Natural Product Research Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
New chemical constituents from the Piper betle Linn. (Piperaceae) Akhtar Atiya, Barij Nayan Sinha & Uma Ranjan Lal To cite this article: Akhtar Atiya, Barij Nayan Sinha & Uma Ranjan Lal (2017): New chemical constituents from the Piper betle Linn. (Piperaceae), Natural Product Research, DOI: 10.1080/14786419.2017.1380018 To link to this article: http://dx.doi.org/10.1080/14786419.2017.1380018
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Published online: 05 Oct 2017.
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Date: 05 October 2017, At: 23:44
Natural Product Research, 2017 https://doi.org/10.1080/14786419.2017.1380018
New chemical constituents from the Piper betle Linn. (Piperaceae) Akhtar Atiyaa, Barij Nayan Sinhab and Uma Ranjan Lala a
ABSTRACT
The phytochemical investigation of chloroform extract from Piper betle var. haldia, Piperaceae, leaves has resulted in the isolation of two new chemical constituents which were identified as 1-n-dodecanyloxy resorcinol (H1) and desmethylenesqualenyl deoxy-cepharadione-A (H4), on the basis of spectroscopic data 1D NMR (1H and 13C) and 2D NMR (1H-1H COSY and HMBC) as well as ESI-MS, FT-IR and HR-ESI-MS analyses. Compounds H1 and H4 showed excellent antioxidant DPPH free radical scavenging activity with IC50 values of 7.14 μg/mL and 8.08 μg/mL compared to ascorbic acid as a standard antioxidant drug with IC50 value of 2.52 μg/mL, respectively. Evaluation of cytotoxic activity against human hepatoma cell line (PLC-PRF-5) showed moderate effect with the GI50 values of 35.12 μg/mL for H1, 31.01 μg/ mL for H4, compared to Doxorubicin® as a standard cytotoxic drug with GI50 value of 18.80 μg/mL.
ARTICLE HISTORY
Received 14 June 2017 Accepted 5 September 2017 KEYWORDS
Piperaceae; Piper betle; chemical constituents; antioxidant; cytotoxic; SAR
OH
O
1-n-dodecanyloxy resorcinol (H1) H
H
H
H
H
CH 2
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Division of Pharmacognosy and Phytochemistry, Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra, India; bDivision of Medicinal Chemistry, Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra, Ranchi, India
O
Piper betle var. haldia leaves (dorsal and ventral view)
O HN
O
desmethylenesqualenyl deoxy-cepharadione-A (H4)
Antioxidant and cytotoxic activity against human hepatoma cell line (PLC-PRF-5)
1. Introduction Piperaceae, a large tropical family, consists of over 10 genera (Sambamurty 2010) and contain more than 1500 species (Alves et al. 2017). Regarding the species of Piperaceae, those from CONTACT Uma Ranjan Lal [email protected], [email protected]; Akhtar Atiya [email protected], atiya_ [email protected] Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2017.1380018. © 2017 Informa UK Limited, trading as Taylor & Francis Group
Downloaded by [University of Connecticut] at 23:44 05 October 2017
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A. ATIYA ET AL.
