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In vitro α-amylase and α-glucosidase inhibition and increased glucose uptake of Morinda citrifolia fruit and scopoletin

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Abstract

Diabetes mellitus is a metabolic disorder and management of blood glucose level is an important strategy in the control of the disease and complications associated with it. Therefore, components that cause uptake of glucose from the bloodstream and inhibitors of carbohydrate hydrolyzing enzymes can be useful in treatment of diabetes and medicinal plants are often used to achieve this aim. Morinda citrifolia fruit (MCF) is used in various countries for treatment of diabetes and the purpose of this study was to investigate the effect of MCF extract and its biomarker scopoletin on glucose uptake in HepG2 cells as well as its inhibitory effect on α-amylase and α-glucosidase. The safe doses for MCF extract and scopoletin were at 1 mg/ml and 0.2 μM, respectively as assessed by MTT assays and these were used for the assays. The extract had glucose uptake of 59.5% which was comparable to the standard metformin whereas the value for scopoletin was 30.6%. The extract had mild inhibitory activity on α-amylase and α-glucosidase with percentage of inhibition at 43.5% and 57%. The biomarker scopoletin showed lower activities at 23.9% and 35.7% for α-amylase and α-glucosidase respectively. Hence, these three activities may possibly be the mechanisms for MCF to exert its antidiabetic activity.
Research J. Pharm. and Tech.8 (2): February 2015
189
ISSN 0974-3618 www.rjptonline.org
RESEARCH ARTICLE
In vitro α-amylase and α-glucosidase inhibition and increased glucose
uptake of Morinda citrifolia fruit and scopoletin
Masitah Khamis1, Fazilah Talib1, Nor Syamira Rosli1, Saravanan Dharmaraj*2, Khamsah
Suryati Mohd1, Sasidharan Srenivasan3, Zubaidi Abdul Latif2, Mahadeva Rao S. Utharkar2
1Faculty of Agriculture, Biotechnology and Food Sciences, Universiti Sultan Zainal Abidin, Tembila Campus,
22200 Besut, Terengganu, Malaysia
2Faculty of Medicine, Universiti Sultan Zainal Abidin, Medical Campus, 20400 Kuala Terengganu, Terengganu,
Malaysia
3Institute for Research in Molecular Medicine (INFORMM), Universiti Sains Malaysia, 11800, Pulau Pinang,
Malaysia
*Corresponding Author E-mail: saravanandharmaraj@unisza.edu.my
ABSTRACT:
Diabetes mellitus is a metabolic disorder and management of blood glucose level is an important strategy in the control
of the disease and complications associated with it. Therefore, components that cause uptake of glucose from the
bloodstream and inhibitors of carbohydrate hydrolyzing enzymes can be useful in treatment of diabetes and medicinal
plants are often used to achieve this aim. Morinda citrifolia fruit (MCF) is used in various countries for treatment of
diabetes and the purpose of this study was to investigate the effect of MCF extract and its biomarker scopoletin on
glucose uptake in HepG2 cells as well as its inhibitory effect on α-amylase and α-glucosidase. The safe dosesforMCF
extract and scopoletin were at 1 mg/ml and 0.2 μM, respectively as assessed by MTT assaysand these were used for
the assays. The extract had glucose uptake of 59.5% which was comparable to the standard metformin whereas the
value for scopoletin was 30.6%. The extract had mild inhibitory activity on α-amylase and α-glucosidase with
percentage of inhibition at 43.5% and 57%. The biomarker scopoletin showed lower activities at 23.9% and 35.7% for
α-amylase and α-glucosidase respectively. Hence, these three activities may possibly be the mechanisms for MCF to
exert its antidiabetic activity.
KEYWORDS: Morinda citrifolia; scopoletin, glucose uptake, glucosidase; amylase
INTRODUCTION:
Diabetic mellitus is a chronic endocrine disorder
characterized by hyperglycemia. The prevalent type 2 DM
occurs in more than 90% of diabetics and is caused by
combination of peripheral insulin resistance and impaired
insulin secretion. This metabolic disorder includes
alterations in carbohydrate, lipid and protein metabolism
and is grouped together under metabolic syndrome with
other lifestyle related diseases. Chronic hyperglycemia of
diabetes often causes long term damage and dysfunction to
various organs, especially eyes and kidneys.
