DEVELOPMENT OF AMINO ACID CONJUGATED VANILLIN COPPER (II) COMPLEXES WITH ANTI-DIABETIC ACTIVITY

Abstract

Diabetes mellitus is a prevalent chronic metabolic disorder characterised by impaired insulin function or resistance. Current treatments for diabetes often face challenges related to efficacy and side effects. This invention is to develop a Copper (II) complex of vanillin and polar non-essential amino acids as an effective anti-diabetic agent with enhanced cellular uptake and bioavailability, which may improve its potential therapeutic efficacy in diabetes management.

Specification

FIELD OF THE INVENTION
This invention presents an advanced therapeutic agent with anti-diabetic activity
exhibiting enhanced cellular uptake and increased bioavailability. Here we
developed a conjugation of vanillin with an amino acid to form a Schiff base
ligand with anti-oxidant and anti-diabetic effect capable of forming a complex
with Cu (II) ions, additionally increasing its cell intake and effectiveness which
improves its potential therapeutic efficacy in diabetes management


BACKGROUND OF THE INVENTION
Diabetes Mellitus (DM) is a chronic metabolic disorder characterized by high
blood glucose levels due to insufficient insulin production or ineffective insulin
use. It affects approximately 530 million adults worldwide. Type 2 diabetes,
which accounts for 90% of DM cases, results from the body's poor glucose
uptake due to ineffective insulin response. Factors contributing to the increasing
prevalence of diabetes include urbanization, longer life expectancy, reduced
physical activity, and lifestyle changes leading to obesity. This underscores the
need for improved treatment methods through research and development.
Vanillin, a component of vanilla, has demonstrated antidiabetic effects,
including antioxidant, anti-inflammatory, and antimicrobial properties. It
improves glucose homeostasis and insulin sensitivity but has low bioavailability
at 7.6%. To enhance bioavailability, vanillin can be supplemented as a ligand
forming complexes with metal ions like copper (Cu), which play roles in
cellular uptake. Amino acids, particularly Glutamine, Arginine, Serine,
Tyrosine and Glutamic acid, can form Schiff bases with vanillin, potentially
enhancing pharmacological properties and enzyme inhibition capabilities
relevant to diabetes. The study explores synthesizing a novel Cu (II) complex of
vanillin/amino acid ligands, aiming to increase cellular uptake and
bioavailability, thereby enhancing anti-diabetic effects. The focus is on the anti-
oxidant and anti-diabetic properties of these complexes, leveraging the diverse
coordination chemistry of Cu ions for improved biological applications. In this
invention we were able to develop a novel anti-diabetic therapeutic method that
could increase effectiveness by enhancing cell uptake and bioavailability in
diabetic patients.

SUMMARY OF THE INVENTION
1. Synthesis of ligands
The Schiff base complex was synthesised by condensation of amino acid with
vanillin in a 1:1 molar ratio using methanol as a solvent according to previously
established protocol. 4 mM of amino acid was dissolved at 60 °C in 15 ml of
100 % methanol, set to a basic pH by adding NaOH at a concentration of 8 mM
to the solution. The solution was stirred on a magnetic stirrer until completely
dissolved. The white colour solution obtained was then used for Gln-AA ligand
and Cu complex synthesis.
Gln-AA ligand was then prepared by adding Vanillin at a concentration of 4
mM to this solution and was refluxed for 1 hr at room temperature. The solution
formed a yellow colour precipitate after the reflux reaction. The precipitate was
filtered through a Whatman filter paper. The residue was collected and dried in
a hot air oven at 50 °C overnight. A yellow colour powder obtained is then
characterised by FTIR, UV-Visible spectroscopy and X-Ray diffraction to
confirm the synthesis of Gln-AA ligand.
2. Synthesis of metal complexes
A- i' H Ki i
The Gln-AA Cu complex was prepared by adding Copper (II) nitrate in the
concentration 1 mM to the methanolic solution of the ligand. This solution was
then refluxed at room temperature for 1 hr until a colour change of the solution
from yellow to blue was observed. The formation of a blue precipitate indicates
the completion of the reflux reaction. The solution was then filtered using a
Whatman filter and the residue was collected and dried overnight at 50 °C in a
hot air oven to obtain a blue colour powder. The presence of Gln-AA Cu
complex in the synthesised material is analysed by FTIR, UV-Visible
spectroscopy and X-Ray diffraction. % yield of the product, for both the ligand
and the Cu complex were calculated by the following equation:
% yield = (actual yield/estimated yield) * 100
actual yield = total weight of the final product (g)
estimated yield
= (mol. wt of final product (g/mol)
* Cone, of substrate (mM))/1000
The ligand and Cu complex was then analysed by pharmacological methods,
namely DPPH antioxidant assay, a-amylase inhibition anti-diabetic assay and
MTT biocompatibility test, for its effect as an anti-diabetic agent.
3. Characterisation Methods
- The success of the synthesis reactions was analysed by FTTR spectroscopy: The
transmittance spectra corresponding to the functional groups present in the
ligand and the Cu complex specifically are obtained using the FTIR
spectrophotometer (ALPHA II, Bruker, Germany). The data were collected
between the wavenumbers ranging from 550 cm-1 to 4000 cm-1. Further,
formation of Cu complex from the ligand was also analysed by UV-Visible
spectroscopy. UV-visible absorption spectra of the Cu Complex between the
wavelengths 200 nm to 900 nm were collected using a Jasco spectrophotometer
(V-730, Jasco, Japan). The analysis of elemental composition was carried out
with an energy-dispersive X-ray (EDX) diffractometer (D8-Advance,
BRUKER, Germany). The crystallinity of the nanoparticles was studied by
using X-Ray diffraction analysis at a voltage of 40 kV and a current of 30 mA
using Cu Ka radiation with a X of 1.5406 A with radiation as an X-ray source.
Its scanning rate was performed at 2° min-1 in the 20 range from 10° to 80°

