INTRODUCTION:

The plant Syzygium cuminii (l) skeel belonging to the family Myrtaceae, native to India (Kirtikar,1975). Commonly it is known as “kala jamun” tree. This indigenous form of medicine uses the active ingredients present in plants for treating diseases (Lewis and Elvin-Lewis, 1977). The jamun tree which is native to India, thrives easily in tropical climates and is found in many parts of our subcontinent. It is also found in South East Asia and Eastern Africa. It is a large evergreen tree which grows widely in the Indo-genetic plains and also in the Cauvery delta of Tamil Nadu, the report showing that oral administration of dried alcoholic extract of the seeds are having anti-hyperglycemic effect and also reduces glycosuria . Thus the available report shows that, very little have been carried on this plant’s root (Indira and Mohan Ram, 1992, Samba-Murthy and Subrahmanyam, 1989). The literature survey emerges, seeds have been used by natives in the treatment of diabetes (Chopra, R.N, 1958). The decoction of the dry leaves of Syzygium cuminii (l) skeel intuitive an anti-hyperglycemic effect (Coimbra, T.C,1992, Mahapatra, P.K. 1985).

The preliminary studies on the anti-hyperglycemic effect of seeds have been found that, they produce hypoglycaemia (Mahapatra, P.K. 1985). The Syzygium cuminii (l) skeel belonging to the family Myrtaceae have been attributed in the Indian folklore medicine system, to possess several medicinal properties (Warrier, P.K, 1996). The bark of the plant is astringent, sweet, refrigerant, carminative, diuretic, digestive, antihelminthic, febrifuge, constipating, stomachic and antibacterial. The fruits and seeds are used to treat diabetes, pharyngitis, spleenopathy, urethrorrhea and ringworm infection. Leucorrhoea, stomachalgia, fever, gastropathy, strangury, and dermopathy (Bhandary, M.J, 1995). It also inhibits the blood discharges in the feces (Rastogi, R.M.1990). The plant possesses acetyl oleanolic acid, triterpenoids, ellagic acid, isoquercitin, quercetin, kaempferol and myricetin in different concentrations (Nikhat F et al 2008). The chemical composition and screening of anti-hyperglycemic activity have been observed, but there is no information about the screening of anti-hyperglycemic activity on isolated molecules and also there is no spectroscopic evidence on this plant. This study is unique in its research finding.

Synonyms of the plant

Hindi : Bahajamana, Jamun

English : Black plum , Jambol, Java plum

Kannada : Nerale

Sanskrit : Surabhipatra

RESULTS AND DISCUSSION.

The crude methanolic extract (8 gm) from the roots of Syzygium cuminii (l) skeel was investigated by chromatographic technique to yield two new compounds:

Fig 1. Structure of a compound 1 and 2 from Syzygium cuminii (l) skeel

Myrecetine glycoside ,([1], code ScReX-5 ) chemically designated as 5-((3,4-dihydroxy-5-(((3,4,5-trihydroxytetrahydro-2-furanyl)methoxy)methyl) tetrahydro -2-furanyl)methoxy)-3,7-dihydroxy-2-(3,4,5-trihydroxyphe nyl)-4H-4-chromenone. ([2], ScReX-6b code) Myrecetine gentobioes, chemically designated as 2-(3,4-dihydroxy-5-((3,4,5-trihydroxy-6-(((3,4,5,6-tetrahydroxy tetrahydro-2H-2-pyranyl)methoxy)methyl)tetrahydro-2H-2pyranyl) methoxy) phenyl)-3,5,7-trihydroxy-4-oxo-4H-chromenium, respectively. Their structures were elucidated by spectroscopic data, especially using NMR and Mass spectroscopic data.The new compounds showed UV absorption band at λmax 251-256 (compound 1), and second compound showed the value at λmax 321-300.

