www.wjpr.net Vol 9, Issue 5, 2020. 1635 OPTIMIZATION AND STANDARDIZATION OF FORMULATION CONTAINING SUPRAMOLECULAR LIPID COMPLEXES OF PSIDIUM GUAJAVA AND ITS BIOACTIVITY EVALUATION Kajal Gupta 1* , Pooja Jaiswal 1 , Tarun Kumar Dasgupta 2 and Priscilla M D’Mello 2 1 Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research, Balanagar, Hyderabad- 500037, India. 2 Department of Pharmacognosy and Phytochemistry, Prin.K.M.Kundnani College of Pharmacy, Colaba, Mumbai-400005, India. ABSTRACT The polyphenol rich extracts of leaves of P.guajava were evaluated for chemo profiling and HPLC with respect to standard quercetin. With the HPLC it was calculated that 0.02% quercetin was present in total extracts. After that the complexation with phospholipids was carried out to convert the polyphenols of extracts of leaves of P.guajava into their bio-absorbable lipophilic fraction which improve their pharmacokinetic profile. The activities were evaluated by lipid peroxidation inhibitory activity and anti-inflammatory activity using various in-vitro and in-vivo model. The superiority of the action of complexes has demonstrated the concepts behind the product development of the herbal raw material. In sum the synergism of the bio activity profile of phyto-constituents is possible by their complexation with phospholipids. These complexes were then formulated into granules for bio-enhancement of this supra molecular phospholipid complexes. The activities of these granules were evaluated by lipid peroxidation inhibitory activity and anti-inflammatory activity using various in-vitro and in-vivo model. INTRODUCTION P. Guajava has a rich ethnomedicinal history. Different parts of the plant are used in various indigenous systems of medicine, primarily for the treatment of GI ailments. Most phytochemical analyses investigated the properties of guava leaf products, revealing more World Journal of Pharmaceutical Research SJIF Impact Factor 8.084 Volume 9, Issue 5, 1635-1655. Research Article ISSN 2277– 7105 Article Received on 10 March 2020, Revised on 31 March 2020, Accepted on 20 April 2020, DOI: 10.20959/wjpr20205-17425 *Corresponding Author Kajal Gupta Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and Research, Balanagar, Hyderabad- 500037, India.
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www.wjpr.net Vol 9, Issue 5, 2020.
Gupta et al. World Journal of Pharmaceutical Research
1635
OPTIMIZATION AND STANDARDIZATION OF FORMULATION
CONTAINING SUPRAMOLECULAR LIPID COMPLEXES OF
PSIDIUM GUAJAVA AND ITS BIOACTIVITY EVALUATION
Kajal Gupta1*
, Pooja Jaiswal1, Tarun Kumar Dasgupta
2 and Priscilla M D’Mello
2
1Department of Pharmaceutical Analysis, National Institute of Pharmaceutical Education and
Research, Balanagar, Hyderabad- 500037, India.
2Department of Pharmacognosy and Phytochemistry, Prin.K.M.Kundnani College of
Pharmacy, Colaba, Mumbai-400005, India.
ABSTRACT
The polyphenol rich extracts of leaves of P.guajava were evaluated for
chemo profiling and HPLC with respect to standard quercetin. With
the HPLC it was calculated that 0.02% quercetin was present in total
extracts. After that the complexation with phospholipids was carried
out to convert the polyphenols of extracts of leaves of P.guajava into
their bio-absorbable lipophilic fraction which improve their
pharmacokinetic profile. The activities were evaluated by lipid
peroxidation inhibitory activity and anti-inflammatory activity using
various in-vitro and in-vivo model. The superiority of the action of
complexes has demonstrated the concepts behind the product
development of the herbal raw material. In sum the synergism of the
bio activity profile of phyto-constituents is possible by their
complexation with phospholipids. These complexes were then formulated into granules for
bio-enhancement of this supra molecular phospholipid complexes. The activities of these
granules were evaluated by lipid peroxidation inhibitory activity and anti-inflammatory
activity using various in-vitro and in-vivo model.
INTRODUCTION
P. Guajava has a rich ethnomedicinal history. Different parts of the plant are used in various
indigenous systems of medicine, primarily for the treatment of GI ailments. Most
phytochemical analyses investigated the properties of guava leaf products, revealing more
World Journal of Pharmaceutical Research SJIF Impact Factor 8.084
Volume 9, Issue 5, 1635-1655. Research Article ISSN 2277– 7105
Article Received on
10 March 2020,
Revised on 31 March 2020,
Accepted on 20 April 2020,
DOI: 10.20959/wjpr20205-17425
*Corresponding Author
Kajal Gupta
Department of
Pharmaceutical Analysis,
National Institute of
Pharmaceutical Education
and Research, Balanagar,
Hyderabad- 500037, India.
