Archives of Natural and Medicinal Chemistry (ISSN: 2577-0195)

Article / Research Article

"Antioxidant flavonoids fromNicotiana Plumbaginifolia Viv.Leaves"

Mohammad Shafiullah Shajib1, Bidyut Kanti Datta1, Rasheduzzaman Chowdhury2*, Mohammad Abdur Rashid2*

 

1Department of Pharmacy, Stamford University Bangladesh, Bangladesh

2Department of Pharmaceutical Chemistry, University of Dhaka, Bangladesh

*Corresponding authors: Rasheduzzaman Chowdhury, Department of Pharmaceutical Chemistry, University of Dhaka, Bangladesh. Tel: +16502506510; Email: chowdhury.rzaman@gmail.com

Mohammad Abdur Rashid, Department of Pharmaceutical Chemistry, University of Dhaka, Bangladesh.Tel: +88029661900 Extn: 8137; Email:rashidma@du.ac.bd

Received Date: 30 April, 2018; Accepted Date: 18May, 2018; Published Date: 24May, 2018.

1.      Abstract

Plant derived phenolics and flavonoids exert profound antioxidant effects due to their ability to scavenge free radicals.Nicotiana plubagnifoliaViv. leaves are known to producelarge quantities of phenolics and flavonoids. Here we report antioxidant activities of amethanol extract of N. plubagnifolialeaves and itsisolates, 3,3',5,6,7,8-hexamethoxy-4',5'-methylenedioxyflavone (1), 3,3',4',5',5,6,7,8-octamethoxyflavone (exoticin) (2), 6,7,4',5'-dimethylenedioxy-3,5,3'-trimethoxyflavone (3) 3,3',4',5,5',8-hexamethoxy-6,7-methylenedioxy-flavone (4) and 5-hydroxy-3,3',6,7,8-pentamethoxy-4',5'-methylenedioxyflavone (5) against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and NO radicals. This is the first report of 5from N. plumbaginifolia. The structure of 5 was determined by using high-field1H and13C-NMR andby comparison with previously reported values.Amongst the tested materials, the extract and compound 5 produced considerable radical scavenging activity against DPPH and NO radicals, which were comparable to the reference standard, ascorbic acid. All extractives were then evaluated fortheir toxicityprofiles by means of brine shrimp lethality assay. While the methanol extract waslethal against brine shrimp nauplii,the isolated compounds were relatively non-toxic. Thus, our findings suggest that the functionalized flavonoids could be suitable candidates for developing antioxidant drugs with minimal or no cytotoxicity.

2.       Keywords: Antioxidant; Cytotoxicity; Nicotiana plubagnifolia; Polymethoxyflavone; Solanaceae

1.                  Introduction

Reactive free radicalsthat are frequently generated in cellscause oxidative damages to biological macromolecules and formvarious toxins. In severe cases, supplementaryantioxidants are required to maintain the free radical contents in the body as part ofthe innate defense mechanism[1].Aberrant regulation of free radicals has been linked to pathogenesis of manydiseasesincluding cancer and inflammation [1,2].However, the therapeutic implications of current synthetic antioxidants are somehow limited[3]. Natural product derived phenolics and flavonoids can potentiallyscavenge free radicals viaH- or electron-transfer with concomitant production of stable radicalsbearing an odd electron[4,5].This newly generated radical is far more stable as the odd electron can delocalize over the aromatic system containingan extended conjugation [6]. In addition, there is evidence that the antioxidant properties of phenolics and flavonoidsoften contribute to anticancer effects[7,8].As part of our continuous impetus in identifying potential antioxidant/ anticancer compounds from natural sources, we investigated Nicotiana plumbaginifoliaViv. (Fam. Solanaceae), a plant that is known to contain phenolics and flavonoids.

N. plumbaginifoliaViv.isan herb found in weedy habitats of Bangladesh andis used in the traditional medicine for the treatment of toothache[9], piles[10], wounds[11], parasitic infection[12], pyrexia[13] and rheumatic swelling (amongst others).Leaves are used in the management of nausea, travel sickness[14], insecticide[15] andvermicide[16].In addition to phenolics and flavonoids, the plant contains a wide range of secondary metabolites including alkaloids, saponins, tannins,glycosides, steroids and terpenoids [14,17-19].