the genus Piper comprises approximately 700 species occur in the American tropics. Another 300 species are found in the Asian tropics, 15 species occur in Africa and up to 40 species are distributed on various island in the tropical Pacific (Kricher 2017). In India the genus Piper contains around 110 species (Parthasarathy et al. 2006). Economically, this genus is the most important of the family Piperaceae due to its edible purposes and medicinal importance (Torres-Pelayo et al. 2016; Alves et al. 2017). Piper betle Linn. (Piperaceae) is an important and popular medicinal plant, widely cultivated in the tropical and subtropical regions in south and south-east Asia (Dammini Premachandra et al. 2014). Its fresh leaves are chewed either alone as breath-freshener (Singh et al. 2009) or in form of ‘betle quid’ (Atiya et al. 2017). It used in the Indian traditional system of medicine (Guha 2006). Their extracts possess several bioactivities (Majumdar et al. 2003; Arambewela et al. 2005; Alam et al. 2013) etc. Previous phytochemical analyses revealed that the leaves of P. betle is very rich source of phenolic compounds, comprising a phenylpropanoid (Evans et al. 1984; Rathee et al. 2006; Ali et al. 2010; Ferreres et al. 2014; Musa et al. 2014). Due to expansion of P. betle varieties cultivation and its increasing consumption in the main India markets, it is necessary to explore the chemical constituents of this plant species (betle). Therefore, this research work aims at exploration of the phytochemical constituents of the chloroform extract of P. betle L. var. haldia leaves afforded one new phenolic analogue: 1-n-dodecanyloxy resorcinol (H1) and a new oxo-aporphine alkaloid analogue: desmethylenesqualenyl deoxy-cepharadione-A (H4). The phytochemical characterisations were done by spectrometric methods using 1D NMR (1H and 13C) and 2D NMR (1H-1H COSY and HMBC), as well as ESI-MS, FT-IR and HR-ESI-MS. The chloroform extract of P. betle L. var. haldia leaves and its isolated compounds have been investigated for their antioxidant DPPH radical scavenging activity and cytotoxic activity against human hepatoma cell line (PLC-PRF-5) using SRB assay. Hepatocellular carcinoma (HCC) is the third leading cause of death and fifth most commonly occurring cancer worldwide (de Oliveria Andrade et al. 2009). It is the most common cancer among men (Poustchi et al. 2010). An increased occurrence of liver cancer in the world is reported in Asia and Africa. Infected with hepatitis B virus (HBV), hepatitis C virus (HCV) and many more are known to cause liver cancer (de Oliveria Andrade et al. 2009; McGlynn and London 2011). There are different treatment options available for HCC (Raza and Sood 2014). Distinctive regimen of chemotherapy has been tried with poor response. However, surgical treatment is the better option in present-day for the treatment of HCC, but not all patients are eligible for it, either due to minimal efficacy or contraindication, this reiterates the need for the new treatment option. Several studies aimed at discovering more molecules for the HCC management and treatment. Whereas a number of chemical constituents are found in natural and dietary products and they had sufficient studies that discussed their roles in HCC (Alnajjar and Elsiesy 2015). Although the benefit of P. betle leaves alone was spread out in present or ancient times, but there has not been vigorous proof of incidence of any side effects. Different kind of experiments were supervised on P. betle leaves extract and recommend that leaves extract did not grow any tumors and no perilous effect when consumed alone. In addition, the extract of P. betle leaves even showed the auspicious effect in terms of reducing the rate magnification of tumor in animal models (Atiya et al. 2017). In recent studies, it revealed that P. betle leaves extract induced a dose- (0.1 to 1 mg/mL) and time-dependent increase of cell
NATURAL PRODUCT RESEARCH
3
toxicity, supporting their application as a novel chemopreventive agent for the treatment of HCC (Wu et al. 2014). Although more studies are needed on the utilisation of P. betle for the treatment of HCC. In this experimental study, the cytotoxic effects against human hepatoma cell line (PLC-PRF-5) showed quite encouraging.
Downloaded by [University of Connecticut] at 23:44 05 October 2017
2. Result and discussion The defatted chloroform extract, which was of good amount, was selected for phytochemical investigation. Classical colorimetric methods were first employed to evaluate the total amounts of phenols and antioxidant activity in the chloroform extract of P. betle var. haldia leaves. The total phenolic content, determined following a Folin-Ciocalteu procedure and calculated as gallic acid equivalents was found to be 153.57 ± 0.39 mg/g of dried leaves (Figure S17). The antioxidant activity was performed by 1, 1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging method, the IC50 values of 11.18 μg/mL for extract and 2.52 μg/ mL for ascorbic acid as standard antioxidant compound (Table S1) (Atiya 2017). Further, the chloroform extract was subjected to thin layer chromatography (TLC) as per conventional one-dimensional ascending method for qualitative and quantitative analysis with solvent system (chloroform: methanol, 9:1). The two major spots were then detected using iodine vapours and a spots were visualised as dark brown in colour (Figure 1). The movement of the active chemical constituents was demonstrated by its Rf (retention factor) values. The Rf values were found to be 0.76 and 0.13, respectively. Furthermore, the chloroform extract was then subjected to silica gel column chromatography afforded two new chemical compounds (H1 and H4). The isolated compounds were then tested for their antioxidant DPPH radical scavenging activity. The IC50 values for compound H1 was 7.14 and 8.08 μg/mL for H4, respectively (Table S1).
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H4
Figure 1. Chemical structures and TLC profile of isolated compounds, H1 and H4 (chloroform: methanol, 9:1) was visualised with the help of iodine vapours.