Received on 22.01.2015 Modified on 30.01.2015
Accepted on 04.02.2015 © RJPT All right reserved
Research J. Pharm. and Tech. 8(2): Feb. 2015; Page 189-193
Lifestyle changes such as exercise is suggested to be helpful
in alleviating the disease[1-3] but worldwide prevalence of
type 2 DM is increasing. This is evident by the estimate of
number of adults with diabetes in 1995 of 135 million and
which was projected to rise to 300 million in 2025[4] . The
projected increase is also seen where in 2010, diabetic
prevalence was calculated to affect 285 million and
estimated to increase to 439 million by 2030[5].
The morbidity of type 2 DM is associated with increased
glucose concentrations and this is often due to postprandial
glucose concentrations. The increase after a meal is caused
by hydrolysis of starch by pancreatic α-amylase as well as
uptake of glucose by intestinal α-glucosidase and therefore,
the strategy for type 2 DM management would be strong
inhibition of pancreatic α-amylase and intestinal α-
glucosidase[6].
Research J. Pharm. and Tech.8 (2): February 2015
190
Plant polyphenols have been reported to possess inhibitory
effect on α-amylase and α-glucosidase as well as increasing
glucose uptake into skeletal muscle and adipocytes[7].
Compounds from plants have also shown beneficiary effect
on glucose uptake in the liver and this is vital as the organ is
an important regulator of plasma glucose level and plays a
key role in glucose metabolism and regulation.
Natural products have always been a source for
development of new drugs even in the present era of
combinatorial chemistry and drugs of plant or microbial
origin account for more than 30% worldwide sales of
natural products. This coupled with the fact that
pharmacological approach using synthetic oral
hypoglycemic cause serious side effects[8-9], makes the
search for alternatives with enhanced therapeutic but
reduced side effects an ever going process.
Morinda citrifolia L. (Rubiaceae) is a small tropical
evergreen tree that is indigenous to Pacific Islands, South
East Asia and other tropical as well as semitropical regions.
It has been used traditionally in folk medicine as a
treatment for diabetes as well as other related diseases[10 -11].
Its fruit contains a variety of natural products. Other than
flavanoids such as rutin, quercetin and kaempferol, other
key markers such as asperulosidic acid, dimethyl morindol
and scopoletin were also detected[12-15].
One of the authors have reported antihyperglycemic[16] and
antihyperlipidemic[17] effect of MCFextract in
streptozotocin-induced diabetic rats. However, the
antidiabetic mechanism responsible for the fruits effect has
not been studied. Therefore, this study would investigate
the effect of MCF extract and scopoletin on glucose uptake
in HepG2 cells. In addition, the in vitro inhibitory effect of
the extract and scopoletin on α-amylase and α-glucosidase
will be studied.
MATERIALS AND METHODS:
Chemicals
Anthrone reagent (C14H10O), Bromo Phenol Blue (BPB),
DMSO and scopoletin were purchased from Nacalai tesque
(Kyoto, Japan). Insulin and metformin were purchased from
Tocris (Bioscience, Bristol, UK). Triple E/Trypsin and
fetal bovine serum (FBS) were from GIBCO (UK).
Dulbecco’s Modified Eagle’s Medium (DMEM), D-glucose
anhydrous, sodium bicarbonate (NaHCO3) and thiazolyl
blue tetrazolium bromide were from SIGMA (UK), whereas
phosphate buffered saline (PBS) tablet was from
Calbiochem®(Darmstadt, Germany). Sodium hydroxide
(NaOH) and Hydrochloric acid (HCI) from Dulchefa
(Netherland). Chloroform, ethanol, ethyl acetate, methanol
and H2SO4were from R&M (Essex, U.K).
Plant materials and preparation of extracts
The leaf and fruits of M. citrifolia were obtained from
Kampung Tok Dor, Besut, Terengganu in September 2012.