4. Antioxidant Assay
The antioxidant activity of the ligand and its Cu complex were analysed
by determining their radical scavenging activity using DPPH assay based on the
previously reported studies. Accordingly, 100 pi of the samples and the control
(ascorbic acid) taken in various concentrations; 200 pg/ml, 400 pg/ml, 600
pg/ml, and 1000 pg/ml, were allowed to react with 500 pi of DPPH (1.6 mg/ml)
prepared in 100% methanol after vigorous mixing and incubation at RT for 60
min in dark conditions. After incubation, the absorbance was recorded at 517
nm. The percentage of radical scavenging activity was determined from average
absorbance using the following equation:
% radical scavenging activity
= [1 - (absorption of sample/absorption of blank)] * 100
5. Anti-Diabetic Assay
Inhibition of a-amylase activity by the samples was analysed to
determine their anti-diabetic properties. 500 pi of the samples and the control
(Acarbose) taken in various concentrations; 100 pg/ml, 200 pg/ml, 300 pg/ml,
and 400 pg/ml, were mixed with 250ul of starch and let to incubate in room temperature for 5 minutes allowing the samples to interact with starch. Then
250 ul of a-amylase was added to the tests and incubated for 15 minutes in 37
°C to catalyse conversion of starch to glucose. After incubation, (3.5 %) DNS A
(3,5- dinitrosalicylic acid) was made to react with glucose by adding 1 ml of it
to the tests and incubating in a water bath at 90 °C for 5 minutes. OD was tested
using ELISA plate reader at 570 nm.
% a - amylase inhibition
= ((OD of control - OD of sample)/OD of sample) * 100
6. Dental Pulp Stem cells (DPSCs) Cell Culture
OSCC (Oral Squamous Carcinoma Cells) which are fibroblast in nature
were cultured in T25 flasks in DMEM (Dulbecco's Modified Eagle Medium)
with 10% FBS (Foetal Bovine Serum) and IX P/S (Penicillin/Streptomycin).
The cells were grown to 80% confluency while the media was changed every 2-
3 days. After reaching 80% confluency the cells were passed and seeded onto
24 well plates at a concentration of 50000 cells/well for biocompatibility
analysis by MTT Assay.
7. Biocompatibility (MTT) Assay
The percentage of cell viability in human OSCC (Oral Squamous
Carcinoma Cells) on treatment with the synthesised ligand and Cu complex at
concentrations 25 pg/ml and 50 pg/ml was analysed against the control
untreated cells cultured in normal growth media by MTT assay adapted from
pre-reported studies. 24 well plates seeded at a cell density of 5000 cells/well
were treated with test groups at 50% confluency and were incubated at 37 °C in
a C02 incubator for 24 hr. After exposure of the cells to the test groups for 24
hr, the treatment media was carefully aspirated. 200 pi of MTT (3-[4,5-
dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) reagent was then added
to all the wells and incubated at 37°C for 4 hr. The MTT reagent was then
aspirated and the wells were washed twice with IX PBS (Phosphate Buffer
Saline) solution. Tetrazolium crystals that are formed due to the mitochondrial
activity of the viable cells are then dissolved by adding 200 pi of DMSO
(Dimethyl Sulphoxide) and OD is tested at 570 nm.
% cell viability = (OD of sample/OD of control) * 100
8. Statistical Analysis
All values are expressed as the mean ± standard error of the mean (SEM) of at
least three independent experiments. A one-way ANOVA (analysis of variance)
was used to test for significant differences, and multiple comparisons were
performed using Scheffe's method. Statistical significance was set at p < 0.05.



WE CLAIM
1. The process consists of adding 1 mM of Copper (II) Nitrate to a mixture
of vanillin and polar non-essential amino acid in the ratio of 1:1.
2. The composition consists of a combination of anti-oxidant and anti-
diabetic agent - vanillin and a Schiff base ligand forming amino acid also
containing anti-diabetic properties. The Schiff base ligand forms a Cu (II)
complex on reaction with copper nitrate.
3. Vanillin exhibits strong anti-diabetic activity in addition to anti-oxidant,
antimicrobial and anti-inflammatory properties.
4. The anti-diabetic activity of vanillin is attributed to its positive effect on
insulin sensitivity in turn effecting glucose metabolism and glucose
homeostasis in the body.
5. Non-essential polar amino acids present a good source for nitrogen moity
which on reacting with carbonyl group present in vanillin is replaced by
an imine group to form a highly reactive Schiff base.
6. Aromatic Schiff bases are capable of strongly reacting with various
enzyme including a-amylase and a-glucosidase further effecting glucose
metabolism.
7. These Schiff base ligands form strong bonds with a central metal ion to
form metal complexes.
8. Cu (II) complexes formed by Van-AA Schiff base ligands form highly
stable compounds with enhanced anti-diabetic properties.
9. Cu has a variety biological applications due to its diverse coordinate
chemistry rendering amino acid conjugated vanillin copper complexes
highly bioavailable.
10. This compound forms as a template for stable natural anti-diabetic agents
with high effectiveness and lesser side effects, leading to better
therapeutic methods for Diabetes Mellitus.





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