FITOTE-03080; No of Pages 8 Fitoterapia xxx (2014) xxx–xxx
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Fitoterapia
New malabaricane triterpenes from the oleoresin of Ailanthus malabarica☆
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journal homepage: www.elsevier.com/locate/fitote
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Prabhakar S. Achanta, Rajesh Kumar Gattu, Adavi Rao V. Belvotagi, Raghuram Rao Akkinepally, Ravi Kumar Bobbala, Appa Rao V.N. Achanta ⁎
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University College of Pharmaceutical Sciences, Kakatiya University, Warangal 506009, Telangana State, India
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a r t i c l e
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Article history: Received 30 September 2014 Accepted in revised form 21 November 2014 Accepted 26 November 2014 Available online xxxx
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Keywords: Ailanthus malabarica Oleoresin Malabaricane type triterpenes Simaroubaceae Halmaddi
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1. Introduction
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Ailanthus malabarica DC (Simaroubaceae) is a medium to tall evergreen rainforest tree found along the Western Ghats of India. Several compounds belonging to different chemical classes such as alkaloids, quassinoids, triterpenoids and nortriterpenoids have been previously reported from different parts of A. malabarica [1–5]. The Oleoresin oozing out of wounded trunk of the tree is aromatic and is called halmaddi in Kannada (Karnataka) and Mattipal in Malayalam (Kerala). Because of its fragrance, it is used in the manufacture of incense sticks and is an article of commerce in India. It is claimed to be useful in dysentery and bronchitis [6]. Earlier phytochemical investigation of this oleoresin yielded a new class of tricyclic triterpenes named as malabaricane triterpenes [7]. There are very few examples of malabaricane-type triterpenes even today. They have been reported from toad stools [8], ferns [9,10], sponges [11,12], and higher plants [13–16]. Mastic
32 33 34 35 36 37 38 39 40 41
13 14 15 16 17
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Ten malabaricane type triterpenes were isolated from the oleoresin of Ailanthus malabarica, out of which six (1–6) were new. For three of the known compounds (7–9), NMR assignments are being reported for the first time. Compound 10, a known one, is a new report from this source. The structures were established by extensive 1D and 2D NMR spectroscopy. The oleoresin and some of the isolates did not possess antimicrobial activity and did not lyse RBCs. © 2014 Published by Elsevier B.V.
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☆ Dedicated to the memory of Dr. Yenamandra Venkateswarlu, Scientist G (IICT, Hyderabad, India) who passed away on the 17th of July, 2013. ⁎ Corresponding author. Tel.: +91 870 2438844; fax: +91 870 2453508. E-mail address: [email protected] (A.R.V.N. Achanta).
appears to be the only resin from which this class of compounds has been reported [16]. This is the first time that the oleoresin is being reinvestigated after its first report five decades ago. We herein report the isolation, structure elucidation of six new (1–6) and spectral data of three known compounds (7–9), all belonging to the class of malabaricane type triterpenoids.
42 43
2. Materials and methods
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2.1. General
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All the solvents used were of laboratory grade. Silica gel for open column chromatography: Normal phase (E. Merck, #230–400) and reversed phase (C-18, Sorbent technologies, USA) silica gel. TLC plates: Normal phase silica gel 60 F254 and reversed phase silica gel 60 RP-18 F254s (both of E. Merck). Melting points were recorded on a Mel-Temp M4125 and were uncorrected. Optical rotations were measured on a Rudolph Autopol V polarimeter. NMR spectra were recorded at 27 °C with a Bruker AMX 500 (1H, 500.13 MHz) with a 5-mm tripleresonance probe equipped with puls-filed z-gradient; d in ppm, J in Hz in CDCl3 or Methanol-D4, NOESY spectra were obtained
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http://dx.doi.org/10.1016/j.fitote.2014.11.022 0367-326X/© 2014 Published by Elsevier B.V.
Please cite this article as: Achanta PS, et al, New malabaricane triterpenes from the oleoresin of Ailanthus malabarica, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.022
44 45 46 47 48
52 53 54 55 56 57 58 59 60 Q6 61
2 t3:1 t3:2 Q1
P.S. Achanta et al. / Fitoterapia xxx (2014) xxx–xxx
Table 1 500 MHz 1H NMR spectral data for compounds 1–9 in (CDCl3)a, b. Hydrogen
1
2
3a
4a
t3:4 t3:5
1 (CH2) 2 (CH2)
1.93 (m), 1.47 (m) 2.51 (ddd, J = 15.7, 9.5, 7.6), 2.45 (ddd, 15.7, 7.7, 4.5) – 1.40 (d, J = 2.7) 1.59 (m), 1.49 (m) 1.58 (m), 1.34 (dd, J = 9.3, 3.1) 1.45 (m) 1.55 (m), 1.28 (m) 1.83 (m), 1.28 (m) 1.63 (m) 1.50 (m), 1.12 (m) 1.50 (m), 1.82 (m) 1.84 (m) 0.91 (s) 0.96 (s) 1.16 (s) 1.74 (m), 1.66 (m) 1.89 (m), 1.82 (m) 3.75 (t, J = 7.3) 1.24 (s) 1.15 (s) 1.11 (s) 1.06 (s) 1.02 (s)
1.95 (m), 1.47 (m) 2.51 (ddd, J = 16.6, 9.5, 7.7), 2.46 (ddd, J = 16.6, 7.7, 4.4) – 1.40 (m) 1.58 (m), 1.50 (m) 1.58 (m), 1.34 (m) 1.45 (m) 1.53 (m), 1.31 (m) 1.86 (m), 1.27 (m) 1.66 (m) 1.50 (m), 1.14 (m) 1.80 (m), 1.51 (m) 1.83 (m) 0.89 (s) 0.94 (s) 1.16 (s) 1.76 (m), 1.63 (m) 1.64 (m), 1.44 (m) 3.42 (dd, J = 10.3, 1.3 Hz) 1.23 (s) 1.18 (s) 1.08 (s) 1.04 (s) 1.00 (s)
1.85 (m), 1.54 (m) 2.60 (ddd, J = 16.2, 10.5, 7.6) 2.41 (ddd, 16.2, 7.2, 3.4) – 1.43 (m) 1.67 (m), 1.59 (m) 1.97 (m), 1.86 (m) 1.45 (m) 1.57 (m), 1.46 (m) 1.89 (m), 1.64 (m) 1.76 (m) 1.92 (m), 1.53 (m) 1.76 (m), 1.36 (m) 3.26 (dd, J = 10.1, 1.2) 1.22 (s) 1.02 (s) 1.14 (s) 1.57 (m), 1.50 (m) 2.14 (m), 2.07 (m) 5.16 (t, J = 7.1) 1.70 (s) 1.65 (s) 1.07 (s) 1.10 (s) 1.03 (s)
1.87 (m), 1.57 (m) 2.62 (ddd, J = 16.3, 11.8, 9.1), 2.44 (ddd, J = 16.3, 7.2, 3.4) – 1.45 (m) 1.68 (m), 1.60 (m) 2.00 (m), 1.88 (m) 1.49 (m) 1.60 (m), 1.48 (m) 1.93 (m), 1.70 (m) 1.76 (m) 1.95 (m), 1.57 (m) 1.80 (m), 1.38 (m) 3.26 (t, br) 1.22 (s) 1.02 (s) 1.14 (s) 1.82 (m), 1.58 (m) 1.80 (m), 1.38 (m) 3.24 (t, br) 1.18 (s) 1.20 (s) 1.07 (s) 1.10 (s) 1.04 (s)
a
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3 (C_O/–OH) 5 (CH) 6 (CH2) 7 (CH2) 9 (CH) 11 (CH2) 12 (CH2) 13 (CH) 15 (CH2) 16 (CH2) 17 (CH) 18 (CH3/_CH2) 19 (CH3) 21 (CH3) 22 (CH2) 23 (CH2) 24 (CH) 26 (CH3) 27 (CH3) 28 (CH3) 29 (CH3) 30 (CH3)
D
t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13 t3:14 t3:15 t3:16 Q2 t3:17 Q3 t3:18 t3:19 t3:20 t3:21 t3:22 t3:23 t3:24 t3:25 t3:26 t3:27
F
t3:3
E
Recorded in methanol-D4. Multiplicity of signals is given in parentheses: s, singlet; d, doublet; t, triplet; m, multiplet; and br, broad; coupling constants (apparent splittings) are reported as numerical values in Hz; eq and ax designate equatorial and axial hydrogen atom, a and b hydrogen below and above the ring plane, respectively; the assignments are based on 500 MHz HSQC–TOCSY and NOESY spectra. c A and B designate H-18 protons trans and cis to C-15, respectively. d Non-stereospecific assignment; the geminal methyl groups exhibit equally intense NOEs to H-24. e These assignments may be interchanged.
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b
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2.2. Plant material
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Oleoresin was hand collected from the wounded trunk of A. malabarica from the forests of Subrahmanya Kukke, South Canara District, Karnataka, India (12°40′N; 75°36′E) in November, 2012. It was identified by Prof. V. S. Raju, Department of Botany, Kakatiya University. A voucher specimen (AVN-HM-12) is being maintained in the natural products laboratory of University College of Pharmaceutical Sciences, Kakatiya University, Warangal.
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2.3. Isolation of phytochemical constituents
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50 g of the oleoresin was extracted with toluene in which it dissolved completely. It was adsorbed onto silica gel (100 g) and flash chromatographed on silica gel (#230–400; 750 g) eluting with hexane:ethylacetate (100–50%) and then with ethylacetate:methanol (50%) later with methanol and methanol:water (50%) all in 1 L quantity. Fractions of ~50 mL were collected and pooled based on TLC pictures which yielded 30 major fractions. While some of the compounds crystallized directly in some of these fractions, the others were isolated by rechromatography as described below.
76 77 78 Q7 79 80 81 82 83 84
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O
C
N
70 71
U
68 69
R
64
with mixing times of 1000 ms. HMBC experiments were optimized for nJC, H = 8 Hz. Mass spectra were recorded on an Agilent 5973i quadrupole mass spectrometer.
