ALPHA GLUCOSIDASE INHIBITORY ACTIVITY FROM Syzigium sp.

Vienna Saraswaty

Research Centre for Chemistry, Indonesian Institute of Sciences

Kampus LIPI - Jalan Cisitu Sangkuriang Gedung 50

Bandung 40135 - INDONESIA

Email : vsaraswaty@gmail.com

ABSTRACT

Objective of this research is to investigate alpha glucosidase inhibitory activity from Syzygium species. 20 of syzygium species was extracted using 70% ethanol and evaporated to be tested for its alpha glucosidase inhibitory activity and resulted that S. cumini and S. syzygioides have a high alpha glucosidase inhibitory activity with IC50 value was 8.71 ppm and 7.48 ppm respectively. Further investigation on some parts of S. cumini plant showed that the highest activity was found  in the cortex with IC50 value of  0.45 ppm. Fractionation of ethanolic extract of S. cumini cortex using n- hexane, dichloromethane, ethyl acetate, n-buthanol and water resulted that the highest yield was in water fraction with IC50 value of 8.15 ppm. Phytochemical screening assay showed that ethanolic extract of S. cumini cortex contained terpenoid, phenol, saponin, tannin and flavonoid.

Key words : Alpha Glucosidase, Syzigium,  inhibitor

ABSTRAK

Tujuan dari penelitian ini adalah untuk mengetahui adanya aktivitas alpha glukosidase dari species Syzigium. Sebanyak 20 species Syzigium di ekstraksi dengan menggunakan etanol 70 % kemudian dipekatkan dengan evaporator vakum kemudian di uji aktivitas inhibisi alpha glukosidase. Hasil pengujian aktivitas alpha glukosidase inhibitor menunjukkan bahwa 2 buah spesies Syzigium menunjukkan aktivitas yang tinggi yaitu pada   S. cumini dan S. syzygioides dengan nilai IC50 sebesar 8,71 ppm and 7,48 ppm berturut-turut. Penelitian lebih lanjut pada beberapa bagian tanaman S. cumini menunjukkan bahwa aktivitas tertinggi ada pada bagian cortex dari tanaman S. cumini dengan nilai IC50 sebesar 0,448 ppm. Hasil fraksinasi dari ekstrak etanol dari bagian korteks     S. cumini dengan menggunakan pelarut n- heksan, diklormetan, etil asetat, n- butanol dan air  menunjukkan bahwa rendemen tertinggi ada pada fraksi air dengan nilai IC50 sebesar 8,15 ppm. Hasil analisa fitokimia menunjukkan adanya senyawa golongan terpenoid, phenol, saponin , tannin dan flavonoid pada ekstrak etanol.

Kata kunci : Alpha Glukosidase, Syzigium, inhibitor

INTRODUCTION

Diabetes mellitus (DM) refers to a number of disorders that share the common feature of elevated blood glucose levels. There are two main subtypes of diabetes i.e. type 1, either autoimmune or idiopathic, and type 2, attributable to insulin resistance, insulin secretion defects, or both. Diabetic type 1 is characterized by deficiency of insulin due to destructive lesions in pancreatic b-cells. It occurs typically in young subjects, but may affect people of any age. Meanwhile Diabetic type 2 comprises about 80% to 90% of all cases. Type 2 is a heterogenous, polygenic disorder resulting from interaction between susceptibility genes and lifestyle/environmental factors.^[1]

A kind of enzyme which has a role in diabetes is Alpha Glucosidase (AGIs) enzyme. This enzyme is responsible for the catalytic cleavage of a glycosidic bond in the digestive process of carbohydrates.^[2] It is produced in the small intestine, so the way to work from the inhibitor is delay intestinal absorption of glucose into the blood This prevention will decreased the sugar level in blood.^[3]

Syzygium is a genus of flowering plants that belongs to the myrtle family, Myrtaceae. The genus comprises about 1100 species, and has a native range that extends from Africa and Madagascar through southern Asia east through the Pacific. Some scientific reported that Syzygium species has potent as diabetic such as S. cumini,^[4,5] S. polyanthum,^[6]  and S. cordatum.^[7]

This paper reported alpha glucosidase inhibitory activity from the syzygium species and its fractions.