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2.1. Isolation and identification of phytochemicals 2.1.1. 1-n-dodecanyloxy resorcinol (H1) The compound H1 was elution of the column with chloroform: methanol (99:1) (fraction 9–15) afforded brownish yellow solid mass of 32.20 mg; ~Rf: 0.76 (chloroform: methanol, 9:1); melting point 80–85 °C. The IR Vmax (KBr): 2928, 2850 (sp3 C-H stretching), 1722 (overtone interaction), 1684 (C=C aromatic), 1459 (C-H scissoring or H–C–H bending), 1364 (C-H methyl rock), 1275, 1187 (C–O or O–C stretching), 1080 (C–OH stretching), 966 (OH bending or CH2 in-plane rock or C–O stretching) and 743 (CH2 rocking) cm−1, respectively. On the basis of ESI-MS spectrum, the molecular weight of H1 has been determined at m/z 279.2 [M + H]+ consistent with molecular formula of 1-n-dodecanyloxy resorcinol, C18H30O2. The molecular formula of compound H1 was also concluded by the HR-ESI-MS gave an [M]+ peak at m/z 278.2254 (calculated for C18H30O2=278.2246) supporting the molecular formula of C18H30O2 and the identity of the sample as compound H1. The 1H NMR spectrum of compound H1, exhibited four one-proton multiplet at δH 7.72 (H-2), 7.56 (H-4), 7.31 (H-6) and 7.01 (H-5), assigned as 1, 3-disubstituted aromatic protons. A two-proton triplet at δH 4.31 (J = 7.3 Hz) was ascribed to oxygenated methylene H2-1ʹ protons. In addition, one three-proton multiplet at δH 0.86 was associated with C-12ʹ methyl protons attached to the methylene C-11ʹ carbon. The remaining methylene protons resonated from δH 1.78–1.25. The basic proton peaks as observed in 1H NMR spectrum of compound H1 are illustrated in Figure S15. The 13C NMR spectrum of H1 showed signals for carbons at δC 130.93 (C-2), 124.45 (C-4), 119.06 (C-5) and 128.83 (C-6), one oxygenated aromatic carbon at δC 167.01 (C-1) and a hydroxyl-substituted carbon at δC 147.06 (C-3) at aromatic region. Another important signals were observed for oxygenated methylene carbon at δC 65.58 (C-1ʹ) and methyl carbon at δC 13.73 (C-12ʹ), respectively. The remaining methylene carbons resonated from δC 38.69 to 14.13. The 1H-1H COSY spectrum of H1 showed correlations of H-4 with H-5 and H-5 with H-6; H2-1′ with H2-2′ and H3-12′ with H2-11′. The HMBC spectrum of H1 exhibited interactions of C-3 with H-5; C-2 with H-4; C-4 with H-6; C-1 with H2-1ʹ; C-1ʹ with H2-3ʹ; C-10ʹ with H3-12ʹ and H2-8ʹ; C-8ʹ with H2-10ʹ; C-6ʹ with H2-8ʹ; C-4ʹ with H2-6ʹ; C-5ʹ with H2-3ʹ (Figure S16). On the basis of above spectral data analysis and literature data (Atiya 2017), the structure H1 has been established as 1-n-dodecanyloxy resorcinol (Figure 1). 2.1.2. Desmethylenesqualenyl deoxy-cepharadione-A (H4) The compound H4, named desmethylenesqualenyl deoxy-cepharadione-A, was elution of the column with chloroform: methanol (99:1) (fraction 55–64) yielding a brownish solid mass, 82.10 mg with a melting point of ˃350°C; ~Rf: 0.13 (chloroform: methanol, 9:1). The IR Vmax (KBr): 2926, 2853 (sp3 C-H stretching), 1730 (C=O stretching), 1642 (C=C stretchings) 1465 (H–C–H bending or H–C–H (oxo) wagging), 1378 (C-H methyl rock or CH3 bending), 1287 (H–C–H (oxo) twisting or C–O stretching), 1122 (C–N stretching), 1074, 966 (C-H bending) and 743 (N-H wag primary amine) cm−1, respectively. On