Herbarium samples are deposited at Universiti Sultan
Zainal Abidin (UniSZA) Herbarium, Faculty of
Agriculture, Biotechnology and Food Sciences, voucher no:
0.00218. The fruits were cut into small pieces, dried in an
oven at temperature of 45C and ground into powder. The
dried fruit powder was macerated for 3 days with methanol
at ratio of 1 g of sample to 10 ml of methanol. The extract
was then filtered and evaporated using rotary evaporator.
Cell culture and treatment
The human liver hepatocellular carcinoma cell line (HepG2
cells) was provided by the Animal cell culture laboratory of
Faculty of Agriculture, Biotechnology and Food Sciences,
UniSZA.The cells were grown in a humidified atmosphere
containing 5% CO2 at 37°C[18]. The cells were routinely
maintained in growth medium which consisted of DMEM
supplemented with 10% heat-inactivated FBS and 0.5%
pens/strep (antibiotic). Proliferating cells were subcultured
into fresh growth medium every 2-3 days. For routine
maintenance, trypsin in phosphate-buffered saline (PBS)
was used as the treatment to detach the cells from the t-flask
and after 5-10 minute exposure at 37°C, the cells were
seeded in growth medium.
Cell viability (MTT assay)
Viability of HepG2 cells after treatment with the plant
extract and standard drug was determined by assaying for
the reduction of 3-(4,5-dimethylthiazol- 2-yl)-2,5-
diphenyltetrazolium bromide (MTT) to formazan. HepG2
cells were seeded in 96 well plates at density of 2 ×106 cells
per well. Cells were inoculated in a volume of 100 μl per
well and 100 μl aliquot of growth media was added to the
free cells. Fresh media containing MCF extract or
scopoletin at indicated concentrations was added 24 hours
after seeding. Control cells were incubated without test
sample and with maintenance medium. The microplates
were incubated at 37°C for a period of 24 hours. The cell
medium was replaced with 100 μl fresh medium per well
containing 0.5 mg/ml MTT and incubated for another 4
hours in the dark. Lastly, 100 μl of isopropanol was added
to solubilize the formazan and its absorption was measured
at 570nm (620 nm as reference) using a micro-plate reader
(Infinite 200 PRO NanoQuant, TECAN).
Glucose uptake assay
Glucose uptake was measured as previously described by
Tandrasasmita et al. (2011)[19]. Briefly, HepG2 were grown
to about 1 × 104 cells/ml in 6 cm diameter plates. When the
cells reached 80% confluency, the cells were washedtwice
with phosphate-buffered saline and incubated with3 ml
glucose solution (10 mg/ml) with or without administration
of MCF extract, metformin, scopoletin and insulin at 37°C
for 10 min. During incubation the cells took up glucose,
enabling free glucose to be measured in the media, from
which the concentration of glucose uptake could then be
determined. Measurements of glucose concentrations were
performed by the reaction of glucose with anthrone in
presence of sulphuric acid.
Research J. Pharm. and Tech.8 (2): February 2015
191
The concentration of glucose taken up by the cells is
described by the equation:
Initial glucose-free glucose within media
%Glucose uptake=-------------------------------------X 100%
Initial glucose
α-Amylase inhibition assay
The assessment of inhibitory effect of MCF extract on α-
amylase activity was measured based on modified method
of Apostolidis et al. (2007)[20]. Sample solutions of 1 mg/ml
of MCF extract or 0.2 μM scopoletinat volume of 500 μl
and 0.2 M phosphate buffer pH 6.9 (500 μl) containing α-
amylase solution (0.5 mg/ml) were incubated at 25°C for 10
min. After preincubation, 500 μl of 1% starch solution in
0.02 M sodium phosphate was added and the reaction
mixture was incubated at 25°C for 10 min. The reaction was
stopped with 1.0 ml of dinitrosalicylic acid (DNS). The
reaction mixture was then incubated in a boiling water bath
for 5 min and allowed to cool to room temperature. The
reaction mixture was then diluted with 10 ml distilled water
and absorbance was read at 540 nm (Infinite 200 PRO
NanoQuant, TECAN). The inhibitory effect of the extract
was compared to standard inhibitor, acarbose.