Compounds 1, 8 & 9: Fractions 8–10 resulted from elution of column with hexane:ethylacetate—80:20. Orthorhombic crystals of 8 (epoxymalabaricol, 1.2 g, 2.4%) formed in these fractions which were collected by filtration. The filtrates were found to contain two common constituents by TLC. They were pooled and rechromatographed on silica gel using hexane: acetone—90:10 to yield 1 (120 mg) and later 9 (800 mg) as a semisolid. Compound 2: Fraction 23 resulted from elution of the column with hexane:ethylacetate—50:50. This was subjected to rechromatography with hexane:acetone—70:30 and later on, reversed phase silica gel (C-18) with methanol:water— 75:25 to yield 2 (170 mg). Compound 3: Fractions 17–21 were obtained by elution of the column with hexane:ethylacetate—60:40. They were mixed and subjected to rechromatography using hexane: acetone—80:20 to yield 3 (1.80 g, 3.6%) as a hygroscopic semisolid. Compound 4: Fractions 26–29 were obtained by elution of column with ethylacetate:methanol—50:50. Rechromatography with hexane:acetone—70:30 yielded 4 (25 mg). Compounds 5 & 6: Fractions 24–25 obtained by elution with ethylacetate were pooled and on chromatography using 90–70% of hexane:acetone to yield 5 (30 mg) and later 6 (15 mg). Compound 7: Fractions 1–5 were obtained by elution of column with hexane:ethylacetate—90:10. Slow evaporation
Please cite this article as: Achanta PS, et al, New malabaricane triterpenes from the oleoresin of Ailanthus malabarica, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.022
85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 Q8 110 111
P.S. Achanta et al. / Fitoterapia xxx (2014) xxx–xxx Table 1 500 MHz 1H NMR spectral data for compounds 1–9 in (CDCl3)a, b. 6a
7
8
9
1.83 (m), 1.55 (m) 2.59 (ddd, J = 15.9, 10.8, 7.6) 2.42 (ddd, J = 15.9, 7.2, 3.4) – 1.31 (d, J = 11.4) 1.68 (m), 1.57 (m) 1.67 (m), 1.21 (m) 1.49 (m) 1.57 (m) 2.08 (m), 1.62 (m) 2.27 (d, J = 9.13) 2.41 (m), 1.86 (m) 1.90 (m), 1.36 (m) 3.31 (d) A: 4.66 (s) B: 4.96 (s)c 1.04 (s) 1.13 (s) 1.81 (m), 1.56 (m) 1.80 (m), 1.36 (m) 3.24 (d) 1.18 (s) 1.19 (s) 1.07 (s) 1.08 (s) 1.09 (s)
1.51 (m), 1.07 (m) 1.68 (m)
1.84 (m), 1.36 (m) 2.59 (ddd, J = 16.1, 10.9, 7.5), 2.41 (ddd, 16.1, 7.0, 3.3) – 1.31 (dd, J = 3.0) 159 (m) 1.76 (m) 1.42 (m) 1.53 (m), 1.42 (m) 1.93 (m), 1.74 (m) 1.75 (m) 1.83 (m), 1.70 (m) 1.83 (m) 3.71 (t, J = 7.1) 1.23d (s) 0.99 (s) 1.21 (s) 1.50 (m), 1.36 (m) 2.08 (m) 5.14 (t, J = 7.1) 1.71 (s) 1.64 (s) 1.11 (s) 1.06 (s) 0.99 (s)
1.81 (m), 1.47 (m) 2.56 (ddd, J = 16.1, 10.7, 7.5), 2.40 (ddd, 16.1, 7.1, 3.4) – 1.35 (m) 1.53 (m), 1.50 (m) 1.73 (m), 1.58 (m) 1.38 (d, J = 6.9) 1.52 (m), 1.40 (m) 1.99 (m), 1.70 (m) 1.78 (m) 1.76 (m), 1.74 (m) 1.93 (m), 1.49 (m) 3.91 (dd, J = 8.5, 2.0) 1.19 (s) 0.94 (s) 1.13 (s) 2.08 (m), 1.50 (m) 2.08 (m), 1.90 (m) 3.81 (dd, J = 7.0, 1.5) 1.22 (s) 1.06 (s) 1.08 (s) 1.03 (s) 0.95 (s)
1.56 (m), 1.03 (m) 1.63 (m), 1.60 (m) 3.20 (dd,J = 10.6, 5.5) 0.73 (d, J = 11.6) 1.62 (m), 1.49 (m) 1.71 (m) 1.33 (m) 1.46 (m), 1.35 (m) 1.89 (m), 1.70 (m) 1.73 (m) 1.82 (m), 1.70 (m) 1.82 (m) 3.67 (t, J = 7.1) 1.19e (s) 0.83 (s) 1.18e (s) 1.48 (m), 1.34 (m) 2.12 (m), 2.04 (m) 5.10 (t, J = 7.0) 1.61 (s) 1.68 (s) 0.97 (s) 0.78 (s) 0.91 (s)
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P
3.15 (dd, J = 11.6, 4.5) 0.68 (d, J = 11.9, 2.1) 1.63 (m), 1.51 (m) 1.60 (m), 1.20 (m) 1.39 (m) 1.52 (m), 1.41 (m) 2.04 (m), 1.62 (m) 2.21 (d, J = 8.16) 2.38 (m), 1.85 (m) 1.87 (m), 1.38 (m) 3.30 (d, J = 10.7) A: 4.66 (s) B: 4.96 (s) 0.92 (s) 1.13 (s) 1.77 (m), 1.54 (m) 1.75 (m), 1.35 (m) 3.25 (d, J = 9.9) 1.18 (s) 1.19 (s) 0.96 (s) 0.80 (s) 1.04 (s)
D
5a
119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136
2.3.5. (17R,20R,24R)-17,20,24,25-tetrahydroxy-14(18)-malabaricen3-one (5) Amorphous powder, [α]25 D + 54° (c0.1, CHCl3); m.p.: 89– 92 °C; HRESIMS m/z 493.38992 [M + H]+, [C30H52O5 + H]+ requires 493.38875; ESIMS m/z 458 [(M − 2H2O) + H]+.
144 145
2.3.6. (17R,20R,24R)-17,20,24,25-tetrahydroxy-14(18)-malabaricen3β-ol (6) Amorphous powder, m.p.: 153–155 °C; HRESIMS m/z 517.38729 [M + Na]+, [C30H54O5Na]+ requires 517.38634; ESIMS m/z 515 [M + Na]+, 457 [(M − 2H2O) + H]+; Optical rotation was not determined due to the small amount of material available.
149 Q9
2.3.7. (14S,17R,20S)-20-hydroxy-14,17-epoxy-24(25)-malabaricen3-one (7) Amorphous powder, [α]25 D + 40°(c0.1, CHCl3); m.p.: 65– 67 °C; ESIMS m/z 481.45, [M + Na]+, [C30H50O3 + Na]+ requires 481.37.
156 157
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2.3.1. (14S,17S,20S,24R)-25-hydroxy-14,17-cyclo-20,24epoxy-malabaricane-3-one (1) Crystalline powder, m.p.: 156–159 °C; HRESIMS m/z 481.36564 [M + Na]+, [C30H50O3Na]+ requires 481.36521; Optical rotation was not determined due to the small amount of material available. ESIMS m/z 459 [M + H]+, 441 [(M − H2O) + H]+.
R
117 118
137
O
116
2.3.4. (14S,17S,20S,24S)-14,17,20,24,25-pentahydroxy-malabarican3-one (4) Amorphous powder, m.p.: 91–94 °C; HRESIMS m/z 533.38140 [M + Na]+, [C30H54O6Na]+ requires 533.38126; ESIMS m/z 493 [(M − H2O) + H]+, 457 [(M − 3H2O) + H]+, optical rotation was not determined due to the small amount of material available.
N C
114 115
of solvent from these fractions resulted in the formation of 7 (10.0 g, 20%) in the form of needles which were collected by filtration. Compound 10: Fractions 13–15 were obtained by elution of the column with hexane:ethylacetate—70:30. They were pooled and subjected to rechromatography with hexane: acetone—95:5 to give 10 (75 mg).
U
112 113
E
C
T
E
t3:1 t3:2
3
2.3.2. (14S,17S,20S,24R)-20,24,25-trihydroxy-14,17-cyclomalabarican-3-one (2) Crystalline powder, [α]25 D + 50° (c0.1, CHCl3); m.p.: 143– 145 °C; HRESIMS m/z 499.37630 [M + Na]+, [C30H52O4Na]+ requires 499.37578; ESIMS m/z 459 [(M − H2O) + H]+, 441 [(M − 2H2O) + H]+. 2.3.3. (14S,17S,20S)-14,17,20-trihydroxy-24(25)-malabaricen-3one (3) Semi solid, [α]25 D + 36°(c 0.28, CHCl3); 499.37597 [M + Na]+, [C30H52O4Na]+ requires 499.37578; ESIMS m/z 459 [(M − H2O) + H]+, 441 [(M − 2H2O) + H]+.
Please cite this article as: Achanta PS, et al, New malabaricane triterpenes from the oleoresin of Ailanthus malabarica, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.022
138 139 140 141 142 143
146 147 148
150 151 152 153 154 155
158 159 160
4 t4:1 t4:2
P.S. Achanta et al. / Fitoterapia xxx (2014) xxx–xxx
Table 2 125 MHz 13C NMR spectral data for compounds 1–9 in (CDCl3)a. 1
2
3a
4a
5a
6a
7
8
9
t4:4 t4:5 t4:6 t4:7 t4:8 t4:9 t4:10 t4:11 t4:12 t4:13 t4:14 t4:15 t4:16 t4:17 t4:18 t4:19 t4:20 t4:21 t4:22 t4:23 t4:24 t4:25 t4:26 t4:27 t4:28 t4:29 t4:30 t4:31 t4:32 t4:33
1 (CH2) 2 (CH2) 3 (C_O/–OH) 4 (C) 5 (CH) 6 (CH2) 7 (CH2) 8 (C) 9 (CH) 10 (C) 11 (CH2) 12 (CH2) 13 (CH) 14 (C) 15 (CH2) 16 (CH2) 17 (CH) 18 (CH3/_CH2) 19 (CH3) 20 (CH2) 21 (CH3) 22 (CH2) 23 (CH2) 24 (CH) 25 (C) 26 (CH3) 27 (CH3) 28 (CH3) 29 (CH3) 30 (CH3)
39.93 34.12 218.08 47.43 55.37 19.67 34.61 40.29 50.16 36.87 22.08 27.39 43.10 50.04 31.45 25.71 49.49 16.37 16.05 86.36 23.58 35.72 26.13 83.33 71.44 27.48d 24.30d 26.73 21.01 15.15
39.89 34.12 218.03 47.44 55.37 19.65 34.54 40.29 49.97 36.85 22.02 27.46 42.60 50.28 31.14 24.92 50.22 16.01 16.35 75.52 25.38 36.84 25.68 78.81 73.16 26.67d 23.36d 26.71 21.03 15.21
39.34 33.71 218.97 47.14 55.02 20.57 36.81 44.18 59.79 36.46 21.02 23.68 59.79 75.40 39.01 25.17 78.25 24.38 14.88 74.17 20.85 37.97 21.65 124.70 130.56 24.47 16.33 20.04 25.70 25.07
39.44 33.72 218.99 47.16c 55.01 20.55 36.80 44.18 58.79 36.45 21.02 23.68 58.78 75.47 39.12 25.13 78.44 24.11 14.89 74.28 20.93 35.10 24.66 79.08 72.57 23.84d 24.11d 20.05 25.67 25.08
38.95 33.72 218.00b 47.13c 55.35 20.22 35.55 44.81 54.31 36.02 20.13 26.93 56.71 155.81 36.42 30.03 77.52 107.90 14.08 74.16 20.58 35.39 24.52 79.03 72.55 24.56d 24.08d 20.08 25.63 23.35
38.68 26.49 78.52 38.40 56.00 18.71 36.45 45.03 55.36 36.24 20.26 27.20 56.78 155.02 36.48 30.04 77.59 107.76 14.75 74.17 20.61 35.34 24.57 79.04 72.56 23.82d 24.08d 27.33 14.60 23.78
39.71 34.20 217.48 47.46 55.53 20.75 36.58 44.08 58.31 36.58 21.15 23.89 59.93 85.66 38.20 26.03 82.27 24.37 15.51 72.78 24.37 37.68 22.22 124.62 131.54 25.70 17.64 26.56 21.15 25.70
39.54 34.18 217.63 47.45 55.14 20.73 36.71 43.95 58.04 36.56 21.33 24.06 60.26 86.36 38.05 28.91 81.10 22.05 15.60 85.40 25.12 31.07 26.57 85.40 71.50 28.07d 25.18d 26.53 21.02 25.82
39.43 27.44 79.30 38.81 56.08 19.54 37.67 44.33 59.28 37.02 21.11 24.19 59.79 85.82 38.31 26.21 82.43 24.29 16.10 72.88 24.29 37.92 22.30 124.77 131.38 25.59 17.59 28.21 15.23 26.12
t4:34 t4:35 t4:36 t4:37
a
c
161
R
2.3.8. (14S,17R,20S,24R)-25-hydroxy-14,17, 20, 24-diepoxymalabarican-3-one (8) Orthorhombic crystals, [α]25 D + 22° (c0.1, CHCl3); m.p.: + + 164 Q10 142–144 °C; ESIMS m/z 497.45, [M + Na] , [C30H50O4 + Na] 165 requires 497.36; 457 [(M − H2O) + H]+.