2.                  Results and Discussion

Repetitive chromatographic purificationled to the isolation of 5 from the leaves of N. plumbaginifolia.Its flavonoid nature was evident from a characteristic dark blue and fluorescent blue colored spot on TLC plate under UV254nm and UV366nm light, respectively and a yellow color spot upon vanillin-H2SO4treatment followed by heating at 110°C.The 13C NMR spectrum (100 MHz, CDCl3) of 5revealed 21 carbon signals that arecharacteristic of a polymethoxyflavone: five methoxyl (-OCH3) at δC 56.7, 60.2, 61.2, 61.7, 62.1, one methylenedioxy (O-CH2-O) at δC 102.2, two methines (-CH) atδC 102.9, 109.2 and thirteen quaternary carbons, including a carbonyl group (C=O) at δC 179.3.The 1H NMR spectrum (400 MHz, CDCl3) of 5displayed signals for five methoxyl groups at δ 3.91 (3H), 3.97 (6H), 4.01 (3H), 4.13 (3H) and a chelated -OH group at 12.36 (1H). The 13C and DEPT spectra revealed that three out of the five -OCH3 were downfield between δC 61.2 - 62.1, suggesting their attachment to quaternary carbons. Comparative analysis of 1H and 13C-NMRof 5 with similar polymethoxyflavones for the substituted ring A enabledassignment of three -OCH3groups [17,20]. The 1H NMR spectrum further showed two aromatic signals at δ 7.54 (1H) and 7.42 (1H) for two meta-coupled protons, which were attributed to H-2' and H-6', respectively.  The 1H NMR signal at δ6.09 integrated for two protons, revealed the presence of a methylenedioxy (O-CH2-O) group which was further supported by the oxygenated methylene carbon at δC 102.0. The remaining two -OCH3 at δ 3.91 (δC60.2) and δ3.99 (δC56.7) wereascribed to C-3 (δC141.6) and C-3' (δC143.6) respectively.

Based on the above analyses, the structure of compound 5 was determined to be 5-hydroxy-3,3',6,7,8-pentamethoxy-4',5'-methylenedioxyflavone, which was previously reported from Polygonum orientale[21]. All the 1H and 13C NMR resonances of the compound wereunambiguously assigned by comparison with the previously reported low resolution data (60 MHz)[21].The melting point of 5 (186-187 °C) was almost identical to that published for 5-hydroxy-3,3',6,7,8-pentamethoxy-4',5'-methylenedioxyflavone[21],which further supported our structure determination. To the best of our knowledge, this is the first report of a complete assignment of high-resolution1H and 13C-NMR of 5-hydroxy-3,3',6,7,8-pentamethoxy-4',5'-methylenedioxyflavone (5) (Figure 1), and the first report fromN. plumbaginifolia.


1 = 3,3',5,6,7,8-hexamethoxy-4',5'-methylenedioxyflavone,

2 = 3,3',4',5',5,6,7,8-octamethoxyflavone (exoticin),

3 = 6,7,4',5'-dimethylenedioxy-3,5,3'-trimethoxyflavone,

4 = 3,3',4',5,5',8-hexamethoxy-6,7-methylenedioxyflavone,

5 = 5-hydroxy-3,3',6,7,8-pentamethoxy-4',5'-methylenedioxyflavone.