Compound 1. Myrecetine glycoside, was obtained as Cream colour solid. Its molecular formula was determined as C₂₆H₂₈O₁₆ by Fab-Mass at [M⁺] m/z 661 fragmentation peak at m/z 599 (30%), 397 (90%). The IR spectrum showed absorption band at 3412cm^-1 (OH), 2932 cm^-1 to 2362 cm^-1 CH₂ and CH₃ stretching of alkane, 1690cm^-1 (C=O group) . The UV spectrum displayed the absorption band at 251,256 indicating presence of flavon in the compound. The ¹H NMR spectrum showed the signals at [ δ_H 0.70 to δ 0.99 -C-R groups of CH₃(methyl) proton(s) J=3.37 Hz H-16, δ 1.02 to δ1.98-C,-O-C,-C=C of α and β CH₃ CH₂ tetrahydropyran and cyclohexane proton(s) J=2.93 Hz H-23, δ 2.00 to δ 2.84 -C,-C=C of α and β CH from cyclohexene proton(s) 3.49 Hz 19 H, δ 3.036 to δ 3.93- O,-C, -O-C,-O-C=C tetrahydropyran of α and β CH and OH proton(s) 3.17 Hz 6 H, δ 7.26 to δ 7.36 -O,-C, -O-C,-O-C=C of 1-benzene 2.05 Hz 12 H

Fig 2 Fragmentation of the compound on in correlation with 1H NMR

Table-1 Phytochemistry of plant extracts.

S.NO	SOLVENT USED	QTY OF RESIDUE	EXTRACTION METHOD	POSITIVE PHYTOCHEMICAL TEST FOR
1.	Pet. Ether	10g	Successive soxhelet extraction	Steroids +ve
2.	Ethyl acetate	19g	Successive soxhelet extraction	Flavanoids +ve
3.	Methanol	30g	Successive soxhelet extraction	Flavanoids +ve Shinod’s test

Table-2 Phytochemical and Chromatographic characterization

S.NO	CHROMATOGRAPHIC RESULTS OF ME			PHYTOCHEMICAL STUDY OF ELUENT
S.NO	Elution ratio	QTY of elute	Code of elute	Rf-value	Physical state	Physical constant	Confirmation tests
1.	C: M (80:20)	1.5gm	ScReX-5	0.5	Cream colour solid	300 ⁰C	Liebermann buchard
2.	C: M (50:50)	3gm	ScReX-6b	0.42	Pure white crystals solid	350 ⁰C	Flavonoids

Table -3 Effect of Syzygium cuminii (L) skeel, roots extract on fasting blood glucose level in alloxan induced diabetic rats

Animal: Albino Rats; Alloxan: 120 mg/kg, i.p.; Extract: p.o.

Group	Treatment	Fasting blood glucose level (mg/dl)
Group	Treatment	Basal value	4^th day	7^th day	10^th day
A.	Normal Control	100.45 ± 3.81	102.81 ± 2.92	99.31 ± 1.74	103.28 ± 3.45
B.	Diabetic Control (Vehicle)	253.9 ± 5.26 (39%)	277.92 ± 5.04 (36%)	299.9 ±5.40 (33%)	315.42±9.74 (32%)
C.	Alloxan + glibenclamide (10 mg/kg)	160.86 ± 6.92 ( 62% )	140.25 ± 7.06*** (72%)	122.18 ± 6.35*** (39%)	115.13 ± 6.20*** (35%)
D.	Alloxan + Ethanolic extract (250 mg/kg)	157.47 ± 5.33 (62%)	137.22 ± 6.66*** (49%)	114.84 ± 9.96*** (38%)	103.06 ± 9.09*** (32%)
E.	Alloxan + Ethanolic extract (500 mg/kg)	147.07 ± 4.13 (57%)	126.22 ± 3.43*** (45%)	112.44 ± 7.36*** (37%)	109.98 ± 6.32*** (34%)

Values are Mean ± S.E.M; n=6; **P <0.01 ,*** P <0.00 vs Diabetic Control

Table-3.1 Effect of Syzygium cuminii (L) skeel, roots extract on body weight in alloxan induced diabetic rats

Animal: Albino Rats ;Alloxan: 120 mg/kg, i.p.; Extract: p.o.