170
O
C
168 169
2.3.9. (14S,17R,20S)-14,17-epoxy-24(25)-malabaricen-3β,20diol (9) Semisolid, [α] 25 D + 34° (c0.28, CHCl3); ESIMS m/z 443.45, [(M − H2O) + H]+, [C30H52O3 − H2O + H]+ requires 443.39.
N
166 167
R
162 163
174
2.4. Biological activity
175 176
U
173
2.3.10. (17S,20R,24R)-17,25-dihydroxy-20,24-epoxy-14(18)malabaricen-3-one (10) Amorphous powder, [α]25 D + 12° (c0.1, CHCl3); m.p.: 135– 137 °C.
171 172
E
T
E
d
Recorded in methanol-D4. Extracted from HMBC. Masked in MeOH peaks. Non-stereospecific assignment of the geminal methyl groups.
O
R O
P
C
b
F
Carbon
D
t4:3
2.4.1. Antimicrobial screening Antimicrobial assays were performed against several bacte177 Q11 ria strains (Pseudomonas aerginosa ATCC BAA 427, P. aerginosa CI 178 15159, Klebsiella pneumonia ATCC 27736, K. pneumonia CI 1876, 179 Escherichia coli ATCC 25922) using an agar disk diffusion assay 180 and a fungal strain (Candida albicans ATCC60193) using broth 181 dilution assay at 100 μg/mL and 250 μg/mL concentrations.
2.4.2. Hemolysis assay Human blood was collected in EDTA containing vacutinaer (2 mg/mL). Plasma and buffy coat were removed from the EDTA-blood by centrifugation at 800 g for 10 min. Later, the erythrocytes were washed three times by using normal saline (0.9%) and resuspended in saline to 5% erythrocyte suspension. The cells were incubated with end-over-end rotation for 1 h at 37 °C in the presence of test compounds (100 μg/mL) and were performed in triplicate. After the incubation, the samples were centrifuged at 800 g for 10 min and the supernatant was transferred to 96 well plates. 2% Triton X-100 (Sigma-Aldrich, St. Louis, USA) served as positive control. The absorbance of hemoglobin released was measured at 540 nm and is expressed as % of Triton X-100 induced hemolysis. The result was calculated by using the formula %Hemolysis = [(absorbance of sample-absorbance of blank) / (absorbance of positive control)] × 100.
182 183
3. Results and discussion
199
HRESI-MS of 1 showed a pseudomolecular ion peak at m/z 481.36072 [M + Na]+ indicating the molecular formula C30H50O3. 13C NMR spectrum contained 30 carbon signals, and an analysis of HSQC spectrum revealed the presence of eight methyls, ten methylenes, five methines and seven quaternary carbons. All the methyls appeared as singlets in the 1H NMR spectrum indicating their attachment to
200 201
Please cite this article as: Achanta PS, et al, New malabaricane triterpenes from the oleoresin of Ailanthus malabarica, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.022
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OH
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9 Fig. 1. Structures of compounds 1–10.
quaternary carbons. Malabaricane [7] carbon skeleton of the ring system (A–C) was established by an analysis of HMBC and NOESY spectra. NOE interactions were observed between H-19, H-29 and H-30, between H-28 and H-5, and H-13 and H-30. HSQC–TOCSY spectra helped in delineating the spin systems. 1 H and 13C NMR data are presented in Tables 1 and 2 respectively and structure is given in Fig. 1. Figures of
compounds with key NOESY correlations in the side chain are given in supporting information. A carbonyl group detected in 13 C NMR spectrum (δ 218.08) was placed at C-3 on the basis of an isolated CH2–CH2 spin system evident in COSY and HSQC– TOCSY. 1H and 13C NMR data coupled with HSQC–TOCSY indicated the presence of substituted cyclobutane and tetrahydrofuran rings in the side chain. The upfield shift of carbon
Please cite this article as: Achanta PS, et al, New malabaricane triterpenes from the oleoresin of Ailanthus malabarica, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.022
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Fig. 3. Effect of the oleoresin of A. malabarica and compounds 1–3, 5, 7–10 on lysis of human RBCs.
an isobutenyl group was attached to C-13 of malabaricane ring system in this molecule. The double bond was placed between C24 and C25, and the hydroxyl groups on C14, C17, and C20 based on HMBC correlations. The relative stereochemistry in 3 was established from NOESY spectrum. NOE correlations were observed between H-17 (δ 3.26) and H-15 (δ 1.92), H-16 (δ 1.36) and H-21 (δ 1.14). H-16 (δ 1.36) correlated with H-15 (δ 1.92), H-18 (δ 1.22) and H-21 (δ 1.14) indicating that all these were cofacial. A proton of shift value δ 1.76 showed correlations with H-16 (δ 1.36), H-17 (δ 3.26), H-18 (δ 1.22) and H-21 (δ 1.14). This proton was deduced to be the β oriented H-13. Thus, the protons with which it correlated were β oriented. A problem arose because one of the H-16s had the same shift value (δ 1.76) as that of H-13, but logical interpretation of the NOESY spectrum solved it. Thus the stereochemistry of the side chain was found to be 14S,17S,20S. 4 was determined to be the 24,25-dihydroxy derivative of 3. It lacked the signals due to doubly bonded carbons in 13C NMR spectrum. Instead, two additional oxygenated carbon signals assigned to C–24 and C–25 appeared at δ 79.08 and 72.57. A pseudomolecular ion peak at m/z 533.38140 corresponding to [M + Na]+ appeared in the mass spectrum of 4. The molecular weight was found to be 510 which was 34 amu higher than that of 3. Correlations were observed in NOESY between H-13 (δ 1.76), H-30 (δ 1.04), and H-18 (δ 1.22) and also between H18, H-15 (δ 1.95), and H-16 (δ 1.38) indicating the protons to be β oriented. H-17 (δ 3.26) showed correlations exclusively with H-16 (δ1.38) and H-21 (δ 1.14). The configuration at C24 was determined from the observation that H-17 (δ 3.26) and H-24 (δ 3.25) had common interactions with H-22 (δ 1.58) and H-23 (δ 1.80), protons that were sandwitched between H-17 and H-24, indicating that H-24 was also β oriented. Thus the stereochemistry in the side chain was determined to be 14S,17S,20S,24S. A pseudomolecular ion peak appeared at m/z 493.38992 [M + H]+ in the HRESI-MS spectrum of 5 compatible with C30H52O5. In contrast to other compounds isolated in this series, there were only seven methyl signals in the 1H NMR spectrum of 5. The 13C NMR spectrum has signals at δ 107.90
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resonance for C-13 (δ 43.10) by about 13–16 ppm, when compared with other compounds reported herein, indicated that the cyclobutane ring was attached to C13 which was confirmed by HMBC. Downfield signals in the 13C NMR spectrum at δ 83.33 and 86.36 indicated the presence of tetrahydrofuran ring which was found to be attached to the cyclobutyl group at C-17. An isopropyloxy group was attached to the tetrahydrofuran ring at C-24. The connectivities were established by HMBC. The relative stereochemistry in the side chain was established by NOE interactions between H-18 (δ 0.91) and H-17 (δ 1.84) and between H-21 (δ 1.16) and H-24 (δ 3.75) indicating that all these were on the same side of the rings and their α orientation was established by their lack of interaction with the β oriented H-13 (δ 1.63). According to Hashimoto et al. isomers with the two largest substituents of the tetrahydrofuran ring in the trans orientation, a hydrogen corresponding to H-24 appeared as a doublet (J = 5.3 and 10.2 Hz), whereas a triplet (J = 7.3 Hz) was observed in the cis diastereomers [17]. H-24 appeared as a triplet (J = 7.3 Hz) at δ 3.76 in the 1 H NMR spectrum of 1 confirming the NOESY results. Based on these results the stereochemistry was determined to be 13S,14S,17S,20S,24R. Structure of 2 was similar to that of 1 up to the cyclobutyl ring. Tetrahydrofuran ring was found to be absent in 2 as there were no downfield oxygenated carbon signals at δ 83.33 and 86.36 in its 13C NMR spectrum. Instead, two hydroxyl bearing carbon signals appeared at δ 75.52 and 78.81 which were assigned to C-20 and C-24 respectively. Thus the side chain in 2 could have resulted from a cascade opening through hydrolysis of the tetrahydrofuran ring present in 1. All the connectivities were confirmed by HMBC. Relative stereochemistry of the side chain in 2 was same as in 1 up to C-20 (NOESY). The configuration at C-24 was arrived at as follows: NOE interaction between the α oriented H-21 (δ 1.16) and H-22 (δ 1.63) indicated the latter to be α oriented. The other H-22 (δ 1.76) which was β oriented correlated with H-24 (δ 3.42) indicating that H-24 was β orientated (Fig. 1). Thus the configuration of the side chain was determined as 14S,17S,20S,24R. A pseudomolecular ion peak appeared at m/z 499.37597 [M + Na]+ in the HRESI-MS spectrum of 3 and its molecular formula was deduced as C30H52O4. An acyclic chain of thirteen carbons containing three hydroxyl groups and terminating into
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P.S. Achanta et al. / Fitoterapia xxx (2014) xxx–xxx
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O
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Fig. 2. Hypothetical formation of various malabaricanes from malabaricol through hydration, dehydration, cyclodehydration, hydrolysis, hydroxylation etc.
