New flavones with antidiabetic activity from Callistemon lanceolatus DC

Fitoterapia 83 (2012) 1623–1627

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New flavones with antidiabetic activity from Callistemon lanceolatus DC Syed Nazreen a, Gurpreet Kaur a, Mohammad Mahboob Alam a, Syed Shafi a, Hinna Hamid a, Mohammad Ali b, Mohammad Sarwar Alam a,⁎ a b

Department of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University) New Delhi-110062, India Department of Phytochemistry and Pharmacognosy, Faculty of Pharmacy, Jamia Hamdard (Hamdard University) New Delhi-110 062, India

a r t i c l e

i n f o

Article history: Accepted 2 June 2012 Accepted in revised form 11 September 2012 Available online 20 September 2012 Keywords: Callistemon lanceolatus DC Myrtaceae Flavones Streptozotocin Antidiabetic

a b s t r a c t Phytochemical investigation of the aerial parts of Callistemon lanceolatus DC (Myrtaceae) led to the isolation of two new flavones characterized as 5,7-dihydroxy-6,8-dimethyl- 4′ -methoxy flavone (1) and 8-(2-hydroxypropan-2-yl)-5-hydroxy-7-methoxy-6-methyl-4′-methoxy flavone (2) along with the seven known phytoconstituents. The structures of new compounds have been established on the basis of chemical and spectral studies and known compounds were compared with the published literature data. The isolated flavones exhibited blood glucose lowering effect in streptozotocin induced diabetic rats. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Medicinal plants are the main source of organic compounds such as polyphenols, tannins, alkaloids, carbohydrates, terpenoids, steroids and flavonoids [1,2]. Callistemon lanceolatus DC (Myrtaceae), commonly called red bottle brush, is an evergreen shrub, indigenous to Queensland, New South Wales and frequently grown throughout India in gardens [3]. The antimicrobial, DPPH scavenging and elastase inhibitory activity of the plant are well reported in the literature [4–7]. Previous phytochemical investigations of the plant have resulted in the isolation of flavonoids, triterpenoids, phloroglucinol derivatives, ellagic acid derivatives and tannins [8–11]. In our earlier studies we have reported the antidiabetic potential of the various fractions obtained from the ethanolic extract of this plant [12]. C. lanceolatus is found to be rich in polyphenols due to which its antidiabetic activity has been anticipated. Keeping in view the biological importance of this plant as an antioxidant and antidiabetic agent [13,14] a bio-activity ⁎ Corresponding author. Tel.: +91 9717927759; fax: +91 11 26059663. E-mail address: [email protected] (M.S. Alam). 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.fitote.2012.09.012

guided phytochemical investigation has been carried out to isolate the polyphenolic constituents from the plant. The present investigation of the aerial parts of the plant deals with the isolation and characterization of two new flavones along with the evaluation of antidiabetic activity.

2. Experimental 2.1. General Melting points were determined on Veego VMP-III and were uncorrected. UV spectra were measured on DV 20 Spectroscan. IR spectra were recorded on Bruker. 1H NMR, 13C NMR and 2D NMR were recorded on a Bruker AM-400 (300, 400 MHz) spectrometer with TMS as the internal standard and chemical shifts are reported in parts per million relative to methanol-d4 (3.30 ppm for 1H and 49.0 ppm for 13C) or CDCl3 (7.27 ppm for 1 H and 77.23 for 13C). Mass spectra were recorded on a Jeol JMS-D 300 instrument fitted with a JMS 2000 data system at 70 eV and is reported in m/z using Argon/Xenon as the FAB gas. All solvents were of analytical grade (Merck). Thin Layer Chromatography (TLC) was performed on precoated plates

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(Silica gel 60 F254, Merck) and Silica gel (60–120 mesh, Merck) was used for column chromatography.

