3, 25.7, 20.4, 20.3 and 19.1) were in the aliphatic region. Strong correlations between two methylene protons of CH2-3 (dH 2.55 and 2.46), CH2-6 (dH 1.99 and 1.25), CH2-7 (dH 3.45 and 2.12), CH2-10 (dH 3.54 and 2.45); CH-5 (dH 1.78) and CH2-6 (dH 1.99 and 1.25), and methine proton H-15 (dH 3.17) of isopropyl group with two methyl protons of H-16 (dH 1.23) and H-17 (dH 1.21) were observed in its COSY spectrum. The 1JCH coupled carbons were assigned from its HSQC spectrum (Table 3). The NOESY spectrum showed correlations between Ha-3 (dH 2.55) with Hb-3 (dH 2.46); Ha-6 (dH 1.99) with Hb-6 (dH 1.25); Ha-7 (dH 3.45) with Hb-7 (dH 2.12); Ha-10 (dH 3.54) with Hb-10 (dH 2.45); and finally methine proton H15 (dH 3.17) of isopropyl group correlated with both methyl groups H-16 (dH 1.23) and H-17 (dH 1.21) (Fig. 4). The assignment of quinone carbonyls determined from its HMBC spectrum. According to the HMBC spectrum, the acetoxyl group was placed at C-12 (Fig. 5). The major HMBC correlations are H-15 (dH 3.17): C-14 (dC 185.5), C-12 (dC 148.3), C-13 (dC 140.5); H-10 (dH 3.54, 2.45): C-11 (dC 179.5), C-8 (dC 147.5), C-9 (dC 138.7); H-7 (dH 3.45, 2.12): C-14 (dC 185.5), C-8 (dC 147.5), C-9 (dC 138.7); H-6 (dH 1.99, 1.25): C-8 (dC 147.5); H-5 (dH 1.78): C-1 (dC 82.6); H-3 (dH 2.55, 2.46): C-2 (dC 171.2), C-4 (dC 33.6), C-19 (dC 32.3) (Fig. 5). The compound 2 may also have anti relation between H-5 and C-1 methyl since there was no correlation observed between H-5 and C-1 methyl in the NOESY spectrum, and the relative configuration is shown in Fig. 1. Thus from the foregoing spectral studies the structure of 2 was elucidated as bharangi quinone (IUPAC name of 2: (4aS,11aR)-8-isopropyl-4,4,11a-trimethyl2,7,10-trioxo-2,3,4,4a,5,6,7,10,11,11a-decahydrobenzo[5,6]cyclohepta[1,2-b]pyran-9-yl acetate). 1
3. Experimental Table 3 1 H–13C COSY (HSQC) 1JCH data of 1 and 2. C/H
3 5 6 7 10 14 15 16 17 18 19 20
1
3.1. General experimental procedures 2
dH
dC
dH
4.63, 4.40 2.55 2.74, 2.67 – 2.35, 2.25 7.34 3.04 1.21 1.20 1.45 1.14 1.10
87.6 50.6 37.9 – 45.2 115.2 27.6 23.1 22.9 20.2 25.9 25.3
2.55, 1.78 1.99, 3.45, 3.54, – 3.17 1.23 1.21 1.26 1.12 0.99
dC 2.46 1.25 2.12 2.45
43.8 57.2 23.1 24.6 39.3 – 25.6 20.4 20.3 19.1 32.3 25.7
Optical rotations were measured on a Horiba high sensitive polarimeter. Spectra were recorded with the following instruments:
Fig. 4. Selected NOE correlations (
) of 2.
K. Satish et al. / Phytochemistry Letters 12 (2015) 129–132
132
O H H
H H
O
(7.0 mg) whereas elution with 15% ethyl acetate in hexane gave compound 2 (7.5 mg).
OAc
O
H H O H H H H
Fig. 5. Selected HMBC correlations (H ! C) of 2.
IR, Perkin Elmer spectrophotometer; UV, Agilent technologies, carry series UV-VIS-NIR spectrophotometer; NMR, Avance 500 MHz; 1H NMR and 13C NMR were recorded in CDCl3 solvent on 500 MHz and 125 MHz spectrometer, respectively, at ambient temperature. Chemical shifts are reported as d values relative to internal CHCl3 d 7.26 or TMS d 0.0 for 1H NMR and CHCl3 d 77.0 for 13C NMR. Mass spectra were recorded for ESI and are given in mass units (m/z). High resolution mass spectra (HRMS) [ESI+] were obtained using either a TOF or a double focusing spectrometer. Column chromatography was carried out using silica gel (60–120 or 100–200 or 230–400 mesh) and the column was eluted with ethyl acetate-hexane and TLC with silica gel GF254. Visualization of the spots on TLC plates was achieved either by UV light or by staining the plates in 5% sulfuric acid in methanol or in methanol–phosphomolybdic acid–sulphuric acid solution and charring on hot plate. 3.2. Plant material
3.3.1. Acetylation of IM To a stirred solution of IM (14.0 mg) in dichloromethane (2 mL) at 0 8C, Et3N (0.02 mL), Ac2O (0.01 mL) and DMAP (catalytic amount) were added and the mixture was stirred for 30 min. After the completion of the reaction, water was added and stirred for 10 min. The residue was extracted with ethyl acetate (1 30 mL) and washed with water and brine. Organic layer was dried over Na2SO4, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give compound 1 (7.0 mg) and compound 2 (7.5 mg) as brown colored semi-solids. 3.4. Neobharangi-d-lactone (1) Brown colored semi-solid; yield: 7.0 mg; ½a25 D þ 17:33 (c 0.15, CHCl3); lmax (methanol): 267 nm (log e 3.60); IR (KBr): nmax 1737, 1687 and 1771 cm1; 1H NMR (500 MHz, CDCl3): see Table 1; 13C NMR (125 MHz, CDCl3): see Table 2; HRMS (ESI): calcd. for C23H31O6 [M+H] + 403.2115, found 403.2113. 3.5. Bharangi quinone (2) Brown colored semi-solid; yield: 7.5 mg; ½a27 D þ 40:0 (c 0.3, CHCl3); lmax (methanol): 274 nm (log e 4.08); IR (KBr): nmax 1773, 1737 and 1658 cm1; 1H NMR (500 MHz, CDCl3): see Table 1; 13C NMR (125 MHz, CDCl3): see Table 2; HRMS (ESI): calcd. for C22H32O6N [M+NH4]+ 406.2229, found 406.2119.
