ISOLATION, SPECTROSCOPIC CHARACTERIZATION OF TWO NOVEL COMPOUNDS OBTAINED FROM LEAF OF SYZYGIUM...

www.wjpps.com

2818

P.Mishra et al. World Journal of Pharmacy and Pharmaceutical Sciences

ISOLATION, SPECTROSCOPIC CHARACTERIZATION OF TWO

NOVEL COMPOUNDS OBTAINED FROM LEAF OF SYZYGIUM

CUMINI (JAMBOLANA) AND ITS STUDY THE

ANTIBACTERIAL ACTIVITY

A.K.Das, N.K. Chaudhary and P.Mishra*

Bioinorganic and Materials Chemistry Research Laboratory, Tribhuvan,

University, M.M.A.M. Campus, Biratnagar. Nepal.

ABSTRACT

Jamun is a very common, large evergreen beautiful tree of Indian

subcontinent. The scientific name of Jamun is Syzygium cumini or

Eugenia jambolana Linn belongs to the family myrtaceae. Syzygium

cumini (S. cumini) (L.) Skeels (jambolan) is one of the widely used

medicinal plants in the treatments of various types’ deseases.The

leaves of Syzygium cumini is considered as an antibacterial and also

used to strengthen the teeth and gums in folklore medicine. In present

investigation, the detailed pharmacognostic study of Syzygium cumini

leaf is carried out to lay down the standards which could be useful in

future experimental studies. The study also includes the antibacterial

activities of isolated novel compounds. The compounds were

characterized by spectral techniques and structure were optimized by

MM2 programme.

Keywords: Jambolana, syzygium cumini, novel, pharmaconostic, optimized, MM2.

1. ITRODUCTION

Herbal medicine or phytomedicine refers to the use of any plant seeds, berries, roots, leaves,

bark or flowers for medicinal purposes .Plants are used medicinally in different countries and

are a source of many potent and powerful drugs [Anand H. et.al, 2013]. Herbal medicines are

promising choice over modern synthetic drugs. They show minimum or no side effects and

are considered to be safe. Generally herbal formulation involves the use of fresh or dried

WWOORRLLDD JJOOUURRNNAALL OOFF PPHHAARRMMAACCYY AANNDD PPHHAARRMMAACCEEUUTTIICCAALL SSCCIIEENNCCEESS

VVoolluummee 22,, IIssssuuee 55,, 22881188--22883300.. RReesseeaarrcchh AArrttiiccllee IISSSSNN 2278 – 4357

Article Received on 23 July 2013,

Revised on 20 August 2013, Accepted on 16 September

*Correspondence for

Author:

* Dr P. Mishra

Bioinorganic and Materials

Chemistry Research

Laboratory, Tribhuvan,

University, M.M.A.M.

Campus, Biratnagar, Nepal.

[email protected],

www.wjpps.com

2819


plant parts. Knowledge of such crude drugs is very important aspect in preparation, safety

and efficacy of the herbal product. Pharmacognosy is a simple and reliable tool, by which

complete information of the crude drug can be obtained [Andre Gordon

et.al.2011].Phytochemical may protect human from various diseases. Phytochemicals are

nonnutritive plant chemicals that have protective or disease preventive properties.

Phytochemicals are basically divided into two groups that are primary and secondary

metabolites according to their functions in plant metabolism. Primary metabolites comprise

of common sugars, amino acids, proteins and chlorophyll while secondary metabolites

consist of alkaloids, flavonoids, tannins and so on.