MATERIAL AND METHODS

Material

Fresh plants was collected from Bogor Botanical Garden (West Java), except S. cumini from Mojokerto (East java). Solvent used for extraction and fractionation process is technical grade solvent (n-hexane, ethyl acetate, dichloromethane, and n- Buthanol,) which were redestillated before use. Alpha glucosidase enzyme and p-nitrophenyl-α-D-glucopyranoside were from SIGMA.Bovine Serum Albumin, Phosphate buffer pH 7.0, Na₂CO₃, and  phytochemical screening reagent were from Merck with p.a grade.

Instrumentation

Instrumentations used in this research are oven blower, eppendorph pipette (2-20 ul and 200-1000 ul), rotary vapour Heidolph (Germany), water bath Memmert, Hitachi U2800 spectrophotometer (Japan), glass ware, macerator, and vacuum pump.

METHODOLOGY

Plants Determination

All plants are determined by Research Centre for Biology at Bogor Botanical Garden, West Java.

Drying process

All fresh plants was dried in blower oven at 40^oC for 3 days then grilled for extraction.^[8,9]

Extraction

All dried plants was macerated using 70% ethanol (3x24 hours) in macerator then the filtrate was evaporated using heidolph rotary vapour at 40^o-50^oC under vacuum.^[8,9]

Fractionation

55,9 grams of S. cumini crude extract was fractionated using liquid-liquid partition. The partition was done using technical grade of n-hexane, dichloromethane, ethyl acetate and n- buthanol as solvent. All fraction then was evaporated using heidolph vacuum rotary vapour at 50^oC.^[10]

Alpha glucosidase inhibition assay

The inhibitory activity of α-glucosidase was performed according to the modified method : 0,25 mg of α-glucosidase enzyme (Saccharomyces sp. From SIGMA) was diluted in Phosphate buffer (pH 7.0) contains 20 mg of Bovine serum albumin. The enzyme solution was diluted 10x before assay. Reagen mixtures are as follow: 250 ul of 20 mM p-nitrophenyl α –D- glucopyranoside and 495 ul of phosphate buffer was mixture with 5 ul of samples in DMSO. The reaction was pre incubated in water bath at 37^oC for 5 minutes, the reaction was begin after addition of 250 ul of enzyme solution and the incubation was continued for 15 minutes. The reaction was stopped by addition of 1 mL 200 mM Na₂CO₃ and the amount of p-nitrophenol resulted was measured using spectrophotometer at λ=400nM. As negative control of samples the mixture of reagent above was used without enzyme and for positive control the mixture of reagent without sample was used. Inhibition activity was calculated using the following formula: [C-S]/C x 100%. Where C is Absorbance of positive control and S is absorbance of sample after reduced by absorbance of negative control of sample. And the IC50 value was defined as the concentration of α-glucosidase  inhibitor that inhibited 50% of α-glucosidase activity.^[8,11,12]

Phytochemical screening

Phytochemical screening assay was performed as described in Harborne.^[8]

RESULT AND DISCUSSION

Alpha glucosidase inhibitory activity of Syzygium Species.

20 Syzigium species, collection of Bogor Botanical Garden were tested for its antidiabetic property through alpha glucosidase inhibitory assay, and the results are as in table 1.  α- glucosidase inhibitory activity is demonstrated by IC50 value (ppm). As reference material (standard) Quercetin was used. Plants tested will be defined as active species or have antidiabetic property if have IC50 value below IC50 of Quercetin as reference material and not active if IC50 value above it (IC50 of Quercetin:22,39 ppm). Tabulated data from Table 1 demonstrated that from 20 Syzygium species, not all of them have antidiabetic property. There are only 12 species from Syzygium sp. which can be defined as active species and others aren’t active. Based on the table, S. cumini and S. syzygoides showed a high alpha glucosidase inhibitory activity with IC50 of 8.71 ppm and 7.48 ppm respectively. Meanwhile S. littorale (Bl) Amsh and S. zeylanicum (L) DC Var. and S. hyntum with IC50 of 23.30 ppm, 24.06 ppm and 23.28 ppm respectively can be stated as fairly active, because their IC50 value near to Quercetin.

Alpha glucosidase inhibitory activity from the part Syzygium cumini plant.