Table 1 1 H and 13C NMR spectral data of compounds 1 and 2. 1

2.2. Plant material The leaves of C. lanceolatus DC were collected from Saket Nursery, New Delhi in March 2010 and authenticated by Dr. H. B. Singh, Taxonomist, National Institute of Science Communication and Information resources (NISCAIR), New Delhi. A voucher specimen (No.1386/188) has been deposited in the author's laboratory. 2.3. Extraction and isolation The air dried and powdered leaves of C. lanceolatus DC were extracted with 95% ethanol in a Soxhlet apparatus. The ethanolic extract was concentrated under reduced pressure to yield a brown viscous mass (550 g). The ethanolic extract was fractionated with petroleum ether (3 × 1.0 L), CHCl3 (3 × 1.0 L), and MeOH (3 × 1.0 L) to furnish petroleum ether fraction (200 g), CHCl3 fraction (150 g) and MeOH fraction (200 g). The CHCl3 fraction was chromatographed over silica gel (60–120 mesh, 1000 g) and eluted with petroleum ether– CHCl3 gradient system from 100:0 to 0:100 to give 4 fractions (Fr. 1–4). Fr.1 (50 g) obtained with petroleum ether–CHCl3 (3:7) was subjected to repeated silica gel column chromatography eluted with n-hexane:EtOAc to give 2 sub fractions (Fr 1a and 1b). Fr. 1a was purified by crystallization with MeOH–acetone to yield pure compound 3 (20 mg). Fr.1b was further column chromatographed and purified by crystallization from DCM:MeOH to yield pure compound 4 (75 mg). Fr. 2 (18 g) obtained with petroleum ether–CHCl3 (2:8) was subjected to column chromatography over silica gel (60–120 mesh) to give 2 sub fractions (Fr. 2a, 2b). Fr. 2a was purified by crystallization with DCM–MeOH to afford compound 5 (15 mg). The supernatant after repeated column chromatography afforded compound 6 (25 mg). Fr. 2b was subjected to CC followed by crystallization with DCM–MeOH afforded compound 1 (95 mg). Fr. 3 (6 g) obtained from petroleum ether–CHCl3 (1:9) was purified by crystallization in MeOH– acetone to give compound 2 (85 mg). Fr.4 (30 g) obtained from CHCl3 (100%) was subjected to repeated CC over silica gel (60–120 mesh) with n-hexane:EtOAc to yield 2 sub fractions (4a, 4b). Fr. 4a (14 g) on further column chromatography followed by purification by crystallization from MeOH yielded pure compounds 7 (102 mg) and 8 (16 mg). Fr.4b (10 g) was purified by crystallization in MeOH-acetone to give compound 9 (10 mg). 2.4. Compound 1 Yellow crystals; m.p. 225–226 °C; UV (MeOH) λmax: 267, 319 nm; IR (KBr) υmax (cm−1): 3510 (OH), 1645 (C_O), 1035 (C\O); 1H and 13C NMR (CDCl3): see Table 1; FAB MS (positive): m/z 313 [M+H]+ (calcd for C18H16O5). 2.5. Compound 2 Yellow crystals; m.p. 176–177 °C; UV (MeOH) λmax: 278, 323 nm; IR (KBr) υmax (cm−1): 3510 (OH), 1703 (C_O), 1012

2

δH, J (Hz)

δC CDCl3

δH, J (Hz)

δC, CDCl3

2 3 4 5

– 6.59 (1H, s) – 13.05 (1H, s, \OH)

161.88 102.88 181.93 159.56

162.33 103.01 182.21 161.55

6 7 8 9 10 1′ 2′&6′

– 10.39 (1H, s, \OH) – – – – 7.87 (2H, d, J = 8.8 Hz) 7.03 (2H, d, J = 8.8 Hz) – 2.36 (3H, s) 2.18 (3H, s,) – – – 3.90 (3H, s)

101.54 162.61 109.53 156.03 103.57 123.21 127.56, 127.81 114.14, 114.56 152.31 8.06 7.71

