world journal of pharmaceutical research 7105

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.



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



1638

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.



1639

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


Mass of granules/ tapped volume

iii. Carr’s index (% compressibility)


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


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

interparticle cohesion.

http://en.wikipedia.org/wiki/Engineering

http://en.wikipedia.org/wiki/Granular_material



1641

Table 2: Angle of repose as an indication of powder flow properties.

Angle of Repose

(Degree) Type of flow

<20 Excellent

20-30 Good

30-34 Passable

>40 Very poor

When bulk granular materials are poured onto a horizontal surface, a conical pile will form.

The internal angle between the surface of the pile and the horizontal surface is known as the

angle of repose and is related to the density, surface area, and coefficient of friction of the

material. Material with a low angle of repose forms flatter piles than material with a high

angle of repose. In other words, the angle of repose is the angle a pile forms with the ground.

Angle of repose or tan Ǿ = Height of the pile / Radius of the cone = 2H/D = H/R

Ǿ = tan -1

[H/R]

Angle of repose close to 250 indicate a very good flow.

More than 50o indicate unsatisfactory flow properties.

Angle of repose can be improved by 0.2% Aerosil.

A graph had been shown from reported literature to understand the relation between Carr’s

index and Angle of Repose of a powder and granules.

Table 3: Relation between Angle of repose and Carr’s index.

Angle of

Repose Carr’s index

25 10

30 15

40 24

45 30

Relation ship between Carr's index

and Angle of Reposey = 0.98x - 14.55

R2 = 0.9973

0

5

10

15

20

25

30

35

0 20 40 60

Angle of Repose

Car

r's

ind

ex

Carr's index

Linear (Carr's

index)

Linear (Carr's

index)

http://en.wikipedia.org/wiki/Cone_(geometry)

http://en.wikipedia.org/wiki/Density

http://en.wikipedia.org/wiki/Coefficient_of_friction



1642

10. Standardization of formulation of phospholipids complexes of P. Guajava by HPLC.

The formulation of phospholipids complexes of P. Guajava was taken for standardization by

HPLC using the method as mentioned above. The quantification of dosage form was done in

terms of quercetin phospholipids complexes.

11. Bio-activity evaluation of formulation of phospholipids complexes of P. Guajava for anti-

inflammatory study.

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. 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

quercetin and formulation of phospholipids complexes of P. guajava extract 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. The paw volumes were measured for 1st, 2

nd, 3

rd, 4

th, 5

th hour respectively by

plethysmometer. The standardized dose of granules of plant phospholipids complexes was

taken which was 100 mg of granules of plant phospholipids complex (equivalent of 69 mg of

plant complex) and compared with the other groups by taking diclofenac sodium as standard.

The percentage of inhibition of edema formation were calculated by using formula.

% of Inhibition = Vc- Vt /Vc x 100

The results were evaluated by analysis of variance (ANOVA) followed by Dunnett’s t-test.

P-value < 0.05 was considered as significant.

RESULTS AND DISCUSSION

1. Preparation of P. Guajava extracts

The yield value was 36.44 gm from 0.8 kg of powdered drugs of leaves of P. Guajava.

Chemical investigation and chemo profiling

Preliminary test was carried out by ferric chloride test where total extracts of P. Guajava was

revealed the presence of polyphenols.

2. Chemo profiling by TLC

Rf value of standard quercetin was compared with the various components of extracts. One of

the components was matched with the standard quercetin to confirm the presence of it in the



1643

total extract of P. Guajava. After inspecting the spot under long UV, the flavonoid peak

appeared as dark band against a light back ground. They are difficult to detect in visible lights

but at high concentration they appear as pale-yellow color. Very useful detection method in

flavonoid analysis is by diphenyl boric acid ethanolamine (NP reagent) complex as spray

reagent (1% in methanol). The strength of this reagent lies in its ability to distinguish between

different substituent patterns in the b-ring of molecules.

Mono substituted produce – green color

2/3 substituent produce – orange and pink color

Substituent group if replaced hydroxyl with methoxy then it became reduced polarity and

showed higher rf value. Sensitivity can be improved further by spraying with 5% ethanolic

solution peg 4000. 1% solution of np in 5% ethanolic peg 4000 gives a comparable result.

