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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Anti-inflammatory flavanol glycosides from Saraca asoca bark ab
a
a
Furkan Ahmad , Laxminarain Misra , Rashi Tewari , Preeti a
c
c
Gupta , Pratikshita Mishra & Rakesh Shukla a
Chemical Sciences Division, CSIR – Central Institute of Medicinal and Aromatic Plants, Lucknow226015, India b
Academy of Scientific and Innovative Research, CIMAP Campus, Lucknow226015, India c
Click for updates
Pharmacology Division, CSIR – Central Drug Research Institute, Sitapur Road, Lucknow226031, India Published online: 23 Mar 2015.
To cite this article: Furkan Ahmad, Laxminarain Misra, Rashi Tewari, Preeti Gupta, Pratikshita Mishra & Rakesh Shukla (2015): Anti-inflammatory flavanol glycosides from Saraca asoca bark, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1023728 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1023728
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [University of Connecticut] at 12:54 11 April 2015
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1023728
SHORT COMMUNICATION Anti-inflammatory flavanol glycosides from Saraca asoca bark Furkan Ahmadab, Laxminarain Misraa*, Rashi Tewaria, Preeti Guptaa, Pratikshita Mishrac and Rakesh Shuklac
Downloaded by [University of Connecticut] at 12:54 11 April 2015
a Chemical Sciences Division, CSIR – Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, India; bAcademy of Scientific and Innovative Research, CIMAP Campus, Lucknow 226015, India; c Pharmacology Division, CSIR – Central Drug Research Institute, Sitapur Road, Lucknow 226031, India
(Received 10 December 2014; final version received 20 February 2015)
Bioactivity guided isolation and characterization
OH O
HO
O OH
O OH Me OH OH
Saraca asoca bark Ayurvedic medicine
Anti-inflammatory
Saraca asoca (Roxb.) de Wilde, a common tree of India, is popularly used in the Ayurvedic and modern herbal systems of medicine for genito-urinary problems of women. Considering the reported antimicrobial or anti-inflammatory effect of S. asoca bark against such infections, we studied the anti-inflammatory activity-guided isolation of active compounds from methanol extract. The methanol extract of bark has yielded 10 compounds out of which 30 -deoxyepicatechin-3-O-b-D -glucopyranoside (6) and 30 deoxycatechin-3-O-a-L -rhamnopyranoside (8) have been found to be in vitro and in vivo active. 30 ,5-Dimethoxy epicatechin (3), 30 -deoxyepicatechin-3-O-b-D glucopyranoside (6), 30 -deoxycatechin-3-O-a-L -rhamnopyranoside (8) and epigallocatechin (9) are being reported for the first time from S. asoca. Keywords: Saraca asoca (Roxb.) de Wild. syn. S. indica Linn; flavan-3-O-glycosides; anti TNF-a activity; anti-inflammatory activity
1. Introduction Saraca asoca (Roxb.) de Wilde syn. S. indica Linn., locally named as Ashoka, is prescribed by Ayurvedic physicians for various diseases, including, gynaecological disorders (Pradhan et al. 2009; Misra 2013). It has been, recently, reported that the MeOH extract reduced paw swelling, increased body weight, reduced the level of lysosomal enzymes, decreased protein-bound carbohydrates, urinary collagen and serum cytokines as well as normalised the histopathology of joints (Saravanan et al. 2011) and did not show any toxicity (Mukhopadhyay & Nath 2011). However, the anti-inflammatory compound could not be identified, so far. Our continued interest in the chemical investigation of Indian medicinal plants for the biologically active natural products (Siddique et al. 2014; Vyry Wouatsa et al. 2013; Ahmad et al. 2015) led to a bioactivity-guided detailed isolation and identification of a number of compounds by using spectroscopic methods, including anti-TNF-a and anti-inflammatory 30 -deoxyepicatechin-3-O-
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
2
F. Ahmad et al.
b-D -glucopyranoside (6) and 30 -deoxycatechin-3-O-a-L -rhamnopyranoside (8) from the methanol extract of S. asoca bark which is discussed in this paper.