– 6.53(1H, s) – 12.81 (1H, brs, \OH) – – – – – – 7.78 (2H, d, J=9.3 Hz) 6.95 (2H, d, J = 7.72 Hz) – 2.31 (3H, s) – 5.48 (1H, s, −OH) 2.13 (3H, s) 2.03 (3H, s) 3.85 (3H, s) 3.82 (3H, s)

3′&5′ 4′ 1′′ 2′′ 1′′′ 2′′′ 3′′′ OMe

– – 55.10

107.99 162.81 107.76 157.42 104.27 122.81 126.94, 126.90 113.52, 113.41 154.89 7.54 – 88.29 7.24 6.25 54.49, 54.86

(C\O); 1H and 13C NMR (CDCl3): see Table 1; FAB MS (positive): m/z 370 [M+H]+ (calcd for C21H22O6). 2.6. Antidiabetic activity The antidiabetic activity was performed in streptozotocin induced diabetic rats as per previously reported method [12]. 2.6.1. Experimental protocol The rats were divided into six groups comprising of five animals each: Group I: Control rats receiving 0.1 M citrate buffer (pH 4.5) Group II: Diabetic controls receiving STZ (60 mg/kg b.w) Group III: Diabetic rats given compound 1 (36 mg/kg b.w) in aqueous solution orally. Group IV: Diabetic rats given compound 2 (36 mg/kg b.w) in aqueous solution orally. Group V: Diabetic rats given glibenclamide (5 mg/kg b.w) in aqueous solution orally. The blood glucose level was checked at 0, 1, 7, and 15 days using Accu-chek glucometer. At the end of the experimental period, the rats were anesthetized and sacrificed by cervical dislocation. Organs (pancreas and liver) were removed for histopathological evaluation. 2.6.2. Statistical analysis Data was analyzed by one way ANOVA followed by Dunnett's ‘t’ test (n=5), ⁎Pb 0.05, ⁎⁎Pb 0.01 significant from diabetic control; ⁎⁎⁎Pb 0.001 extremely significant from diabetic control; ns— not significant.

S. Nazreen et al. / Fitoterapia 83 (2012) 1623–1627

H

H

H H

H

H

1625

H

O

C

C

C

H H

H O

HO

O CH3

H

O

H3 C

H

H

H

OH

O H

H

H

H C

H

H

H

OH

H H

C

H

H

O

HO

O

(1)

(2)

Cosy-

HMBC-

HSQC-

Fig. 1. COSY, HSQC and HMBC correlations of 1 and 2.

3. Results and discussion

at 267 and 319 nm were typically of substituted flavones [15]. The FAB MS displayed a molecular ion peak at m/z 313 [M+ H]+ consistent with molecular formula C18H16O5 of a flavone that was supported by its IR and NMR data. The IR spectrum revealed absorption bands at 3510 (\OH), 1645 (C_O) and 1035 (C\O) cm−1 respectively. The 1H NMR

3.1. Structure elucidation Compound 1 was isolated as yellow crystals and gave a positive Shinoda test for flavonoids. Its UV absorption maxima 3'

2"

2'

CH3 HO

8

7

O

2

9 6

H3C

1"

OH

H3C

CH3

4'

2'''

3'

CH3

H 3C

O

4'

8

7

O

2

1'

6

3

H 3C 1"

O

CH3

5'

9

4

O

2'

1'"

5'

1'

OH

6'

10 5

3'''

O

6'