Rf value of standard quercetin was found to be 0.6

3. Chemo profiling by HPLC

The HPLC using C18 column with above mentioned mobile phase with PDA detector

detected standard quercetin at retention time 32.2 mins. The total extract of P. Guajava

revealed quercetin peak distinctly at same retention time as that of standard. Standard

Quercetin was taken at 1 µg/ml. 10 µg of Standard Quercetin had given 92460 peak areas.

The extracts were prepared 10µg/ml. The peak area was obtained from this is 2311.5(FIG-2)

The quantification of quercetin in total extracts of P. Guajava was .02%w/w

Fig 1: Chromatogram of standard quercetin.

Sr. No. Retention time Area % Area Total area

1. 32.221 2311.5 100 2311.5



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4. Preparation of phospholipids complex of quercetin and extract of P. guajava 5gm of the

complexes was obtained after vacuum drying.

5. Determination of total polyphenol content of leaf extracts of P. guajava Total polyphenol

content was determined by the gallic acid equivalent methods by establishing a standard

curve of gallic acid.

Concentration of gallic acid

in micrograms/ml Absorbance

0.2 0.1565

0.4 0.3225

0.6 0.4795

0.8 0.6395

1 0.7867

Fig 2: A standard Gallic acid curve.

Total polyphenol content was found to be in hydrolyzed extracts was 0.0494% w/w by GAE

method.

6. 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. As per the reported literature the complexes of

phospholipids mask the major aromatic ring proton of the flavonoids compare to its free

from. Spectral study of both 1h NMR and 13c NMR had proved the formation of the

complexes. Similarly, the extracts were compared to its complexed form.

a) Characterization by 1H NMR

Complexation with phospholipids changes the solubility of the flavonoids like quercetin and

extract containing them therefore converting them in to more lipophilic one so the complexes

can be evaluated by NMR spectroscopy by solubilizing them in to deuterated chloroform.



1645

The spectroscopic characteristic of the complexes was appreciably different than their

individual form. The proton spectra of the complexes are characterized by a strong

broadening or disappearance of the signals of the flavonoids and the polar head of

phosphatidylcholine, which is choline NMe3 group. The signals due to terminal methyl

groups (about 0.9 ppm) and the methylene’s (about 1.4- 3.0) of the lipidic chain became

broader with loss of the fine structure.[6,7]

b) Characterization by 13C NMR

The C13NMR the signals due to the lipidic chain are still observed together with a broadened

NMe3quartet about 55 ppm. The polar head signal of lipid moiety, as well as the lines of the

parent compounds, disappears as consequences of the complexation which strongly modify

the molecular mobility. The complexes were characterized by strong lipophilic, resulting in

increased bioavailability and activity. Both the complexes of quercetin and the extracts were

characterized by similar fashion.



1646



1647

7. UV Analysis of the Quercetin and its Phospholipid Complexes

The complexes were evaluated by UV. The quercetin produced three distinct maxima where

the complex form of quercetin exhibited one distinct peak ensuring the process of

complexation. The plant phospholipids complexes were taken and observed. They were

super- imposable with the quercetin complex further ensuring the evidence of complexation.

Figure-8: UV spectra of the quercetin.

Fig 9: UV spectra of the quercetin-phospholipids complex.



1648

Fig 10: UV spectra of the quercetin-phospholipids complex and plant-phospholipid

complex.

8. Chromatographic evaluation of phospholipids complexes of quercetin and p. Guajava

extracts

A HPLC method was developed to evaluate phospholipids complexes of quercetin and

extracts of p. Guajava. The retention time of complexes was lesser than the retention time of

free quercetin reflects the reduction of polarity upon complexation of quercetin.

I. Standardization of phospholipids complexes of P. guajava extracts by HPLC.

Quantification of quercetin complex in complexes of P. Guajava extracts were carried out by

HPLC methods and results were reported bellow.

As per the HPLC data of standard quercetin-Phospholipids complex 10µg quercetin-

Phospholipids complex showed Peak area = 1305799. As per HPLC data of Phospholipids

complex of P. Guajava 10µg Plant-Phospholipids complex showed Peak area = 890225.So as

per the calculation 10 µg Plant-Phospholipids complex contain 10 X 890225 / 1305799 = 6.8

µg of Quercetin Phospholipids complex. The percentage of quercetin-Phospholipids complex

in Plant-Phospholipids complex is 68% Remaining part of Phospholipids complexes may

contain complexes of other polyphenols present in the extracts. This may contribute their role

in the pharmacological activity of total plant phospholipids complexes.