Syzygium cumini (L.) is belonging to the family Myrtaceae [Ayyanar et.al.2013]. Large trees

cultivated throughout Indian subcontinent including subtropical belt of Nepal for the edible

fruits (Black Plum) and are reported to contain vitamin C, gallic acid, tannins, anthocyanins,

includes cyanidin, petunidin, malvidinglucoside and other components [Baliga MS et.al

2013]. The juice of unripe fruits is used for preparing vinegar that is considered to be a

stomachic, carminative and diuretic. The ripe fruits are used for making preserves, squashes

and jellies. The fruits are astringent. A wine is prepared from the ripe fruits in different part

of Indian subcontinent. Leaves have been used in traditional medicine as a remedy for

diabetes mellitus in many countries as ethanomedicin.The leaves are also used to strengthen

the teeth and gums, to treat leucorrhoea, stomachalgia, fever, gastropathy, strangury,

dermopathy [Bandhopadya SS et.al 2012], constipation and to inhibit blood discharges in the

faeces [Chauthe et.al. 2012]. The berry is oblong in shape with dark purple skin and whitish

aril. The major anthocyanin being found in the berries are cyanidin-diglucoside, ptunidin-

diglucoside, delphinidin-diglucoside and malvidin-diglucoside [Devlar R.V. et.al.2012]. It

has been reported to possessed several bioactivities against ailment of diabetic. In the present

study, isolation spectral study of isolated compounds and determination of bimetals available

in leaves of Syzygium cumini (jambolana).

2. MATERIALS AND METHODS

All the chemicals used in this project were of analytical grade which obtained from Merck.

The leaf of syzygium cumini(jambolana) were collected from Botanical garden of

Department of Botany,M.M.A.M.Campus ,Biratnagar.The leaf were dried at laboratory

temperature in dark cabinet for moisture freed from the leaves then followed the following

www.wjpps.com

2820


procedure[Raghavendra et.al]. The stoichiometric analyses(C, H and N) of the complexes

were performed using Elementar vario EL III (Germany) model. Metal contents were

estimated on an AA-640-13 Shimadzu flame atomic absorption spectrophotometer in solution

prepared by decomposing the respective complex in hot concentrated HNO3. Their IR spectra

were recorded on Perkins–Elmer FTIR spectrophotometer in KBr and polyethylene pellets. 1H NMR spectra were recorded in DMSO-d6 solvent on a Bruker Advance 400 instrument

.The structure were optimized by MM2 computer software programme.

Extraction procedure

2.1. PREPARATION OF CRUDE EXTRACTS

The air-dried leaves of S. cumini were pulverized into powdered form. The dried powder (0.5

g) was extracted by soaking with methanol (Me-OH), and methylene chloride (Me-Cl)

separately using orbital shaker for 48 hrs at room temperature [Ghosh A.et.al 2013] T.he

extracts were filtered through Whatman No.1 filter paper. Residues were re-extracted twice

with fresh aliquots of the same solvents. Solvents from the combined extracts were

evaporated using a vacuum rotary evaporator [Ibrahim et.al.2001]. After solvent evaporation

in a rotary evaporator, the methanolic extract (ME) was further fractionated through solvent–

solvent partitioning to obtain different fractions. The four solvents used for solvent–solvent

partitioning to cover the range from high to low polarity were water, ethyl acetate,

chloroform, and n-hexane. The ME and its four water (WtF), ethyl acetate (EaF), chloroform

(CfF) and n-hexane (HxF) fractions were stored in an electronic dry cabinet protected from

light with aluminum foil after solvent evaporation and the resulting residues were used for the

analyses outlined below.

2.3. HPLC ANALYSIS OF PHENOLIC COMPOUNDS

The contents of phenolic compounds in leaf extracts of S. cumini were determined by HPLC,

performed with an Agilent 1100 diode array detector system equipped with a quaternary

pump. The analyses were carried out on a Hypersil ODS column (4.6 mm × 250 mm, 2.5 µm)

column. Extracts were filtered through a 0.45 µm filter before use. Gradient B in A according

to the elution profile 0-3 min 2% B (isocratic), 3-20 min 2%-25% B (linear gradient), 20-25

min 25%-35% B (linear gradient), (A) water (0.05% TFA), (B) CH3CN (0.05% TFA); flow

rate 1 mL/min; volume injected 10 µL; temperature 22 °C; detection 280 nm.The extracted

samples were collected for spectral analysis.

www.wjpps.com

2821


2.4. ISOLATION OF BIOMETALS

Leaf sample(10) gram dried were ignite in Muffle furnace at 4000C and then collect white ash

in a beaker treated with concentrated nitric acid(10 ml) diluted in 250 ml volumetric flask for

analyze metal ion by AAS PerkinElmer.