The plant extract and the isolated compounds (1-5) were then tested for their ability to scavenge the DPPH and NO radicals. The methanol extract and compound 5producedstatistically significant (p< 0.05) DPPH and NO radical scavenging activity compared to the control. In both assays, they produced concentration dependent effects(Figure 2).In DPPH radical assay, the extract and compound 5 exhibited 84 and 83% scavenging at 400μg/mlwith theIC50values of 14.3 and 90.7µg/ml, respectively. As compared to 5,compounds 1, 2, 3 and 4produced61, 73, 40% and 62% scavenging effect at 400μg/ml(Figure 2A)withsubstantially higherIC50 valuesof 297.0 (1), 177.6 (2), 212.5 (4)μg/ml(Figure 2B), respectively.IC50 for3could not be measured as itproduced <50 % inhibition of DPPH radical at the maximum tested dose.The standard(ascorbic acid) produced >95% scavenging effect at 50-400 µg/ml (Figure 2A) withan IC50value of 8.7μg/ml, which was comparableto that of the methanol extract (Figure 2B).As shown in figure 2C, the extract and compound 5 showed strong NO scavenging activity of 91 and 90%, respectively which was comparable to the standard, ascorbic acid (97% at 400 µg/ml).Compounds1, 2,3 and 4exhibitedmaximumNO scavenging effect of 68, 53, 54 and 72%, respectively. The lowest IC50value of NO scavenging was again shown by the extract (11.6µg/ml), which was comparable to the ascorbic acid (4.8 µg/ml). Compound 5revealed significantly (p< 0.05) lower IC50value (22µg/ml) in NO scavenging assay compared to other isolated compounds(Figure 2D). It has beenprecedented that -OH groups significantly increasethe anti-radical potentiality of flavonoids possibly via enhancing H-transfer to free radicals whereas -OCH3 groups reduce radical scavenging ability[22].However, care should be taken to interpret the in vitro dataas one of the caveats using the flavonoids is cell permeability. In fact, it is likely that -OCH3 may facilitate cell permeability, which may undergo metabolic deprotection to produce free -OH in cells. Thus, although the presence of an additional -OH group in compound 5apparently contributes togreater activityin our in vitro assayscompared to other isolated flavonoids, further studies are required to validate the results in cells.

1 = 3,3',5,6,7,8-hexamethoxy-4',5'-methylenedioxyflavone,

2 = 3,3',4',5',5,6,7,8-octamethoxyflavone (exoticin),

3 = 6,7,4',5'-dimethylenedioxy-3,5,3'-trimethoxyflavone,

4 = 3,3',4',5,5',8-hexamethoxy-6,7-methylenedioxyflavone,

5 = 5-hydroxy-3,3',6,7,8-pentamethoxy-4',5'-methylenedioxyflavone.

Because antioxidant compounds are often toxic to cells, we evaluated the extract and the isolates for their cytotoxicity using a simple Brine Shrimp Lethality (BSL) assay[23].The plant extract was substantially lethal to brine shrimp nauplii with an LC50value of 8.6µg/ml(Table 1), as compared tothe standard drug, vincristine sulfate (1.4 µg/ml). The isolated compounds (1-5) were relativelynon-toxic against the organism as evident by significantly higher LC50 values ranging from 66.2 to 75.5 µg/ml(Table 1). The observation that the methanol extract was substantially more toxic compared to the isolated compounds imply that there are potentially more cytotoxic principles in the N. plumbaginifolialeaves that we were unable to isolate.

Data are presented as mean ± SEM (n = 3). *p< 0.05, compared to 1, 2, 3, 4 and 5 treatments. MENP = methanol extract of N. plumbaginifolialeaves.

1 = 3,3',5,6,7,8-hexamethoxy-4',5'-methylenedioxyflavone,

2 = 3,3',4',5',5,6,7,8-octamethoxyflavone (exoticin),

3 = 6,7,4',5'-dimethylenedioxy-3,5,3'-trimethoxyflavone,

4 = 3,3',4',5,5',8-hexamethoxy-6,7-methylenedioxyflavone,

5 = 5-hydroxy-3,3',6,7,8-pentamethoxy-4',5'-methylenedioxyflavone.

3.                 Conclusion

Our results suggest the occurrences of considerable antioxidant principles in the N. plumbaginifolialeaves including the polymethoxoyflavones. In particular, compound 5demonstrated promising antioxidant properties presumably due to its ability fortransferringhydrogen(s) efficiently. The polymethoxoyflavones are unusually rare amongst plants, occurring only in a few genera includingPolygonum and Murraya, suggesting a unique biosynthetic pathway.Thus, the observation thatN. plumbaginifolia leaves arerich in polymethoxyflaovens warrantsfurther investigations for their potential chemotaxonomic relationships with PolygonumandMurraya. Because these compounds have the potential to scavenge radicals, further research can be directed towards comprehensive Structure-Activity Relationship (SAR)studies aimed at developing polymethoxyflavone-based antioxidants. While we primarily focused on identifyingantioxidant flavonoids, the N. plumbaginifolialeaves may also serve as a potential source of cytotoxic compounds that will likelyfind ways forward to anticancer drug development.

