- Email: [email protected]
Two new tetrahydroisoquinoline analogs from Indian medicinal plant Annona squamosa
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 5 1 0 e5 1 5
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Original Article
Two new tetrahydroisoquinoline analogs from Indian medicinal plant Annona squamosa Jayendra*, Y. Kumar, S. Sadish Kumar Chemistry Division, Department of Pharmacy, I.T.S Paramedical (Pharmacy) College, Muradnagar, Ghaziabad, Uttar Pradesh 201206, India
article info
abstract
Article history:
Objective: To investigate the bioactive principles obtained from chloroform extract of Indian
Received 25 May 2013
medicinal plant Annona squamosa.
Accepted 29 June 2013
Methods: The successively extracted twigs of A. squamosa using various solvents were
Available online 17 July 2013
analyzed for their antiulcer activity and finally two new compounds are isolated and characterized based on bioassay guided fractionation and isolation.
Keywords:
Results: Isolated compounds (compound no. 1 & 2) were examined for Hydrogen Potassium
Annona squamosa
ATPase activity. When compared to the standard drug omeprazole, both compounds are
Antisecretory activity
showing better activity.
Tetrahydroisoquinoline analogs
Conclusions: The results obtained justify the traditional usage of A. squamosa for their antisecretory activity. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Annona squamosa L. belongs to the family Annonaceae. It is a widely used Indian medicinal plant for the cure of deadly disease, diabetes.1 In recent decades, a great no. of chemical and pharmacological studies have been done on A. squamosa L.2 More than fifty compounds including cyclopeptides, Annonins, linear acetogenins, hydroxylated bistetrahydrofuran moieties, alkaloids, as well as other trace elements have been found in the plant.3e5 Studies show that A. squamosa L. and its active principals possess wide pharmacological actions including antidiabetic, antioxidative, antirheumatic, antilipidemic and insecticide.6e10 A fraction of total alkaloid from roots exhibits antihypertensive, antispasmodic, antihistaminic and bronchodilator properties. Leaves contain cardiotonic alkaloids, quinoline, squamone, and bullatacinone were selectively cytotoxic to human breast carcinoma.
Two new compounds have been isolated & are reported in this paper which are 5-((6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)methyl)-2-methoxybenzene-1,3-diol and (1R,3S )-6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoq uinoline-1,3-diol. These compounds are found to be antiulcer in nature. The isolated compounds were evaluated for their activity on Hydrogen Potassium ATPase enzyme and were compared with the omeprazole as the standard drug. Activity was found to be quite comparable.
2.
Methods
2.1.
Chemicals and reagents
All chemicals used were of analytical grade.
* Corresponding author. Tel.: þ91 7503712661. E-mail addresses: [email protected], [email protected] ( Jayendra). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.06.022
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j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 5 1 0 e5 1 5
Table 1 e Column chromatography chloroform fraction. Fraction 01e09 10e17 18e24 25e31 32e47 48e64 65e78 79e84 85e91 92e104
2.2.
Eluent (hexane:CHCl3:MeOH)
Weight (g)
100:00:00e85:15:00 75:25:00 50:50:00 25:75:00 00:100:0 00:95:05 00:85:15 00:75:25 00:70:30 00:60:40
12.00 11.20 7.30 9.50 6.50 13.5 9.00 4.40 8.50 7.50
Preparation of plant extract
Table 3 e 1H and 13C NMR (300 and 75 MHz) data of compound no. 2 in CDCl3. dH (J in Hz)
dC
3.54 (1H, m, J ¼ 6.8) e 3.55 (H, m, J ¼ 6.6) 2.94 (2H, d, J ¼ 6.3) 7.61 (1H, s) e e 6.64 (1H, s) e e 3.93 (3H, s) 3.92 (3H, s) 3.14 (3H, s)
56 e 56 27.7 110.7 151.8 148.1 109.4 122.2 131.7 56.3 56.2 35.3
Position dH 1 2 3 4 5 6 7 8 9 10 6-OCH3 7-OCH3 2-NCH3
Twigs of A. squamosa (6.0 Kg) were shade dried and finely powdered and placed for maceration with ethanol (18 L) and were kept at room temperature for 48 h. The macerated material was collected. This process of extraction was repeated for five times, till the plant material was extracted exhaustively. The total extract concentrated at 40e45 C and weighed. The extract weighed 520 g (8.66%). Ethanolic extract (500 g) was taken and triturated with n-hexane (250 ml 15), the hexane fraction concentrated under low pressure at 40 C. After trituration with hexane the residue was triturated with chloroform (250 ml 15), chloroform soluble fraction was evaporated under low pressure; weight of fraction obtained 95 g. After trituration with chloroform, residue was then kept in distilled water (2 L) and then it was fractionated with Aq. saturated n-butanol (500 ml 10). This fraction was concentrated low pressure at 50 C (15 g). Aqueous fraction also concentrated under low pressure at 45e50 C (20 g).