The plant material of P. herbacea (Kingdom: Plantae; Phylum: Magnoliophyta; Class: Magnoliate; Order: Lamiales; Family: Lamiaceae; Sub-Family: Verbenaceae; Genus: Premna or (Pygmacopremna); Species: herbacea) was collected from Chintapalli forest area, Visakhapatnam (District), A.P., India in June 2009 and identity was confirmed by Dr. P. Padma Rao, Centre for Homeopathic standardization unit, Hyderabad. The specimen voucher No. 1596 is kept in the herbarium of the Pharmacognosy Division of Andhra University, Visakhapatnam, Andhra Pradesh.
We thank Dr. Lakshmi kantam, Director, CSIR-IICT, Hyderabad and Dr. V.J. Rao, Head, Crop Protection Chemicals Division (Organic-II) for constant encouragement and financial support. The author (K.S.) thanks CSIR, New Delhi, India for the award of senior research fellowship.
3.3. Extraction and isolation
Appendix A. Supplementary data
P. herbacea air died root nodules (8.0 kg) were powdered and then successively extracted with n-hexane (90 L) and ethyl acetate (90 L) in a Soxhlet apparatus. The extracts were concentrated under reduced pressure to afford n-hexane extract (50 g) and ethyl acetate extract (130 g). The ethyl acetate extract (130 g) was chromatographed on silica gel eluted with hexane followed by increasing polarity of eluents resulted 44 fractions of 250 mL each. The eluates were combined based on TLC gave eight fractions (EA1–EA8). Fraction EA4 (20 g) was subjected to silica gel column chromatography [60–120 mesh, 1800 g, column was prepared using hexane] eluted with hexane followed by increasing polarity with ethyl acetate yielded five fractions (EA 4-1–EA 4-5). Fraction EA4-3 (400 mg) on further purification by column chromatography over silica gel (100–200 mesh) elution with 10% ethyl acetate resulted the isolation of known compounds neobharangin (3) (12 mg) and elution with 15% ethyl acetate bharangin (4) (200 mg). Fraction EA4-4 (250 mg) on further purification by column chromatography over silica gel (230–400 mesh) elution with 12% ethyl acetate resulted the isolation of known compound (4) (90 mg) whereas elution with 15% ethyl acetate gave inseparable mixture designated as IM (14.0 mg). Successful acetylation of IM gave the mixture of two spots exclusively on TLC which were separated by column chromatography over silica gel [100–200 mesh] elution with 12% ethyl acetate in hexane gave compound 1
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2015.03.010.
Acknowledgments
References Ambasta, S.P., 1996. The Useful Plants of India. Publications and Information Directorate, CSIR, New Delhi, India, pp. 489. Krishnamurthi, A., 1969. The Wealth of India. Publication and Information Directorate, CSIR, New Delhi VIII, 239. Nayar, R.C., Yognarsimha, S.N., Subramanyam, K., 1976. Pharmacognosy of a local market sample of bharangin: P. herbacea. Indian J. Pharm. 38, 39–44. Sankaram, A.V.B., Marthandamurthy, M., 1991. Pygmacone, a phenalenone derivative of diterpenoid origin from P. herbacea. Phytochemistry 30, 359–360. Sankaram, A.V.B., Marthandamurthy, M., Bhaskraiah, K., Narasimharao, G.L., Subrahmanyam, M., Shoolery, J.N., 1988a. Bharangin, a novel diterpenoid quinonemethide from P. herbacea (Roxb.) Moldenke. Tetrahedron Lett. 29, 245–248. Sankaram, A.V.B., Bhaskraiah, K., Marthandamurthy, M., Subrahmanyam, M., 1988b. Bharanginin, a novel heterocyclic ortho-quinone from P. herbacea (Roxb.) Moldenke. Tetrahedron Lett. 29, 4881–4884. Sankaram, A.V.B., Bhaskraiah, K., Marthandamurthy, M., Subrahmanyam, M., 1989. Isobharangin, a new biogenetically significant diterpenoid quinonemethide from P. herbacea (Roxb.) Moldenke. Tetrahedron Lett. 30, 867–868. Satish, K., Vishnuvardhan, M.V.P.S., Lakshma Nayak, V., Srihari, G., Subramanyam, M., Prabhakar Rao, T., Ramakrishna, S., Ravikumar, K., Sridhar, B., Marthanda Murthy, M., 2011. Cytotoxic diterpenoid quinonemethides from the roots of P. herbacea. Bioorg. Med. Chem. Lett. 21, 4581–4584. Srihari, G., Vishuvardhan, M.V.P.S., Satish, K., Subramanyam, M., Raju, T.V., Prabhakar, T., Marthandamurthy, M., 2011. Two new diterpenoids from the root nodules of P. herbacea. Phytochem. Lett. 4, 109–112.