4.                  Materials and Methods

4.1.              Chemicals and Reagents

Except for sodium chloride (Merck Co., Darmstadt, Germany), ammonium molybdate (Merck, Mumbai, India) and sodium nitroprusside (Loba Chemie, Mumbai, India), all solvents and reagents including n-Hexane, chloroform, methanol, toluene, ethyl acetate, sulfuric acid, phosphoric acid, DPPH (2,2-Diphenyl-1-picrylhydrazyl), vanillin, dimethyl sulfoxide (DMSO), ascorbic acid, sulfanilamide, N-(1-Naphthyl)ethylenediamine dihydrochloride were from BDH (U.K.) or Sigma (St. Louis, MO, USA) Chemicals. Vincristine sulfate was a gift from Beacon Pharmaceuticals Limited, Bangladesh.

4.2.              Extraction and Isolation of Compounds 1-5

Plant collection and extraction of N. plumbaginifoliaViv. leaves were conducted as described [17]. Successive chromatographic purification yielded twelve fractions (F1- F12) of which fraction 12(4.58 g) lead to the isolation of 1-5. Compounds (1-4), which we have reported previously were identified as  3,3',5,6,7,8-hexamethoxy-4',5'-methylenedioxyflavone (1), 3,3',4',5',5,6,7,8-octamethoxyflavone (exoticin) (2), 6,7,4',5'-dimethylenedioxy-3,5,3'-trimethoxyflavone (3) and 3,3',4',5,5',8-hexamethoxy-6,7-methylenedioxyflavone (4) [17]. Compound 5 (17.4 mg), which is a new report from N. plumbaginifolia, was isolated from the same fraction (F12) using preparative TLC (mobile phase, toluene:ethyl acetate, 4:1, Rf= 0.91) on repeated development. The compound was re-crystalized from CHCl3-MeOH (98:2) as pale-yellow needles.

4.3.              5-Hydroxy-3,3',6,7,8-pentamethoxy-4',5'-methylenedioxyflavone(5):

Pale-yellow needles; m.p. 186-187 °C; 1H NMR (400 MHz, CDCl3): δ(ppm) 12.36 (1H, s, 5-OH), 7.54 (1H, d, J= 1.2 Hz, H-2'), 7.42 (1H, d, J = 1.6 Hz, H-6'), 6.09 (2H, s, 4', 5'-O-CH2-O-); 4.13 (3H, s, 7-OMe), 4.01 (3H, s, 8-OMe), 3.99 (6H, s, 6-OMe, 3'-OMe), 3.91 (3H, s, 3-OMe); 13C NMR (100 MHz, CDCl3): δ(ppm) 179.3 (C-4), 155.4 (C-2), 153.0 (C-7), 149.2 (C-5), 149.1 (C-5'), 147.0 (C-9), 144.8 (C-6), 143.6 (C-3'), 141.6 (C-3), 139.3 (C-4'), 138.0 (C-8), 124.6 (C-1'), 114.1 (C-10), 109.2 (C-2'), 102.9 (C-6'), 102.2 (4', 5'-O-CH2-O-), 62.1 (8-OMe), 61.7 (6-OMe), 61.2 (7-OMe), 60.2 (3-OMe), 56.7 (3'-OMe).

4.4.              NMR Spectroscopy and Melting Point Determination

1H and 13C NMR of compound 5 were recorded in CDCl3 with respect to the residual solvent at 400 and 100 MHz on AVANCE DRX 400 and ASCENDTM 400 (Avance III HD NanoBay), Bruker, Germany, respectively. Melting point of 5 was recorded on a Stuart SMP30 melting point apparatus. The plateau temperature was set at 300 °C and ramp rate was set at 0.5 °C/min.

4.5.              DPPH Free Radical Scavenging Assay

The scavenging of DPPH free radicals by the crude extract or compounds (1-5) was evaluated as described [24]. Briefly, 1.6 mg of standard (ascorbic acid), plant extract or isolates was dissolved in methanol and diluted to obtain the concentrations of 1.5625 - 400 µg/ml. An 0.1mM DPPH solution was prepared in methanol. 2 ml of DPPH solution was then added to 2 ml of tested materials. The mixture was mixed well and left for 30 min in a dark area at the room temperature. The final absorbance of the mixture was measured at 517 nm using a spectrophotometer (HACH, DR 5000™, Shanghai, China) with respect to DPPH blank solution. Each experiment was carried out in triplicate. The inhibition of DPPH free radicals was measured as percent (%) using the following equation:


The concentration required for scavenging 50% of DPPH free radicals (IC50) was determined from the % of inhibition.