α-Glucosidase inhibition assay
The α-glucosidase method was determined according to the
method of Apostolidis et al. (2007)[20]. In brief, 50 μl of the
test samples and 100 μl of 0.1 M phosphate buffer (pH 6.9)
containing yeast α-glucosidase solution (1.0 U/ml) were
preincubated in 96 well plates at 25°C for 10 min. After
incubation, 50 μl of 5 mM pNPG solution in 0.1 M
phosphate buffer (pH 6.9) was added to each well and the
reaction mixtures were incubated at 25°C for 5 min. The
absorbance of the reaction mixtures was recorded with a
micro-plate reader at 405 nm (Infinite 200 PRO
NanoQuant, TECAN) before and after incubation with
pNPG solution and compared to that of the control which
had 50 μl buffer solutions instead of test samples. The
experiments were performed in triplicate and the α-
glucosidase inhibitory activity was expressed as percentage
inhibition. Acarbose was prepared in distilled water at 1.0
mg/ml concentration and used as positive control.
Statistical analysis
All the data points are mean values ± standard error. Where
appropriate, statistical analysis were performed using one-
way analysis of variance (ANOVA) to treat difference
between mean while Tukey’s multiple comparison test with
P 0.05 was taken as significant. The software employed
for statistical analysis was SPSS.
RESULTS:
Cell viability by MTT assay
Cell viability by MTT assay was used to assess the safe
dose of MCF extract and scopoletin for monitoring glucose
uptake with HepG2 cells.It is important to use the safe dose
as we do not want the extract or compound to be harmful to
the tested cells. The safe dose was identified at the
concentration of which the cells viability is about 80% and
the concentration for safe dose of MCF extract was
1.0mg/ml while scopoletin was 0.2 µM.
In vitro α-amylase inhibition study
The ethanolic extract of MCFat concentration of 1 mg/ml
showed mild inhibition of amylase but its percentage of
inhibition of 43.5% was higher than that for 0.2 μM
scopoletin at 23.9% as well as that for the 1 mg/ml standard
acarbose at 35.2%. The percentage values for inhibition of
amylase by ethanolic extract of MCF, acarbose and
scopoletin are shown in Fig. 1.
Figure 1 Percentage inhibition of ethanol extract of MCF,
scopoletin and acarbose on α-amylase in vitro. Values represent
mean ± SEM of triplicate tests. Bars with different letters are
significantly different (p<0.05).
In vitro α-glucosidase inhibition study
The ethanolic extract of MCFat concentration of 1 mg/ml
showed stronginhibition of α-glucosidase and its percentage
of inhibition of 57% was comparable to that of the standard
acarbose at concentration of 1.0 mg/ml (Percentage of
inhibition of 57.7%). However, the percentage of inhibition
for0.2 μM scopoletin at concentration was lower than both
of these and its value was only 35.7%. The values for
percentage of inhibition by α-glucosidase are shown in
Fig. 2.
Figure 2 Percentage inhibition of ethanol extract of MCF,
scopoletin and acarbose on α-glucosidase in vitro. Values represent
mean ± SEM of triplicate tests. Bars with different letters are
significantly different (p<0.05).
Research J. Pharm. and Tech.8 (2): February 2015
192
In vitro glucose uptake study
The effect on glucose uptake by 1 mg/ml of MCF extract,
0.2 μM scopoletin as well as the two positive controls of
metformin and insulin were studied in vitro using HepG2
cells and the results show that insulin had the highest
activity. Its percentage of glucose uptake of 69.3% was
higher than both of metformin as well as for the MCF
extract. The values of 59.5% for the fruit extract and 58.2%
for metformin were not statistically different from each
other but they were higher than that of scopoletin, which
value was only 30.6%. The percentage values for glucose
uptake by 1 mg/ml of ethanolic extract of MCF, 0.2 μM
scopoletin, 100 μg/ml metformin and 1 IU/ml insulin are
shown in Fig. 3.
Figure 3 Percentage of glucose uptake by ethanol extract of MCF,
scopoletin, metformin and insulin in HepG2 cells. Values represent
mean ± SEM of triplicate tests. Bars with different letters are
significantly different (p<0.05).