3.0. RESULTS AND DISCUSSIONS

The present study was carried out on the leaf samples revealed the presence of novel

chemical constituents. Satisfactory results of elemental analysis (Table 1) and spectral studies

revealed that the complexes were of good purity. Various attempts to obtain the single

crystals have so far been unsuccessful. X-ray diffraction studies indicate crystalline nature of

the isolated compounds. The complexes were soluble in polar solvents.

Table 1.Color, reaction yield and elemental analysis of complexes

Empirical formula Color Yield

(%)

Analysis: found (calculated)(%)

C H N M.P.0C

C43H42N2O19

Molecular Weight:

890.80

white 65 57.27 4.86 3.15 35

(57.98) ( 4.75 ) (3.14)

C71H64N4O18S

Molecular Weight:

1293.35

Pale

yellow

60 65.91 4.97 4.34 S=2.14 65

65.93 4.99 4.33 S =2.48

Table 2. For metals

SN Parameters method Unit Results

1 Iron(Fe) AAS mg/100gm 15.45

2. Sodium(Na) AAS mg/100gm 307.21

3 Potassium(K) mg/100gm 72.05

4 Zinc(ZN) AAS mg/100gm 4.25

5 Total ( Cr) AAS mg/100gm 0.75

6 Arsenic(As) AAS mg/100gm <0.005

7 Cobalt(Co) AAS mg/100gm <0.005

www.wjpps.com

2822


3.1. ASSIGNMENT OF VIBRATIONAL SPECTRA

Novel isolated compounds absorptions ν[( 3180 cm-1NH stretchingAr),3019 aromatic

ring,2855 aldehyde,1668 CO stretching 1591, 10amine and C-C aromatic ring,1511,130 OH

group,1153 NH aromatic amine,1200 CO stretching]cm-1 and 1135 glycosidic linkage for

compound 1. ν[3330(m),3029(m) CH alkene stretching,1686 (s)C=O conjugated

ring,1521(s)N-O,1351(s)OH,C-SH1430,1130 C-O-C ] cm-1glycoside linkage for compound

2.

Figure 1.FTIR spectra novel compound1 [C43H42N2O19 ]

Figure 2.FT-IR spectra of novel compound 2.[ C71H64N4O18S]

www.wjpps.com

2823


3.2. 1H N M R

The 1HNMR spectra of the isolated compounds in a DMSO-d6 solvent of the compounds

show well-resolved signals Figure 3 and 4:1HNMR spectrum of complex (I).The N-H protons

of amine, which would have undergone very rapid exchange with the solvent, appear as quite

broad ragged singlet doublet multiplet and t he various assignments of 1HNMR of the

compounds are summarized in table3.Chemical shift are in ppm from TMS & multiplicity in

parentheses (bd, broad; d, doublet; m, multiplet).

Table 3.1HNMR of compounds

Compounds δ (ppm)

C43H42N2O19 [3.0(s),15 1H,OH,1.58-16(d).7,AR-OH COOH(m),10 1H(m),2H,2.15-6.88,Ar-

H(d),7H,1.29-2.64(bd)2H7.357.5 ArH]

C71H64N4O18S 8HOH3.58-16.77(s),23HCH3.65-6.58(m),2HCH2,199(s),2H-CH310.96-1.18(m)

Figure 3.1HNMR Compound 1. [C43H42N2O19 ]

Figure 4.Compound 2[C71H64N4O18S]

www.wjpps.com

2824


3.3. TOF–MS SPECTRA

Mass spectrometry has been successfully used to investigate molecular species[MH]+ in

solution .The molecular ion peaks of the compound have been used to confirm the proposed

formula .The pattern of the mass spectrum gives an impression of the successive degradation

of the target compound with the series of peaks corresponding to the various fragments. Their

intensity gives an idea of stability of fragments. The fragmentation of compounds m/z:

890.24 (100.0%), 891.24 (48.5%), 892.24 (14.8%), 893.25 (3.5%) for compound 1 and m/z:

