Identification of new phytoconstituents and antimicrobial activity in stem bark of Mangifera indica (L.)

Journal of Pharmaceutical and Biomedical Analysis 105 (2015) 150–155

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Identification of new phytoconstituents and antimicrobial activity in stem bark of Mangifera indica (L.) Ruchi Singh a , S.K. Singh b , R.S. Maharia c , A.N. Garg d,∗ a

Department of Oriental Studies, Dev Sanskrit University, Haridwar 249411, India Department of Botany, DAV College, Kanpur 208001, India Department of Chemistry, Indian Institute of Technology, Roorkee 247667, India d Institute of Nuclear Science and Technology, Amity University, Sector 125, Noida 201313, India b c

a r t i c l e

i n f o

Article history: Received 23 September 2014 Received in revised form 5 December 2014 Accepted 9 December 2014 Available online 18 December 2014 Keywords: Mangifera indica Chemical constituents Thin layer chromatography Column chromatography GC–MS

a b s t r a c t Mangifera indica, commonly called mango or amra belonging to a family of Anacardiaceae, is an important medicinal plant widely used in a variety of Ayurvedic preparations. Extract of its bark, leaves, flowers and kernels are being extensively used for curing various chronic diseases. Mango wood is used in yagya as base fire through which medicated smoke is generated. Three new compounds have been isolated from methanolic and hexane extracts of stem bark: 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl)ester and 9,12-tetradecadiene-1-ol-acetate from the hexane extract and 3-chloroN-(2-phenylethyl) propanamide from the methanolic extract. These were first separated by thin layer chromatography and later in a silica gel column and identified by characteristic infrared bands corresponding to respective functional groups. The compounds were further confirmed on the basis of GC–MS fragmentation pattern after comparing the data with NIST mass spectral database. All three compounds exhibited antimicrobial activity due to triterpenoids and flavonoids. Elemental analyses by INAA show it to be enriched in essential nutrient elements such as Ca, Fe, K, Mn and Zn which all play an important role in enzymatic processes. © 2014 Elsevier B.V. All rights reserved.

1. Introduction The mango is native to South and Southeast Asia from where it has been distributed worldwide to become one of the most cultivated fruits in the tropics. Mango belongs to the family Anacardiaceae, order Rutales and genus Mangifera. It is one of the most popular tropical fruit bearing trees in the world with global production exceeding 30 million tonnes [1]. It contains mangiferin, a pharmacologically active flavonoid-natural xanthone C-glycoside with antioxidant activity [2]. If fully ripe, mango is high in vitamin A (␤ carotene – a cancer fighting agent), vitamin C, and vitamin B1 , B2 , potassium, iron and fibre. On the contrary unripe mangoes have oxalic, citric, malic, and tartaric and succinic acids resulting in its sour taste [3]. Extract of Mangifera indica have been reported to possess antiviral, antibacterial, analgesic, anti-inflammatory and immuno-modulatory activities [4], in vitro ant amoebic activity [5], interesting ␣-amylase and ␣-glycosidase inhibitory activities [6] and cardio toxic and diuretic properties [7].

Scartezzini and Speroni [7] and Ross [8] have reviewed medicinal importance including antioxidant activity of different constituents of M. indica. Bark is reported to contain protocatechic acid, catechin, mangiferin, alanine, glycine, ␥-amino-butyric acid, kinic acid, shikimic acid and the tetra cyclic triterpenoids cycloart-24-en-3␤, 26-diol, 3 keto dammar-24 (E)-en-20 S, 26-diol, C-24 epimers of cycloart-25-en-3␤, 24, 27-triol and cycloartan-3␤, 24,27-triol [9]. Present work was undertaken to isolate and identify new organic compounds from the hexane and alcoholic extracts of M. indica L. The compounds were identified by infrared spectral and gas chromatography–mass spectrometry (GC–MS) techniques. The antibacterial activities of both the extracts have also been studied. Also its elemental contents were determined by instrumental neutron activation analysis (INAA). 2. Materials and methods 2.1. General

∗ Corresponding author. Tel.: +91 0120 2658861. E-mail address: [email protected] (A.N. Garg). http://dx.doi.org/10.1016/j.jpba.2014.12.010 0731-7085/© 2014 Elsevier B.V. All rights reserved.

All the solvents such as methanol (MOH), dichloromethane (CH2 Cl2 , DCM), chloroform (CHCl3 , CFM), carbon tetrachloride

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151

Fig. 1. Flow sheet for the separation of various constituents from mango wood.