the basis of ESI-MS spectrum, the molecular formula of H4 has been determined at m/z 695 [M]+ consistent with molecular formula of desmethylenesqualenyl deoxy-cepharadione-A, C47H69O3 N. The molecular formula of compound H4 was also concluded by the HR-ESI-MS gave an [M]+ peak at m/z 695.5281 (calculated for C47H69O3 N = 695.5277) supporting the molecular formula of C47H69O3 N and the identity of the sample as compound H4. The fragmentation process of the molecule H4 was given in Figure S11. The 1H NMR spectrum of H4 exhibited a
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5
two-proton doublet at δH 3.26 (J = 6.5 Hz) was ascribed to oxygenated methylene H2-27′ protons. Two one-proton multiplets at δH 5.02 and 5.08 were accounted to vinylic H-12′ and H-13′ protons. Seven three-proton doublets at 0.98 (J = 6.1 Hz), 0.95 (J = 6.0 Hz), 0.93 (J = 6.2 Hz), 0.91 (J = 6.3 Hz), 0.88 (J = 5.8 Hz), 0.85 (J = 6.1 Hz) and 0.83 (J = 6.3 Hz) were accounted to secondary C-1′, C-24′, C-25′, C-26′, C-28′, C-23 and C-29′ methyl protons, respectively, all are attached to saturated carbons. Further, a one-proton multiplet at δH 2.01 was ascribed to methine H-14′ proton adjacent to the oxygenated methylene protons (H2-27′). The remaining methine and methylene protons resonated from δH 1.22 to 2.06 and 1.22 to 1.18 (H-2′ to H2-11′ and H2-15′ to H-22′). In addition, the presence of an oxo-methylene protons at δH 4.28 (J = 5.5, 5.5 Hz, H2-1) which was assigned to C-8 position, whereas one-proton doublet at δH 4.09 (J = 5.5 Hz) assigned to anomeric H-2 proton of the cepharadione-A moiety. Further, two one-proton singlet at δH 6.70 (H-4) and 6.78 (H-14) were observed together with five one-proton multiplet at δH 7.90 (H-12), 7.75 (H-10), 7.58 (H-11), 7.32 (H-13) and 2.31 (H-18) in the aromatic region of cepharadione-A moiety. The basic proton peaks as observed in 1H NMR spectrum of compounds H4 are illustrated in Figure S15. The 13C NMR spectrum of H4 showed signals for 29 carbons of desmethylenesqualenyl unit and presence of 18 carbons of cepharadione-A unit. The important signals appeared for the oxygenated methylene carbon at δC 65.55 (C-27′), anomeric carbon at δC 72.40 (C-2), vinylic carbons at δC 115.59 (C-12′) and 115.23 (C-13′) and methyl carbons at δC 13.67 (C-1′), 14.06 (C-23′), 13.90 (C-24′), 22.63 (C-25′), 19.69 (C-26′), 19.12 (C-28′), 19.10 (C-29′). The remaining methylene and methine carbons resonated from δC 31.37 to 30.14 and 27.67 to 30.51. In addition, the ketone (C=O) group at C-17 resonated at δC 167.72. The 1H-1H COSY spectrum of H4 showed correlations of H-10 with H-11; H-12′ with H-13′; H3-1′ with H-2′. The HMBC spectrum of H4 exhibited interactions of H-10 with C-8, C-9; H-15 with C-17; H-2 with C-27′; H-1 with C-27′; H-13 with C-15; H2-27′ with C-4, C-2; H2-15′ with C-2; H3-1′ with C-2′; H2-21′ with C-29′; H2 -17′ with C-28′; H2-9′ with C-26′; H2-5′ with C-25′; H2-7′ with C-9′; H2-3′ with C-5′; H2-15′ with C-17′; H2-19′ with C-21′ (Figure S16). On the basis of spectral data analysis, the structure of H4 has been established as desmethylenesqualenyl deoxy cepharadione-A or 2-(2ʹ, 6ʹ, 10ʹ, 18ʹ, 22ʹ-pentamethyl-tricos-22ʹ-sn-27ʹ-oxymethylene) 18-deoxy-cepharadione-A (Figure 1).