5
H3CO
9
4 3
6 7 H3CO
8
10
1
N
2 CH3
HO
H3CO OH
5-((6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-1yl)methyl)-2-methoxybenzene-1,3-diol Fig. 1 e Structure of compound no. 1.
Table 2 e 1H and 13C NMR (300 and 75 MHz) data of compound no. 1 in CDCl3. Position 1 2 3-a 3-a0 4-a 4-a0 5 6 7 8 9 10 C-a C-a0 C-10 C-20 ,60 C-30 ,50 C-40 6-OCH3 7-OCH3 40 -OCH3 2-NCH3
dH (J in Hz)
dC
3.68 (1H, dd, J ¼ 7.5, 5.1) e 3.24e3.12 (1H, m) 2.90e2.73 (1H, m) 2.64e2.59 (1H, m) 2.90e2.73 (1H, m) 6.57 (1H, s) e e 6.02 (1H, s) e e 3.24e3.12 (1H, m) 2.90e2.73 (3H, m) e 7.03 (2H, d, J ¼ 8.4) e e 3.85 (3H, s) 3.79 (3H, s) 3.57 (3H, s) 2.64e2.59 (3H, m)
65.1 e 46.9 e 25.5 e 111.3 147.4 146.4 111.3 125.9 129.3 40.4 e 132.1 130.9 118.9 158.1 55.9 55.6 55.4 42.7
3.
Results
Repeated column chromatography was done on chloroform fraction in order to isolate the two new compounds viz. 5((6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl) methyl)-2-methoxybenzene-1,3-diol and (1R,3S )-6,7-dimethoxy2-methyl-1,2,3,4-tetrahydroisoquinoline-1,3-diol. Melting point for compound no. 1 is 194e196 C, molecular formula is C20H25NO5, m/z obtained at 360.17. Compound no. 2 which is
5
H3CO
4
OH
9
6
3
7
2 10
H3CO
1
8
N CH3
OH
(1R,3S)-6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline-1,3-diol
Fig. 2 e Structure of compound no. 2.
512
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characterized as (1R,3S )-6,7-dimethoxy-2-methyl-1,2,3,4tetrahydroisoquinoline-1,3-diol has a melting point range of 124e126 C, molecular formula is C12H17NO4, m/z obtained at 240.13. The chloroform fraction (95.0 g) was chromatographed on silica gel (60e120 mesh, 900 g), using hexane with increasing amount of chloroform and methanol as eluent. Fractions of 500 ml each were taken and similar fractions were combined after monitoring TLC. First two fractions are oil containing, which have no resolved spot on TLC (Table 1). Repeated column chromatography of fraction (85e90) with (Hexane:CHCl3:MeOH: 00:70:30) yielded compound no. 1 & fraction (92e104) with (Hexane:CHCl3:MeOH: 00:60:40) yielded compound no. 2. 1H NMR & 13C NMR data for compound no. 1 is given in Table 2 and 1H NMR & 13C NMR data for compound no. 2 is provided in Table 3. Fig. 3 e HMBC correlations of compound no. 1.
Fig. 4 e HMBC correlations of compound no. 2.
4.
Discussion
4.1.