Group	Treatment	Body weight of the animal (g)
Group	Treatment	Basal value	4^th day	7^th day	10^th day
A.	Normal Control	210.50 ± 2.74	220.0 ± 2.77	220.60 ± 2.79	210.90 ± 2.85
B.	Diabetic Control (Vehicle)	150.49 ± 2.88 (71%)	149.01 ± 2.58(67%)	151.00 ± 2.50(68%)	145.01 ± 1.71(69%)
C.	Alloxan + glibenclamide (10 mg/kg)	206.50 ± 2.84 (98%)	203.00 ± 2.64* (92%)	196.50 ± 2.02* (93%)	192.00 ± 1.90* (91%)
D.	Alloxan + ME (250 mg/kg)	205.50 ± 2.39* (97%)	198.00 ± 2.15* (90%)	191.50 ± 1.89* (86%)	182.50 ± 1.44* (86%)
E.	Alloxan + ME (500 mg/kg)	209.50 ± 2.39* (97%)	203.00 ± 2.15* (90%)	209.50 ± 1.89* (86%)	200.10 ± 1.34* (86%)

Values are Mean ± S.E.M; n=6; **P <0.01 ,*** P <0.00 vs normal Control

Table -4 Effect of treatment of ScReX- 6b on fasting blood glucose level in alloxan induced diabetic rats

Animal: Albino Rats; Alloxan: 120 mg/kg, i.p. ;Extract: p.o.

Group	Treatment	Fasting blood glucose level (mg/dl)
Group	Treatment	Basal value	4^th day	7^th day	10^th day
A.	Normal Control	91.44 ± 2.98	93.64 ± 2.77	96.30 ± 1.99	89.88 ± 2.89
B.	Diabetic Control (Vehicle)	251.90 ± 5.00 (36% )	284.36 ± 5.71( 32% )	289.92 ±5.58( 33% )	284.30 ± 7.29( 31% )
C.	Alloxan + glibenclamide (5 mg/kg)	160.86 ± 5.81 ( 63% )	140.25 ± 7.06*** (49%)	122.18 ± 6.35*** (42%)	105.13 ± 6.20*** (36%)
D.	Alloxan + ScReX- 6b (250 mg/kg)	130.41 ± 5.27 ( 51% )	120.04 ± 6.59** ( 42% )	110.84 ± 7.76*** ( 38% )	99.47 ± 6.19*** ( 36% )
E.	Alloxan + ScReX- 6b (500 mg/kg)	121.63 ± 4.18 ( 51% )	119.12 ± 9.46** ( 42% )	109.99 ± 8.96*** ( 38% )	102.47 ± 9.88*** ( 36% )

Values are Mean ± S.E.M; n=6; **P <0.01, *** P <0.00 vs Diabetic Control

Table -4.1 Effect of treatment of ScReX- 6b on body weight in alloxan induced diabetic rats

Animal: Albino Rats; Alloxan: 120 mg/kg, i.p.; Extract: p.o.

Group	Treatment	Body weight of the animal (g)
Group	Treatment	Basal value	4^th day	7^th day	10^th day
A.	Normal Control	200.93 ± 2.54	202.08 ± 2.09	204.80 ± 2.42	208.64 ± 2.90
B.	Diabetic Control (Vehicle)	201.40 ± 2.61(100% )	170.71 ± 2.87( 85% )	155.34 ± 2.34( 75%)	143.44 ± 1.65( 68%)
C.	Alloxan + glibenclamide (5 mg/kg)	206.50 ± 2.84 ( 103% )	203.00 ± 2.64* ( 100% )	196.50 ± 2.02* ( 98% )	192.00 ± 1.90* ( 92% )
D.	Alloxan + Screx-6b (250 mg/kg)	206.67 ± 2.64 ( 103% )	215.12 ± 2.62* ( 106% )	200.98 ± 1.79* ( 98% )	203.54 ± 1.86* ( 95% )
E.	Alloxan + Screx-6b (500 mg/kg)	199.67 ± 2.64 ( 103% )	200.12 ± 2.62* ( 106% )	201.98 ± 1.79* ( 98% )	203.54 ± 1.86* ( 95% )

Values are Mean ± S.E.M; n=6; **P <0.01 ,*** P <0.00 vs normal Control

Method of isolation

The ScS methanolic extract (ME) was selected Due to the complicated constituents and pharmacological diversities of plants, in vivo bioassay guided fractionation has been effectively applied to screen the biological activities, that contribute important indications for investigating the characteristics of active components(Warrier et al 1996) the ME of ScS selected for chromatographic isolation, the results of isolation were in Table-2

Screening of pharmacological activity for crude methanolic extract

The Alloxan induced diabetic mellitus and insulin deficiency lead to increase in blood glucose level, When Syzygium cuminii (L) skeel roots extract was administered to diabetic rats, hypoglycaemia was observed after 4^th day, the maximum effect have been observed at 7^th and 10^th day of treatments. From the results it is assumed that the root extract could be responsible for stimulation of insulin release and observed restoration of blood glucose level. Furthermore decreased blood glucose lowering effect of the extract in Alloxan induced diabetic rats could also possibly due to increased peripheral glucose utilization.