2.2. In vitro cytotoxic activity The SRB method requires only simple equipment and inexpensive reagents and allows a large number of samples to be tested within a few days. The SRB assay is, therefore, an efficient and highly cost-effective method for screening. According to Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), India, GI50 value of = 20 μg/mL or
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: http://www.tandfonline.com/loi/gnpl20
New chemical constituents from the Piper betle Linn. (Piperaceae) Akhtar Atiya, Barij Nayan Sinha & Uma Ranjan Lal To cite this article: Akhtar Atiya, Barij Nayan Sinha & Uma Ranjan Lal (2017): New chemical constituents from the Piper betle Linn. (Piperaceae), Natural Product Research, DOI: 10.1080/14786419.2017.1380018 To link to this article: http://dx.doi.org/10.1080/14786419.2017.1380018
View supplementary material
Published online: 05 Oct 2017.
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=gnpl20 Download by: [University of Connecticut]
Date: 05 October 2017, At: 23:44
Natural Product Research, 2017 https://doi.org/10.1080/14786419.2017.1380018
New chemical constituents from the Piper betle Linn. (Piperaceae) Akhtar Atiyaa, Barij Nayan Sinhab and Uma Ranjan Lala a
ABSTRACT
The phytochemical investigation of chloroform extract from Piper betle var. haldia, Piperaceae, leaves has resulted in the isolation of two new chemical constituents which were identified as 1-n-dodecanyloxy resorcinol (H1) and desmethylenesqualenyl deoxy-cepharadione-A (H4), on the basis of spectroscopic data 1D NMR (1H and 13C) and 2D NMR (1H-1H COSY and HMBC) as well as ESI-MS, FT-IR and HR-ESI-MS analyses. Compounds H1 and H4 showed excellent antioxidant DPPH free radical scavenging activity with IC50 values of 7.14 μg/mL and 8.08 μg/mL compared to ascorbic acid as a standard antioxidant drug with IC50 value of 2.52 μg/mL, respectively. Evaluation of cytotoxic activity against human hepatoma cell line (PLC-PRF-5) showed moderate effect with the GI50 values of 35.12 μg/mL for H1, 31.01 μg/ mL for H4, compared to Doxorubicin® as a standard cytotoxic drug with GI50 value of 18.80 μg/mL.
ARTICLE HISTORY
Received 14 June 2017 Accepted 5 September 2017 KEYWORDS
Piperaceae; Piper betle; chemical constituents; antioxidant; cytotoxic; SAR
OH
O
1-n-dodecanyloxy resorcinol (H1) H
H
H
H
H
CH 2
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Division of Pharmacognosy and Phytochemistry, Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra, India; bDivision of Medicinal Chemistry, Department of Pharmaceutical Sciences and Technology, Birla Institute of Technology, Mesra, Ranchi, India
O
Piper betle var. haldia leaves (dorsal and ventral view)
O HN
O
desmethylenesqualenyl deoxy-cepharadione-A (H4)
Antioxidant and cytotoxic activity against human hepatoma cell line (PLC-PRF-5)
1. Introduction Piperaceae, a large tropical family, consists of over 10 genera (Sambamurty 2010) and contain more than 1500 species (Alves et al. 2017). Regarding the species of Piperaceae, those from CONTACT Uma Ranjan Lal [email protected], [email protected]; Akhtar Atiya [email protected], atiya_ [email protected] Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2017.1380018. © 2017 Informa UK Limited, trading as Taylor & Francis Group
Downloaded by [University of Connecticut] at 23:44 05 October 2017
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A. ATIYA ET AL.