Downloaded by [University of Connecticut] at 12:54 11 April 2015
2. Results and discussion Methanol extract of S. asoca bark after chromatographic separations yielded leucopelargonidin (1), leucocyanidin (2), 30 ,5-dimethoxy epicatechin (3), epicatechin (4), catechin (5), 30 deoxyepicatechin-3-O-b-D -glucopyranoside (6), lyoniside (7), 30 -deoxycatechin-3-O-a-L rhamnopyranoside (8), epigallocatechin (9) and gallocatechin (10) (Figure S1). Out of these isolated compounds, 3, 6, 8 and 9 are being reported from S. asoca for the first time with 6 and 8 (Figure 1) showing dose-dependent significant anti-TNF-a and anti-inflammatory activities. The structure of compounds 1 –5, 7, 9 and 10 were confirmed by comparing with spectroscopic data in the literature (Hetter et al. 1985; Morimoto et al. 1985; Davis et al. 1996; Sadhu et al. 2007). Structure of 6 and 8 was elucidated by spectroscopic methods. The IR and mass spectra of 6 showed spectral data as given in Experimental (see Supplementary material). Its 1H NMR spectrum showed two doublets at d 6.28 and 6.07 (1H each) with a meta coupling (J ¼ 3.0 Hz) along with another set of two double doublets (J ¼ 8.5, 2.5 Hz and J ¼ 8.5 and 2.5 Hz, 2H each) at d 6.78 and 7.31, respectively. The 1H NMR spectrum also showed a doublet (J ¼ 6.0 Hz) at d 4.85 which coupled with a triple doublets (J ¼ 8.0, 6.0, 5.0 Hz) at d 4.18. The latter ddd further coupled with two doublets (J ¼ 16.0, 5.0 and 16.0, 8.0) at d 2.80 and 3.00, respectively. These signals clearly indicated that the compound belongs to an epicatechin nucleus having substitutions at C-5, C-7 in A ring and C-40 in B ring. The lower coupling constant (J ¼ 6.0 Hz) between the protons at C-3 and C-2 indicated that the substitution in C ring at these carbons are cis as in case of epi-catechin (Morimoto et al. 1985). The signals of H-2, H-3 and H-4 showed standard 1H – 1H COSY correlations among them, sequentially. In its 13C NMR spectrum, typical signals for epi-catechin type of structure were also present. In addition, its 1H and 13C NMR spectra showed signals for a glucoside with a doublet (J ¼ 7.2 Hz) at d 4.87 and d 102.4, respectively, for an anomeric proton and carbon. The larger coupling constant of the anomeric proton clearly indicated that the glucoside is b-linked to the epicatechin nucleus. Since the anomeric proton at d 4.87 showed correlation with C-3 at 67.1 in its HMBC spectrum, it was evident that the glucoside is attached at C-3. These data clearly established that compound 6 is a 3-O-b-D -glucoside. On acetylation, 6 gave 6a having typical signals of a tetra acetate glucoside.
R
O
HO
OH
OR1 OH O
HO
3' 8
HO
O
HO
7
R2
OH
OH
O1
10
3
4
O O
(4) R,R1 = H; R2 = H (9) R,R1 = H; R2 = OH OH
OH MeO
OH
O
HO
OH O
OH
OH
O
HO OMe
O
HO
O HO
OH
OH
O OH
Me
OH
(8) OH
MeO
OMe OH
(7)
Figure 1. Isolated compounds from S. asoca bark.