Table 1. showed that S. cumini is one of the active species with IC50 of 8.71 ppm. In order to know which part can be used for antidiabetic treatment the following parts of S. cumini were tested i.e. cortex, old fruit, old seed, young seed and leaves. The results of active part was tabulated in Table 2. From Table 2. it was found that the most active part from  S. cumini plant is the cortex with IC50 of 0.45 ppm followed by young seed, leaves and young fruit with IC50 of  9.77 ppm, 17.4 ppm and 2.78 ppm respectively. The old seed and old fruit are not active due the IC50 above 22.39 ppm. Based on those values, it is confirmed that not all of the part of S. cumini plant needed for the treatment of diabetic. The active parts recommended for antidibetic treatment are cortex, young fruit, young seeds and leaves. The old seed and old fruit are not recommended.

Table 1. Alpha glucosidase inhibitory activity from some Syzygium Species

No	Species	Inhibition concentration of 50% (IC50) (ppm)	Active/not active
1	S. samarangense	72.03	Not active
2	S. hyntum	23.28	Active
3	S. sumatranum	13.99	Active
4	S. cumini	8.71	Active
5	S. reinwardtianum	33.60	Not active
6	S. pseudoformosum	10.47	Active
7	S. paucifunctatum	12.69	Active
8	S. racemosum (Bl.) DC	15.92	Active
9	S. palembaniceum	13.41	Active
10	S. jambos (L) Alst.	17.39	Active
11	S. littorale (Bl) Amsh	23.30	Active
12	S. polycephalum	37.74	Not active
13	S. Cf. palembanicum	44.04	Not active
14	S. pyenathum Merr & Perry	20.85	Active
15	S. syzygioides	7.48	Active
16	S. clarathum (Korth) Merr & Perry	17.77	Active
17	S. boxifolium (Wild) Gaertn	39.69	Not active
18	S. zeylanicum (L) DC Var.	24.06	active
19	S. polycephaloides (C.B Rob) Merr	43.01	Not active
20	S. suringarianum (Koord & Val) Amshoft	29.09	Not active
21	Quercetin (standard)	22.39

Yield and Alpha glucosidase inhibitory activity from the fraction of  Syzygium cumini cortex.

In order to have information about distribution of bioactive compounds further extraction and fractionation was conducted to S. cumini cortex as the most active part. Liquid-liquid partition was performed to separate inactive and active compound. Step wise polarization was used during fractionation using n-hexane, dichloromethane, ethyl acetate and n- buthanol respectively as solvent and the results are as in table 3.

Table 2. Alpha glucosidase inhibitory activity from parts of S. cumini plant

No	Plants’s part	Inhibition concentration of 50% (IC50) (ppm)	Active/Not active
1	Cortex	0.45	Active
2	Old fruit	345.27	Not active
3	Young fruit	21.78	Active
4	Old seed	29.11	Not active
5	Young seed	9.77	Active
6	Leaves	17.40	Active

Table 3. Yield and Alpha glucosidase inhibitory activity from the fraction of  Syzygium cumini cortex

No	Fraction	Yield  from 55,9 gr of ethanolic extract (%)	IC50 (ppm)
1	n-hexane	0.50	897.10
2	Dichloromethane	0.90	44.29
3	Ethyl acetate	3.72	10.88
4	n- butanol	18.40	17.87
5	Water	32.38	8.15

Table 3 showed that the highest yield of compounds distribution was in water followed by n-buthanol, ethyl acetate, dichloromethane and n-hexane with yield of 32.38 %, 18.4%, 3.72%, 0.9% and 0.5 % respectively. Those values showed that the majority of compounds are polar compound due to the high yield in n-buthanol and water fraction. From IC50 values it is confirmed that the bioactive compounds are distributed in water, n- buthanol and ethyl acetate fraction with IC50 of 8.15 ppm, 17.87 ppm and 10.88 ppm respectively. N- hexane fraction didn’t demonstrate distribution of bioactive compound due IC50 value of 897.10 ppm. It can be understand because the yield of n-hexane fraction is very small i.e. only 0.5% compared to other fractions and commonly the hexane fraction contain lipid and ester compound. In other hand ethyl acetate fraction with IC50 values of 10.88 ppm showed a higher alpha glucosidase inhibitory activity than n-buthanol fraction, it means that the semipolar compounds also one of bioactive compound. 

Phytochemical screening assay

In order to prove there is a correlation between phytochemical constituent and antidiabetic activity, phytochemical screening assay of crude ethanolic extract from S. cumini cortex was conducted and the results as figured in Table 4. The table showed the presence of terpenoid, phenol,saponin, tannin and flavonoid in crude ethanolic extract. Those chemical constituents are in the line with previous investigation which found that the stem bark of Eugenia jambolana (another name of S. cumini)  contains  terpenoids betulinic acid, eugenin, friedelin,^[13] phenolic acids, elagitannins and quercetin.^[14] Because the majority of bioactive constituent distributed in polar fraction, it is expected that quercetin and phenolic compounds as the polar compound may responsible for its alpha glucosidase inhibitory activity.