the genus Piper comprises approximately 700 species occur in the American tropics. Another 300 species are found in the Asian tropics, 15 species occur in Africa and up to 40 species are distributed on various island in the tropical Pacific (Kricher 2017). In India the genus Piper contains around 110 species (Parthasarathy et al. 2006). Economically, this genus is the most important of the family Piperaceae due to its edible purposes and medicinal importance (Torres-Pelayo et al. 2016; Alves et al. 2017). Piper betle Linn. (Piperaceae) is an important and popular medicinal plant, widely cultivated in the tropical and subtropical regions in south and south-east Asia (Dammini Premachandra et al. 2014). Its fresh leaves are chewed either alone as breath-freshener (Singh et al. 2009) or in form of ‘betle quid’ (Atiya et al. 2017). It used in the Indian traditional system of medicine (Guha 2006). Their extracts possess several bioactivities (Majumdar et al. 2003; Arambewela et al. 2005; Alam et al. 2013) etc. Previous phytochemical analyses revealed that the leaves of P. betle is very rich source of phenolic compounds, comprising a phenylpropanoid (Evans et al. 1984; Rathee et al. 2006; Ali et al. 2010; Ferreres et al. 2014; Musa et al. 2014). Due to expansion of P. betle varieties cultivation and its increasing consumption in the main India markets, it is necessary to explore the chemical constituents of this plant species (betle). Therefore, this research work aims at exploration of the phytochemical constituents of the chloroform extract of P. betle L. var. haldia leaves afforded one new phenolic analogue: 1-n-dodecanyloxy resorcinol (H1) and a new oxo-aporphine alkaloid analogue: desmethylenesqualenyl deoxy-cepharadione-A (H4). The phytochemical characterisations were done by spectrometric methods using 1D NMR (1H and 13C) and 2D NMR (1H-1H COSY and HMBC), as well as ESI-MS, FT-IR and HR-ESI-MS. The chloroform extract of P. betle L. var. haldia leaves and its isolated compounds have been investigated for their antioxidant DPPH radical scavenging activity and cytotoxic activity against human hepatoma cell line (PLC-PRF-5) using SRB assay. Hepatocellular carcinoma (HCC) is the third leading cause of death and fifth most commonly occurring cancer worldwide (de Oliveria Andrade et al. 2009). It is the most common cancer among men (Poustchi et al. 2010). An increased occurrence of liver cancer in the world is reported in Asia and Africa. Infected with hepatitis B virus (HBV), hepatitis C virus (HCV) and many more are known to cause liver cancer (de Oliveria Andrade et al. 2009; McGlynn and London 2011). There are different treatment options available for HCC (Raza and Sood 2014). Distinctive regimen of chemotherapy has been tried with poor response. However, surgical treatment is the better option in present-day for the treatment of HCC, but not all patients are eligible for it, either due to minimal efficacy or contraindication, this reiterates the need for the new treatment option. Several studies aimed at discovering more molecules for the HCC management and treatment. Whereas a number of chemical constituents are found in natural and dietary products and they had sufficient studies that discussed their roles in HCC (Alnajjar and Elsiesy 2015). Although the benefit of P. betle leaves alone was spread out in present or ancient times, but there has not been vigorous proof of incidence of any side effects. Different kind of experiments were supervised on P. betle leaves extract and recommend that leaves extract did not grow any tumors and no perilous effect when consumed alone. In addition, the extract of P. betle leaves even showed the auspicious effect in terms of reducing the rate magnification of tumor in animal models (Atiya et al. 2017). In recent studies, it revealed that P. betle leaves extract induced a dose- (0.1 to 1 mg/mL) and time-dependent increase of cell
NATURAL PRODUCT RESEARCH
3
toxicity, supporting their application as a novel chemopreventive agent for the treatment of HCC (Wu et al. 2014). Although more studies are needed on the utilisation of P. betle for the treatment of HCC. In this experimental study, the cytotoxic effects against human hepatoma cell line (PLC-PRF-5) showed quite encouraging.
Downloaded by [University of Connecticut] at 23:44 05 October 2017
2. Result and discussion The defatted chloroform extract, which was of good amount, was selected for phytochemical investigation. Classical colorimetric methods were first employed to evaluate the total amounts of phenols and antioxidant activity in the chloroform extract of P. betle var. haldia leaves. The total phenolic content, determined following a Folin-Ciocalteu procedure and calculated as gallic acid equivalents was found to be 153.57 ± 0.39 mg/g of dried leaves (Figure S17). The antioxidant activity was performed by 1, 1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging method, the IC50 values of 11.18 μg/mL for extract and 2.52 μg/ mL for ascorbic acid as standard antioxidant compound (Table S1) (Atiya 2017). Further, the chloroform extract was subjected to thin layer chromatography (TLC) as per conventional one-dimensional ascending method for qualitative and quantitative analysis with solvent system (chloroform: methanol, 9:1). The two major spots were then detected using iodine vapours and a spots were visualised as dark brown in colour (Figure 1). The movement of the active chemical constituents was demonstrated by its Rf (retention factor) values. The Rf values were found to be 0.76 and 0.13, respectively. Furthermore, the chloroform extract was then subjected to silica gel column chromatography afforded two new chemical compounds (H1 and H4). The isolated compounds were then tested for their antioxidant DPPH radical scavenging activity. The IC50 values for compound H1 was 7.14 and 8.08 μg/mL for H4, respectively (Table S1).