1292.39 (100.0%), 1293.40 (78.2%), 1294.40 (34.5%), 1295.40 (11.1%), 1294.39 (5.7%),

1295.39 (3.6%), 1296.40 (2.7%), 1293.39 (2.3%), 1296.41 (1.5%) for compound 2.The mass

spectra is given below:

Figure 5.Mass spectra of compound1

Figure 6.Mass spectra of compound2

www.wjpps.com

2825


3.4. 3D - MOLECULAR MODELING

3D molecular modeling of the proposed structure of the complexes was performed using

CsChem3D Ulta -11 program package. The correct stereochemistry was assured through the

manipulation and modification of the molecular coordinates to obtain reasonable low energy

molecular geometries. The optimized structures of the complexes were performed by MM2

programme contained CS chem. Office programme. The potential energy of the molecule was

the sum of the following terms: E = Estr + Eang + Etor + Evdw + Eoop + Eele.Where all E’s

represent the energy values corresponding to the given types of interaction. The subscripts

str, ang, tor, vdw,oop and ele denote bond stretching, angle bonding, torsion deformation, van

der waals interactions, out of plain bending and electronic interaction, respectively[Lawrence

et.al. 2013].From the above discussion the structure of isolated compounds obtained from

leaf of jambolana to be proposed as

Figure7: Optimized structure of compound 1. [Steric energy 21.23 kcal]

www.wjpps.com

2826


Figure8: Asymmetry structure of (3S,6S)-2-((3R,4R,5S,6R)-6-(6,10-diamino-3,7,9,11-

tetrahydroxy-13,15-dioxo-5a,8a,12a,13,14a,15-hexahydroxantheno[2,3-b]xanthen-1-

yloxy)-4-hydroxy-5-methyltetrahydro-2H-pyran-3-yloxy)-5-hydroxy-6-

methyltetrahydro-2H-pyran-3-yl 3,6-dihydroxy-4-oxocyclohexa-1,5-dienecarboxylate

Figure 9: Optimized structure of compound 2.[Steric energy 35.564 kcal]

www.wjpps.com

2827


Figure 9 : Asymmetric structure of (2S,3R,4S,5S,6S)-2,3-diamino-6-(5-(14a-carboxy-

1,6,12,18-tetrahydroxy-19-mercapto-1,2,9,13-tetramethyl-17-oxo-1,10b,14a,16a,17,22a

hexahydroanthra[2,1-a]benzo[l]pentacen-21-yloxy)-3-(2,4-diamino-5-hydroxy-3-

methylphenyl)-8-methyl-9-oxo-9,9a-dihydro-4aH-xanthen-1-yloxy)-2,4,5-trihydroxy-5-

methyltetrahydro-2H-pyran-3-carboxylic acid.

3.5. ANTIMICROBIAL EVALUATION OF COMPLEXES

Antibacterial activity was carried out for the synthesized complex as well as the parent drug.

Table 3 shows the MIC value for the synthesized compound MC novel compounds and

physical admixture against various Gram-positive as well as Gram-negative micro organisms

(Fig. 6). A significant increase in antibacterial activity (p < 0.05 Tukeys test) was observed

for BC. As the MIC value for ligands as well as M-Physical admixture was similar or nearly

the same (p > 0.05 Tukeys test), clearly indicating that the isolated compounds resulted in a

significant increase in the activity of nove compounds. Transport of quinolones across the

bacterial cytoplasm membrane is strongly pH dependant [Omar R. et.al 2012,2013]

Uncharged quinolone species are responsible for diffusion through cytoplasm membranes

BC (2), remaining an unionized species at neutral or higher pH values would be transported

into higher concentration in the microorganisms leading to lowering of MIC of BC in

comparison to compounds physical mixture member of studies have indicated the

antimicrobial activities of many heavy metals due to their effects on iron uptake by bacteria .