(CCl4 , CTC), n-hexane (C6 H14 , HEX), benzene (C6 H6 , BZ) and ethyl acetate (CH3 COOC2 H5 , EAC) were distilled before use. Silica gel containing 13% CaSO4 as binder (SRL, Mumbai) was used as adsorbent for thin layer chromatography (TLC). Silica Gel-G (Merck, Mumbai) 60–120 mesh was used for column chromatography. Antibacterial standards tetracycline (Sigma–Aldrich, Taufkirchen, Germany) were included as antimicrobial standards in each assay. Infrared spectra in the range 4000–600 cm−1 were recorded in KBr pellet on a Thermo Nicolet (Nexus, USA) FT-IR spectrometer. 2.2. Plant material and extracts The dried stems (about 1 cm dia) of M. indica were collected from the local gardens of Shantikunj, Haridwar. These were thoroughly washed with doubly distilled water to remove any dirt and other surface contaminants. Finally, these were dried at 80 ◦ C in an oven for 24 h and crushed to homogeneous powder (80 mesh). 500 g air-dried powder was first extracted with 600 mL nhexane in a soxhlet for 48 h. The solvent was removed and the residue (15 g) was kept aside. The extracted powder was dried in air and re-extracted with methanol in soxhlet for 30 h. Again, the solvent was removed and the residue (13 g) was collected. Both the extracts were stored at 4 ◦ C in a refrigerator for further use.

2.3. Separation of organic constituents TLC separations were carried out on a glass plate (5 cm × 20 cm) coated with 0.5 cm thick silica gel. A spot of organic extract in a solvent was put and after drying the plate was developed in several chambers containing solvent(s) with increasing polarity where the components have different solubility. After drying the plate in oven at ∼80◦ C was then kept in iodine chamber (glass) for development of spots. A circular spot with no tailing, indicative of pure compound, was used for calculating Rf value. The n-hexane extract was subjected to column chromatography in a 2 cm × 45 cm column (made from Borosil glass) fitted with sintered frit and filled with 60–120 mesh Silica Gel-G (Merck, Mumbai). Afterwards elution was done at atmospheric pressure with solvents of their increasing polarity (HEX > CTC > BZ > DCM > CFM > EAC > MOH) respectively. The fractions were collected and the BZ fraction was subjected to TLC using solvent mixture of increasing proportion of EAC and BZ. Two distinct spots were observed in BZ:EAC (6:1) mixture corresponding to Rf = 0.56 and 0.43. These were separated by column chromatography, dried over water bath and finally marked as A (3.6 mg) and B (2.9 mg) respectively. Similarly MOH extract was checked by TLC using different solvent mixtures but only BZ:CTC (1:1) mixture showed five distinct spots corresponding to Rf = 0.21, 0.36, 0.52, 0.77, and

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R. Singh et al. / Journal of Pharmaceutical and Biomedical Analysis 105 (2015) 150–155 Table 1 Infrared spectral and GC–MS fragmentation assignments for organic constituent A.

O

O OH

O .

IR spectral band

Fragments from GC–MS −1

Wave numbers (cm

)

3442 2930 1625 and 1567 1631 1383

Fig. 2. Gas chromatogram and mass fragmentation lines for constituent A.

0.84 respectively. Finally, only the fraction with Rf = 0.36 could be separated by column chromatography and marked as C (2.2 mg). Complete separation scheme is shown as flow sheet in Fig. 1. The compounds were identified by characteristic IR frequencies and gas chromatography–mass spectrometry (GC–MS). GC–MS were recorded using Perkin-Elmer Clarus-500 gas chromatograph (66 cm × 72 cm) coupled with a mass spectrometer equipped with an HP5-MS column (5%-phenyl-methylsiloxane, 30 m × 0.32 mm, 0.25 ␮m film thickness). Helium served as carrier gas at a flow rate of 1 mL/min. One microlitre of each sample was injected at 250 ◦ C. The oven was programmed to heat from 50 ◦ C to a maximum temperature of 250 ◦ C at a rate of 10 ◦ C min−1 . The oven was then held at 250 ◦ C for a 3 min post run. The interface, which kept the capillary column end into the ion source block, was maintained at 280 ◦ C. The mass spectrometer is fitted with a quadrupole prefilter assembly. The detector consists of a common dynode, phosphor plate and photomultiplier tube. The Turbo Mass software v.4.4 is preloaded into the system. The compounds were further confirmed by their mass fragmentation pattern and comparing the individual spectra data with those of built in NIST chemical library 2.0 database. A typical gas chromatogram and mass spectral lines formed after fragmentation for the compound A are shown in Fig. 2. 2.4. Antimicrobial bioassay A disc-diffusion assay was used to evaluate antimicrobial activity. All microorganisms were obtained from the Microbial Type Culture Collection (MTCC) and stored at −85 ◦ C. Microorganisms