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Gupta et al. World Journal of Pharmaceutical Research
1636
than 20 isolated compounds, including alkaloids, anthocyanins, carotenoids, essential oils,
fatty acids, lectins, phenols, saponins, tannins, triterpenes, and vitamin C. Psidium guajava
budding leaves (PBL) are characteristically enriched in polyphenolics. The main active
constituent in the plant is quercetin.
Since their high hydrophilic character was responsible for their poor pharmacokinetic profile
the attempt had been made to convert them in to more lipophilic and easy absorbable
therapeutic entity after complexation with natural phospholipids. The complexes were
confirmed by various spectral studies and standardized by chromatographic techniques. Their
(complexes) bioactivity evaluation was carried out on in-vitro and in-vivo model by
comparing them with standard drugs. Optimization of formulation of preparation containing
plant phospholipids complexes. Standardization of optimized formulation of plant
phospholipids complexes. Bioactivity studies of optimized formulation of plant phospholipids
complexes. The activity of formulation of plant phospholipids complexes was given a
necessary conclusion about bio enhancement of the activity after complexation.
MATERIAL AND METHOD[8]
Leaves of P. guajava were taken from a local source from Mumbai Andheri (E) and
authenticated at laboratory. Quercetin and phospholipids were obtained from Sigma Aldrich.
All the solvents used were technical grade. For the animal experimentation work the prior
approval had been taken from the Animal Ethics Institution Committee.
1. Preparation of P. guajava extracts
Dried and powdered leaves of P. guajava were extracted by using ethanol in Soxhlet
extraction apparatus. Extracts were taken for vacuum drying for one day to get powder
extract. The extracts were hydrolyzed by mixing with equal volume of 2 M HCL and refluxed
on a water bath for 30 minutes. The mixture is cooled and liberated aglycones were extracted
with ethyl acetate.
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2. Chemical investigation of P. guajava extracts and their chemo profiling
A preliminary chemical investigation was carried out by Ferric chloride test and chemo
profiling were carried out by HPLC method.
a) Phytochemical chemo profiling by TLC
Mobile phase used for detecting flavonoids in the extract of P. guajava was combination of
ethyl acetate: formic acid: acetic acid: water 100:11:11:27 ratios. The spots were developed
on silica plate over 35 minutes and inspected under long UV by using a detecting reagents
Diphenyl boric acid ethanolamine (NP reagent).
b) Phytochemical Chemo profiling by HPLC
Chemo profiling of total extract were done by JASCO-HPLC using a C18 column with
mobile phase Acetonitrile: Acetic acid in ratio of 1: 0.2. PDA detector was used to detect
quercetin in total extract. The column was washed by a combination phase of acetonitrile:
methanol: acetic acid for 1 hour before treatment. The flow rate was adjusted 1ml/min.
3. Total polyphenol content determination of P. Guajava
i. Gallic acid equivalent method
The amounts of total polyphenols in the extracts were determined according to the Prussian
blue method using 1% gum acacia and 85% phosphoric acid as a color stabilizer.[4]
To 0.1 ml
of sample solutions, 1 ml of 0.016 M Potassium Ferricyanide (K3Fe (CN)6) was added
followed immediately by 1 ml of 0.02 M Ferrous Chloride (fecl3) in 0.1 N HCl. The contents
were mixed well and kept at room temperature for 15 min. This was followed by addition of
5 ml of stabilizer containing water, 85% Phosphoric Acid (H3PO4) and 1% gum acacia in
volume proportions of 3:1:1. The contents were vortexed and the color density was measured
at 700 nm against a reagent blank consisting of all of the reagents except the polyphenols
using Shimadzu UV/Vis spectrophotometer-1601. The total polyphenol contents were
calculated as % w/w of gallic acid equivalents.
4. Preparation of phospholipids complexes of quercetin and P. guajava extracts
Weighed quantity of quercetin was taken in to suitable solvent with phospholipids as per
reported literature.[1]
The complexes were purified by precipitating with non-solvent and
vacuum dried to get pure form. In similar way extract of P. Guajava was taken for
complexation with phospholipids. Complexation was carried out in RBF with the help of
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reflux condenser. Phospholipids complexes of quercetin and extracts of P. guajava were
prepared as per reported literature.[2]
5. Characterization of complexes of phospholipids
Phospholipids complexes of quercetin and extracts of P. guajava were taken for the
characterization by 1H NMR and 13C NMR. Both the complexes were also evaluated by
simple UV method using Shimadzu UV/Vis spectrophotometer-1601.