DISCUSSION:
The MCF has been used for treating diabetes mellitus but
its mechanism of action for alleviating blood glucose is not
established although leaf extract from another species from
the same genus in Africa has been reported to inhibit α-
amylase and α-glucosidase[21]. Therefore, in our study we
evaluated the effect of inhibition of both enzymes in
addition to the in vitro evaluation of glucose uptake by the
fruitextract as well as the biomarker, scopoletin. Often
glucose uptake for diabetic studies are carried out using
adipocytes[22-24] and differentiated skeletal muscle cells[25-26]
but our experiment utilized HepG2 cells as they have
similar physiological function to normal hepatocytes[27] and
also they are stable during many passages. Considerable
previous studies have used HepG2 cell to monitor glucose
uptake[28-29]. Glucose uptake in our study was determined by
measuring the differences in concentration of glucose in
media before and after incubation with test compounds.
Prior to evaluating the effect of plant extracts and
compounds, viability studies were carried out using the
MTT assay. The MTT assay which measures the activity of
the mitochondrial reductase enzyme[30] is an estimate of the
number of viable cells and these studies help in eliminating
the cytotoxic doses of plant extracts and for determining the
precise range of concentrations of test samples for further
study.The safety dose is often ascertained as the dose which
gives viability of about 80%[31-32] and the value of this for
MCF extract was 1.2 mg/kg whereas for scopoletin was
0.2μM.
The therapeutic approach in using M. citrifolia extract to
treat diabetes is suggested to decrease post-prandial
hyperglycemia. Firstly, this is achieved by increased
glucose uptake into the liver. The result of our study shows
that MCF extract increases glucose uptake in HepG2 cells.
This is the first study that reports this mechanism for extract
of this species as earlier study by Nguyen and co-workers[33]
reported increased glucose uptake in adipocytes by isolated
compounds from M. citrifolia. The compounds were
episesamin 2,6-dicatechol, lirioresinol B, lirioresinol B
dimethyl ether, and ursolic acid, and did not include the
predominant biomarker, scopoletin.
Secondly, the decrease in postprandial hyperglycemia is
achieved by hindered absorption of glucose by inhibition of
the carbohydrate hydrolyzing enzymes in the digestive
organs. The enzymes that are affected are α-amylase, that
catalyses the breakdown of starch to maltose and finally to
glucose, as well as α-glucosidase, present in the small
intestine and catalyzing the breakdown and absorption of
complex sugars.Examples of such inhibitors in clinical use
are acarbose, miglitol and voglibose but they do have side
effects[34].
The present study indicates that ethanolic extract of MCF
possesses inhibitory effect on glucosidase and amylase.
However, scopoletin which is the major biomarker in
ethanolic extract of M. citrifolia possesses only mild
activity on inhibition of both these enzymes. Various
studies have shown the presence of flavonoids such as rutin,
kaempferol and quercetin in fruits of M. citrifolia. These
compounds are suggested to be responsible for this activity
as they have been shown to possess inhibitory effect on
amylase[35-36] and glucosidase[37-40] in in vitro studies.
CONCLUSION:
This study investigated the potential antidiabetic effect of
M. citrifolia with focus on increased glucose uptakeas well
as inhibition of α-amylase and α-glucosidase, which would
be beneficial by reducing hyperglycemia. The ethanolic
extract of the fruit showed these three activities and at the
concentration of the safe dose, they were higher for the fruit
than the biomarker scopoletin. In conclusion, the results
from this study give scientific support to theuse of M.
citrifolia in traditional medicine for the treatment ofdiabetes
and show, for the first time, the potential role ofα-
glucosidase and α-amylase inhibition as well as increased
glucose uptake by hepatocytes in its activity.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
Research J. Pharm. and Tech.8 (2): February 2015
193
ACKNOWLEDGEMENTS:
The authors graciously acknowledge the financial backing
of Ministry of Higher Education, Malaysia for granting
Dr. Mahadeva Rao the research grant for the execution of
this work (FRGS/1/2012/SKK03/UNISZA/02/01).
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... Some studies have been employed to inhibit those proteins for achieving average blood glucose concentration and improving insulin performance [18][19][20] . Thus, targeting ɑ-amylase, DPP4, and PTP1B have favourable results in preventing DM advancement [21][22][23][24][25] . Nevertheless, there were no studies for understanding the role of bioactive compounds in DL to inhibit those proteins and regulate DM conditions. ...