Assignment

m/z

Assignments

␯O H ␯C H ␯C O , carboxylate ␯C C ␯C O

278 167 149 113 57

C16 H22 O4 + C8 H7 O4 + C8 H5 O3 + C8 H17 + C4 H9 +

were inoculated on nutrient agar (Microxpress Ltd.) in petri dishes for purity evaluations prior to use in the bioassay. Stock solutions of the plant extracts and the antimicrobial agents were prepared in dimethylsulfoxide (DMSO). Sterile discs (6 mm dia; Microxpress Ltd.) saturated with extract (25 ␮L) were placed on the surface of each inoculated plate (70 mm; Borosil). The agar plates were prepared by spreading the bacterial strains separately over nutrient agar plates, impregnated with different test extracts (25 ␮L/disc) were then placed on the surface of seeded agar plate. The plates were then incubated at 37 ◦ C in bacteriological incubator (Yorco Scientific Instruments Ltd.) for 24 h. Antibacterial activity was evaluated by measuring the zones of inhibition in mm. Inhibition zones with dia < 12 mm were considered as having low antibacterial activity and between 12 and 16 mm were considered moderately active [10]. 2.5. Elemental analysis by instrumental neutron activation analysis (INAA) Approximately 100 mg each of powdered wood sample and reference materials (Peach leaves, SRM 1547 and Mixed Polish Herbs, MPH-2) were accurately weighed, packed in aluminium foil and irradiated for 6 h at ∼1012 n cm2 s−1 in the CIRUS reactor of Bhabha Atomic Research Centre (BARC), Mumbai. The gamma activities of the activation products of the sample and RMs were assayed after suitable cooling time using a Compton suppressed gamma spectrometer consisting of HPGe detector (40% efficiency and 2.0 keV resolution at 1332 keV of 60 Co) coupled to a PC based 8k MCA. Gamma ray spectra were recorded and analyzed using PHAST software at BARC. The peak areas under characteristic gamma rays of various isotopes were used for calculating elemental concentrations by the relative method of INAA as described earlier [11]. 3. Results and discussion 3.1. Chemical analysis Three new compounds two from HEX extract and one from MOH extract were separated and identified by characteristic IR frequencies and GC–MS fragmentation lines after comparing the data with NIST mass spectral database [12]. The compounds identified are 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl)ester (A); 9,12tetradecadiene,1-ol,acetate (B) and 3-chloro-N-(2-phenylethyl) propanamide (C), last one being used as antimalarial [13]. It may be noted that A is an allelopathic compound that reduces the need

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Table 2 Infrared spectral and GC–MS fragmentation assignments for organic constituent B.

O

O . IR spectral band

Fragments from GC–MS

Wave numbers (cm−1 )

Assignment

m/z

Assignments

2921 2853 1707 1643 1461 1380

␯CH2 ␯C H ␯C O (ester) ␯C O ␦C H (methylene) ␯C O

252 192 163 149 121 107 95 81 67 55

C16 H28 O2 + C14 H24 + C12 H19 + C11 H17 + C9 H13 + C8 H11 + C7 H11 + C6 H9 + C5 H7 + C4 H7 +

for weed management in other crops [14]. This compound has also been reported from our laboratory in curry leaves (Murraya koenigi) [15]. A was a cloudy liquid identified as 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl)ester from the IR spectral bands and mass spectral lines listed in Table 1. A band for hydroxyl group (␯ OH ) at 3442 cm−1 can be observed indicating the presence of carboxylic acid OH group. The other prominent peaks were seen at 2930 cm−1 (C H stretching), 1625 cm−1 and 1567 cm−1 (C Ocarboxylate ), 1631 cm−1 (C C stretching) and 1383 cm−1 (C O stretching). The mass spectrum in Fig. 2 indicates a molecular ion peak at m/z 278 corresponding to C16 H22 O4 + and a base peak at m/z 149 corresponding to C8 H5 O3 + . The other prominent peaks at m/z 167, 113 and 57 correspond to molecular fragments C8 H7 O4 + , C8 H17 + and C4 H9 + respectively. B was a colourless liquid identified as 9,12-tetradecadiene-1-olacetate from IR and mass spectral data listed in Table 2. The typical diagnostic band for ester carbonyl group (>C O) was observed at 1707 cm−1 with other prominent bands at 2853 cm−1 ( CH3 stretching), 2921 cm−1 ( CH2 stretching), 1461 cm−1 (methylene

