In Vitro Anti-diabetic Properties of Methanolic Extract of
Thymus vulgaris Using ?-glucosidase and ?-amylase Inhibition
Assay and Determination of its Bioactive
Chemical Compounds
Ali Kadhim Aljarah1, Imad Hadi Hameed2
1Department of Biology, College of Science for women, University of Babylon, Hillah City, Iraq,
2Biomedical Science Department, University of Babylon, College of Nursing, Hillah City, Iraq
ABSTRACT
The objectives of our research were analysis of the bioactive chemical metabolite products and determination
In vitro anti-diabetic properties. The results of anti-diabetic activity produced by Thymus vulgaris showed
that the volatile compounds were effective to ?-glucosidase and ?-amylase inhibition. The purification of
compounds produced by Thymus vulgaris can be useful. Twenty seven bioactive compounds were identified in
the methanolic extract of Thymus vulgaris. Gas chromatography – mass spectrum analysis analysis of Thymus
vulgaris revealed the existence of the Erythritol , ?-Terpinene, Octan-2-one , 3,6-dimethyl- , DL-Leucine,
N-glycyl-, Trans-2-Caren-4-ol , Dithiocarbamate , S-methyl-,N-(2-methyl-3-oxobutyl)- , 5-Caranol, trans,
trans-(+)- , Phenol,2-methyl-5-(1-methylethyl)- , Phenol ,2-methyl-5-(1-methylethyl)-, acetate , (5?)Pregnane-
3,20?-diol, 14?,18?-[4-methyl-3-oxo(1-oxa-4-, ?-Bisabolene , 1-Chloro-1-n-decyloxy-1-silacyclopentane,
7-epi-trans-sesquisabinene hydrate , (-)-Spathulenol , Caryophyllene oxide, 1-Heptatriacotanol , Ppropiolic
acid , 3-(1-hydroxy-2-isopropyl-5-methylcyclohexyl)- , Naphthalene,1,2,3,4,4a,5,6,7-octahydro-4a-methyl-,
Paromomycin, Gibberellic acid, Phytol , 2,7-Diphenyl-1,6-dioxopyridazino[4,5:2’,3’]pyrrolo[4’,5’-d]
pyrida, Androst-4,6-dien-3,11,17-trione, 9-mercapto- , Ricinoleic acid , 4a,10a-Methanophenenthren-9?-ol,
11-syn-bromo-1,2,3,4,4a and (+)-?- Tocopherol , O-methyl.
Keywords: Thymus vulgaris, Anti-diabetic, ?-amylase, GC-MS, ?-glucosidase, Secondary metabolites.
Corresponding author:
Imad Hadi Hameed.
Biomedical Science Department, University of Babylon,
College of Nursing, Hillah City, Iraq;
Phone number: 009647716150716;
E-mail: imad_dna@yahoo.com
INTRODUCTION
Thyme is one of flowering plant in the family of
Lamiaceae, native to southern Europe and western
Mediterranean 1, 2. Growing to fifteen to thirteen cm
(6–12 in) tall by forty cm (16 in) wide, it is a bushy,
and clusters of purple or pink flowers in early summer.
P-cymene, myrcene, borneol, and linalool are Thyme
essential oil. Many compounds in the composition of
the bioactive chemical compounds (essential oil) are
antioxidants that act in secondary products response and
other oxidant species. These responses are promoted
by toxins produced by pathogenic bacteria and fungi.
The British Herbal Pharmacopoeia classifies these
species as a bioactive medicinal plants for its use it
mentions bronchial catarrh, bronchitis, sore throats,
and whooping cough 3-6. Diabetes mellitus is a disorder
that affects the metabolism of protein, carbohydrate,
and fat. Diabetes are in three forms. Type 1, type 2,
and gestational diabetes are the three main types of
diabetes. ?-glucosidase are responsible for the analysis
of oligo- and/or disaccharides to monosaccharides.
Therefore these enzymes leads to a decrease the level
of blood glucose, because the form of carbohydrates
(monosaccharides) are absorbed through the mucosal
border in the small intestine 7-10. ?-amylase enzyme
which is dependable for the collapse of starch to more
DOI Number: 10.5958/0976-5506.2018.00241.3
Indian Journal of Public Health Research & Development, March 2018, Vol. 9, No. 3 389
uncomplicated sugars (glucose , maltose maltotriose and
dextrin). Acarbose are inhibitors or controller currently
in clinical use and miglitol which inhibit glycosidases
such as ?-glucosidase and ?-amylase 11, 12. The aims
of our study were evaluation In vitro anti-diabetic
properties and screening bioactive chemical products of
Thymus vulgaris.