10

3

5

4

OH

HO

O

1

3

2

OH

HO O O OH H H OH H OH

H O O

HO

6

O

4

5

H3 C

OH

CH3

HO O

CH3

CH3

O

COOH

OH OH HO

7

CH 3 HO H3 C

CH3

8 Fig. 2. Structure of isolated compounds from C. lanceolatus DC.

OH

O

9

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δC 101.54, 162.61, 159.56 (C-6, 7, 5); δH 2.18 (H-2″) to δC 109.53, 162.61, 156.03 (C-8, 7, 9) and δH 3.90 (4′-OMe)/δC 152.31(C-4′). The 1H–1H COSY spectrum of 1 showed interactions of H-2′ with H-3′ and H-6′; H-3′ with H-2′ and H-5′. The HSQC spectrum of 1 showed correlations of H-3 (δH 6.59) with C-3 (δC 102.88); H3-1″ (δH 2.36) with C-1″ (δC 8.06); H3-2″ (δH 2.18) with C-2″ (δC 7.71); and ring B protons with the respective carbons (Fig. 1). On the basis of these evidences, the structure of 1 has been elucidated as 6,8-dimethyl-5,7-dihydroxy 4′-methoxy flavone. Compound (2) obtained as yellow crystals gave positive Shinoda test for flavonoids. Its UV absorption maxima at 278 and 323 nm were typically of a flavone [15]. The FAB MS displayed a molecular ion peak at m/z 370 [M+ H]+ consistent with molecular formula C21H22O6 of a flavone that was supported by its IR and NMR data. The IR spectrum revealed absorption bands at 3510 (\OH), 1703 (C_O) and 1012 (C\O) cm−1 respectively. The 1H NMR spectrum showed the presence of four aromatic protons (ring B) forming an AA′XX′ system at δH 7.78 (2H, d, J = 9.3 Hz, H-2′, H-6′) and δH 6.95 (2H, d, J = 7.72 Hz, H-3′, H-5′). The long-range 1H– 13C correlation of δH 6.53 (H-3) to δC 162.33, 122.81, 182.21, 104.27 (C-2, 1′, 4, 10) is indicative of an aromatic proton at C-3. There were no additional signals in the aromatic region indicating that A ring of flavone was substituted. It also showed the signal for two methoxyl groups (δH 3.85, s) and (δH 3.82, s) which was confirmed by 13C NMR signal at δC 54.49 and δC 54.86. The attachment of two methoxy groups at C-7 and C-4′ was evident from the long range correlations (HMBC) observed between δH 3.85 (7-OMe)/δC 162.81, 107.99, 107.76 (C-7, 6, 8) and δH 3.82 (4′-OMe)/δC 154.89 (C-4′). The presence of a hydroxyl group at C-5 and methyl group at C-6 was based on the long range 1H– 13C correlations between δH 12.81 (5-OH)/δC 161.55, 107.99, 104.27 (C-5, 6, 10) and δH 2.31 (H-1″) to δC 107.99, 162.81, 161.55 (C-6, 7, 5). The HMBC correlations of δH 2.13, 2.03

Fig. 3. Antidiabetic activity of new flavones (1 and 2) in streptozotocin induced diabetic rats. Data is analyzed by one way ANOVA followed by Dunnett's ‘t’ test and expressed as mean±SEM from five observations; ⁎⁎⁎ indicates Pb 0.001, ⁎⁎ indicates Pb 0.01 and ⁎ indicates Pb 0.05 vs diabetic control.

spectrum showed the presence of four aromatic protons (ring B) forming an AA′XX′ system at δH 7.87 (2H, d, J = 8.8 Hz, H-2′, H-6′) and δH 7.03 (2H, d, J =8.8 Hz, H-3′, H-5′). Three singlets observed at δH 13.05 (s), δH 10.39 (s) and δH 6.59 (s) were assigned to 5-OH, 7-OH and H-3 on the basis of their long range correlations between δH 13.05 (5-OH)/δC 159.56, 101.54, 103.57 (C-5, 6, 10); δH 10.39 (7-OH)/δC 162.61, 101.54, 109.53 (C-7, 6, 8) and δH 6.59 (H-3) to δC 161.88, 123.21, 181.93, 103.57 (C-2, 1′, 4, 10) which indicated a 5,7 dihydroxylated pattern for ring A of flavone skeleton. The appearance of three singlets at δH 2.36, 2.18 and 3.90 in 1H NMR and δC 8.06, 7.71 and 55.10 in the 13C NMR indicated the presence of two methyl and one methoxy groups respectively. The assignment of two methyl groups at C-6 and C-8 and methoxy group at C-4′ was based on their long range correlations (HMBC) observed between δH 2.36 (H-1″) to