Please cite this article as: Achanta PS, et al, New malabaricane triterpenes from the oleoresin of Ailanthus malabarica, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.022
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side chain in 8 (up to C-20) was identical to that of malabaricol (7) in which stereochemistry of the side chain was determined as 14S,17R,20S by X-ray diffraction [20]. A stereostructure for compound 8 had been reported earlier though there was no mention of how it was arrived at [7]. NMR data with assignments is being reported for the first time. 9 has been identified as the known compound malabaricanediol [7]. NOESY study revealed that the hydroxyl at C-3 was β-oriented. The relative stereochemistry of the side chain was same as in malabaricol (7). Thus, the stereochemistry of the side chain was determined as 14S,17R,20S. NMR data of the compound is being reported for the first time. 10 was isolated earlier from Caloncoba echinata [15], and is being reported for the first time from the oleoresin of A. malabarica. 1H and 13C NMR data of compounds 1–9 are given in Tables 1 and 2 respectively and structures of compounds 1–10 are given in Fig. 1. The present investigation resulted in the isolation of structurally related malabaricane triterpenes with novel structural features. 1 and 2 have cyclobutyl ring in the side chain and 3–6 were polyhydroxylated. Our literature survey reveals that compounds 1–6 are new. Yield-wise, malabaricol (7) was the chief constituent of the oleoresin (20% w/w) followed by 3 (3.6% w/w) and epoxymalabaricol (8, 2.4% w/w). The other compounds proved to be minor constituents. Whether the minor metabolites are formed from the major metabolite malabaricol or it is the other way round is a subject to be investigated. However it can be said that their formation involves reactions like hydration, dehydration, cyclodehydration, hydrolysis and hydroxylation. Their hypothetical formation from malabaricol is shown in Fig. 2. Triterpenes of the malabaricane skeleton are rare and constitute a small group. The oleoresin of A. malabarica and compounds 1–3, 5, 7–10 were tested for their antimicrobial activity on P. aerginosa, K. pneumonia, E. coli, C. albicans in view of the traditional claims of halmaddi resin to treat bronchitis and dysentery. None of them exhibited considerable activity, i.e., an inhibition zone N0.5 mm was not found at tested (100 and 250 μg/mL) concentrations. The compounds were also tested for their toxicity to RBCs and all the compounds (except 10) were considerably less toxic compared to the reference standards polymyxin (4.99%) and ampicillin (4.46%) at 100 μg/mL (Fig. 3). 10 was more toxic (4.92%) and its activity was comparable to that of polymyxin. It was reported earlier to have antiplasmodial activity but also caused transformation of erythrocytes into stomatocytes [21]. Interestingly, 5 which is structurally related to 10 (opening of epoxy ring in 10 results in 5) was found to be least toxic (1.84%).
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Acknowledgments
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PSA thanks CSIR, New Delhi for Senior Research Fellowship (No. 09/384(159)/2013. EMR-I) and ARVNA thanks AICTE for the award of Emeritus Fellowship (No. 06/AICTE/RIFD(Policy4th)/EF(01)/2012–13). Principal, UCPSc is thanked for his encouragement. Mukesh Pasupuleti, Vishnu Sravan, Rama Rao and Raghavachary are thanked for their help. Authors thank Dr. Gayathri Withers for providing NMR spectra. NMR instrumentation at Carenegie Mellon University was partially supported by NSF (CHE-0130903 and CHE-1039870). Authors thank Prof. GA
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and 155.81, characteristic of an exo methylene group. It also showed four oxygenated carbon signals. A side chain 323 resembling the one present in 4 with a double bond between 324 C-14 and C-18 was found attached to C-13 in this compound. 325 Though 13C NMR spectrum of 5 was not properly obtained, 326 all the connectivities could be established through HSQC, 327 HSQC–TOCSY and HMBC. The relative stereochemistry of 328 the side chain in 5 was determined by a study of its NOESY 329 spectrum. H-13 (δ 2.27) showed interaction with H-15 (δ 2.41) 330 indicating that this hydrogen was β-oriented. The α-oriented 331 H-15 (δ 1.86) showed a correlation with H-17 (δ 3.31) and this 332 in turn showed interaction with H-16 (δ 1.36) and H-21 (δ 333 1.13) indicating all have α-orientation. While H-24 (δ 3.25) 334 showed only three interactions with protons on one side of 335 the chain i.e., with H-22 (δ1.56), H-23 (δ1.80), and H-26 (δ 336 1.18), H-17 (δ 3.31), being centrally located in the side chain, 337 showed five interactions with protons on either side 338 i.e., with H-15 (δ 1.86), H-16 (δ 1.36), H-21 (δ1.13) H-22 339 (δ1.56) and H-23 (δ1.80). The common interaction of H-17 340 and H-24 with protons that were sandwitched between 341 them i.e., H-22 (δ 1.56) and H-23 (δ 1.80), established that 342 H-24 was also α-oriented. Thus the stereochemistry of the 343 side chain was determined to be 17R,20R,24R. 344 Structure of 6 was similar to that of 5. The 13C NMR 345 spectrum of 6 lacked a signal due to a carbonyl group. 346 Instead, there was an additional peak at δ 78.52 indicating 347 that the keto group at C-3 present in 5 was reduced to a 348 secondary alcohol group in 6. The C-3 hydroxyl was β349 oriented as confirmed by the NOE interactions between H-3 350 and H-28 and H-28 and H-5. Stereochemistry of the side 351 chain was same as in 5. Both 5 and 6 resemble the known 352 compound 10 in having a double between C14–C18. 353 7 was identified as the known compound malabaricol, 354 reported previously from the oleoresin of A. malabarica. Its 355 structure was established earlier through extensive chemi356 cal degradation [7,18,19]. Its stereochemistry was confirmed 357 by X-ray diffraction studies [20]. Though 13C NMR data was 358 given for this compound, assignments were not made [11]. 359 Even the latest report on the identification of malabaricol and 360 epoxymalabaricol isolated from the heartwood of Ailanthus 361 excelsa does not contain the NMR data and assignments [21]. 362 NMR study of 8, though a known compound, proved quite 363 interesting. It was identified as epoxymalabaricol reported 364 previously from the oleoresin of A. malabarica. NOESY study 365 revealed that the stereochemistry in the side chain for 8 was 366 identical to that of 1. The absolute stereochemistry of the side 13 367 Q12 chain in 8 was determined as 14S,17R,20S,24R. The C NMR 368 spectra of compounds with a 24R-configuration show for the C369 26 and C-27 methyls' different (ca 3 ppm) chemical shifts [14], 370 due to a strong hydrogen bond between the tertiary hydroxyl 371 group and the tetrahydrofuran oxygen. By contrast, in the 13C 372 NMR spectra of the 24S-series the two methyls exhibit very 373 close values (b1 ppm). In 8 the difference was 2.9 ppm sup374 porting R configuration at C-24. A malabaricane triterpene 375 glucoside isolated from Adesmia aconcaguensis has a side chain 376 similar to the one present in 8. However, the two methyls, C-26 377 and C-27, exhibited very close 13C NMR values (b 1 ppm) and 378 so C-24 was assigned S configuration. Also 13C NMR values 379 differed from 8, especially at C17 and C20. The configuration of 380 the side chain was described as 14R,17R,20R,24S [14]. It is 381 worth mentioning here that the relative stereochemistry of the
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Please cite this article as: Achanta PS, et al, New malabaricane triterpenes from the oleoresin of Ailanthus malabarica, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.022
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NMR spectra, figures of compounds with key NOESY correlations are given in supporting information. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fitote.2014.11. 022.
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References
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[1] Joshi BS, Kamat VN, Gawad DH. Some β-carboline alkaloids of Ailanthus malabarica DC. Heterocycles 1977;7:193. [2] Khan SA, Fozdar BI. Quassinoids from Ailanthus malabarica. Indian J Chem Sect B 1982;21:1133–4. [3] Joshi BS, Kamat VN, William Pelletier S, Go K, Bhandary K. The structure of ailanthol, a new triterpenoid from Ailanthus malabarica DC. Tetrahedron Lett 1985;26:1273–6. [4] Aono H, Koike K, Kaneko J, Ohmoto T. Alkaloids and quassinoids from Ailanthus malabarica. Phytochemistry 1994;37:579–84. [5] Hitotsuyanagi Y, Ozeki A, Choo CY, Chan KL, Itokawa H, Takeya K. Malabanones A and B, novel nortriterpenoids from Ailanthus malabarica DC. Tetrahedron 2001;57:7477–80. [6] Drury H. The useful plants of India. 2nd Ed. Dehradun: International Book Distributors; 1978. p. pp. 23. [7] Chawla A, Dev S. A new class of triterpenoids from Ailanthus malabarica DC derivatives of malabaricane. Tetrahedron Lett 1967;8:4837–43.
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[8] Sontag B, Fröde R, Bross M, Steglich W. Chromogenic triterpenoids from Cortinarius fulvoincarnatus, C. sodagnitus and related toadstools (Agaricales). Eur J Org Chem 1999;1999:255–60. [9] Ageta H, Arai Y. Chemotaxonomy of ferns, 3. Triterpenoids from Polypodium polypodioides. J Nat Prod 1990;53:325–32. [10] Masuda K, Shiojima K, Ageta H. Fern constituents 3: two new malabaricatrienes isolated from Lemmaphyllum microphyllum var. obovatum. Chem Pharm Bull 1989;37:1140–2. [11] Ravi BN, Wells RJ, Croft KD. Malabaricane triterpenes from a Fijian collection of the sponge Jaspis stellifera. J Org Chem 1981;46: 1998–2001. [12] Ravi B, Wells R. Malabaricane triterpenes from a great barrier reef collection of the sponge Jaspis stellifera. Aust J Chem 1982;35:39–50. [13] Jakupovic J, Eid F, Bohlmann F, El-Dahmy S. Malabaricane derivatives from Pyrethrum santolinoides. Phytochemistry 1987;26:1536–8. [14] Faini F, Castillo M, Torres R, Monache GD, Gacs-Baitz E. Malabaricane triterpene glucosides from Adesmia aconcaguensis. Phytochemistry 1995; 40:885–90. [15] Ziegler HL, Christensen J, Olsen CE, Sittie AA, Jaroszewski JW. New dammarane and malabaricane triterpenes from Caloncoba echinata. J Nat Prod 2002;65:1764–8. [16] Marner F-J, Freyer A, Lex J. Triterpenoids from gum mastic, the resin of Pistacia lentiscus. Phytochemistry 1991;30:3709–12. [17] Hashimoto M, Kan T, Nozaki K, Yanagiya M, Shirahama H, Matsumoto T. Total syntheses of (+)-thyrsiferol, (+)-thyrsiferyl 23-acetate, and (+)venustatriol. J Org Chem 1990;55:5088–107. [18] Sobti RR, Dev S. A direct correlation of (+)-malabaricol with (+)ambreinolide. Tetrahedron Lett 1968;9:2215–7. [19] Dev S. Biogenetic concepts in terpene structure elucidation. Pure Appl Chem 1979;51. [20] Paton WF, Paul IC, Bajaj AG, Dev S. The structure of malabricol. Tetrahedron Lett 1979;20:4153–4. [21] Srinivas PV, Rao RR, Rao JM. Two new tetracyclic triterpenes from the heartwood of Ailanthus excelsa Roxb. Chem Biodivers 2006;3:930–4.