6. Standardization of phospholipids complexes of P. guajava extracts by HPLC
The complexes of P. guajava extracts were taken for the quantification by HPLC in terms of
the quercetin phospholipids complex. The method for HPLC was same as stated above. The
peak area was taken in to consideration to evaluate the concentration of complexes in the
sample.
7. Bio-activity evaluation of phospholipid complexes of quercetin and P. Guajava extracts
The activities of both the complexes were evaluated by various in vivo and in-vitro model.
a) Evaluation of Lipid Peroxidation Inhibitory Activity[3]
Lipid peroxidation inhibitory activity of phospholipids complexes of P. Guajava extracts and
quercetin were carried out and compared with their free form. The lipid peroxidation was
generated by Fenton reaction and measured as thiobarbituric acid reactive substances
(TBARS) as per reported literature.[4]
b) Preparation of Mice liver homogenates
Mice of 40-50 g fed on a standard laboratory diet and supplied water ad libitum. The liver
was excised, perfused and homogenized with 120 mm KCL, 50mm phosphate buffer, pH 7.4
(1:10 w/v).
c) Ferrous ascorbate induced lipid peroxidation scavenging
Principle of Fenton reaction was used to generate the free radicals in liver homogenates after
addition of sample in to it. After 30 minutes of incubation the reaction was terminated by
using 0.67% Thiobarbituric acid in 50% acetic acid. The mixture was heated in a water bath
at 850C
for 30 minutes and in boiling water bath to complete the reaction. The intensity of
pink color complex formed was measured at 535 nm by UV spectrophotometer.
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The percentage inhibition of lipid-peroxidation was calculated as per the following formula: -
Inhibition (%) = (control – test) X 100/ control
The results were evaluated by Analysis of variance (ANOVA) followed by Dunnett’s t-test.
P-Value < 0.05 was considered as significant
8. Evaluation of anti - inflammatory activity[5]
Anti-inflammatory activity was carried out by winter et al method. Healthy Wister albino rats
of either sex weighing about 150-250 gm were used and starved overnight. To ensure
uniform hydration the rats received water ad libitum.
a) Grouping of the animal
The rats were grouped in to six (each contain eight rats of both sex). They are control (with
tween 80%), standard (diclofenac sodium), group treated with P. guajava extract, quercetin
and phospholipids complexes of P. guajava extract and quercetin respectively. Carrageenan
(.1 ml of 1% in normal saline) was injected in the right paw to all groups one hour after the
administration of vehicle, standard, marker, extracts, and complexes by using a catheter.
b) Paw volume measurement
The paw volumes were measured for 1st, 2
nd, 3
rd, 4
th, 5
th hour respectively by plethysmometer
at two different dose (100 mg/kg and 50mg/kg) levels. The percentage of inhibition of edema
formation were calculated by using formula.
% of inhibition = Vc- Vt /Vc x 100
Vc = paw volume of control
Vt = paw volume of test
The results were evaluated by analysis of variance (ANOVA) followed by Dunnett’s t-test.
P-value < 0.05 was considered as significant.
9. Formulation development of phospholipids complexes of P. Guajava
A suitable oral solid dosage forms were designed by optimizing the formulation on the basis
of preformulation studies. The excipients used were lactose, MCC, and Aerosil in different
ratio. Optimization of formulation was carried by evaluating various parameters like Bulk
density, Tapped density, Carr’s index, Hausner ratio and angle of repose. The optimized
granules were incorporated in to hard gelatin capsule.
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i. Bulk density
This was calculated by formula
Mass of granules/ untapped volume
ii. Tapped density
This was calculated by formula
Mass of granules/ tapped volume
iii. Carr’s index (% compressibility)
This was calculated by formula
Tap density – Bulk density X 100
Tap density
Table 1: The following values of Carr’s index are indicating the complementary flow
properties.
Carr’s index Type of flow
5-15% Excellent
12-16% Good
18-21% Fair
23-28% Poor
35-38% Very poor
iv. Hausner ratio
This was calculated by formula
Tap density/ Bulk density
A low Hausner ratio indicates that granules were in good flow properties. A Hausner ratio of
less than 1.25 (equivalent to 20% Carr’s) indicates good flow. While greater than 1.5
(equivalent to 33% Carr’s) indicates poor flow.
v. Angle of repose.
The angle of repose (sometimes incorrectly confused with the 'Angle of Internal Friction') is
an engineering property of granular materials. The angle of repose is the maximum angle of a
stable slope determined by friction, cohesion and the shapes of the particles. It has been an
indirect methods of quantifying powder flowability, because of their relationship with