... Other dominant polyphenol compound, rutin, also identified 39 . Phenolic acid has been proved to exhibits an antidiabetic nature, particularly by inhibiting ɑ-amylase 21,23,24,[40][41][42] . Therefore, this result discover a wide potential of DL as anti-diabetic agent. ...
... Regulating glucose metabolism and insulin performance are the key factors in diabetes management 21,24,43,44 . An enzyme called ɑ-amylase plays a vital role in glucose metabolism from dietary intake 45 . ...
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... [31,36] MCFE have been reported to inhibit alpha-amylase and alpha-glucosidase on uptake of glucose in HepG2 cells in vitro and the presence of flavonoids such as rutin, kaempferol, and quercetin contributed to the effect. [37] Imbalance of oxidative and antioxidant defense potential has been noted with L-arginine administration. [32,38] In the present exploration, pancreatitis induced by L-arginine demonstrated an increase in the oxidative stress by increasing Which was observed as increased levels of MDA, MPO, and Nitrite due to peroxidation of membrane lipids and activation of NOS. ...
... Dussossoy et al. revealed that the administration of MCFE has inhibitory effects on nitric oxide and prostaglandin E 2 contributing to its anti-inflammatory effects. [43] CRP and LDH have been well associated with progression of inflammation in many diseases and are accounted for the prognosis of the disease in AP. [37,44] The administration of L-arginine increased the levels of CRP and LDH signifying the infiltration of inflammatory mediators and oxygen deprivation. In contrast to this, MCFE and melatonin administration showed a beneficial effect by the reduction of CRP and LDH contributing its anti-inflammatory effects [42] where the administration of Noni juice reported to reduce the levels of CRP, triglycerides, low-density lipoprotein, and homocysteine in smoking patients. ...
... Compounds from plants have also shown the beneficiary effect on glucose uptake in the liver and this is vital as the organ is an important regulator of plasma glucose level and plays a key role in glucose metabolism and regulation. 12 α-Amylase Inhibition Assay: It is the common method to estimate the amount of reducing sugars in different types of test samples. In this assay, we can measure the amount of reducing sugar generated after-treatment of the test solution with the α-amylase enzyme. ...
... α-amylase inhibition activity= [(Ac− As)/Ac] × 100, where Ac denotes the absorbance of the control reaction (containing all reagents except the sample), and As denotes the absorbance of the sample α-glucosidase inhibition assay: α-glucosidase inhibition of the extracts was measured with 1mg/mL 4-nitrophenyl-α-D-glucopyranoside and αglucosidase(0.3U) 23 . After incubating at 37 ºC for 30 min, reaction was stopped by the addition of 50 mM sodium hydroxide, and the absorbance was recorded at 405 nm. ...
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Aims: This study was designed to investigate the enzyme inhibitory and antidiabetic activity for the constituents isolated from Dillenia indica. Methods: The leaves of D. indica were extracted with methanol and subjected to fractionation and chromatographic separation, which led to the isolation of seven compounds: betulinic acid (1), n-heptacosan-7-one (2), n-nonatriacontan-18-one (3), quercetin (4), β sitosterol (5), stigmasterol (6), and stigmasteryl palmitate (7). Among these isolates, compounds 1, 4, 5, and 6 were evaluated for in vitro enzyme inhibition and compounds 4, 5 and 6 were evaluated for antidiabetic activity in streptozotocin-nicotinamide induced diabetic mice. Results: Compounds 1, 4, 5, and 6 showed 47.4, 55.2, 48.8, and 44.3% α -amylase inhibition, respectively, and 52.2, 78.2, 52.5, and 34.2% α -glucosidase inhibition, respectively, at the dose of 50 µg/kg. Compounds 4, 5 and 6 also showed significant (∗P < 0.05) antidiabetic activity in streptozotocin-nicotinamide induced diabetic mice at the dose of 10 mg/kg. Conclusion: These results provide evidence that Dillenia indica might be a potential source of antidiabetic agents.