C H bending) and 1380 cm−1 (C O stretching). A molecular ion peak was observed at m/z 252 corresponding to C16 H28 O2 + and a base peak at 55 corresponding to C4 H7 + . Other fragmentation peaks were observed at m/z, 67, 81, 95, 107, 121, 149, 163 and 192 corresponding to various fragments as listed Table 2. Possible fragmentation scheme in accordance with the observed mass spectral lines for the compound is shown in Fig. 3. C was a colourless, sticky solid and its IR spectrum gave prominent bands at 3770 cm−1 corresponding to the amide (␯N H ), 2925 and 2845 cm−1 due to ␯C H ( CH2 ), 1680 cm−1 due to carbonyl (>C O) group and the bands at 3424 and 1620 may be ascribed to benzene ring as listed in Table 3. This compound gave a molecular ion peak at m/z 211 and base peak at m/z 104 corresponding to C11 H14 ONCl+ and C8 H8 + respectively. Other prominent fragmentation lines were observed at m/z 175, 148, 120, 105, 104, 91, 77, 63, 65, 51 and 49 and their assignments are listed in Table 3. Recent reports have suggested that Vimang of stem bark of mango exhibits anthelminthic and anti-allergic activities [16] which in turn may be correlated with their organic constituents that may help in developing an understanding its pharmacological action.

Table 3 Infrared spectral and GC–MS fragmentation assignments for organic constituent C.

3.2. Antimicrobial assay

O

Cl

NH

. IR spectral band

Fragments from GC–MS −1

Wave numbers (cm 3770 2925 2845 1680 3424 1620

)

Assignment

m/z

Assignments

␯N ␯C ␯C ␯C ␯C ␯C

211 213 175 148 120 105 104 91 77 63 51

C11 H14 ONCl+ C11 H14 ONCl+ C11 H13 ON+ C9 H10 ON+ C8 H10 N+ C8 H9 + C8 H8 + C7 H7 + C6 H5 + C2 H4 Cl+ CH2 Cl+

H H H

( CH2 ) ( CH2 )

O H C

(aromatic) (aromatic)

Antimicrobial activities of the HEX and MOH extracts were evaluated against human pathogens Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus subtilis, Salmonella typhi. Tetracycline was employed as a positive standard. It is observed that the antibacterial activity of plant extracts increased with increasing concentration of crude extracts. The extracts showed prominent antibacterial activity against gram negative (S. typhi, E. coli, P. aeruginosa) and gram positive (S. aureus, B. subtilis) bacteria. Among the tested plant extracts, HEX extract of M. indica showed highest antibacterial activity of 16 mm inhibition zone against P. aeruginosa as shown in Table 4. Minimum inhibition concentration (MIC) of HEX extract against the tested organisms varied between 1.25 mg/mL and 2.50 mg/mL while that of MOH extract ranged between 3.26 mg/mL and 4.88 mg/mL. The standard tetracycline exhibited MIC values varying between 0.013 mg/mL and 0.50 mg/mL. The results indicated that standard antibiotic tetracycline has stronger activity than the two plant extracts as shown in Table 5. The antimicrobial activity of the extracts may be attributed to the presence of phytoconstituents such as triterpenoids, flavonoids and tannins. It is well reported in literature that the plants containing triterpenoids exhibit

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R. Singh et al. / Journal of Pharmaceutical and Biomedical Analysis 105 (2015) 150–155