OH OH
(10)
6" 5"
1" 2" RO
(6) R = H (6a) R = Ac
(3) R,R1 = Me; R2 = H
5'
6'
2
C
4'
B
1'
OH
(5)
OH
OR
(1) R = H (2) R = OH
5
OH
OH
9
A
6 OH
OH
2'
OH
OH
OR 4" OR OR 3"
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A perusal of literature showed that 6 was earlier isolated from Cassia fistula (Morimoto et al. 1988) having comparable spectral data. Therefore, the structure of 6 was confirmed as 30 -deoxyepicatechin-3-O-b-D -glucopyranoside. Compound 8 showed similar IR and NMR spectral data as in case of 6 except the NMR values for protons at C-3, C-100 , C-600 . H-3 appeared at d 3.95 as ddd, J ¼ 5, 8 and 8 Hz indicating the possibility of a reversed stereochemistry (b-glycosidation) at C-3 as compared to compound 6. H-100 appeared at d 5.50 with lower coupling constant as broad singlet indicating an a-Olinkage to C-3. It also showed the presence of a doublet (J ¼ 5.7 Hz) at d 1.59 for a methyl clearly indicating the presence of a rhamnosyl sugar. Its 13C NMR also supported 3-O-a-L rhamnosyl substitution at C-3 (Experimental, Supplementary material; Shi et al. 2010). A perusal of literature showed that these data match well (except the chemical shift variations due to pyridine-d5 solvent) with the values reported for (þ )-afzelchin-3-O-a-L -rhamnopyranoside (Drewes et al. 1992) (Figure 1). The anti-TNF-a activity of compounds 6, 8 and the corresponding extract (methanol) was tested on whole-blood assay along with standard drug pentoxifylline. The methanol extract (100 mg/mL) as such showed an inhibition of 26%, while pure 6 and 8 showed 26 –34% and 17– 26% activity, respectively, at 50, 100 and 200 mg/mL. The activity was slightly enhanced when concentration was increased to 200 mg/mL (Table S1). Both 6 and 8 also showed significant antiinflammatory activity in rats. Earlier, Su et al. (2002) have found promising COX activities in 8 as indicative of anti-inflammatory activity. The methanol extract of S. asoca bark has been studied for carrageenan-induced rat paw oedema. When the difference in the volume of developed oedema was calculated, the percent inhibition of the oedema between the control group and the extracts/compounds-treated group showed that the methanol extract showed a maximum of 17% inhibition while compounds 6 and 8 showed 52% and 33%, respectively, at 5 h. Ibuprofen a standard drug showed maximum 68% anti-inflammatory activity (Table S2). Therefore, it was concluded that 6 and 8 are somewhat comparable with the standard drug ibuprofen (Table S2). This is the first time that an in vivo activity is being reported for compounds 6 and 8. The bark of S. asoca extract has earlier been studied for anti-inflammatory activity (Preethi & Krishnakumar 2011; Saravanan et al. 2011) but the active compounds could not be identified.
3. Conclusion Ten isolated compounds from S. asoca bark have been characterised by using spectroscopic methods, and 6 and 8 have been found to be anti-inflammatory, substantiating the traditional use of S. asoca bark in gynaecological problems of women.
Supplementary material Experimental details relating to this paper are available online.
Acknowledgements The authors are thankful to DBT, New Delhi for awarding a research grant to carry out this work and Prof. Vasudeva R., College of Forestry, Sirsi (University of Agricultural Sciences, Dharwad), India for providing the plant material. Director CSIR-CIMAP, Lucknow and CSIR-CDRI, Lucknow are thanked for providing the facilities.
Disclosure statement No potential conflict of interest was reported by the authors.