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Since the approval of Noni juice as a novel food by the European Commission in 2003, products derived from Noni fruit (Morinda citrifolia, Rubiaceae) are becoming increasingly popular as food supplements [1]. While the knowledge on constituents of Noni fruit has considerably increased over recent years, quantitative data on Noni secondary metabolites remain scarce and the chemical composition of commercial products distributed mainly via internet is poorly established. In the present study, TLC profiles of commercial Noni juices and capsules were compared. Chromatographic markers such as 3-methyl-1,3-butanediol typically found in Noni juices were identified. The presence of sorbic acid (E200) was also revealed in one Noni juice declared as additive-free. In order to obtain quantitative data on the composition of Noni products, an HPLC-MS method has been developed and validated, which enables the quantification of various Noni constituents, including iridoid glucosides, scopoletin, rutin, fatty acid glucosides and anthraquinones. The separation is performed on a C-18 column with a gradient of acetonitrile in water containing 0.1% formic acid. Detection is carried out with ESI-MS in the negative ion mode. The method was applied to the analysis of various commercial juices and capsules. Significant differences were observed between the samples. Asperulosidic acid, deacetylasperulosidic acid and rutin were present in all products analysed, but their concentrations differed greatly between the products. The fatty acid glucosides noniosides B and C [2], as well as scopoletin, present in the fruit powder, were only detected in some commercial preparations. The mutagenic anthraquinone alizarin which has been reported from roots and leaves was not detected in the investigated samples. References: [1] Potterat, O., Hamburger, M. (2007) Planta Med. 73: 191. [2] Dalsgaard, P.W. et al. (2006) Planta Med. 72: 1322.
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Diabetes mellitus has emerged as a major healthcare problem in India. Management of postprandial plasma glucose (PPG) level is important to prevent the complications associated with type-2 diabetes. Considering paucity of studies motivated us to compare the effect of Acarbose, Miglitol and Voglibose on postprandial hyperglycemia and HbA1C. It was single blind, randomized, parallel group, comparative, prospective clinical trial on 90 diabetes type 2 patients defined as post prandial plasma glucose (PPG) levels more than 200 mg % and glycosylated haemoglobin more than 7 % at visit 1. Glycosylated hemoglobin (p=0.78) and post prandial blood glucose (p=0.61) was reduced more by Voglibose than Miglitol and Acarbose. Though this finding is not statistically significant, adverse effect profile was better with Voglibose (6.66%) than Miglitol (16.66%) and Acarbose(33.33%). Present study recommends use of Voglibose looking at its efficacy and safety profile as preferential choice in the management of postprandial hyperglycaemia in treatment of type-2 diabetes mellitus.
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Background: In South Asian countries, stems of the plant, Tinnospora crispa, Linn (TC) are often used as a folk medicine in the treatment of type 2 diabetes mellitus. Though TC's antiglycemic activity has been demonstrated in diabetic rats, the mechanism for its action has not yet been elucidated. Objective: To investigate the effect of TC's aqueous extract (TCA) on glucose transport activity in skeletal muscle cell line. Materials and methods: A skeletal muscle cell line, L6 myoblasts, was used for this study. The myoblasts grown to the stage of fused myotubes were pre-incubated with and without TCA for 24 hours. Then, a 2-[ 3H]-deoxy-D-glucose (2-DG) uptake test was made in a 24-well plate for 10 minutes. In the downstream transport regulation studies, the TCA pre-incubated cells was either treated or untreated with specific inhibitors of the PI3-Kinase (wortmannin) and p38 MAP-Kinase (SB203580) pathways prior to the uptake test. All studies were carried out in triplicate with a minimum of three independent experiments. Results were expressed as mean±SE and compared with student's t test for a level of significance at p<0.05. Results: TCA at 4 mg/mL significantly enhances glucose uptake of L6 myotubes in dose and time dependent manner with the half maximum effects at 24 hours (196.60±11.09%, p<0.05). The effect was completely abolished by a cytoskeletal blockade (10 μM of cytochalasin B), supportive of active glucose transport activity. Both wortmannin and SB203580 have no effect on the TCA-stimulated glucose uptake. Conclusion: Tinospora crispa enhances glucose transport of L6 myotubes in an insulin-independent pathway in a time- and dose-dependent manner.