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Cordell and Prof. Alessandra Braca for their helpful discussions and valuable suggestions.
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Please cite this article as: Achanta PS, et al, New malabaricane triterpenes from the oleoresin of Ailanthus malabarica, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.022
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Contents lists available at ScienceDirect
Fitoterapia
New malabaricane triterpenes from the oleoresin of Ailanthus malabarica☆
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journal homepage: www.elsevier.com/locate/fitote
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Prabhakar S. Achanta, Rajesh Kumar Gattu, Adavi Rao V. Belvotagi, Raghuram Rao Akkinepally, Ravi Kumar Bobbala, Appa Rao V.N. Achanta ⁎
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University College of Pharmaceutical Sciences, Kakatiya University, Warangal 506009, Telangana State, India
Q52Q4
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a r t i c l e
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Article history: Received 30 September 2014 Accepted in revised form 21 November 2014 Accepted 26 November 2014 Available online xxxx
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Keywords: Ailanthus malabarica Oleoresin Malabaricane type triterpenes Simaroubaceae Halmaddi
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1. Introduction
26 27
Ailanthus malabarica DC (Simaroubaceae) is a medium to tall evergreen rainforest tree found along the Western Ghats of India. Several compounds belonging to different chemical classes such as alkaloids, quassinoids, triterpenoids and nortriterpenoids have been previously reported from different parts of A. malabarica [1–5]. The Oleoresin oozing out of wounded trunk of the tree is aromatic and is called halmaddi in Kannada (Karnataka) and Mattipal in Malayalam (Kerala). Because of its fragrance, it is used in the manufacture of incense sticks and is an article of commerce in India. It is claimed to be useful in dysentery and bronchitis [6]. Earlier phytochemical investigation of this oleoresin yielded a new class of tricyclic triterpenes named as malabaricane triterpenes [7]. There are very few examples of malabaricane-type triterpenes even today. They have been reported from toad stools [8], ferns [9,10], sponges [11,12], and higher plants [13–16]. Mastic
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Ten malabaricane type triterpenes were isolated from the oleoresin of Ailanthus malabarica, out of which six (1–6) were new. For three of the known compounds (7–9), NMR assignments are being reported for the first time. Compound 10, a known one, is a new report from this source. The structures were established by extensive 1D and 2D NMR spectroscopy. The oleoresin and some of the isolates did not possess antimicrobial activity and did not lyse RBCs. © 2014 Published by Elsevier B.V.
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☆ Dedicated to the memory of Dr. Yenamandra Venkateswarlu, Scientist G (IICT, Hyderabad, India) who passed away on the 17th of July, 2013. ⁎ Corresponding author. Tel.: +91 870 2438844; fax: +91 870 2453508. E-mail address: [email protected] (A.R.V.N. Achanta).
appears to be the only resin from which this class of compounds has been reported [16]. This is the first time that the oleoresin is being reinvestigated after its first report five decades ago. We herein report the isolation, structure elucidation of six new (1–6) and spectral data of three known compounds (7–9), all belonging to the class of malabaricane type triterpenoids.
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2. Materials and methods
49
2.1. General
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All the solvents used were of laboratory grade. Silica gel for open column chromatography: Normal phase (E. Merck, #230–400) and reversed phase (C-18, Sorbent technologies, USA) silica gel. TLC plates: Normal phase silica gel 60 F254 and reversed phase silica gel 60 RP-18 F254s (both of E. Merck). Melting points were recorded on a Mel-Temp M4125 and were uncorrected. Optical rotations were measured on a Rudolph Autopol V polarimeter. NMR spectra were recorded at 27 °C with a Bruker AMX 500 (1H, 500.13 MHz) with a 5-mm tripleresonance probe equipped with puls-filed z-gradient; d in ppm, J in Hz in CDCl3 or Methanol-D4, NOESY spectra were obtained
51
http://dx.doi.org/10.1016/j.fitote.2014.11.022 0367-326X/© 2014 Published by Elsevier B.V.
Please cite this article as: Achanta PS, et al, New malabaricane triterpenes from the oleoresin of Ailanthus malabarica, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.022
44 45 46 47 48
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P.S. Achanta et al. / Fitoterapia xxx (2014) xxx–xxx
Table 1 500 MHz 1H NMR spectral data for compounds 1–9 in (CDCl3)a, b. Hydrogen
1
2
3a
4a
t3:4 t3:5
1 (CH2) 2 (CH2)
1.93 (m), 1.47 (m) 2.51 (ddd, J = 15.7, 9.5, 7.6), 2.45 (ddd, 15.7, 7.7, 4.5) – 1.40 (d, J = 2.7) 1.59 (m), 1.49 (m) 1.58 (m), 1.34 (dd, J = 9.3, 3.1) 1.45 (m) 1.55 (m), 1.28 (m) 1.83 (m), 1.28 (m) 1.63 (m) 1.50 (m), 1.12 (m) 1.50 (m), 1.82 (m) 1.84 (m) 0.91 (s) 0.96 (s) 1.16 (s) 1.74 (m), 1.66 (m) 1.89 (m), 1.82 (m) 3.75 (t, J = 7.3) 1.24 (s) 1.15 (s) 1.11 (s) 1.06 (s) 1.02 (s)
1.95 (m), 1.47 (m) 2.51 (ddd, J = 16.6, 9.5, 7.7), 2.46 (ddd, J = 16.6, 7.7, 4.4) – 1.40 (m) 1.58 (m), 1.50 (m) 1.58 (m), 1.34 (m) 1.45 (m) 1.53 (m), 1.31 (m) 1.86 (m), 1.27 (m) 1.66 (m) 1.50 (m), 1.14 (m) 1.80 (m), 1.51 (m) 1.83 (m) 0.89 (s) 0.94 (s) 1.16 (s) 1.76 (m), 1.63 (m) 1.64 (m), 1.44 (m) 3.42 (dd, J = 10.3, 1.3 Hz) 1.23 (s) 1.18 (s) 1.08 (s) 1.04 (s) 1.00 (s)
1.85 (m), 1.54 (m) 2.60 (ddd, J = 16.2, 10.5, 7.6) 2.41 (ddd, 16.2, 7.2, 3.4) – 1.43 (m) 1.67 (m), 1.59 (m) 1.97 (m), 1.86 (m) 1.45 (m) 1.57 (m), 1.46 (m) 1.89 (m), 1.64 (m) 1.76 (m) 1.92 (m), 1.53 (m) 1.76 (m), 1.36 (m) 3.26 (dd, J = 10.1, 1.2) 1.22 (s) 1.02 (s) 1.14 (s) 1.57 (m), 1.50 (m) 2.14 (m), 2.07 (m) 5.16 (t, J = 7.1) 1.70 (s) 1.65 (s) 1.07 (s) 1.10 (s) 1.03 (s)
1.87 (m), 1.57 (m) 2.62 (ddd, J = 16.3, 11.8, 9.1), 2.44 (ddd, J = 16.3, 7.2, 3.4) – 1.45 (m) 1.68 (m), 1.60 (m) 2.00 (m), 1.88 (m) 1.49 (m) 1.60 (m), 1.48 (m) 1.93 (m), 1.70 (m) 1.76 (m) 1.95 (m), 1.57 (m) 1.80 (m), 1.38 (m) 3.26 (t, br) 1.22 (s) 1.02 (s) 1.14 (s) 1.82 (m), 1.58 (m) 1.80 (m), 1.38 (m) 3.24 (t, br) 1.18 (s) 1.20 (s) 1.07 (s) 1.10 (s) 1.04 (s)
a
O
R O
P
3 (C_O/–OH) 5 (CH) 6 (CH2) 7 (CH2) 9 (CH) 11 (CH2) 12 (CH2) 13 (CH) 15 (CH2) 16 (CH2) 17 (CH) 18 (CH3/_CH2) 19 (CH3) 21 (CH3) 22 (CH2) 23 (CH2) 24 (CH) 26 (CH3) 27 (CH3) 28 (CH3) 29 (CH3) 30 (CH3)
D
t3:6 t3:7 t3:8 t3:9 t3:10 t3:11 t3:12 t3:13 t3:14 t3:15 t3:16 Q2 t3:17 Q3 t3:18 t3:19 t3:20 t3:21 t3:22 t3:23 t3:24 t3:25 t3:26 t3:27
F
t3:3
E
Recorded in methanol-D4. Multiplicity of signals is given in parentheses: s, singlet; d, doublet; t, triplet; m, multiplet; and br, broad; coupling constants (apparent splittings) are reported as numerical values in Hz; eq and ax designate equatorial and axial hydrogen atom, a and b hydrogen below and above the ring plane, respectively; the assignments are based on 500 MHz HSQC–TOCSY and NOESY spectra. c A and B designate H-18 protons trans and cis to C-15, respectively. d Non-stereospecific assignment; the geminal methyl groups exhibit equally intense NOEs to H-24. e These assignments may be interchanged.
62 63
E
C
T
b
65
2.2. Plant material
66 67
72 73
Oleoresin was hand collected from the wounded trunk of A. malabarica from the forests of Subrahmanya Kukke, South Canara District, Karnataka, India (12°40′N; 75°36′E) in November, 2012. It was identified by Prof. V. S. Raju, Department of Botany, Kakatiya University. A voucher specimen (AVN-HM-12) is being maintained in the natural products laboratory of University College of Pharmaceutical Sciences, Kakatiya University, Warangal.