Iron is a co-factor for many essential enzymes [Shrivastava S. 2013] Potent antimicrobial

activity of BC would result out of a combination of higher transport of the complexes through

www.wjpps.com

2828


the cell membrane and iron limitation into the cells of the bacteria [Safdar ME

et.al.2013].Table8.. MIC values (mg/l) for (a) compound 1(b) compound 2

Table.4. MIC values (mg/l) for (a) compound 1(b) compound 2

Organism used (stain no.) Minimum inhibit concentration in mg/l ±standard division P-value(Turkeys test)

Compound 1 Compound 2

Escherichia coli(ATCC25922) 0.041 ± 0.004 0.0256 ± 0.00789 0.021

Klebsiells pneumonia(NTCC 10320)0.071± 0.002885 0.071±0.0043 0.025

Bacillus(NTCC8241) 0.0251±0.004 0.035 ±0.0432 0.03

S.aureus(ATCC8241) 0.061±0.00678 0.0457±0.00564 0.036

Staphylococcus epidemidis ( ATCC 12228(0.034±0.0125 0.0231±0.000456 0.142

CONCLUSIONS

Jambolan is widely used by the traditional healers for the treatment of various diseases

especially diabetes and related complications. The plant has many important compounds

which confer the most of the characteristics of the plant. Most pharmacological works on

diabetes were carried out with seeds but the pharmacological potential of the other parts of

the plant is required to explore in detail. Similarly, not many works are there with

pharmacological actions of phytochemical constituents of jambolan. Based on these facts, the

authors hope that this review highlights the role of jambolan in various treatments and

recommend that further phytochemical and clinical research should be done on this

traditional medicinal plant for the discovery of safer drugs. The leaves of syzygium cumini

are using as ethanomedicine for the ailment of diabetes, gastronomical and control blood

pressure as well as external ointment.

ACKNOWLEDGMENT

The authors would like to acknowledge USIC University of Delhi and SEAMN-MMA

Environmental Laboratory for the analytical support.

www.wjpps.com

2829


REFERENCES

1. Anand H, Misro MM, Sharma SB, Prakash S.,Cytoprotective effects of fruit pulp of

Eugenia jambolana on H 2 O 2 -induced oxidative stress and apoptosis in rat Leydig cells

in vitro.Andrologia. 2013 Jun;45(3):145-57. doi: 10.1111/j.1439-0272.2012.01323.x.

2. Anand H, Misro MM, Sharma SB, Prakash S. RNA as a tool to delineate pathway

channelization in H(2)O(2) induced apoptosis of primary Leydig cells in vitro.Apoptosis.

2012 Nov;17(11):1131-43. doi: 10.1007/s10495-012-0749-7.

3. André Gordon, Elvira Jungfer, Bruno Alexandre da Silva, José Guilherme S. Maia, and

Friedhelm Marx , Phenolic Constituents and Antioxidant Capacity of Four Underutilized

Fruits from the Amazon Region, J. Agric. Food Chem., 2011, 59 (14), pp 7688–7699

4. Ayyanar M, Subash-Babu P, Ignacimuthu S.,Syzygium cumini (L.) Skeels., a novel

therapeutic agent for diabetes: folk medicinal and pharmacological evidences.

Complement The J. Med. 2013 Jun;21(3):232-43. doi: 10.1016/j.ctim.2013.03.004.

5. Ayyanar M, Subash-Babu P.Syzygium cumini (L.) Skeels: A review of its phytochemical

constituents and traditional uses. Asian Pac J Trop Biomed. 2012 Mar;2(3):240-6. doi:

10.1016/S2221-1691(12)60050-

6. Baliga MS, Fernandes S, Thilakchand KR, D'souza P, Rao S.,Scientific validation of the

antidiabetic effects of Syzygium jambolanum DC (black plum), a traditional medicinal

plant of India.J Altern Complement Med. 2013 Mar;19(3):191-7. doi:

10.1089/acm.2011.0752.

7. Bandyopadhyay SS, Ghosh D, Micard V, Sinha S, Chatterjee UR, Ray B.,Structure,

fluorescence quenching and antioxidant activity of a carbohydrate polymer from Eugenia

jambolana.Int J Biol Macromol. 2012 Jul-Aug;51(1-2):158-64. doi:

10.1016/j.ijbiomac.2012.04.004.