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Figure 1. Chemical structures and TLC profile of isolated compounds, H1 and H4 (chloroform: methanol, 9:1) was visualised with the help of iodine vapours.
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A. ATIYA ET AL.
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2.1. Isolation and identification of phytochemicals 2.1.1. 1-n-dodecanyloxy resorcinol (H1) The compound H1 was elution of the column with chloroform: methanol (99:1) (fraction 9–15) afforded brownish yellow solid mass of 32.20 mg; ~Rf: 0.76 (chloroform: methanol, 9:1); melting point 80–85 °C. The IR Vmax (KBr): 2928, 2850 (sp3 C-H stretching), 1722 (overtone interaction), 1684 (C=C aromatic), 1459 (C-H scissoring or H–C–H bending), 1364 (C-H methyl rock), 1275, 1187 (C–O or O–C stretching), 1080 (C–OH stretching), 966 (OH bending or CH2 in-plane rock or C–O stretching) and 743 (CH2 rocking) cm−1, respectively. On the basis of ESI-MS spectrum, the molecular weight of H1 has been determined at m/z 279.2 [M + H]+ consistent with molecular formula of 1-n-dodecanyloxy resorcinol, C18H30O2. The molecular formula of compound H1 was also concluded by the HR-ESI-MS gave an [M]+ peak at m/z 278.2254 (calculated for C18H30O2=278.2246) supporting the molecular formula of C18H30O2 and the identity of the sample as compound H1. The 1H NMR spectrum of compound H1, exhibited four one-proton multiplet at δH 7.72 (H-2), 7.56 (H-4), 7.31 (H-6) and 7.01 (H-5), assigned as 1, 3-disubstituted aromatic protons. A two-proton triplet at δH 4.31 (J = 7.3 Hz) was ascribed to oxygenated methylene H2-1ʹ protons. In addition, one three-proton multiplet at δH 0.86 was associated with C-12ʹ methyl protons attached to the methylene C-11ʹ carbon. The remaining methylene protons resonated from δH 1.78–1.25. The basic proton peaks as observed in 1H NMR spectrum of compound H1 are illustrated in Figure S15. The 13C NMR spectrum of H1 showed signals for carbons at δC 130.93 (C-2), 124.45 (C-4), 119.06 (C-5) and 128.83 (C-6), one oxygenated aromatic carbon at δC 167.01 (C-1) and a hydroxyl-substituted carbon at δC 147.06 (C-3) at aromatic region. Another important signals were observed for oxygenated methylene carbon at δC 65.58 (C-1ʹ) and methyl carbon at δC 13.73 (C-12ʹ), respectively. The remaining methylene carbons resonated from δC 38.69 to 14.13. The 1H-1H COSY spectrum of H1 showed correlations of H-4 with H-5 and H-5 with H-6; H2-1′ with H2-2′ and H3-12′ with H2-11′. The HMBC spectrum of H1 exhibited interactions of C-3 with H-5; C-2 with H-4; C-4 with H-6; C-1 with H2-1ʹ; C-1ʹ with H2-3ʹ; C-10ʹ with H3-12ʹ and H2-8ʹ; C-8ʹ with H2-10ʹ; C-6ʹ with H2-8ʹ; C-4ʹ with H2-6ʹ; C-5ʹ with H2-3ʹ (Figure S16). On the basis of above spectral data analysis and literature data (Atiya 2017), the structure H1 has been established as 1-n-dodecanyloxy resorcinol (Figure 1). 2.1.2. Desmethylenesqualenyl deoxy-cepharadione-A (H4) The compound H4, named desmethylenesqualenyl deoxy-cepharadione-A, was elution of the column with chloroform: methanol (99:1) (fraction 55–64) yielding a brownish solid mass, 82.10 mg with a melting point of ˃350°C; ~Rf: 0.13 (chloroform: methanol, 9:1). The IR Vmax (KBr): 2926, 2853 (sp3 C-H stretching), 1730 (C=O stretching), 1642 (C=C stretchings) 1465 (H–C–H bending or H–C–H (oxo) wagging), 1378 (C-H methyl rock or CH3 bending), 1287 (H–C–H (oxo) twisting or C–O stretching), 1122 (C–N stretching), 1074, 966 (C-H bending) and 743 (N-H wag primary amine) cm−1, respectively. On the basis of ESI-MS spectrum, the molecular formula of H4 has been determined at m/z 695 [M]+ consistent with molecular formula of desmethylenesqualenyl deoxy-cepharadione-A, C47H69O3 N. The molecular formula of compound H4 was also concluded by the HR-ESI-MS gave an [M]+ peak at m/z 695.5281 (calculated for C47H69O3 N = 695.5277) supporting the molecular formula of C47H69O3 N and the identity of the sample as compound H4. The fragmentation process of the molecule H4 was given in Figure S11. The 1H NMR spectrum of H4 exhibited a