Structure elucidation of compound no. 1
Compound no. 1 (Fig. 1) was obtained as yellow crystalline compound, mp 194e196 C. It gave positive dragendorff test indicating its alkaloidal nature. It showed molecular ion peak at m/z ¼ 361.17 [M þ H]þ in ESI-MS mass spectrum corresponding to molecular formula C20H25NO5 which confirmed by 1H (Fig. 5), 13C (Fig. 6) and DEPT spectra. In 1H NMR spectrum (Table 2) a set of isolated protons of H-5 and H-8 as AX system were appeared at dH 6.57 (1H, s) and 6.02 (1H, s). A set of A2B2 protons appeared at dH 7.03 (d, J ¼ 8.4 Hz, 2H), due to H-20 ,60 and
Fig. 5 e 1H NMR of compound no. 1.
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 5 1 0 e5 1 5
Fig. 6 e
13
C NMR of compound no. 1.
Fig. 7 e 1H NMR of compound no. 2.
513
514
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 5 1 0 e5 1 5
6.83 (d, J ¼ 8.7 Hz, 2H, s). A doublet of doublet appeared at dH 3.68, due to H-1. One multiplet of two proton count appeared between the range at dH 3.24e3.12, due to H-a and H-3 and another multiplet of three proton count resonated at dH 2.90e2.73, were due to H-a0 H-3, H-4. Three singlets appeared at dH 3.85, 3.79, 3.57, were due to methoxy attached to aromatic ring. NeCH3 and one H-4 proton were merged and appeared as multiplet at dH 2.64e2.59 of four proton count. 13C NMR and Dept spectra (Table 2) indicated that 20 carbons of the molecule were present as four methyls, six methines, three methylenes, one aliphatic methine and six quaternary carbon atoms assignable to compound no. 1. Comparatively downfield shift of C-1 and C-3, at dC 65.1 and 46.9 in aliphatic region prove their vicinity to nitrogen atom. Position of three methoxy and a nitrogen attached methyl were assigned by HMBC spectrum analysis (Fig. 3).
4.2.
Structure elucidation of compound no. 2
Compound no. 2 (Fig. 2) was isolated as yellow crystalline compound, mp 124e126 C. It gave positive dragendorff test indicating its alkaloidal nature. It showed molecular ion peak at m/z 241.14 [M þ H]þ in ESI-MS mass spectrum corresponding to molecular formula C12H17NO4which confirmed by 1H (Fig. 7), 13C (Fig. 8) and DEPT spectra. In 1H NMR (Table 3) spectrum a set of isolated protons as AX system appeared at dH 7.61 (1H, s, H-5) and 6.64 (1H, s, H-8). A comparatively downfield triplet at 3.55 (2H, m, J ¼ 6.6 Hz), which indicated vicinity
Fig. 8 e
13
Table 4 e Effect of active compounds, isolated from Annona squamosa, compound no. 1 & compound no. 2, (10e100 mg/ml) each and control drug omeprazole (10e50 mg/ml) on Hydrogen Potassium ATPase. Treatment (mg/ml)
Compound no. 1 Compound no. 2 Omeprazole
Hyd. Pot. ATPase inhibition IC50 (mg/ml) 101.22 55.4 29.34
95% confidence of IC50 limit Lower limit
Upper limit
93.55 51.21 26.84
119.23 61.54 32.47
of nitrogen atom and another triplet appeared at 2.94 (2H, d, J ¼ 6.3 Hz), which was due to H-4 protons. Three signals each having three proton count at 3.93, 3.92, 3.14 denoted by two methoxy moieties and one nitrogen attached methyl. 13C NMR (Fig. 8) and Dept spectra (Table 3) indicated that 12 carbons of the molecule were present as three methyls, two methylenes, two methines and five quaternary carbon atoms assignable to compound no. 2. Position of to methoxy and one methyl were fixed by HMBC spectrum analysis (Fig. 4).
4.3.
Activity of compounds
Compound no. 1 (10e50 mg/ml) & compound no. 2 (10e50 mg/ ml) was able to inhibit the gastric Hydrogen Potassium ATPase activity in comparison to omeprazole with an IC50 value of
C NMR of compound no. 2.
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 5 1 0 e5 1 5
101.22 mg/ml & 55.4 mg/ml respectively. Positive control used during experiment was omeprazole (10e50 mg/ml) and it was able to reduce the enzyme activity with an IC50 value of 30.24 mg/ml (Table 4).