4
F. Ahmad et al.
Downloaded by [University of Connecticut] at 12:54 11 April 2015
References Ahmad F, Misra LN, Gupta VK, Darokar MP, Prakash O, Khan F, Shukla R. 2015. Isolation of (þ)-pinitol from Saraca asoca and in-silico possible mechanism of its synergistic effect with b-lactam antibiotics. J Asian Nat Prod Res. Communicated. Davis AL, Cai Y, Davies AP, Lewis JR. 1996. 1H and 13C NMR assignments of some green tea polyphenols. Magn Reson Chem. 34:887–890. doi:10.1002/(SICI)1097-458X(199611)34:11,887:AID-OMR995. Drewes SE, Taylor CW, Cunningham AB. 1992. (þ )-Afzelechin 3-rhamnoside from Cassipourea gerrardii. Phytochemistry. 31:1073–1075. doi:10.1016/0031-9422(92)80229-8. Hetter W, Britsch L, Forkmann G, Grisebach H. 1985. Leucoanthocyanidins as intermediate in anthocyanidin biosynthesis in flowers of Matthiola incana R. Br. Planta. 163:191–196. Misra LN. 2013. Traditional phytomedicinal systems, scientific validations and current popularity as nutraceuticals. Int J Trad Nat Med. 2:27–75. Morimoto S, Nonaka G, Chen R, Nishioka I. 1988. Tannins and related compounds. lxi. Isolation and structures of novel bi- and triflavanoids from the leaves of Cassia fistula L. Chem Pharma Bull. 36:39– 47. doi:10.1248/cpb.36.39. Morimoto S, Nonaka GI, Nishioka I, Ezaki N, Takizawa N. 1985. Tannins and related compounds. xxix. Seven new methyl derivatives of flavan-3-ols and a 1,3-diarylpropan-2-ol from Cinnamomum cassia, C. obtusifolium and Lindera umbellata var. membranacea. Chem Pharm Bull. 33:2281–2286. doi:10.1248/cpb.33.2281. Mukhopadhyay MK, Nath D. 2011. Phytochemical screening and toxicity of Saraca asoca bark methanolic extract. Int J Phytomed. 3:498–505. Pradhan P, Joseph L, Gupta V, Chulet R, Arya H, Verma R, Bajpai A. 2009. Saraca asoca (Ashoka): A review. J Chem Pharma Res. 1:62– 71. Preethi MF, Krishnakumar K. 2011. Anti-inflammatory activity of the bark of Saraca indica Linn. Pharmacology. 2:657–662. Online. Sadhu SK, Khatun A, Phattanawasin P, Ohtsuki T, Ishibashi M. 2007. Lignan glycosides and flavonoids from Saraca asoca with antioxidant activity. J Nat Med. 61:480–482. doi:10.1007/s11418-007-0182-3. Saravanan S, Babu NP, Pandikumar P, Ignacimuthu S. 2011. Therapeutic effect of Saraca asoca (Roxb.) Wilde on lysosomal enzymes and collagen metabolism in adjuvant induced arthritis. Inflammopharmacology. 19:317–325. doi:10.1007/s10787-011-0091-7. Shi LS, Liao YR, Su MJ, Lee AS, Kuo PC, Damu AG, Kuo SC, Sun HD, Lee KH, Wu TS. 2010. Cardiac glycosides from Antiaris toxicaria with potent cardiotonic activity. J Nat Prod. 73:1214–1222. doi:10.1021/np9005212. Siddique AA, Joshi P, Misra LN, Sangwan NS, Darokar MP5. 2014. 5,6-de-epoxy-5-en-7-one-17-hydroxy withaferin A, a new cytotoxic steroid from Withania somnifera L. Dunal leaves. Nat Prod Res. 28:392–398. doi:10.1080/ 14786419.2013.871545. Su BN, Cuendet M, Hawthorne ME, Kardono LBS, Riswan S, Fong HHS, Mehta RG, Pezzuto JM, Kinghorn AD. 2002. Constituents of the bark and twigs of Artocarpus dadah with cyclooxygenase inhibitory activity. J Nat Prod. 65:163–169. doi:10.1021/np010451c. Vyry Wouatsa NA, Misra RLN, Venkatesh Kumar R, Darokar MP, Tchoumbougnang F. 2013. Zantholic acid, a new monoterpenoid from Zanthoxylum zanthoxyloides. Nat Prod Res. 27:1994 –1998. doi:10.1080/14786419.2013. 811662.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Anti-inflammatory flavanol glycosides from Saraca asoca bark ab
a
a
Furkan Ahmad , Laxminarain Misra , Rashi Tewari , Preeti a
c
c
Gupta , Pratikshita Mishra & Rakesh Shukla a
Chemical Sciences Division, CSIR – Central Institute of Medicinal and Aromatic Plants, Lucknow226015, India b
Academy of Scientific and Innovative Research, CIMAP Campus, Lucknow226015, India c
Click for updates
Pharmacology Division, CSIR – Central Drug Research Institute, Sitapur Road, Lucknow226031, India Published online: 23 Mar 2015.