8. Chauthe SK, Sharma RJ, Aqil F, Gupta RC, Singh IP.,Quantitative NMR: an applicable

method for quantitative analysis of medicinal plant extracts and herbal products.,

Phytochem Anal. 2012 Nov-Dec; 23(6):689-96. doi: 10.1002/pca.2375.

9. Devkar RV, Pandya AV, Shah NH.,Protective role of Brassica olerecea and Eugenia

jambolana extracts against H₂O₂ induced cytotoxicity in H9C2 cells. Food Funct. 2012

Aug;3(8):837-43. doi: 10.1039/c2fo00001f.

10. Ghosh A, Jana K, Ali KM, De D, Chatterjee K, Ghosh D., Corrective role of Eugenia

jambolana on testicular impairment in streptozotocin-induced diabetic male albino rat: An

www.wjpps.com

2830


approach through genomic and proteomic study.Andrologia. 2013 Mar 24. doi:

10.1111/and.12081.

11. Ibrahim I Mahmoud, Mohamed S.A Marzouk, Fatma A Moharram, Mohamed R El-

Gindi, Amel M.K Hassan, Acylated flavonol glycosides from Eugenia jambolana leaves

Original, Phytochemistry, Volume 58, Issue 8, December 2001, Pages 1239-1244

12. Lawrence O. Arot Manguro, Samuel Otieno Wagai, Peter Lemmen Flavonol and iridoid

glycosides of Ajuga remota aerial parts ,Phytochemistry, Volume 67, Issue 8, April 2006,

Pages 830-837.

13. Lidiane Silva, Evelyn D., Jairo P., E.C.de A. Bessa, Effect of Solanum paniculatum leaf

extract on food consumption, fertility and carbohydrate metabolism of Bradybaena

similaris snail. Journal of Natural Products,6:168-176, (2013):

14. Omar R, Li L, Yuan T, Seeram NP.,α-Glucosidase inhibitory hydrolyzable tannins from

Eugenia jambolana seeds.J Nat Prod. 2012 Aug 24;75(8):1505-9.

15. Raed Omar, Liya Li, Tao Yuan, and Navindra P. Seeram, α-Glucosidase Inhibitory

Hydrolyzable Tannins from Eugenia jambolana Seeds, J. Nat. Prod., 2012, 75 (8), pp

1505–1509.

16. Raghavendra BS, Prathibha KP, Vijayan VA.,Synergistic effect of Eugenia jambalaya

Linn. and Solidago canadensis Linn. leaf extracts with deltamethrin against the dengue

vector Aedes aegypti Linn. at Mysore.Environ Sci Pollut Res Int. 2013 Jun;20(6):3830-5.

doi: 10.1007/s11356-012-1308-z.

17. Rizvi SI, Mishra N.,Traditional Indian medicines used for the management of diabetes

mellitus.,J .Diabetes Res. 2013;2013:712092. doi: 10.1155/2013/712092.

18. Russell J. Molyneux, Stephen T. Lee, Dale R. Gardner, Kip E. Panter, Lynn F. James,

Medicinal chemistry and pharmacology of genus Tripterygium (Celastraceae)

,Phytochemistry, Volume 68, Issue 6, March 2007, Pages 732-766

19. Safdar ME, Tanveer A, Khaliq A, Naeem MS, Ahmad S.,Tree species as a potential

source of bio-herbicides for controlling Parthenium hysterophorus L.Nat Prod Res. 2013

Aug 20.

20. Srivastava S, Chandra D.,Pharmacological potentials of Syzygium cumini: a review. J Sci

Food Agric. 2013 Jul;93(9):2084-93. doi: 10.1002/jsfa.6111. Epub 2013 May 15.

ISOLATION, SPECTROSCOPIC CHARACTERIZATION OF TWO NOVEL COMPOUNDS OBTAINED FROM LEAF OF SYZYGIUM...

Documents

Transcript of ISOLATION, SPECTROSCOPIC CHARACTERIZATION OF TWO NOVEL COMPOUNDS OBTAINED FROM LEAF OF SYZYGIUM...