Oral treatment with crude methanolic extract of ScS roots (250 and 500 mg/kg b.w.) causes’ reduction of blood glucose level in alloxon induced diabetic rat model. The 10 day study parameters as follows for the crude extract (n=6, p<0.00) (-54.41 to –36.63 g/dl, respectively) compared to diabetic control group (Table-3) ( methanolic extract at a dose of 250 mg/kg b.w. and 500 mg/kg b.w. reduced the elevated level of blood glucose from 157.47 to103.06 and 147.07 to110.44 g/dl), respectively. Glibenclamide (3 mg/kg b.w) also produced a significant reduction in blood glucose levels compared to control group (160.86 to115.13 P<0.01).

Screening of pharmacological activity for isolated constituent

The in vivo bioassay guided ME extract was selected by author to isolation and identification the secondary metabolites, on the basis of phytochemical pharmacological and spectroscopic evidence the ScReX-6b was the responsible molecule for antidiabetic activity, the molecule lead to greater degree of hypoglycemic activity when comparable with glibenclamide, 3mg/kg bw a standard hypoglycemic drug, in alloxan induced diabetic rate model at 4^th ,7^th and 10^th days (140.25,122.18 and 105 g/dl P<0.00) on compared with normal. But the excellent results have been observed on oral treatment of ScReX-6b on 4^th, 7th and 10^th days at 250 and 500mg/kg bw (120.09, 110.84 and 99.47g/dl and secondly 119.12, 109.99 and 102.47 g/dl P<0.00) when compared with standard. The results of the study as given in the table (Table-4 )

A marked rise in fasting blood glucose level observed in diabetic control compare to normal control rats. ME of ScS, roots (at 250 and 500 mg/kg) exhibited a dose dependent significant anti hyperglycemic activity on 4th, 7th and 10th day post treatment. The extract dose of 250 mg/kg also caused reduction in blood glucose when compared with reference standard of Glibenclamide. Simultaneously the body weight in alloxan induced diabetic rat was measured which showed significant reduction during 10 days, when compared with normal control group. Alloxan mediated body weight reduction was significantly reversed by the methanolic extract in dose dependant fashion (at 250 500 mg/kg bw).

CONCLUSION:

Diabetes is a global problem and every year a considerable amount of foreign exchange is involved in import of antidiabetic drugs. The utilization of indigenous drug resources with the collaboration of local industry will minimize the expenditure incurred on the purchase of foreign drugs. The extensive literature survey on the economic importance of indigenous plants, efforts should be focused on to identify the active compounds responsible for the anti-hyperglycemic activities from Syzygium cuminii (L) skeel (ScS) belonging to the family Myrtaceae. It can be concluded on the above research parameters that ScReX-6b might possess both pancreatic and extra-pancreatic mechanisms in its antidiabetic action and such apparent dual pancreatic and extra-pancreatic actions of plant’s root molecule, would be more advantageous to the existing oral antidiabetic mono-therapy.

To give a successful ending to this entitled research topic, the authors are curiously curio to develop the Retrosynthetic approach to synthesis the molecule and develop a pharmaceutical dosage form in order to contribute the simple task in the area of research and development.

ACKNOWLEDGEMENT:

It gives me immense pleasure and delightful movement to put into words all my gratitude to the management of KTC and specially Abdul Razak H, the director of AIKTC new Panvel, Mumbai, to listen me and providing me the facilities during the study period and giving a valuable suggestions unknowingly to mould me to become a respectful teacher cum researcher of excellent institution, without their support it is impossible to complete the task.

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Received on 31.07.2013 Modified on 16.08.2013

Asian J. Research Chem. 6(10): October 2013; Page 920-925