74
2.3. Isolation of phytochemical constituents
75
50 g of the oleoresin was extracted with toluene in which it dissolved completely. It was adsorbed onto silica gel (100 g) and flash chromatographed on silica gel (#230–400; 750 g) eluting with hexane:ethylacetate (100–50%) and then with ethylacetate:methanol (50%) later with methanol and methanol:water (50%) all in 1 L quantity. Fractions of ~50 mL were collected and pooled based on TLC pictures which yielded 30 major fractions. While some of the compounds crystallized directly in some of these fractions, the others were isolated by rechromatography as described below.
76 77 78 Q7 79 80 81 82 83 84
R
O
C
N
70 71
U
68 69
R
64
with mixing times of 1000 ms. HMBC experiments were optimized for nJC, H = 8 Hz. Mass spectra were recorded on an Agilent 5973i quadrupole mass spectrometer.
Compounds 1, 8 & 9: Fractions 8–10 resulted from elution of column with hexane:ethylacetate—80:20. Orthorhombic crystals of 8 (epoxymalabaricol, 1.2 g, 2.4%) formed in these fractions which were collected by filtration. The filtrates were found to contain two common constituents by TLC. They were pooled and rechromatographed on silica gel using hexane: acetone—90:10 to yield 1 (120 mg) and later 9 (800 mg) as a semisolid. Compound 2: Fraction 23 resulted from elution of the column with hexane:ethylacetate—50:50. This was subjected to rechromatography with hexane:acetone—70:30 and later on, reversed phase silica gel (C-18) with methanol:water— 75:25 to yield 2 (170 mg). Compound 3: Fractions 17–21 were obtained by elution of the column with hexane:ethylacetate—60:40. They were mixed and subjected to rechromatography using hexane: acetone—80:20 to yield 3 (1.80 g, 3.6%) as a hygroscopic semisolid. Compound 4: Fractions 26–29 were obtained by elution of column with ethylacetate:methanol—50:50. Rechromatography with hexane:acetone—70:30 yielded 4 (25 mg). Compounds 5 & 6: Fractions 24–25 obtained by elution with ethylacetate were pooled and on chromatography using 90–70% of hexane:acetone to yield 5 (30 mg) and later 6 (15 mg). Compound 7: Fractions 1–5 were obtained by elution of column with hexane:ethylacetate—90:10. Slow evaporation
Please cite this article as: Achanta PS, et al, New malabaricane triterpenes from the oleoresin of Ailanthus malabarica, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.022
85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 Q8 110 111
P.S. Achanta et al. / Fitoterapia xxx (2014) xxx–xxx Table 1 500 MHz 1H NMR spectral data for compounds 1–9 in (CDCl3)a, b. 6a
7
8
9
1.83 (m), 1.55 (m) 2.59 (ddd, J = 15.9, 10.8, 7.6) 2.42 (ddd, J = 15.9, 7.2, 3.4) – 1.31 (d, J = 11.4) 1.68 (m), 1.57 (m) 1.67 (m), 1.21 (m) 1.49 (m) 1.57 (m) 2.08 (m), 1.62 (m) 2.27 (d, J = 9.13) 2.41 (m), 1.86 (m) 1.90 (m), 1.36 (m) 3.31 (d) A: 4.66 (s) B: 4.96 (s)c 1.04 (s) 1.13 (s) 1.81 (m), 1.56 (m) 1.80 (m), 1.36 (m) 3.24 (d) 1.18 (s) 1.19 (s) 1.07 (s) 1.08 (s) 1.09 (s)
1.51 (m), 1.07 (m) 1.68 (m)
1.84 (m), 1.36 (m) 2.59 (ddd, J = 16.1, 10.9, 7.5), 2.41 (ddd, 16.1, 7.0, 3.3) – 1.31 (dd, J = 3.0) 159 (m) 1.76 (m) 1.42 (m) 1.53 (m), 1.42 (m) 1.93 (m), 1.74 (m) 1.75 (m) 1.83 (m), 1.70 (m) 1.83 (m) 3.71 (t, J = 7.1) 1.23d (s) 0.99 (s) 1.21 (s) 1.50 (m), 1.36 (m) 2.08 (m) 5.14 (t, J = 7.1) 1.71 (s) 1.64 (s) 1.11 (s) 1.06 (s) 0.99 (s)
1.81 (m), 1.47 (m) 2.56 (ddd, J = 16.1, 10.7, 7.5), 2.40 (ddd, 16.1, 7.1, 3.4) – 1.35 (m) 1.53 (m), 1.50 (m) 1.73 (m), 1.58 (m) 1.38 (d, J = 6.9) 1.52 (m), 1.40 (m) 1.99 (m), 1.70 (m) 1.78 (m) 1.76 (m), 1.74 (m) 1.93 (m), 1.49 (m) 3.91 (dd, J = 8.5, 2.0) 1.19 (s) 0.94 (s) 1.13 (s) 2.08 (m), 1.50 (m) 2.08 (m), 1.90 (m) 3.81 (dd, J = 7.0, 1.5) 1.22 (s) 1.06 (s) 1.08 (s) 1.03 (s) 0.95 (s)
1.56 (m), 1.03 (m) 1.63 (m), 1.60 (m) 3.20 (dd,J = 10.6, 5.5) 0.73 (d, J = 11.6) 1.62 (m), 1.49 (m) 1.71 (m) 1.33 (m) 1.46 (m), 1.35 (m) 1.89 (m), 1.70 (m) 1.73 (m) 1.82 (m), 1.70 (m) 1.82 (m) 3.67 (t, J = 7.1) 1.19e (s) 0.83 (s) 1.18e (s) 1.48 (m), 1.34 (m) 2.12 (m), 2.04 (m) 5.10 (t, J = 7.0) 1.61 (s) 1.68 (s) 0.97 (s) 0.78 (s) 0.91 (s)
F
R O O
P
3.15 (dd, J = 11.6, 4.5) 0.68 (d, J = 11.9, 2.1) 1.63 (m), 1.51 (m) 1.60 (m), 1.20 (m) 1.39 (m) 1.52 (m), 1.41 (m) 2.04 (m), 1.62 (m) 2.21 (d, J = 8.16) 2.38 (m), 1.85 (m) 1.87 (m), 1.38 (m) 3.30 (d, J = 10.7) A: 4.66 (s) B: 4.96 (s) 0.92 (s) 1.13 (s) 1.77 (m), 1.54 (m) 1.75 (m), 1.35 (m) 3.25 (d, J = 9.9) 1.18 (s) 1.19 (s) 0.96 (s) 0.80 (s) 1.04 (s)
D
5a
119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136
2.3.5. (17R,20R,24R)-17,20,24,25-tetrahydroxy-14(18)-malabaricen3-one (5) Amorphous powder, [α]25 D + 54° (c0.1, CHCl3); m.p.: 89– 92 °C; HRESIMS m/z 493.38992 [M + H]+, [C30H52O5 + H]+ requires 493.38875; ESIMS m/z 458 [(M − 2H2O) + H]+.
144 145
2.3.6. (17R,20R,24R)-17,20,24,25-tetrahydroxy-14(18)-malabaricen3β-ol (6) Amorphous powder, m.p.: 153–155 °C; HRESIMS m/z 517.38729 [M + Na]+, [C30H54O5Na]+ requires 517.38634; ESIMS m/z 515 [M + Na]+, 457 [(M − 2H2O) + H]+; Optical rotation was not determined due to the small amount of material available.
149 Q9
2.3.7. (14S,17R,20S)-20-hydroxy-14,17-epoxy-24(25)-malabaricen3-one (7) Amorphous powder, [α]25 D + 40°(c0.1, CHCl3); m.p.: 65– 67 °C; ESIMS m/z 481.45, [M + Na]+, [C30H50O3 + Na]+ requires 481.37.
156 157
R
2.3.1. (14S,17S,20S,24R)-25-hydroxy-14,17-cyclo-20,24epoxy-malabaricane-3-one (1) Crystalline powder, m.p.: 156–159 °C; HRESIMS m/z 481.36564 [M + Na]+, [C30H50O3Na]+ requires 481.36521; Optical rotation was not determined due to the small amount of material available. ESIMS m/z 459 [M + H]+, 441 [(M − H2O) + H]+.
R
117 118
137
O
116
2.3.4. (14S,17S,20S,24S)-14,17,20,24,25-pentahydroxy-malabarican3-one (4) Amorphous powder, m.p.: 91–94 °C; HRESIMS m/z 533.38140 [M + Na]+, [C30H54O6Na]+ requires 533.38126; ESIMS m/z 493 [(M − H2O) + H]+, 457 [(M − 3H2O) + H]+, optical rotation was not determined due to the small amount of material available.
N C
114 115
of solvent from these fractions resulted in the formation of 7 (10.0 g, 20%) in the form of needles which were collected by filtration. Compound 10: Fractions 13–15 were obtained by elution of the column with hexane:ethylacetate—70:30. They were pooled and subjected to rechromatography with hexane: acetone—95:5 to give 10 (75 mg).
U
112 113
E
C
T
E
t3:1 t3:2
3
2.3.2. (14S,17S,20S,24R)-20,24,25-trihydroxy-14,17-cyclomalabarican-3-one (2) Crystalline powder, [α]25 D + 50° (c0.1, CHCl3); m.p.: 143– 145 °C; HRESIMS m/z 499.37630 [M + Na]+, [C30H52O4Na]+ requires 499.37578; ESIMS m/z 459 [(M − H2O) + H]+, 441 [(M − 2H2O) + H]+. 2.3.3. (14S,17S,20S)-14,17,20-trihydroxy-24(25)-malabaricen-3one (3) Semi solid, [α]25 D + 36°(c 0.28, CHCl3); 499.37597 [M + Na]+, [C30H52O4Na]+ requires 499.37578; ESIMS m/z 459 [(M − H2O) + H]+, 441 [(M − 2H2O) + H]+.