To cite this article: Furkan Ahmad, Laxminarain Misra, Rashi Tewari, Preeti Gupta, Pratikshita Mishra & Rakesh Shukla (2015): Anti-inflammatory flavanol glycosides from Saraca asoca bark, Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2015.1023728 To link to this article: http://dx.doi.org/10.1080/14786419.2015.1023728
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Downloaded by [University of Connecticut] at 12:54 11 April 2015
Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2015 http://dx.doi.org/10.1080/14786419.2015.1023728
SHORT COMMUNICATION Anti-inflammatory flavanol glycosides from Saraca asoca bark Furkan Ahmadab, Laxminarain Misraa*, Rashi Tewaria, Preeti Guptaa, Pratikshita Mishrac and Rakesh Shuklac
Downloaded by [University of Connecticut] at 12:54 11 April 2015
a Chemical Sciences Division, CSIR – Central Institute of Medicinal and Aromatic Plants, Lucknow 226015, India; bAcademy of Scientific and Innovative Research, CIMAP Campus, Lucknow 226015, India; c Pharmacology Division, CSIR – Central Drug Research Institute, Sitapur Road, Lucknow 226031, India
(Received 10 December 2014; final version received 20 February 2015)
Bioactivity guided isolation and characterization
OH O
HO
O OH
O OH Me OH OH
Saraca asoca bark Ayurvedic medicine
Anti-inflammatory
Saraca asoca (Roxb.) de Wilde, a common tree of India, is popularly used in the Ayurvedic and modern herbal systems of medicine for genito-urinary problems of women. Considering the reported antimicrobial or anti-inflammatory effect of S. asoca bark against such infections, we studied the anti-inflammatory activity-guided isolation of active compounds from methanol extract. The methanol extract of bark has yielded 10 compounds out of which 30 -deoxyepicatechin-3-O-b-D -glucopyranoside (6) and 30 deoxycatechin-3-O-a-L -rhamnopyranoside (8) have been found to be in vitro and in vivo active. 30 ,5-Dimethoxy epicatechin (3), 30 -deoxyepicatechin-3-O-b-D glucopyranoside (6), 30 -deoxycatechin-3-O-a-L -rhamnopyranoside (8) and epigallocatechin (9) are being reported for the first time from S. asoca. Keywords: Saraca asoca (Roxb.) de Wild. syn. S. indica Linn; flavan-3-O-glycosides; anti TNF-a activity; anti-inflammatory activity
1. Introduction Saraca asoca (Roxb.) de Wilde syn. S. indica Linn., locally named as Ashoka, is prescribed by Ayurvedic physicians for various diseases, including, gynaecological disorders (Pradhan et al. 2009; Misra 2013). It has been, recently, reported that the MeOH extract reduced paw swelling, increased body weight, reduced the level of lysosomal enzymes, decreased protein-bound carbohydrates, urinary collagen and serum cytokines as well as normalised the histopathology of joints (Saravanan et al. 2011) and did not show any toxicity (Mukhopadhyay & Nath 2011). However, the anti-inflammatory compound could not be identified, so far. Our continued interest in the chemical investigation of Indian medicinal plants for the biologically active natural products (Siddique et al. 2014; Vyry Wouatsa et al. 2013; Ahmad et al. 2015) led to a bioactivity-guided detailed isolation and identification of a number of compounds by using spectroscopic methods, including anti-TNF-a and anti-inflammatory 30 -deoxyepicatechin-3-O-
*Corresponding author. Email: [email protected] q 2015 Taylor & Francis
2
F. Ahmad et al.
b-D -glucopyranoside (6) and 30 -deoxycatechin-3-O-a-L -rhamnopyranoside (8) from the methanol extract of S. asoca bark which is discussed in this paper.