5.
Conclusion
A. squamosa is known for its different types of medicinal properties, but still a lot of work is required to establish its antiulcer activity. In our present work, we have tested antiulcer activity of ethanolic extract of A. squamosa whole plant and have established a better antiulcer activity. The results obtained are comparable to standard drug omeprazole. Isolated compounds (compound no. 1 & 2) were tested for Hydrogen Potassium ATPase activity & they are showing a very good antiulcer activity.
Conflicts of interests All authors have none to declare.
Acknowledgments The author gratefully acknowledges the expert guidance of Dr. Y. Kumar and Dr. S. Sadish Kumar for their valuable suggestions. Author also acknowledges the necessary platform & financial assistance for research provided by I.T.S Paramedical (Pharmacy) College, Muradnagar, Ghaziabad.
515
references
1. Andrade EHAA, Zoghbi MDB, Maia JGS, Fabricius H, Marx F. Chemical characterization of the fruit of Annona squamosa L. occurring in the Amazon. J Food Compos Anal. 2001;14:227e232. 2. Araya H, Sahai M, Singh S, et al. Squamocin-O(1) and squamocin-O(2), new adjacent bis-tetrahydrofuran acetogenins from the seeds of Annona squamosa. Phytochemistry. 2002;61:999e1004. 3. Bhakuni DS, Tewari S, Dhar MM. Aporphine alkaloids of Annona squamosa. Phytochemistry. 1972;11:1819e1822. 4. Baumik PK, Mukherjee B, Juneau JP, Bhacca NS, Mukherjee R. Alkaloids from leaves of Annona squamosa. Phytochemistry. 1979;18:1584e1586. 5. Fujimoto Y, Eguchi T, Kakinuma K, Ikekava N, Sahai M, Gupta Y. Squamocin, a new cytotoxic bis-tetrahydrofuran containing acetogenin from Annona squamosa. Chem Pharm Bull. 1988;36:4802e4806. 6. Hopp DC, Alali FQ, Gu ZM, Mclaughlin JL. Three new bioactive bis-adjacent THF-ring acetogenins from the bark of Annona squamosa. Bioorg Med Chem. 1998;6:569e575. 7. Janssen Richard HAM, Lousberg Robert JJC, Wijkens P, Kruk C, Theuns HG. Assignment of 1H and 13C NMR resonances of some isoquinoline alkaloids. Phytochemistry. 1989;28(10):2833e2839. 8. Kaleem M, Medha P, Ahmed QU, Asif M, Bano B. Beneficial effects of Annona squamosa extract in streptozotocin-induced diabetic rats. Singapore Med J. 2008;49:800e804. 9. Kotkar HM, Mendki PS, Sadan SVGS, Jha SR, Upasani SM, Maheshwari VL. Antimicrobial and pesticidal activity of partially purified flavonoids of Annona squamosa. Pest Manag Sci. 2002;58:33e37. 10. Lturriga-Vasquez P, Miquel R, Lvorra MD, Cassels BK. Simplified tetrandrine congeners as possible antihypertensive agents with a dual mechanism of action. J Nat Prod. 2003;66:954e957.
Available online at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/jopr
Original Article
Two new tetrahydroisoquinoline analogs from Indian medicinal plant Annona squamosa Jayendra*, Y. Kumar, S. Sadish Kumar Chemistry Division, Department of Pharmacy, I.T.S Paramedical (Pharmacy) College, Muradnagar, Ghaziabad, Uttar Pradesh 201206, India
article info
abstract
Article history:
Objective: To investigate the bioactive principles obtained from chloroform extract of Indian
Received 25 May 2013
medicinal plant Annona squamosa.
Accepted 29 June 2013
Methods: The successively extracted twigs of A. squamosa using various solvents were
Available online 17 July 2013
analyzed for their antiulcer activity and finally two new compounds are isolated and characterized based on bioassay guided fractionation and isolation.
Keywords:
Results: Isolated compounds (compound no. 1 & 2) were examined for Hydrogen Potassium
Annona squamosa
ATPase activity. When compared to the standard drug omeprazole, both compounds are
Antisecretory activity
showing better activity.