4.6.              NO Scavenging Assay

The assay was conducted as described [25]. Briefly, 1.6 mg of plant extract, isolates (1-5) or standard (ascorbic acid) was dissolved in methanol to obtain solutions ranging from 1.5625-400 µg/ml. 1 ml of sodium nitroprusside (5 mM) was then thoroughly mixed with 4 ml of each solution and incubated for 2 h at 30°C. The final absorbance of the mixture was measured at 550 nm followed by the addition of 1.2 ml of Griess reagent (1% sulfanilamide, 0.1% naphthylene diamine dihydrochloride in 2% H3PO4). IC50 values were calculated from % scavenging of NO radicals. The assay was done in triplicates.

4.7.              Brine Shrimp Lethality (BSL) Assay

The assay was conducted as described [23]. Briefly, 1 mg of the methanol extract, its isolates (1-5) or standard (vincristine sulfate) was dissolved in 60 µl of DMSO. For each of standard, control and experimental groups, ten brine shrimp nauplii were taken in a graduated test tube containing 5 mL simulated sea water. 30 µL of the test solution was then added to the test tubes leading to final concentrations ranging from 100 - 0.195 µg/ml. 30 µL DMSO served as a control. Following 24h of the transfer, each test tube was observed for the number of survived brine shrimp nauplii. The concentration of each sample responsible for the 50% lethality of the brine shrimp nauplii (LC50) was determined by Probit analysis. A mean LC50 value was obtained by performing the experiment in triplicate.

4.8.              Statistical Analysis

Data are presented as mean ± SEM (n = 3). IC50values were determined using GraphPad Prism 6.05, USA. The multiple comparison between the groups were determined from Tukey’s post-hoc test using Statistical Package for Social Sciences (SPSS) software (Version 22, IBM Corporation, USA), where p< 0.05 was considered as statistically significant. 


Figure1: Chemical structures of compounds 1-5.



Figure 2: Antioxidant activity of the methanol extract of N.plumbaginifolia(MENP) and its isolated compounds (1-5). Panels (A) and (B) show % scavenging effect and IC50 values using DPPH free radical scavenging assay as indicated. Note,IC50value for 3 could not be measured as it displayed<50 % inhibition of DPPH radical at the maximum tested dose.Panels (C) and (D) depict % scavenging effect and IC50 values using NO scavenging assay. Data are presented as mean ± SEM (n = 3). *p< 0.05 compared to control for the compound 1,2, 3, 4 and 5 treatments.AA = ascorbic acid,


Treatment

LC50 (µg/ml)

Vincristine sulfate

1.42 ± 0.48*

MENP

8.55 ± 1.65*

1

74.92 ± 0.95

2

69.29 ± 3.09

3

72.78 ± 1.43

4

75.50 ± 2.22

5

66.23 ± 3.09

Table 1: Cytotoxicity of the methanol extract and its isolated compound (1-5) in brine shrimp lethality assay.



1.       Droge W (2002) Free radicals in the physiological control of cell function. Physiol Rev82: 47-95.

2.       Maeda H and Akaike T (1998) Nitric oxide and oxygen radicals in infection, inflammation, and cancerBiochemistry63: 854-865.

3.       Moure A, Cruz JM, Franco D, Domı́nguez JM, Sineiro J, et al.(2001) Natural antioxidants from residual sources. Food Chem 72: 145-171.

4.       LeopoldiniM, Russo N, Toscano M (2011) The molecular basis of working mechanisms of natural polyphenolic antioxidants. Food Chem125:288-306.

5.       Rice-Evans C (2001) Flavonoid antioxidants. Curr Med Chem 8: 797-807.

6.       WrightJS, JohnsonER, DiLabioGA (2001)Predicting the activity of phenolic antioxidants: Theoretical method, analysis of substituent effects, and application to major families of antioxidants. J Am Chem Soc 123:1173-1183.