Downloaded by [University of Connecticut] at 12:54 11 April 2015
2. Results and discussion Methanol extract of S. asoca bark after chromatographic separations yielded leucopelargonidin (1), leucocyanidin (2), 30 ,5-dimethoxy epicatechin (3), epicatechin (4), catechin (5), 30 deoxyepicatechin-3-O-b-D -glucopyranoside (6), lyoniside (7), 30 -deoxycatechin-3-O-a-L rhamnopyranoside (8), epigallocatechin (9) and gallocatechin (10) (Figure S1). Out of these isolated compounds, 3, 6, 8 and 9 are being reported from S. asoca for the first time with 6 and 8 (Figure 1) showing dose-dependent significant anti-TNF-a and anti-inflammatory activities. The structure of compounds 1 –5, 7, 9 and 10 were confirmed by comparing with spectroscopic data in the literature (Hetter et al. 1985; Morimoto et al. 1985; Davis et al. 1996; Sadhu et al. 2007). Structure of 6 and 8 was elucidated by spectroscopic methods. The IR and mass spectra of 6 showed spectral data as given in Experimental (see Supplementary material). Its 1H NMR spectrum showed two doublets at d 6.28 and 6.07 (1H each) with a meta coupling (J ¼ 3.0 Hz) along with another set of two double doublets (J ¼ 8.5, 2.5 Hz and J ¼ 8.5 and 2.5 Hz, 2H each) at d 6.78 and 7.31, respectively. The 1H NMR spectrum also showed a doublet (J ¼ 6.0 Hz) at d 4.85 which coupled with a triple doublets (J ¼ 8.0, 6.0, 5.0 Hz) at d 4.18. The latter ddd further coupled with two doublets (J ¼ 16.0, 5.0 and 16.0, 8.0) at d 2.80 and 3.00, respectively. These signals clearly indicated that the compound belongs to an epicatechin nucleus having substitutions at C-5, C-7 in A ring and C-40 in B ring. The lower coupling constant (J ¼ 6.0 Hz) between the protons at C-3 and C-2 indicated that the substitution in C ring at these carbons are cis as in case of epi-catechin (Morimoto et al. 1985). The signals of H-2, H-3 and H-4 showed standard 1H – 1H COSY correlations among them, sequentially. In its 13C NMR spectrum, typical signals for epi-catechin type of structure were also present. In addition, its 1H and 13C NMR spectra showed signals for a glucoside with a doublet (J ¼ 7.2 Hz) at d 4.87 and d 102.4, respectively, for an anomeric proton and carbon. The larger coupling constant of the anomeric proton clearly indicated that the glucoside is b-linked to the epicatechin nucleus. Since the anomeric proton at d 4.87 showed correlation with C-3 at 67.1 in its HMBC spectrum, it was evident that the glucoside is attached at C-3. These data clearly established that compound 6 is a 3-O-b-D -glucoside. On acetylation, 6 gave 6a having typical signals of a tetra acetate glucoside.
R
O
HO
OH
OR1 OH O
HO
3' 8
HO
O
HO
7
R2
OH
OH
O1
10
3
4
O O
(4) R,R1 = H; R2 = H (9) R,R1 = H; R2 = OH OH
OH MeO
OH
O
HO
OH O
OH
OH
O
HO OMe
O
HO
O HO
OH
OH
O OH
Me
OH
(8) OH
MeO
OMe OH
(7)
Figure 1. Isolated compounds from S. asoca bark.