Tetrahydroisoquinoline analogs
Conclusions: The results obtained justify the traditional usage of A. squamosa for their antisecretory activity. Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
1.
Introduction
Annona squamosa L. belongs to the family Annonaceae. It is a widely used Indian medicinal plant for the cure of deadly disease, diabetes.1 In recent decades, a great no. of chemical and pharmacological studies have been done on A. squamosa L.2 More than fifty compounds including cyclopeptides, Annonins, linear acetogenins, hydroxylated bistetrahydrofuran moieties, alkaloids, as well as other trace elements have been found in the plant.3e5 Studies show that A. squamosa L. and its active principals possess wide pharmacological actions including antidiabetic, antioxidative, antirheumatic, antilipidemic and insecticide.6e10 A fraction of total alkaloid from roots exhibits antihypertensive, antispasmodic, antihistaminic and bronchodilator properties. Leaves contain cardiotonic alkaloids, quinoline, squamone, and bullatacinone were selectively cytotoxic to human breast carcinoma.
Two new compounds have been isolated & are reported in this paper which are 5-((6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl)methyl)-2-methoxybenzene-1,3-diol and (1R,3S )-6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoq uinoline-1,3-diol. These compounds are found to be antiulcer in nature. The isolated compounds were evaluated for their activity on Hydrogen Potassium ATPase enzyme and were compared with the omeprazole as the standard drug. Activity was found to be quite comparable.
2.
Methods
2.1.
Chemicals and reagents
All chemicals used were of analytical grade.
* Corresponding author. Tel.: þ91 7503712661. E-mail addresses: [email protected], [email protected] ( Jayendra). 0974-6943/$ e see front matter Copyright ª 2013, JPR Solutions; Published by Reed Elsevier India Pvt. Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jopr.2013.06.022
511
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 5 1 0 e5 1 5
Table 1 e Column chromatography chloroform fraction. Fraction 01e09 10e17 18e24 25e31 32e47 48e64 65e78 79e84 85e91 92e104
2.2.
Eluent (hexane:CHCl3:MeOH)
Weight (g)
100:00:00e85:15:00 75:25:00 50:50:00 25:75:00 00:100:0 00:95:05 00:85:15 00:75:25 00:70:30 00:60:40
12.00 11.20 7.30 9.50 6.50 13.5 9.00 4.40 8.50 7.50
Preparation of plant extract
Table 3 e 1H and 13C NMR (300 and 75 MHz) data of compound no. 2 in CDCl3. dH (J in Hz)
dC
3.54 (1H, m, J ¼ 6.8) e 3.55 (H, m, J ¼ 6.6) 2.94 (2H, d, J ¼ 6.3) 7.61 (1H, s) e e 6.64 (1H, s) e e 3.93 (3H, s) 3.92 (3H, s) 3.14 (3H, s)
56 e 56 27.7 110.7 151.8 148.1 109.4 122.2 131.7 56.3 56.2 35.3
Position dH 1 2 3 4 5 6 7 8 9 10 6-OCH3 7-OCH3 2-NCH3
Twigs of A. squamosa (6.0 Kg) were shade dried and finely powdered and placed for maceration with ethanol (18 L) and were kept at room temperature for 48 h. The macerated material was collected. This process of extraction was repeated for five times, till the plant material was extracted exhaustively. The total extract concentrated at 40e45 C and weighed. The extract weighed 520 g (8.66%). Ethanolic extract (500 g) was taken and triturated with n-hexane (250 ml 15), the hexane fraction concentrated under low pressure at 40 C. After trituration with hexane the residue was triturated with chloroform (250 ml 15), chloroform soluble fraction was evaporated under low pressure; weight of fraction obtained 95 g. After trituration with chloroform, residue was then kept in distilled water (2 L) and then it was fractionated with Aq. saturated n-butanol (500 ml 10). This fraction was concentrated low pressure at 50 C (15 g). Aqueous fraction also concentrated under low pressure at 45e50 C (20 g).
5
H3CO
9
4 3
6 7 H3CO
8
10
1
N
2 CH3
HO
H3CO OH
5-((6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-1yl)methyl)-2-methoxybenzene-1,3-diol Fig. 1 e Structure of compound no. 1.