Please cite this article as: Achanta PS, et al, New malabaricane triterpenes from the oleoresin of Ailanthus malabarica, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.022
138 139 140 141 142 143
146 147 148
150 151 152 153 154 155
158 159 160
4 t4:1 t4:2
P.S. Achanta et al. / Fitoterapia xxx (2014) xxx–xxx
Table 2 125 MHz 13C NMR spectral data for compounds 1–9 in (CDCl3)a. 1
2
3a
4a
5a
6a
7
8
9
t4:4 t4:5 t4:6 t4:7 t4:8 t4:9 t4:10 t4:11 t4:12 t4:13 t4:14 t4:15 t4:16 t4:17 t4:18 t4:19 t4:20 t4:21 t4:22 t4:23 t4:24 t4:25 t4:26 t4:27 t4:28 t4:29 t4:30 t4:31 t4:32 t4:33
1 (CH2) 2 (CH2) 3 (C_O/–OH) 4 (C) 5 (CH) 6 (CH2) 7 (CH2) 8 (C) 9 (CH) 10 (C) 11 (CH2) 12 (CH2) 13 (CH) 14 (C) 15 (CH2) 16 (CH2) 17 (CH) 18 (CH3/_CH2) 19 (CH3) 20 (CH2) 21 (CH3) 22 (CH2) 23 (CH2) 24 (CH) 25 (C) 26 (CH3) 27 (CH3) 28 (CH3) 29 (CH3) 30 (CH3)
39.93 34.12 218.08 47.43 55.37 19.67 34.61 40.29 50.16 36.87 22.08 27.39 43.10 50.04 31.45 25.71 49.49 16.37 16.05 86.36 23.58 35.72 26.13 83.33 71.44 27.48d 24.30d 26.73 21.01 15.15
39.89 34.12 218.03 47.44 55.37 19.65 34.54 40.29 49.97 36.85 22.02 27.46 42.60 50.28 31.14 24.92 50.22 16.01 16.35 75.52 25.38 36.84 25.68 78.81 73.16 26.67d 23.36d 26.71 21.03 15.21
39.34 33.71 218.97 47.14 55.02 20.57 36.81 44.18 59.79 36.46 21.02 23.68 59.79 75.40 39.01 25.17 78.25 24.38 14.88 74.17 20.85 37.97 21.65 124.70 130.56 24.47 16.33 20.04 25.70 25.07
39.44 33.72 218.99 47.16c 55.01 20.55 36.80 44.18 58.79 36.45 21.02 23.68 58.78 75.47 39.12 25.13 78.44 24.11 14.89 74.28 20.93 35.10 24.66 79.08 72.57 23.84d 24.11d 20.05 25.67 25.08
38.95 33.72 218.00b 47.13c 55.35 20.22 35.55 44.81 54.31 36.02 20.13 26.93 56.71 155.81 36.42 30.03 77.52 107.90 14.08 74.16 20.58 35.39 24.52 79.03 72.55 24.56d 24.08d 20.08 25.63 23.35
38.68 26.49 78.52 38.40 56.00 18.71 36.45 45.03 55.36 36.24 20.26 27.20 56.78 155.02 36.48 30.04 77.59 107.76 14.75 74.17 20.61 35.34 24.57 79.04 72.56 23.82d 24.08d 27.33 14.60 23.78
39.71 34.20 217.48 47.46 55.53 20.75 36.58 44.08 58.31 36.58 21.15 23.89 59.93 85.66 38.20 26.03 82.27 24.37 15.51 72.78 24.37 37.68 22.22 124.62 131.54 25.70 17.64 26.56 21.15 25.70
39.54 34.18 217.63 47.45 55.14 20.73 36.71 43.95 58.04 36.56 21.33 24.06 60.26 86.36 38.05 28.91 81.10 22.05 15.60 85.40 25.12 31.07 26.57 85.40 71.50 28.07d 25.18d 26.53 21.02 25.82
39.43 27.44 79.30 38.81 56.08 19.54 37.67 44.33 59.28 37.02 21.11 24.19 59.79 85.82 38.31 26.21 82.43 24.29 16.10 72.88 24.29 37.92 22.30 124.77 131.38 25.59 17.59 28.21 15.23 26.12
t4:34 t4:35 t4:36 t4:37
a
c
161
R
2.3.8. (14S,17R,20S,24R)-25-hydroxy-14,17, 20, 24-diepoxymalabarican-3-one (8) Orthorhombic crystals, [α]25 D + 22° (c0.1, CHCl3); m.p.: + + 164 Q10 142–144 °C; ESIMS m/z 497.45, [M + Na] , [C30H50O4 + Na] 165 requires 497.36; 457 [(M − H2O) + H]+.
170
O
C
168 169
2.3.9. (14S,17R,20S)-14,17-epoxy-24(25)-malabaricen-3β,20diol (9) Semisolid, [α] 25 D + 34° (c0.28, CHCl3); ESIMS m/z 443.45, [(M − H2O) + H]+, [C30H52O3 − H2O + H]+ requires 443.39.
N
166 167
R
162 163
174
2.4. Biological activity
175 176
U
173
2.3.10. (17S,20R,24R)-17,25-dihydroxy-20,24-epoxy-14(18)malabaricen-3-one (10) Amorphous powder, [α]25 D + 12° (c0.1, CHCl3); m.p.: 135– 137 °C.
171 172
E
T
E
d
Recorded in methanol-D4. Extracted from HMBC. Masked in MeOH peaks. Non-stereospecific assignment of the geminal methyl groups.
O
R O
P
C
b
F
Carbon
D
t4:3
2.4.1. Antimicrobial screening Antimicrobial assays were performed against several bacte177 Q11 ria strains (Pseudomonas aerginosa ATCC BAA 427, P. aerginosa CI 178 15159, Klebsiella pneumonia ATCC 27736, K. pneumonia CI 1876, 179 Escherichia coli ATCC 25922) using an agar disk diffusion assay 180 and a fungal strain (Candida albicans ATCC60193) using broth 181 dilution assay at 100 μg/mL and 250 μg/mL concentrations.
2.4.2. Hemolysis assay Human blood was collected in EDTA containing vacutinaer (2 mg/mL). Plasma and buffy coat were removed from the EDTA-blood by centrifugation at 800 g for 10 min. Later, the erythrocytes were washed three times by using normal saline (0.9%) and resuspended in saline to 5% erythrocyte suspension. The cells were incubated with end-over-end rotation for 1 h at 37 °C in the presence of test compounds (100 μg/mL) and were performed in triplicate. After the incubation, the samples were centrifuged at 800 g for 10 min and the supernatant was transferred to 96 well plates. 2% Triton X-100 (Sigma-Aldrich, St. Louis, USA) served as positive control. The absorbance of hemoglobin released was measured at 540 nm and is expressed as % of Triton X-100 induced hemolysis. The result was calculated by using the formula %Hemolysis = [(absorbance of sample-absorbance of blank) / (absorbance of positive control)] × 100.
182 183
3. Results and discussion
199
HRESI-MS of 1 showed a pseudomolecular ion peak at m/z 481.36072 [M + Na]+ indicating the molecular formula C30H50O3. 13C NMR spectrum contained 30 carbon signals, and an analysis of HSQC spectrum revealed the presence of eight methyls, ten methylenes, five methines and seven quaternary carbons. All the methyls appeared as singlets in the 1H NMR spectrum indicating their attachment to
200 201
Please cite this article as: Achanta PS, et al, New malabaricane triterpenes from the oleoresin of Ailanthus malabarica, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.022
184 185 186 187 188 189 190 191 192 193 194 195 196 197 198
202 203 204 205 206
P.S. Achanta et al. / Fitoterapia xxx (2014) xxx–xxx
OH
HO
3
O
29
1 4 28
30
13
9 10
H
5
H
8 7
6
25
H 24 O 20 21 18 H 17 14
OH
23
H
H
16 15
H
O
H
2
1
OH
OH
H
H
O
H
P
HO
HO
H
T
6
H
OH
E
O H
R
H
C
5
H
OH
H
H
E
HO
H
O O
O N C
U
HO
207 208 209 210 211 212 213
H
O
H
OH
H O H
H
H H
8
R
7
OH
D
OH
H
HO
HO
OH
O
OH
4
3
H
OH
OH
H
H
H
O
HO
HO
HO
O
OH
H
22
R O O
2
12
11
19
27
F
26
5
26
OH 2
H
3
O
H
29
1 4
12
11
19
9 10 5
30
H 6
13 8 7
22
18 16 14
H
15
21 17
HO H
23
20 24
O
25
OH 27
H
28
10
9 Fig. 1. Structures of compounds 1–10.
quaternary carbons. Malabaricane [7] carbon skeleton of the ring system (A–C) was established by an analysis of HMBC and NOESY spectra. NOE interactions were observed between H-19, H-29 and H-30, between H-28 and H-5, and H-13 and H-30. HSQC–TOCSY spectra helped in delineating the spin systems. 1 H and 13C NMR data are presented in Tables 1 and 2 respectively and structure is given in Fig. 1. Figures of
compounds with key NOESY correlations in the side chain are given in supporting information. A carbonyl group detected in 13 C NMR spectrum (δ 218.08) was placed at C-3 on the basis of an isolated CH2–CH2 spin system evident in COSY and HSQC– TOCSY. 1H and 13C NMR data coupled with HSQC–TOCSY indicated the presence of substituted cyclobutane and tetrahydrofuran rings in the side chain. The upfield shift of carbon
Please cite this article as: Achanta PS, et al, New malabaricane triterpenes from the oleoresin of Ailanthus malabarica, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.022
214 215 216 217 218 219 220
242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263
F O R O
P
240 241
Fig. 3. Effect of the oleoresin of A. malabarica and compounds 1–3, 5, 7–10 on lysis of human RBCs.