MATERIAL AND METHOD
Gas chromatography – Mass Spectrum analysis
Thymus vulgaris Gas chromatography –
Mass Spectrum analysis were follow out in a Gas
chromatography system (Agilent 7890A series, USA).
The flow rate of the carrier gas, helium (He) was set
to beat 1 mL min?1, split ratio was 1:50. The injector
temperature was adjusted at 250?C, while the detector
temperature was fixed to280?C. Performance of data
of mass spectrum was conducted NIST, USA. The
database consists of more than 62,000 patterns of
known compounds 13-16. The spectrum of the extract was
matched with the spectrum of the known components
stored in the NIST library.
In Vitro ?-glucosidase Inhibition Assay
1 mg of ?-glucosidase was melt in one hundred ml
of phosphate buffer (pH 6.8).
Two hundred ?l ?-glucosidase were added to one
hundred ?l of (2, 4, 8, 10, 15 ?g/ml) sample extracts then
the mixture was incubated at 37?C for 20 min 17-19. Then
one hundred ?l 3mM p-nitrophenyl ?-D-glucopyranoside
(p-NPG) was added to the mixture and incubated at 37
?C for 10 min. By the addition of 2ml Na2CO3 0.1M The
reaction was terminated and the ?-glucosidase efficiency
was fixed spectro-photometrically at 405 nm on spectrophotometer
UV-VIS.
In Vitro ?-amylase Inhibition Assay
Two hundred ?l porcine pancreatic amylase was
added to one hundred ?l of (2, 4, 8, 10, 15 ?g/ml)
Thymus vulgaris extract. Then one hundred ?l (1%)
starch thaw was added and incubated at 37 ?C for ten
min. The reaction watered with 2.2 ml and absorbance
was read at 540 nm 20,21. For each concentration, blank
tubes were prepared by replacing the enzyme solution
with two hundred ?L in distilled water.
Positive monitoring of ?-amylase and
?-glucosidase inhibitor
Positive control of ?-glucosidase inhibitor
and ?-amylase was Acarbose 22-24. Inhibit 50% of
?-glucosidase and ?-amylase activity under the
examination conditions was defined as the IC50 value.
Percentage inhibition (I %) was studied by:
I % = (Ac-As)/Ac X 100
As is the absorbance of the sample and Ac is the
absorbance of the control.
Table 1: Bioactive compounds detected in Thymus vulgaris extract.
Molecular Exact Mass
Phytochemical compound RT (min) Weight
Serial
No.
1. Erythritol 3.430 122 122.057909
2. ?-Terpinene 4.500 136 136.1252
3. Octan-2-one , 3,6-dimethyl- 5.009 156 156.151415
4. DL-Leucine , N-glycyl- 5.112 188 188.116093
5. Trans-2-Caren-4-ol 5.318 152 152.120115
6. Cis-p-Mentha-2,8-dien-1-ol 5.484 152 152.120115
7. Dithiocarbamate , S-methyl-,N-(2-methyl-3-oxobutyl)- 5.656 191 191.043856
8. 5-Caranol , trans , trans-(+)- 5.833 154 154.135765
9. Phenol,2-methyl-5-(1-methylethyl)- 6.297 150 150.1044655
390 Indian Journal of Public Health Research & Development, March 2018, Vol. 9, No. 3
10. Phenol ,2-methyl-5-(1-methylethyl)-, acetate 7.567 192 192.115029
(5?)Pregnane-3,20?-diol , 14?,18?-[4-methyl-3-oxo(1- 8.540 489 489.309038
11. oxa-4-
12. ?-Bisabolene 8.992 204 204.1878
13. 1-Chloro-1-n-decyloxy-1-silacyclopentane 9.204 276 276.16762
14. 7-epi-trans-sesquisabinene hydrate 9.335 222 222.198365
15. (-)-Spathulenol 9.867 220 220.182715
16. Caryophyllene oxide 9.942 220 220.182715
17. 1-Heptatriacotanol 10.348 536 536.58962
Ppropiolic acid , 3-(1-hydroxy-2-isopropyl-5- 11.979 224 224.141245
18. methylcyclohexyl)-
19. Naphthalene ,1,2,3,4,4a,5,6,7-octahydro-4a-methyl- 13.386 150 150.140851
20. Paromomycin 12.448 615 615.296303
21. Gibberellic acid 14.302 346 346.141638
22. Phytol 15.029 296 296.307917
2,7-Diphenyl-1,6-dioxopyridazino[4,5:2’,3’] 15.183 355 355.106924
23. pyrrolo[4’,5’-d]pyrida
24. Androst-4,6-dien-3,11,17-trione , 9-mercapto- 15.990 330 330.128965
25. Ricinoleic acid 16.985 298 298.250795