OH OH
(10)
6" 5"
1" 2" RO
(6) R = H (6a) R = Ac
(3) R,R1 = Me; R2 = H
5'
6'
2
C
4'
B
1'
OH
(5)
OH
OR
(1) R = H (2) R = OH
5
OH
OH
9
A
6 OH
OH
2'
OH
OH
OR 4" OR OR 3"
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A perusal of literature showed that 6 was earlier isolated from Cassia fistula (Morimoto et al. 1988) having comparable spectral data. Therefore, the structure of 6 was confirmed as 30 -deoxyepicatechin-3-O-b-D -glucopyranoside. Compound 8 showed similar IR and NMR spectral data as in case of 6 except the NMR values for protons at C-3, C-100 , C-600 . H-3 appeared at d 3.95 as ddd, J ¼ 5, 8 and 8 Hz indicating the possibility of a reversed stereochemistry (b-glycosidation) at C-3 as compared to compound 6. H-100 appeared at d 5.50 with lower coupling constant as broad singlet indicating an a-Olinkage to C-3. It also showed the presence of a doublet (J ¼ 5.7 Hz) at d 1.59 for a methyl clearly indicating the presence of a rhamnosyl sugar. Its 13C NMR also supported 3-O-a-L rhamnosyl substitution at C-3 (Experimental, Supplementary material; Shi et al. 2010). A perusal of literature showed that these data match well (except the chemical shift variations due to pyridine-d5 solvent) with the values reported for (þ )-afzelchin-3-O-a-L -rhamnopyranoside (Drewes et al. 1992) (Figure 1). The anti-TNF-a activity of compounds 6, 8 and the corresponding extract (methanol) was tested on whole-blood assay along with standard drug pentoxifylline. The methanol extract (100 mg/mL) as such showed an inhibition of 26%, while pure 6 and 8 showed 26 –34% and 17– 26% activity, respectively, at 50, 100 and 200 mg/mL. The activity was slightly enhanced when concentration was increased to 200 mg/mL (Table S1). Both 6 and 8 also showed significant antiinflammatory activity in rats. Earlier, Su et al. (2002) have found promising COX activities in 8 as indicative of anti-inflammatory activity. The methanol extract of S. asoca bark has been studied for carrageenan-induced rat paw oedema. When the difference in the volume of developed oedema was calculated, the percent inhibition of the oedema between the control group and the extracts/compounds-treated group showed that the methanol extract showed a maximum of 17% inhibition while compounds 6 and 8 showed 52% and 33%, respectively, at 5 h. Ibuprofen a standard drug showed maximum 68% anti-inflammatory activity (Table S2). Therefore, it was concluded that 6 and 8 are somewhat comparable with the standard drug ibuprofen (Table S2). This is the first time that an in vivo activity is being reported for compounds 6 and 8. The bark of S. asoca extract has earlier been studied for anti-inflammatory activity (Preethi & Krishnakumar 2011; Saravanan et al. 2011) but the active compounds could not be identified.
3. Conclusion Ten isolated compounds from S. asoca bark have been characterised by using spectroscopic methods, and 6 and 8 have been found to be anti-inflammatory, substantiating the traditional use of S. asoca bark in gynaecological problems of women.
Supplementary material Experimental details relating to this paper are available online.
Acknowledgements The authors are thankful to DBT, New Delhi for awarding a research grant to carry out this work and Prof. Vasudeva R., College of Forestry, Sirsi (University of Agricultural Sciences, Dharwad), India for providing the plant material. Director CSIR-CIMAP, Lucknow and CSIR-CDRI, Lucknow are thanked for providing the facilities.
Disclosure statement No potential conflict of interest was reported by the authors.