Table 2 e 1H and 13C NMR (300 and 75 MHz) data of compound no. 1 in CDCl3. Position 1 2 3-a 3-a0 4-a 4-a0 5 6 7 8 9 10 C-a C-a0 C-10 C-20 ,60 C-30 ,50 C-40 6-OCH3 7-OCH3 40 -OCH3 2-NCH3
dH (J in Hz)
dC
3.68 (1H, dd, J ¼ 7.5, 5.1) e 3.24e3.12 (1H, m) 2.90e2.73 (1H, m) 2.64e2.59 (1H, m) 2.90e2.73 (1H, m) 6.57 (1H, s) e e 6.02 (1H, s) e e 3.24e3.12 (1H, m) 2.90e2.73 (3H, m) e 7.03 (2H, d, J ¼ 8.4) e e 3.85 (3H, s) 3.79 (3H, s) 3.57 (3H, s) 2.64e2.59 (3H, m)
65.1 e 46.9 e 25.5 e 111.3 147.4 146.4 111.3 125.9 129.3 40.4 e 132.1 130.9 118.9 158.1 55.9 55.6 55.4 42.7
3.
Results
Repeated column chromatography was done on chloroform fraction in order to isolate the two new compounds viz. 5((6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinolin-1-yl) methyl)-2-methoxybenzene-1,3-diol and (1R,3S )-6,7-dimethoxy2-methyl-1,2,3,4-tetrahydroisoquinoline-1,3-diol. Melting point for compound no. 1 is 194e196 C, molecular formula is C20H25NO5, m/z obtained at 360.17. Compound no. 2 which is
5
H3CO
4
OH
9
6
3
7
2 10
H3CO
1
8
N CH3
OH
(1R,3S)-6,7-dimethoxy-2-methyl-1,2,3,4-tetrahydroisoquinoline-1,3-diol
Fig. 2 e Structure of compound no. 2.
512
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 5 1 0 e5 1 5
characterized as (1R,3S )-6,7-dimethoxy-2-methyl-1,2,3,4tetrahydroisoquinoline-1,3-diol has a melting point range of 124e126 C, molecular formula is C12H17NO4, m/z obtained at 240.13. The chloroform fraction (95.0 g) was chromatographed on silica gel (60e120 mesh, 900 g), using hexane with increasing amount of chloroform and methanol as eluent. Fractions of 500 ml each were taken and similar fractions were combined after monitoring TLC. First two fractions are oil containing, which have no resolved spot on TLC (Table 1). Repeated column chromatography of fraction (85e90) with (Hexane:CHCl3:MeOH: 00:70:30) yielded compound no. 1 & fraction (92e104) with (Hexane:CHCl3:MeOH: 00:60:40) yielded compound no. 2. 1H NMR & 13C NMR data for compound no. 1 is given in Table 2 and 1H NMR & 13C NMR data for compound no. 2 is provided in Table 3. Fig. 3 e HMBC correlations of compound no. 1.
Fig. 4 e HMBC correlations of compound no. 2.
4.
Discussion
4.1.
Structure elucidation of compound no. 1
Compound no. 1 (Fig. 1) was obtained as yellow crystalline compound, mp 194e196 C. It gave positive dragendorff test indicating its alkaloidal nature. It showed molecular ion peak at m/z ¼ 361.17 [M þ H]þ in ESI-MS mass spectrum corresponding to molecular formula C20H25NO5 which confirmed by 1H (Fig. 5), 13C (Fig. 6) and DEPT spectra. In 1H NMR spectrum (Table 2) a set of isolated protons of H-5 and H-8 as AX system were appeared at dH 6.57 (1H, s) and 6.02 (1H, s). A set of A2B2 protons appeared at dH 7.03 (d, J ¼ 8.4 Hz, 2H), due to H-20 ,60 and
Fig. 5 e 1H NMR of compound no. 1.
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 5 1 0 e5 1 5
Fig. 6 e
13
C NMR of compound no. 1.
Fig. 7 e 1H NMR of compound no. 2.