4a,10a-Methanophenenthren-9?-ol , 11-syn-bromo- 18.931 292 292.046276
26. 1,2,3,4,4a
27. (+)-?- Tocopherol , O-methyl- 22.793 430 430.38108
RESULTS AND DISCUSSION
Identification of biochemical compounds
Analysis of component was done in extract of
Thymus vulgaris, shown in Table 1. Chromatogram
gas chromatography – mass spectrum analysis of
Thymus vulgaris extract showed the presence of twenty
seven major peaks were determined as follows. All
peaks were determined to be Erythritol , ?-Terpinene ,
Octan-2-one , 3,6-dimethyl- , DL-Leucine , N-glycyl- ,
Trans-2-Caren-4-ol , Dithiocarbamate , S-methyl-,N-
(2-methyl-3-oxobutyl)- , 5-Caranol , trans , trans-(+)- ,
Phenol,2-methyl-5-(1-methylethyl)- , Phenol ,2-methyl-
5-(1-methylethyl)-, acetate , (5?)Pregnane-3,20?-diol
, 14?,18?-[4-methyl-3-oxo(1-oxa-4- , ?-Bisabolene
, 1-Chloro-1-n-decyloxy-1-silacyclopentane , 7-epitrans-
sesquisabinene hydrate , (-)-Spathulenol ,
Cont... Table 1: Bioactive compounds detected in Thymus vulgaris extract.
Caryophyllene oxide , 1-Heptatriacotanol , Ppropiolic
acid , 3-(1-hydroxy-2-isopropyl-5-methylcyclohexyl)-
, Naphthalene ,1,2,3,4,4a,5,6,7-octahydro-4a-methyl- ,
Paromomycin , Gibberellic acid , Phytol , 2,7-Diphenyl-
1,6-dioxopyridazino[4,5:2’,3’]pyrrolo[4’,5’-d]pyrida
, Androst-4,6-dien-3,11,17-trione , 9-mercapto- ,
Ricinoleic acid , 4a,10a-Methanophenenthren-9?-ol
, 11-syn-bromo-1,2,3,4,4a and (+)-?- Tocopherol ,
O-methyl.
In vitro ?-glucosidase inhibition assay
Methanol, ethanol and aqueous extract of Thymus
vulgaris showed notable anti-diabetic activities.
Secondary metabolite of methanolic extract of Thymus
vulgaris was active In vitro ?-glucosidase inhibition.
?-glucosidase inhibition had been distributed according
to alcoholic extract of Thymus vulgaris (Methanol,
Indian Journal of Public Health Research & Development, March 2018, Vol. 9, No. 3 391
ethanol and aqueous) in different concentrations (Con.
4 ?g/ml, 8 ?g/ml, 15 ?g/ml, 20 ?g/ml, and 15 ?g/ml)
were (4.35, 22.04, 30.77, 43.13 and 51.08) respectively
in methanolic extract, and (4.03, 19.12, 27.89, 39.12
and 46.58) respectively in ethanolic extract, and (3.51,
18.37, 25.71, 36.94 and 44.31) respectively in aqueous
extract, and (16.11, 44.16, 53.02, 63.70 and 70.21)
respectively in Acarbose, as a control.
In vitro ?-Amylase inhibition method
Secondary metabolite of methanol, ethanol and
aqueous extract of Thymus vulgaris were used for test
of anti-diabetic activities in different concentrations
(Con. 4 ?g/ml, 8 ?g/ml, 15 ?g/ml, 20 ?g/ml, and 15 ?g/
ml). ?-amylase inhibition recorded (6.39, 11.47, 17.01,
22.93 and 24.07) respectively in methanolic extract,
and (5.97, 10.02, 16.05, 21.27 and 23.05) respectively
in ethanolic extract, and (4.94, 9.88, 15.69, 20.44 and
22.63) respectively in aqueous extract, and (12.37,
25.16, 36.08, 44.97 and 56.13) respectively in Acarbose,
as a control.
CONCLUSION
Twenty seven bioactive chemical compounds have
been detected from Thymus vulgaris extract by gas
chromatogram mass spectrometry (GC-MS). In vitro
antifungal and antibacterial evaluation of secondary
metabolite products of Thymus vulgaris forms a primary
platform for further phyto-chemical and pharmacological
evaluation for the expansion of new useful antimicrobial
compounds.
Financial Disclosure: There is no financial
disclosure.
Conflict of Interest: None to declare.
Ethical Clearance: In our research, all protocols
were approved under the Department of Biology, College
of Science for women, University of Babylon, Hillah
city, Iraq and all methods were carried out in accordance
with approved guidelines.
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