1649

Fig 11: HPLC Chromatogram of quercetin- phospholipids complex.

Minutes

0 5 10 15 20 25 30 35 40 45 50 55

Volts

0.00

0.01

0.02

0.03

Fig 12: HPLC Chromatogram of plant phospholipids complex.

9. Bio-activity evaluation of complexes

A) Evaluation of lipid peroxidation inhibitory activity

Both the complexes of p. Guajava extract and quercetin were found to be capable of reducing

the thio-barbituric acid reactive substances (TBARS) formation better than their free form

with an ic50 value of 16.86µg/ml and 13.09µg/ml. These results were evaluated by comparing

with standard curcumin which showed the ic50 value of 49.33µg/ml (table-!). This suggested

the phospholipids complexes of p. Guajava extract and quercetin possesses better free radical

scavenging and antioxidant activity in vitro than its free form.



1650

Table 4: Evaluation of lipid peroxidation inhibition activity by TBARS method.

Material Ic50 µg/µl Quercetin 24.32

Quercetin phospholipids complex 16.86

P. Guajava extract 18.94

Phospholipids complexes of p.

Guajava extract 13.09

Curcumin 49.33

A comparative result had been shown in the following graph which concludes the superiority

of plant phospholipids complexes over the standard curcumin and its free form.

24.32484076

18.942116.8677

13.0947

49.33

0

10

20

30

40

50

60

Quercetin Polyphenols Quercetin

complex

Plant

complex

Curcumin

Fig 13: Comparative study of lipid peroxidation inhibitory activity.

10. Evaluation of anti-inflammatory activity

The complexes of p. Guajava extract exhibited significant reduction of paw edema[6,7]

1.2(±0.01637) ** in rat at third hour compare to control 2.5(±0.0378) ** and standard

diclofenac sodium 0.85(±0.0189) ** at 100 mg/kg dose at third hour. The bio enhanced

activities of complexes significantly support the concepts of the method of complexation

which was the conversion of hydrophilic flavonoids in to their lipophilic phospholipid

complexed form.



1651

Table 5: Evaluation of anti-inflammatory activity by rat paw edema methods.

Evaluation of Anti-Inflammatory Activity by Rat Paw edema (by Winter et al Method) were

presented in terms of % reduction value and compared with different groups at the third hour.

( Diclofenac

Na)

Quercetin

Polyphenols

Quercetin

complex

Plant

complex

0

10

20

30

40

50

60

% reduction of paw oedemaof different group

% re

duct

ion

valu

e

Fig 14: (A) % Reduction of paw edema at 50 mg/kg dose.



1652

( Diclofenac

Na),

48.48(±.0091)*

Quercetin,

36(±0.01637)**

Polyphenols,

40(±0.0378)**

Quercetin

complex,

45.6(±0.0548)**

Plant complex,

52(±0.0183)**

0

10

20

30

40

50

60

% reduction of paw oedema

of different group

% r

edu

ctio

n v

alu

e

Fig 15: (B) % Reduction of paw edema at 100mg/kg dose.

11. Formulation development of phospholipids complexes of P. Guajava

After confirming the enhanced therapeutic potential of plant phospholipids complex of P.

guajava, was taken into formulation development. The formulation was optimized on the

basis of preformulation parameters. The correct proportion of starch and aerosol in the

formulation of plant phospholipids complexes were set the satisfactory preformulation

criteria for further studies (which were depicted in the table-5). 5 gm of formulation was

taken for each study. The final formulation selected on the basis of good pre-formulation

index. The optimized formulation was exhibited the satisfactory Carr’s index, Hausner ratio,

and Angle of repose.

Table 6: Formulation and evaluation components of phospholipids complex granules.

Sr

No. Diluent

Preformulation Evaluation

Bulk

density

Tap

density

Carr’s

Index

Hausner’s

Ratio

Angle of

Repose

1 400mg MCC 0.517 1.142 50 2 52.51

2 200mg MCC+0.2mg Starch

Paste 0.35 0.445 21.34 1.27 40.08


Paste+0.2%Aerosil 0.416 0.5 16.8 1.20 36.24


Paste+0.1%Aerosil 0.714 0.833 14.28 1.16 21.15

Standardization of formulation of phospholipids complexes of P. guajava by HPLC.

The formulation of phospholipids complexes of P. guajava was taken for standardization by

HPLC using the method as mentioned above. The quantification of dosage form was done in

terms of quercetin phospholipids complexes.