Table 4. Phytochemical screening assay of ethanolic extract from S. cumini cortex.

Chemical constituent	Present/Absent
Terpenoid	Present
Triterpenoid	Absent
Saponin	Present
Flavonoid	Present
Phenol	Present
Steroid	Absent
Tannin	Present

CONCLUSION

It is concluded that species from Syzygium sp. which have a high alpha glucosidase inhibitory activity are S. cumini and  S. Syzygoides. It is also found that the most active part from S. cumini plant is the cortex of bark and the bioactive compound can be classified as polar compound.

ACKNOWLEDGEMENT

This work was supported by the Grant from Integrated Research fellowship from Indonesia’s  Research and Technology Ministry. I also are grateful to DR. Lenny Sutedja and Uun Sundawati for their technical support.

REFERENCE

1.        Markku Laakso,2008, Epidemiology of Type 2 Diabetes, in Textbook of Type 2 Diabetes, Informa HealthCare, New York, London.

2.        Park H, Kyo Yeol Hwang, Young Hoon Kim, Kyung Hwan Oh, Jae Yeon Lee, and Keun Kim, 2008, Discovery and biological evaluation of novel a-glucosidase   inhibitors with in vivo antidiabetic effect, Bioorganic & Medicinal Chemistry Letters 18, p.3711–3715

3.        Cheng Alice Y.Y. and Josse R. G., 2004, Intestinal absorption inhibitors for type 2 diabetes mellitus: prevention and treatment, Drug Discovery Today: Therapeutic Strategies Volume 1, No. 2, p. 201-206

4.        Mir Qurat Y, Ali M., and Alam P, 2009, Lignan derivatives from the stem bark of Syzygium cumini (L.) Skeels, Journal of Natural Product Research, Volume 23, Issue 5 March 2009 , p. 422 - 430

5.        Rafiullah, M.R.M.et. al., 2006,  Antidiabetic Activity of Some Indian Medicinal Plants, Pharmaceutical Biology (Formerly International Journal of Pharmacognosy), Volume 44, Number 2, March 2006 , p. 95-99

6.        Anggadireja K, Elin Y, Sodiro I, 1998, Study of antidiabetic activity from water extract of salam leaves (Syzygium polyanthum (Wight) Walp.,) and the fractions on white Male ddY mice, thesis ITB. Retrieved from http://bahan- alam.fa.itb.ac.id/detail. php?id =282 on May, 27 2010. (in Indonesian)

7.     Musabayane, C.T., Mahlalela N., Shode F.O. and Ojewole J.A.O., 2005, Effects of Syzygium cordatum (Hochst.) [Myrtaceae] leaf extract on plasma glucose and hepatic glycogen in streptozotocin-induced diabetic rats, Journal of Ethnopharmacology,Volume 97, Issue 3, 21 March 2005, p. 485-490

8.        Sutedja L, 2005, Bioprospecting Tumbuhan Asli Indonesia sebagai Sediaan Fitofarmaka Antidiabetes, Laporan Riset Unggulan Terpadu Bidang Lingkungan, Kementrian Riset dan Teknologi RI, LIPI.

9.        Colegate, S. M., and Molyneux, R. J. 1993. Bioactive Natural Products, Detection, Isolation and Structural Determination. CRC Press., Inc. USA

10.    Harborne, J.B. 1987. Metode Fitokimia. Penerbit ITB. Bandung.

11.    Zhi-yun Du et.al. 2006, α-Glucosidase inhibition of natural curcuminoids and curcumin analogs, European Journal of Medicinal Chemistry, Volume 41. p.213-218

12.    Anam K, R.M. Widharna and D. Kusrini, 2009, α-glucosidase inhibitory activity of Terminalia species, International Journal of Pharmacology, p. 277-280.

13.    Sengupta, P., & Das, P.B. (1965). Terpenoids from the stem bark of Eugenia jambolana. Journal of Indian Chemical Society, Volume 42, p. 255–258

14.    Bhatia, I.S., & Bajaj, K.L. (1975). Chemical constituents of the seeds and bark of Syzygium cumini. Planta Medica, Volume 28, p.346–352.