H 3C

H3C

CH HC

CH3

CH2 H2 C

HC C H CH3

C H2

H2 C

C H2

H2 O C C C H 2 H2

CH2

CH

HC

C H

CH2 HC

C H

149

CH3

H C C H

CH

CH

HC

H2 C C H

121

HC

HC

CH2

C H

CH

CH2 HC

H C C H

107

55

CH2

HC

67

C H

95

CH2

CH3

CH C H

CH2 HC

CH

CH3 H C

CH

163

CH3

CH3

CH

H 3C

192

CH HC

HC

CH2 HC C H

CH3

252

CH

HC

H2 C

HC

O

C

CH

CH HC

CH CH

CH2 HC CH

81 Fig. 3. Fragmentation scheme of organic constituent B in accordance with the mass spectral lines.

antimicrobial activity [17]. Cushnie and Lamb [18] have reviewed the antimicrobial activity of flavonoids. Also tannins extracted from Rhizophora appicurata bark have shown antimicrobial activity [19]. Thus, the antibacterial activity of the extracts may be attributed to the presence of various phytoconstituents present in the extracts. 3.3. Elemental contents INAA data of mango wood shows major constituents of K (2.68 ± 0.05%), Ca (1.72 ± 0.12%) and Cl (0.21 ± 0.0.01%) including trace amounts of Mg (520 ± 80 ␮g/g), Na (313 ± 16 ␮g/g), Fe (237 ± 21 ␮g/g), Mn (38.9 ± 1.1 ␮g/g), Zn (33.9 ± 2.1 ␮g/g), Cr (0.97 ± 0.07 ␮g/g), Cu (2.39 ± 0.39 ␮g/g) and V (1.72 ± 011 ␮g/g). These were all comparable with those of neem bark [11] which

Table 5 Minimum inhibitory concentration (MIC) of plant extracts. Bacterial species

MIC (mg/mL)

Escherichia coli Staphylococcus aureus Pseudomonas aeruginosa Bacillus subtilis Salmonella typhi

Hexane extract

Methanol extract

Tetracycline

1.25 1.40 1.00 2.50 2.0

4.88 3.56 3.26 4.20 4.63

0.031 0.025 0.013 0.50 0.25

is also widely used in Indian medicine system. Besides ultra trace amounts of Se (90 ± 10 ng/g), Co (57.5 ± 3.5 ng/g), Hg (59.7 ± 1.3 ng/g) and As (230 ± 20 ng/g) were also observed. It is well reported that several mineral elements are nutritionally essential whereas Hg and As are environmental pollutants [20].

Table 4 Zone of inhibition (in mm) of bacterial species in two M. indica extracts. Hexane extract of M. indica (mg/mL) Bacterial species Escherichia coli Staphylococcus aureus Pseudomonas aeruginosa Bacillus subtilis Salmonella typhi

10 9 11.5 12 10 9

20 11.5 12 12.5 11.5 10

30 13 13 14 12 11

Methanol extract of M. indica (mg/mL) 40 14.4 14 14.7 13 12

50 15.5 15 16 15.3 12.8

10 8.2 9 8 10 11

20 9 9.8 9 11 11.5

30 10.4 11 10 11.5 12.3

40 11 12 11.2 12.2 13

50 12 13.9 12 13 14.1

R. Singh et al. / Journal of Pharmaceutical and Biomedical Analysis 105 (2015) 150–155

Since mango wood is enriched in Ca, Fe, Mn, Zn etc., its medicinal properties may be attributed to these elements which may remain complexed with organic ligands to make them bio available and cure ailments. 4. Conclusion Three new compounds have been separated from methanol and hexane extracts of mango wood by thin layer and column chromatography and identified by IR and GC–MS studies. One of these is reported to be antimalarial and another one is used for weed management. Both the extracts exhibit antimicrobial activity which may be exploited for developing new antibiotics. Elemental analyses by INAA suggest the wood to be enriched in Ca, K, Mn, Fe, and Zn all essential nutrients. It is suggested that these elements may remain complexed with organic ligands enabling them to be bioavailable to the human system. Acknowledgements The authors gratefully acknowledge Professors M.R. Maurya and R.K. Dutta, Department of Chemistry, Indian Institute of Technology, Roorkee for providing laboratory facilities. We also thank Dr R. Acharya, Radiochemistry Division, BARC, Mumbai for irradiation and counting facilities. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.jpba.2014.12.010. References [1] C.J. Barreto, M.T.S. Trevisan, E. Hull, G. Erben, S.E. De Brito, B. Pfundstein, G. Wurtele, B. Spiegelhaler, W.R. Owen, Characterization and quantization of polyphenolic compounds in bark, kernel, leaves and peel of mango (Mangifera indica L.), J. Agric. Food Chem. 56 (2008) 5599–5610. [2] F.R. Ferriera, I.B. Valentim, E.L.C. Ramones, M.T.S. Trevisan, C.O. Azar, F.P. Cruz, F.C. Abreu, M.O.F. Goulart, Antioxidant activity of the mangiferin inclusion complex with ␤-cyclodextrin, LWT-Food Sci. Technol. 51 (2013) 129–134.

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