4
F. Ahmad et al.
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References Ahmad F, Misra LN, Gupta VK, Darokar MP, Prakash O, Khan F, Shukla R. 2015. Isolation of (þ)-pinitol from Saraca asoca and in-silico possible mechanism of its synergistic effect with b-lactam antibiotics. J Asian Nat Prod Res. Communicated. Davis AL, Cai Y, Davies AP, Lewis JR. 1996. 1H and 13C NMR assignments of some green tea polyphenols. Magn Reson Chem. 34:887–890. doi:10.1002/(SICI)1097-458X(199611)34:11,887:AID-OMR995. Drewes SE, Taylor CW, Cunningham AB. 1992. (þ )-Afzelechin 3-rhamnoside from Cassipourea gerrardii. Phytochemistry. 31:1073–1075. doi:10.1016/0031-9422(92)80229-8. Hetter W, Britsch L, Forkmann G, Grisebach H. 1985. Leucoanthocyanidins as intermediate in anthocyanidin biosynthesis in flowers of Matthiola incana R. Br. Planta. 163:191–196. Misra LN. 2013. Traditional phytomedicinal systems, scientific validations and current popularity as nutraceuticals. Int J Trad Nat Med. 2:27–75. Morimoto S, Nonaka G, Chen R, Nishioka I. 1988. Tannins and related compounds. lxi. Isolation and structures of novel bi- and triflavanoids from the leaves of Cassia fistula L. Chem Pharma Bull. 36:39– 47. doi:10.1248/cpb.36.39. Morimoto S, Nonaka GI, Nishioka I, Ezaki N, Takizawa N. 1985. Tannins and related compounds. xxix. Seven new methyl derivatives of flavan-3-ols and a 1,3-diarylpropan-2-ol from Cinnamomum cassia, C. obtusifolium and Lindera umbellata var. membranacea. Chem Pharm Bull. 33:2281–2286. doi:10.1248/cpb.33.2281. Mukhopadhyay MK, Nath D. 2011. Phytochemical screening and toxicity of Saraca asoca bark methanolic extract. Int J Phytomed. 3:498–505. Pradhan P, Joseph L, Gupta V, Chulet R, Arya H, Verma R, Bajpai A. 2009. Saraca asoca (Ashoka): A review. J Chem Pharma Res. 1:62– 71. Preethi MF, Krishnakumar K. 2011. Anti-inflammatory activity of the bark of Saraca indica Linn. Pharmacology. 2:657–662. Online. Sadhu SK, Khatun A, Phattanawasin P, Ohtsuki T, Ishibashi M. 2007. Lignan glycosides and flavonoids from Saraca asoca with antioxidant activity. J Nat Med. 61:480–482. doi:10.1007/s11418-007-0182-3. Saravanan S, Babu NP, Pandikumar P, Ignacimuthu S. 2011. Therapeutic effect of Saraca asoca (Roxb.) Wilde on lysosomal enzymes and collagen metabolism in adjuvant induced arthritis. Inflammopharmacology. 19:317–325. doi:10.1007/s10787-011-0091-7. Shi LS, Liao YR, Su MJ, Lee AS, Kuo PC, Damu AG, Kuo SC, Sun HD, Lee KH, Wu TS. 2010. Cardiac glycosides from Antiaris toxicaria with potent cardiotonic activity. J Nat Prod. 73:1214–1222. doi:10.1021/np9005212. Siddique AA, Joshi P, Misra LN, Sangwan NS, Darokar MP5. 2014. 5,6-de-epoxy-5-en-7-one-17-hydroxy withaferin A, a new cytotoxic steroid from Withania somnifera L. Dunal leaves. Nat Prod Res. 28:392–398. doi:10.1080/ 14786419.2013.871545. Su BN, Cuendet M, Hawthorne ME, Kardono LBS, Riswan S, Fong HHS, Mehta RG, Pezzuto JM, Kinghorn AD. 2002. Constituents of the bark and twigs of Artocarpus dadah with cyclooxygenase inhibitory activity. J Nat Prod. 65:163–169. doi:10.1021/np010451c. Vyry Wouatsa NA, Misra RLN, Venkatesh Kumar R, Darokar MP, Tchoumbougnang F. 2013. Zantholic acid, a new monoterpenoid from Zanthoxylum zanthoxyloides. Nat Prod Res. 27:1994 –1998. doi:10.1080/14786419.2013. 811662.