1653

Minutes

0 5 10 15 20 25 30 35 40 45 50 55

Volts

0.00

0.01

0.02

0.03

Fig 16: Standardization of Formulation of Quercetin phospholipids complex through

HPLC methods.

Name Retention time Area

Channel A 30.28 901025

12. Standardization of Formulation of Quercetin phospholipids complex

As per the HPLC data of standard quercetin-Phospholipids complex

10µg quercetin-Phospholipids complex showed Peak area = 1305799

As per HPLC data of Phospholipids complex of P. guajava

50µg of formulation of Plant-Phospholipids complex showed

Peak area = 901025

So as per the calculation

50 µg formulation of Plant-Phospholipids complex contain

50 X 901025 = 34.5 µg of Plant-Phospholipids complex

1305799

The percentage of Plant-Phospholipids complex in formulation of Plant-Phospholipids

complex was

34.5 X 2 = 69%

Therefore, 69mg of Plant-Phospholipids complex present in 100 mg of plant phospholipids

granules.

Therefore, 100mg Plant-Phospholipids complex was present in 144.92 mg of granules of

plant phospholipids complex.

145mg of granules of plant phospholipids complex can be equivalent to 100 mg plant

phospholipids complexes.



1654

But the activity was carried out at 100 mg granules of plant phospholipids complex to

compare the activity with the 100 mg of plant phospholipid complex.

13. Bio-activity evaluation of formulation of phospholipids complexes of P. guajava for anti-

inflammatory study

The granules of weighed quantity of phospholipids complexes of P. guajava extract exhibit

significant reduction of paw edema by 45.45% in rat at third hour compare to standard

diclofenac sodium (48.48%).

Table 7: Percent reduction of rat paw edema upon treatment with granules.

Name of the

group

(n = 8)

Dose

In

mg/kg

% reduction of paw edema

at different hour intervals(±SEM)

1

st 2

nd 3

rd 4

th 5

th 6

th

Standard

(Diclofenac Na) 40

29.23

(±0.0189)

34.78

(±.0091)

48.48

(±.0091) **

36.61

(±0.0183)

31.57

(±0.01637)

28.57

(±0.01637)

Quercetin 50 13.84

(±0.0189)

17.39

(±0.0378)

25.45

(±0.0548) **

22.53

(±0.0378)

10.52

(±0.0548)

7.14

(±0.0548)

100 6.66

(±0.0189)

21.05

(±.0091)

36

(±0.01637) **

17.64

(±.0091)

14.28

(±.0091) ___

Plant extracts 50 23.07

(±0.0378)

23.91

(±0.0183)

33.33

(±0.0183) **

26.05

(±0.0183)

14.73

(±0.01637)

14.28

(±0.01637)

100 16

(±0.0183)

24.73

(±.0091)

40

(±0.0378) **

23.52

(±0.0548)

10.25

(±0.0378) ___

Quercetin complex 50 29.33

(±0.0378)

29.34

(±0.0378)

39.39

(±0.0548) **

33.09

(±0.0378)

20

(±.0091)

21.42

(±0.01637)

100 20

(±0.0548)

31.57

(±0.0548)

45.60

(±0.0548) **

29.41

(±0.0183)

21.42

(±0.0183) ___

Formulation of

plant

complex(Granules)

100 33.84

(±0.0183)

33.69

(±.0091)

45.45

(±0.0183) **

43.66

(±.0091)

36.84

(±.0091) ___

** p- value < .01

CONCLUSION

The summary of the project entitled product development from an herbal raw material can be

composed in the following fashion.

The polyphenol rich extracts of leaves of p. Guajava were evaluated for chemo profiling by

talc and HPLC with respect to standard quercetin. The total polyphenol content was

determined by gallic acid equivalent methods which revealed 0.0494% of polyphenol in total

extracts. With the HPLC it was calculated that .02% quercetin was present in total extracts.

After that the complexation with phospholipids was carried out to convert the polyphenols of



1655

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 from an herbal raw material. The plant complexes were taken for further

formulation development. The formulations were optimized on the basis of preformulation

parameters. Then the optimized formulation was evaluated for its bioactivity profile by in

vivo method to certify the originality of the claims.

In conclusion it can be stated that the intervention of polyphenolic synergism can also

influence the bio activity profile of phyto molecules after their complexation with

phospholipids.

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