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NATURAL PRODUCT RESEARCH
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two-proton doublet at δH 3.26 (J = 6.5 Hz) was ascribed to oxygenated methylene H2-27′ protons. Two one-proton multiplets at δH 5.02 and 5.08 were accounted to vinylic H-12′ and H-13′ protons. Seven three-proton doublets at 0.98 (J = 6.1 Hz), 0.95 (J = 6.0 Hz), 0.93 (J = 6.2 Hz), 0.91 (J = 6.3 Hz), 0.88 (J = 5.8 Hz), 0.85 (J = 6.1 Hz) and 0.83 (J = 6.3 Hz) were accounted to secondary C-1′, C-24′, C-25′, C-26′, C-28′, C-23 and C-29′ methyl protons, respectively, all are attached to saturated carbons. Further, a one-proton multiplet at δH 2.01 was ascribed to methine H-14′ proton adjacent to the oxygenated methylene protons (H2-27′). The remaining methine and methylene protons resonated from δH 1.22 to 2.06 and 1.22 to 1.18 (H-2′ to H2-11′ and H2-15′ to H-22′). In addition, the presence of an oxo-methylene protons at δH 4.28 (J = 5.5, 5.5 Hz, H2-1) which was assigned to C-8 position, whereas one-proton doublet at δH 4.09 (J = 5.5 Hz) assigned to anomeric H-2 proton of the cepharadione-A moiety. Further, two one-proton singlet at δH 6.70 (H-4) and 6.78 (H-14) were observed together with five one-proton multiplet at δH 7.90 (H-12), 7.75 (H-10), 7.58 (H-11), 7.32 (H-13) and 2.31 (H-18) in the aromatic region of cepharadione-A moiety. The basic proton peaks as observed in 1H NMR spectrum of compounds H4 are illustrated in Figure S15. The 13C NMR spectrum of H4 showed signals for 29 carbons of desmethylenesqualenyl unit and presence of 18 carbons of cepharadione-A unit. The important signals appeared for the oxygenated methylene carbon at δC 65.55 (C-27′), anomeric carbon at δC 72.40 (C-2), vinylic carbons at δC 115.59 (C-12′) and 115.23 (C-13′) and methyl carbons at δC 13.67 (C-1′), 14.06 (C-23′), 13.90 (C-24′), 22.63 (C-25′), 19.69 (C-26′), 19.12 (C-28′), 19.10 (C-29′). The remaining methylene and methine carbons resonated from δC 31.37 to 30.14 and 27.67 to 30.51. In addition, the ketone (C=O) group at C-17 resonated at δC 167.72. The 1H-1H COSY spectrum of H4 showed correlations of H-10 with H-11; H-12′ with H-13′; H3-1′ with H-2′. The HMBC spectrum of H4 exhibited interactions of H-10 with C-8, C-9; H-15 with C-17; H-2 with C-27′; H-1 with C-27′; H-13 with C-15; H2-27′ with C-4, C-2; H2-15′ with C-2; H3-1′ with C-2′; H2-21′ with C-29′; H2 -17′ with C-28′; H2-9′ with C-26′; H2-5′ with C-25′; H2-7′ with C-9′; H2-3′ with C-5′; H2-15′ with C-17′; H2-19′ with C-21′ (Figure S16). On the basis of spectral data analysis, the structure of H4 has been established as desmethylenesqualenyl deoxy cepharadione-A or 2-(2ʹ, 6ʹ, 10ʹ, 18ʹ, 22ʹ-pentamethyl-tricos-22ʹ-sn-27ʹ-oxymethylene) 18-deoxy-cepharadione-A (Figure 1).
2.2. In vitro cytotoxic activity The SRB method requires only simple equipment and inexpensive reagents and allows a large number of samples to be tested within a few days. The SRB assay is, therefore, an efficient and highly cost-effective method for screening. According to Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), India, GI50 value of = 20 μg/mL or