7.       Ren W, Qiao Z, Wang H, Zhu L, Zhang L (2003) Flavonoids: promising anticancer agents. Med Res Rev23: 519-534.

8.       Huang WY, Cai YZ, Zhang Y (2009) Natural phenolic compounds from medicinal herbs and dietary plants: potential use for cancer prevention. Nutr Cancer 62: 1-20.

9.       Devi AD, Devi OI, Singh TC, Singh EJ (2014) A study of aromatic plant species especially in Thoubal district, Manipur, Northeast India. Int J Sci Res Pub 4: 1-12.

10.    Bhowmik R, Saha MR, Rahman MA, Islam MAU(2015) Ethnomedicinal survey of plants in the Southern District Noakhali, Bangladesh. Bangladesh Pharm J 17: 205-214.

11.    Dangwal LR, Sharma A, Rana CS (2010) Ethnomedicinal plants of the Garhwal Himalaya used to cure various diseases: a case study. New York SciJ3: 28-31.

12.    Rao PK, Hasan SS, Bhellum BL, Manhas RK (2015) Ethnomedicinal plants of Kathua district, J&K, India. JEthnopharmacol171: 12-27.

13.    Sharma R, Manhas RK, Magotra R (2012) Ethnoveterinary remedies of diseases among milk yielding animals in Kathua, Jammu and Kashmir, India. JEthnopharmacol141: 265-272.

14.    Singh KP, Daboriya V, Kumar S, Singh S (2010) Antibacterial activity and phytochemical investigations on Nicotiana plumbaginifolia viv.(wild tobacco). Romanian J Biol Plant Biol 55: 135-142.

15.    Rothe SP (2012) Exotic medicinal plants from West Vidarbha region of Maharashtra. Adv Res Pharm Bio2: 33-40.

16.    Kulkarni S, Kulkarni DK, Deo AD, Pande AB, Bhagat RL (2014) Use of ethno-veterinary medicines (EVM) from Vidarbha region (MS) India. Biosci Discov 5: 180-186.

17.    Shajib MS, Datta BK, Sohrab MH, Rashid MA, Nahar L, et al.(2017) highly oxygenated flavonoids from the leaves of Nicotiana plumbaginifolia (Solanaceae). Rec Nat Prod11: 568-572.

18.    Shajib MS, Rashid RB, Ming LC, Islam S, Sarker MMR, et al. (2018) Polymethoxyflavones from Nicotiana plumbaginifolia(Solanaceae) exert antinociceptive and neuropharmacological effects in mice. Front. Pharmacol.9: 85.

19.    Singh S, Khanna NM, Dhar MM (1974) Solaplumbin, a new anticancer glycoside from Nicotiana plumbaginifolia. Phytochemistry 13: 2020-2022.

20.    Machida K, Osawa K (1989) On the flavonoid constituents from the peels of Citrus hassaku Hort. ex Tanaka. Chem Pharm Bull 37: 1092-1094.

21.    Kuroyanagi M, Fukushima S (1982) Highly oxygenated flavonoids from Polygonum orientale. Chem Pharm Bull 30: 1163-1168.

22.    Heim KE, Tagliaferro AR, Bobilya DJ (2002) Flavonoid antioxidants: chemistry, metabolism and structure-activity relationships.J Nutr Biochem13: 572-584.

23.    Meyer BN, Ferrigni NR, Putnam JE, Jacobsen LB, Nichols DJ,et al.(1982) Brine shrimp: a convenient general bioassay for active plant constituents. Planta Med45: 31-34.

24.    Islam S, Shajib MS, Ahmed T (2016) Antinociceptive effect of methanol extract of Celosia cristata Linn. in mice. BMC Complement. Altern Med 16: 400.

25.    Alisi CS, Onyeze GOC (2008) Nitric oxide scavenging ability of ethyl acetate fraction of methanolic leaf extracts of Chromolaena odorata(Linn.). African J Biochem Res2: 145-150.


Citation: Shajib MS, Datta BK, Chowdhury R, Rashid MA (2018) Chemical Substance and Human Health. Arch Nat Med Chem ANMC-117. DOI: 10.29011/ANMC-117.000017