513
514
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 5 1 0 e5 1 5
6.83 (d, J ¼ 8.7 Hz, 2H, s). A doublet of doublet appeared at dH 3.68, due to H-1. One multiplet of two proton count appeared between the range at dH 3.24e3.12, due to H-a and H-3 and another multiplet of three proton count resonated at dH 2.90e2.73, were due to H-a0 H-3, H-4. Three singlets appeared at dH 3.85, 3.79, 3.57, were due to methoxy attached to aromatic ring. NeCH3 and one H-4 proton were merged and appeared as multiplet at dH 2.64e2.59 of four proton count. 13C NMR and Dept spectra (Table 2) indicated that 20 carbons of the molecule were present as four methyls, six methines, three methylenes, one aliphatic methine and six quaternary carbon atoms assignable to compound no. 1. Comparatively downfield shift of C-1 and C-3, at dC 65.1 and 46.9 in aliphatic region prove their vicinity to nitrogen atom. Position of three methoxy and a nitrogen attached methyl were assigned by HMBC spectrum analysis (Fig. 3).
4.2.
Structure elucidation of compound no. 2
Compound no. 2 (Fig. 2) was isolated as yellow crystalline compound, mp 124e126 C. It gave positive dragendorff test indicating its alkaloidal nature. It showed molecular ion peak at m/z 241.14 [M þ H]þ in ESI-MS mass spectrum corresponding to molecular formula C12H17NO4which confirmed by 1H (Fig. 7), 13C (Fig. 8) and DEPT spectra. In 1H NMR (Table 3) spectrum a set of isolated protons as AX system appeared at dH 7.61 (1H, s, H-5) and 6.64 (1H, s, H-8). A comparatively downfield triplet at 3.55 (2H, m, J ¼ 6.6 Hz), which indicated vicinity
Fig. 8 e
13
Table 4 e Effect of active compounds, isolated from Annona squamosa, compound no. 1 & compound no. 2, (10e100 mg/ml) each and control drug omeprazole (10e50 mg/ml) on Hydrogen Potassium ATPase. Treatment (mg/ml)
Compound no. 1 Compound no. 2 Omeprazole
Hyd. Pot. ATPase inhibition IC50 (mg/ml) 101.22 55.4 29.34
95% confidence of IC50 limit Lower limit
Upper limit
93.55 51.21 26.84
119.23 61.54 32.47
of nitrogen atom and another triplet appeared at 2.94 (2H, d, J ¼ 6.3 Hz), which was due to H-4 protons. Three signals each having three proton count at 3.93, 3.92, 3.14 denoted by two methoxy moieties and one nitrogen attached methyl. 13C NMR (Fig. 8) and Dept spectra (Table 3) indicated that 12 carbons of the molecule were present as three methyls, two methylenes, two methines and five quaternary carbon atoms assignable to compound no. 2. Position of to methoxy and one methyl were fixed by HMBC spectrum analysis (Fig. 4).
4.3.
Activity of compounds
Compound no. 1 (10e50 mg/ml) & compound no. 2 (10e50 mg/ ml) was able to inhibit the gastric Hydrogen Potassium ATPase activity in comparison to omeprazole with an IC50 value of
C NMR of compound no. 2.
j o u r n a l o f p h a r m a c y r e s e a r c h 7 ( 2 0 1 3 ) 5 1 0 e5 1 5
101.22 mg/ml & 55.4 mg/ml respectively. Positive control used during experiment was omeprazole (10e50 mg/ml) and it was able to reduce the enzyme activity with an IC50 value of 30.24 mg/ml (Table 4).
5.
Conclusion
A. squamosa is known for its different types of medicinal properties, but still a lot of work is required to establish its antiulcer activity. In our present work, we have tested antiulcer activity of ethanolic extract of A. squamosa whole plant and have established a better antiulcer activity. The results obtained are comparable to standard drug omeprazole. Isolated compounds (compound no. 1 & 2) were tested for Hydrogen Potassium ATPase activity & they are showing a very good antiulcer activity.
Conflicts of interests All authors have none to declare.
Acknowledgments The author gratefully acknowledges the expert guidance of Dr. Y. Kumar and Dr. S. Sadish Kumar for their valuable suggestions. Author also acknowledges the necessary platform & financial assistance for research provided by I.T.S Paramedical (Pharmacy) College, Muradnagar, Ghaziabad.
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