an isobutenyl group was attached to C-13 of malabaricane ring system in this molecule. The double bond was placed between C24 and C25, and the hydroxyl groups on C14, C17, and C20 based on HMBC correlations. The relative stereochemistry in 3 was established from NOESY spectrum. NOE correlations were observed between H-17 (δ 3.26) and H-15 (δ 1.92), H-16 (δ 1.36) and H-21 (δ 1.14). H-16 (δ 1.36) correlated with H-15 (δ 1.92), H-18 (δ 1.22) and H-21 (δ 1.14) indicating that all these were cofacial. A proton of shift value δ 1.76 showed correlations with H-16 (δ 1.36), H-17 (δ 3.26), H-18 (δ 1.22) and H-21 (δ 1.14). This proton was deduced to be the β oriented H-13. Thus, the protons with which it correlated were β oriented. A problem arose because one of the H-16s had the same shift value (δ 1.76) as that of H-13, but logical interpretation of the NOESY spectrum solved it. Thus the stereochemistry of the side chain was found to be 14S,17S,20S. 4 was determined to be the 24,25-dihydroxy derivative of 3. It lacked the signals due to doubly bonded carbons in 13C NMR spectrum. Instead, two additional oxygenated carbon signals assigned to C–24 and C–25 appeared at δ 79.08 and 72.57. A pseudomolecular ion peak at m/z 533.38140 corresponding to [M + Na]+ appeared in the mass spectrum of 4. The molecular weight was found to be 510 which was 34 amu higher than that of 3. Correlations were observed in NOESY between H-13 (δ 1.76), H-30 (δ 1.04), and H-18 (δ 1.22) and also between H18, H-15 (δ 1.95), and H-16 (δ 1.38) indicating the protons to be β oriented. H-17 (δ 3.26) showed correlations exclusively with H-16 (δ1.38) and H-21 (δ 1.14). The configuration at C24 was determined from the observation that H-17 (δ 3.26) and H-24 (δ 3.25) had common interactions with H-22 (δ 1.58) and H-23 (δ 1.80), protons that were sandwitched between H-17 and H-24, indicating that H-24 was also β oriented. Thus the stereochemistry in the side chain was determined to be 14S,17S,20S,24S. A pseudomolecular ion peak appeared at m/z 493.38992 [M + H]+ in the HRESI-MS spectrum of 5 compatible with C30H52O5. In contrast to other compounds isolated in this series, there were only seven methyl signals in the 1H NMR spectrum of 5. The 13C NMR spectrum has signals at δ 107.90
D
238 239
T
236 237
C
234 235
E
232 233
R
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resonance for C-13 (δ 43.10) by about 13–16 ppm, when compared with other compounds reported herein, indicated that the cyclobutane ring was attached to C13 which was confirmed by HMBC. Downfield signals in the 13C NMR spectrum at δ 83.33 and 86.36 indicated the presence of tetrahydrofuran ring which was found to be attached to the cyclobutyl group at C-17. An isopropyloxy group was attached to the tetrahydrofuran ring at C-24. The connectivities were established by HMBC. The relative stereochemistry in the side chain was established by NOE interactions between H-18 (δ 0.91) and H-17 (δ 1.84) and between H-21 (δ 1.16) and H-24 (δ 3.75) indicating that all these were on the same side of the rings and their α orientation was established by their lack of interaction with the β oriented H-13 (δ 1.63). According to Hashimoto et al. isomers with the two largest substituents of the tetrahydrofuran ring in the trans orientation, a hydrogen corresponding to H-24 appeared as a doublet (J = 5.3 and 10.2 Hz), whereas a triplet (J = 7.3 Hz) was observed in the cis diastereomers [17]. H-24 appeared as a triplet (J = 7.3 Hz) at δ 3.76 in the 1 H NMR spectrum of 1 confirming the NOESY results. Based on these results the stereochemistry was determined to be 13S,14S,17S,20S,24R. Structure of 2 was similar to that of 1 up to the cyclobutyl ring. Tetrahydrofuran ring was found to be absent in 2 as there were no downfield oxygenated carbon signals at δ 83.33 and 86.36 in its 13C NMR spectrum. Instead, two hydroxyl bearing carbon signals appeared at δ 75.52 and 78.81 which were assigned to C-20 and C-24 respectively. Thus the side chain in 2 could have resulted from a cascade opening through hydrolysis of the tetrahydrofuran ring present in 1. All the connectivities were confirmed by HMBC. Relative stereochemistry of the side chain in 2 was same as in 1 up to C-20 (NOESY). The configuration at C-24 was arrived at as follows: NOE interaction between the α oriented H-21 (δ 1.16) and H-22 (δ 1.63) indicated the latter to be α oriented. The other H-22 (δ 1.76) which was β oriented correlated with H-24 (δ 3.42) indicating that H-24 was β orientated (Fig. 1). Thus the configuration of the side chain was determined as 14S,17S,20S,24R. A pseudomolecular ion peak appeared at m/z 499.37597 [M + Na]+ in the HRESI-MS spectrum of 3 and its molecular formula was deduced as C30H52O4. An acyclic chain of thirteen carbons containing three hydroxyl groups and terminating into
OH
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Fig. 2. Hypothetical formation of various malabaricanes from malabaricol through hydration, dehydration, cyclodehydration, hydrolysis, hydroxylation etc.
Please cite this article as: Achanta PS, et al, New malabaricane triterpenes from the oleoresin of Ailanthus malabarica, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.022
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side chain in 8 (up to C-20) was identical to that of malabaricol (7) in which stereochemistry of the side chain was determined as 14S,17R,20S by X-ray diffraction [20]. A stereostructure for compound 8 had been reported earlier though there was no mention of how it was arrived at [7]. NMR data with assignments is being reported for the first time. 9 has been identified as the known compound malabaricanediol [7]. NOESY study revealed that the hydroxyl at C-3 was β-oriented. The relative stereochemistry of the side chain was same as in malabaricol (7). Thus, the stereochemistry of the side chain was determined as 14S,17R,20S. NMR data of the compound is being reported for the first time. 10 was isolated earlier from Caloncoba echinata [15], and is being reported for the first time from the oleoresin of A. malabarica. 1H and 13C NMR data of compounds 1–9 are given in Tables 1 and 2 respectively and structures of compounds 1–10 are given in Fig. 1. The present investigation resulted in the isolation of structurally related malabaricane triterpenes with novel structural features. 1 and 2 have cyclobutyl ring in the side chain and 3–6 were polyhydroxylated. Our literature survey reveals that compounds 1–6 are new. Yield-wise, malabaricol (7) was the chief constituent of the oleoresin (20% w/w) followed by 3 (3.6% w/w) and epoxymalabaricol (8, 2.4% w/w). The other compounds proved to be minor constituents. Whether the minor metabolites are formed from the major metabolite malabaricol or it is the other way round is a subject to be investigated. However it can be said that their formation involves reactions like hydration, dehydration, cyclodehydration, hydrolysis and hydroxylation. Their hypothetical formation from malabaricol is shown in Fig. 2. Triterpenes of the malabaricane skeleton are rare and constitute a small group. The oleoresin of A. malabarica and compounds 1–3, 5, 7–10 were tested for their antimicrobial activity on P. aerginosa, K. pneumonia, E. coli, C. albicans in view of the traditional claims of halmaddi resin to treat bronchitis and dysentery. None of them exhibited considerable activity, i.e., an inhibition zone N0.5 mm was not found at tested (100 and 250 μg/mL) concentrations. The compounds were also tested for their toxicity to RBCs and all the compounds (except 10) were considerably less toxic compared to the reference standards polymyxin (4.99%) and ampicillin (4.46%) at 100 μg/mL (Fig. 3). 10 was more toxic (4.92%) and its activity was comparable to that of polymyxin. It was reported earlier to have antiplasmodial activity but also caused transformation of erythrocytes into stomatocytes [21]. Interestingly, 5 which is structurally related to 10 (opening of epoxy ring in 10 results in 5) was found to be least toxic (1.84%).
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Acknowledgments
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PSA thanks CSIR, New Delhi for Senior Research Fellowship (No. 09/384(159)/2013. EMR-I) and ARVNA thanks AICTE for the award of Emeritus Fellowship (No. 06/AICTE/RIFD(Policy4th)/EF(01)/2012–13). Principal, UCPSc is thanked for his encouragement. Mukesh Pasupuleti, Vishnu Sravan, Rama Rao and Raghavachary are thanked for their help. Authors thank Dr. Gayathri Withers for providing NMR spectra. NMR instrumentation at Carenegie Mellon University was partially supported by NSF (CHE-0130903 and CHE-1039870). Authors thank Prof. GA
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and 155.81, characteristic of an exo methylene group. It also showed four oxygenated carbon signals. A side chain 323 resembling the one present in 4 with a double bond between 324 C-14 and C-18 was found attached to C-13 in this compound. 325 Though 13C NMR spectrum of 5 was not properly obtained, 326 all the connectivities could be established through HSQC, 327 HSQC–TOCSY and HMBC. The relative stereochemistry of 328 the side chain in 5 was determined by a study of its NOESY 329 spectrum. H-13 (δ 2.27) showed interaction with H-15 (δ 2.41) 330 indicating that this hydrogen was β-oriented. The α-oriented 331 H-15 (δ 1.86) showed a correlation with H-17 (δ 3.31) and this 332 in turn showed interaction with H-16 (δ 1.36) and H-21 (δ 333 1.13) indicating all have α-orientation. While H-24 (δ 3.25) 334 showed only three interactions with protons on one side of 335 the chain i.e., with H-22 (δ1.56), H-23 (δ1.80), and H-26 (δ 336 1.18), H-17 (δ 3.31), being centrally located in the side chain, 337 showed five interactions with protons on either side 338 i.e., with H-15 (δ 1.86), H-16 (δ 1.36), H-21 (δ1.13) H-22 339 (δ1.56) and H-23 (δ1.80). The common interaction of H-17 340 and H-24 with protons that were sandwitched between 341 them i.e., H-22 (δ 1.56) and H-23 (δ 1.80), established that 342 H-24 was also α-oriented. Thus the stereochemistry of the 343 side chain was determined to be 17R,20R,24R. 344 Structure of 6 was similar to that of 5. The 13C NMR 345 spectrum of 6 lacked a signal due to a carbonyl group. 346 Instead, there was an additional peak at δ 78.52 indicating 347 that the keto group at C-3 present in 5 was reduced to a 348 secondary alcohol group in 6. The C-3 hydroxyl was β349 oriented as confirmed by the NOE interactions between H-3 350 and H-28 and H-28 and H-5. Stereochemistry of the side 351 chain was same as in 5. Both 5 and 6 resemble the known 352 compound 10 in having a double between C14–C18. 353 7 was identified as the known compound malabaricol, 354 reported previously from the oleoresin of A. malabarica. Its 355 structure was established earlier through extensive chemi356 cal degradation [7,18,19]. Its stereochemistry was confirmed 357 by X-ray diffraction studies [20]. Though 13C NMR data was 358 given for this compound, assignments were not made [11]. 359 Even the latest report on the identification of malabaricol and 360 epoxymalabaricol isolated from the heartwood of Ailanthus 361 excelsa does not contain the NMR data and assignments [21]. 362 NMR study of 8, though a known compound, proved quite 363 interesting. It was identified as epoxymalabaricol reported 364 previously from the oleoresin of A. malabarica. NOESY study 365 revealed that the stereochemistry in the side chain for 8 was 366 identical to that of 1. The absolute stereochemistry of the side 13 367 Q12 chain in 8 was determined as 14S,17R,20S,24R. The C NMR 368 spectra of compounds with a 24R-configuration show for the C369 26 and C-27 methyls' different (ca 3 ppm) chemical shifts [14], 370 due to a strong hydrogen bond between the tertiary hydroxyl 371 group and the tetrahydrofuran oxygen. By contrast, in the 13C 372 NMR spectra of the 24S-series the two methyls exhibit very 373 close values (b1 ppm). In 8 the difference was 2.9 ppm sup374 porting R configuration at C-24. A malabaricane triterpene 375 glucoside isolated from Adesmia aconcaguensis has a side chain 376 similar to the one present in 8. However, the two methyls, C-26 377 and C-27, exhibited very close 13C NMR values (b 1 ppm) and 378 so C-24 was assigned S configuration. Also 13C NMR values 379 differed from 8, especially at C17 and C20. The configuration of 380 the side chain was described as 14R,17R,20R,24S [14]. It is 381 worth mentioning here that the relative stereochemistry of the
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Please cite this article as: Achanta PS, et al, New malabaricane triterpenes from the oleoresin of Ailanthus malabarica, Fitoterapia (2014), http://dx.doi.org/10.1016/j.fitote.2014.11.022
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NMR spectra, figures of compounds with key NOESY correlations are given in supporting information. Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.fitote.2014.11. 022.
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Cordell and Prof. Alessandra Braca for their helpful discussions and valuable suggestions.
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