(A)

(B) Fig. 4. Histopathological changes in pancreas (A) and liver (B) of diabetic rats after treatment with compounds 1, 2 and glibenclamide. (A)— Histopathology report of rat pancreas. STZ treated groups showing reduce islet of Langerhans. Compound 1, 2 and glibenclamide treated groups showing recovery of islet of Langerhans. (B)— Histopathology report of rat liver showing normal arrangement of hepatocytes in the centrizonal area (Control). Compound 1, 2 and glibenclamide treated groups showing normal arrangement of cells in the liver lobule and normal arrangement of hepatocytes in the centrizonal area. Strepto (STZ) treated groups showing perivenular inflammatory infiltration filling over the sinusoidal vacoulation of the hepatocyte nuclei.

S. Nazreen et al. / Fitoterapia 83 (2012) 1623–1627

(H-2‴, H-3‴) to 88.29, 107.76 (C-1‴, C-8); δH 5.48 (1‴-OH) to 88.29, 107.76, 7.24, 6.59 (C-1‴, C-8, C-2‴, C-3‴) indicated the attachment of side chain at C-8 (Fig. 1). The 1H–1H COSY spectrum of 2 showed correlations of H-2′ with H-3′ and H-6′; and H-3′ with H-2′ and H-5′. The HSQC spectrum of 2 exhibited that H-3 (δH 6.53) interacted with C-3 (δC 103.01); H3-1″ (δH 2.31) with C-1″ (δC 7.54), H3-2‴(δH 2.13) with C-2‴(δC 7.24) and H3- 3‴(δH 2.03) with C-3‴(δC 6.25) (Fig. 1). On the basis of the above data, the structure of 2 was elucidated as 5-hydroxy-8-(2-hydroxypropan-2-yl)-7-methoxy-6-methyl-4′methoxy flavone. The structures of known compounds were identified by comparing their spectroscopic data with literature values. They were identified as α-amyrin (3) [16], β-sitosterol (4) [17], 3-epiursolic acid acetate (5) [18], urs-12-en-3β-ol-β-Dglucopyranoside (6) [19], betulinic acid (7) [20], oleanolic acid (8) [21] and kaempferol (9) [22] (Fig. 2). 3.2. Antidiabetic activity Compounds 1 and 2 were tested against streptozotocin induced diabetic rats. It was found that compounds 1 and 2 showed significant blood glucose lowering effect compared to standard glibenclamide (Fig. 3). The compounds have exerted the blood glucose lowering by the regeneration of pancreatic islets and probably increase insulin release in streptozotocininduced diabetic rats [23,24]. The bioavailabilty of flavonoids have been reported in the literature [25,26]. However there are reports that C-alkylated flavonoids, which are relatively rare class of compounds, is rapidly absorbed after oral administration. This is in sharp contrast to the generally poor oral bioavailability of flavonoids [27,28]. The histopathological examination revealed extensive alterations in pancreas and liver of STZ-induced diabetic rats. STZ was found to induce a significant damage to islets of langerhans of pancreas. Diabetic rats showed markedly reduced islet cells, which were restored to near normal upon treatment with compounds 1, 2 and glibenclamide (Fig. 4). The liver of diabetic rat showed perivenular inflammatory infiltration filling over the sinusoidal vacoulation of the hepatocyte nuclei. The pathological changes observed in STZ-induced diabetes appeared to get close to normal after treatment with compounds 1, 2 and glibenclamide. The antidiabetic activities of compounds 1 and 2 isolated from C. lanceolatus support its use in the treatment of diabetes in folk medicine and thus this plant could be a potential candidate for the development of novel antidiabetic agents. Acknowledgments The authors thank Dr G N Qazi, Vice Chancellor, Jamia Hamdard for providing necessary research facilities to the department and University Grants Commission for providing the financial assistance. The author also thanks Central Drug Research Institute (CDRI), India for recording the 1H NMR and mass spectra of the compounds. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.fitote.2012.09.012.

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