1.
INTRODUCTION:
Rheumatoid arthritis is an autoimmune disease
which could lead to severe joint damage and disability. The goal of treatment
for rheumatoid arthritis patients are to eliminate symptoms, slow disease
progression, and optimize quality-of-life [1]. Therefore, various strategies
for treatment of rheumatoid arthritis include non-steroidal anti-inflammatory
drugs or anti-rheumatic drugs. Nevertheless, enduring risks of these drugs i.e.
cardiovascular complications, hematologic toxicity, gastrointestinal ulcers, pulmonary
toxicity, nephrotoxicity, cirrhosis, and diarrhoea requires continuous
monitoring [2]. Consequently, herbal treatments are considered as safer
alternative for management of rheumatoid arthritis [3,4]. Petroleum ether
extract of Trigonella foenum-graecum (TFG) has been found to have anti-inflammatory and anti-arthritic activities due to
affluent content of linolenic acid [5-7]. Therefore, TFG ether extract can be topically used
for local inflammation, myalgia, arthritis and gout. In this investigation, Trigonella foenum-graecum ether extract phytosomes were developed by thin-film hydration technique using L-α-Phosphatidylcholine and cholesterol followed
by optimization using central
composite design to determine optimized composition and processing conditions [8].
Furthermore, optimized phytosomes were incorporated into semi-solid dosage form i.e. cream
which could generate superior therapeutic effect owing to enhanced permeability
and skin retention potential of phytosomes without producing any adverse effect [2,9-11].
2.
MATERIALS AND
METHOD:
Trigonella foenum-graecum powdered extract was procured from NJP Healthcare Pvt. Ltd. Gujarat. L-α-Phosphatidylcholine was procured
from Himedia, Mumbai. Cholesterol,
potassium dihydrogen phosphate and sodium hydroxide were procured from Loba
Chemicals Private Limited, Mumbai, India. All other ingredients employed were
of analytical grade.
2.1.
Preparation of Trigonella foenum-graecum Ether Extract:
Accurately weighed 10g of Trigonella foenum-graecum powdered extract was drenched in 50ml petroleum ether with constant
stirring for 3 hrs and subsequent filtration and evaporation of solvent. Trigonella foenum-graecum ether extract was analyzed for presence of linolenic acid using UV spectrophotometer at λmax
of 233nm [5].
2.2.
Experimental
Design:
Three factors central composite design
response surface methodology was employed using Design-Expert software (Trial Version 11.1.2.0,
Stat-Ease Inc., MN). Independent and response variables for
fabrication of Trigonella
foenum-graecum ether
extract phytosomes (TFG-PH) and
20 batches central composite
design (CCD) layout have been
depicted in Table 1 and 2, respectively [12-15].
2.4.
Evaluation of Response
Variables (Y1-Y2) of TFG-PH:
Ultracentrifugation method was employed to conclude % entrapment
efficiency of TFG-PH. In brief; TFG-PHs was centrifuged for 90 minutes at15000
rpm via cooling centrifuge apparatus followed by separation of supernatant
and analysis by UV spectrophotometer at 233 nm. % entrapment efficiency was
calculated using following equation:
DT-
DS
% Entrapment efficiency, % w/w =---------------- X 100
-------Eq. (1)
DT
Where, DT and DS are theoretical and detected amount of
linolenic acid, respectively. Percent yield (Y2) was calculated as
weight percentage of phytosomes recovered from each experimental, with respect
to initial total weight of phosphatidylcholine, cholesterol and TFG extract using
following equation [18-19].
Total weight of phytosomes
% Yield, w/w= -------------------------------------------
X 100 -------Eq. (2)
Initial weight of TFC extract and
lipids
2.5. Statistical and Response Surface Analysis by Design-Expert Software:
Quadratic equations were generated by
regressions analysis and three-dimensional response surface plots (3-D) and
corresponding two-dimensional contour plots (2-D) were created via
graphs tool of Design-Expert software to examine outcome of independent variables
on response parameters [20-22].
2.6. Search for Optimized TFG-PH by Numerical
Optimization Method:
Optimal values of composition and process variables
for production of TFG-PH were obtained employing Design-expert® 11.1.2.0
software [20]. To obtain graphical outlook of region analogous to maximum overall
desirability function, three-dimensional response surface graph and related
contour plot were created for desirability coefficient.
2.7.
Manufacturing of
Topical Phytosomes-Based Cream of Optimized TFG-PH:
Briefly, melt the beeswax (5% w/v) and hard
paraffin (5% w/v) along with liquid paraffin (30% w/v) at 70°C with gentle
mixing. Dissolve the sodium borate (0.1% w/v) in distilled water and heat up
solution to 70°C. Furthermore, dissolve glycerin (9% w/v) as humectants and
propyl paraben (0.2% w/v) as preservative to heated aqueous phase. Add mangosteen
and erythrosine to aqueous phase as fragrance and coloring agent, respectively.
Subsequently, both the phases were maintained at room temperature (~40°C) and
optimized TFG-PH (10 % w/v) was added to oil phase with
gentle stirring. Afterwards, aqueous phase was slowly added to oil phase with
moderate agitation preferably mechanically until the cream has thickened to set
[23].
2.8. Physical Evaluation of Topical
Phytosomes-Based Cream of Optimized TFG-PH:
2.8.1. Organoleptic Characteristics:
Topical phytosomes-based cream of optimized
TFG-PH was tested by visual observation for physical appearance, colour and phase
separation. Homogeneity and texture were evaluated on the basis of consistency
of cream and presence of coarse particles through pressing little amount of cream
between index finger and thumb [24,25].
2.8.2.
Spreadability
and pH Value:
Spreadability of cream was evaluated from spreading
diameter of 1 g of cream between two horizontal glass plates of
10 cm × 20 cm dimensions. The standard weight imposed upon upper plate
was 25 g for 1 minute [26]. The pH was measured by pH meter (361, Systronics, India) calibrated through standard buffer
solutions (pH 4, 7, and 10) [24,27].
2.8.3.
Viscosity Measurement:
Brookfield rotational digital viscometer
model DV-II with LV-spindle # 64 at 6 rpm was used to determine viscosity of cream. Approximately 50 g cream was used for
measurement which was maintained at temperature of 25°C during the
measurements. All measurements were taken in triplicate and represented as mean
± SD [24,27].
2.8.4.
Thermodynamic Stability
Study:
Thermodynamic stability study was performed
by heating-cooling cycle and centrifugation test. Six cycles between refrigerator temperature (4°C) and
45°C with storage at each temperature of 6 hrs were conducted, and examined for
physical stability. For centrifugation test, formulations were centrifuged at
3,500 rpm for 30 min, and observed for any phase separation [24,27,28].
2.9. STATISTICAL ANALYSIS:
Statistical analysis of polynomial equations was
performed by application of analysis of variance (ANOVA) tool in Design-Expert
software trial version 11.1.2.0. Statistical difference (p < 0.05)
was considered significant.
3. RESULTS AND DISCUSSION:
3.1. Statistical and Response Surface Analysis of Response Parameters (Y1-Y2):
Second order polynomial model generated for % entrapment efficiency (Y1) and % yield (Y2)
by Design-Expert software
(Trial Version 11.1.2.0, Stat-Ease Inc., MN) through multiple regression analysis has been depicted below:
% Entrapment efficiency = 69.75 + 9.21 X1 + 0.5191 X2 + 1.61 X3 + 1.19 X1X2 +
0.5488 X1X3- 1.06 X2X3 - 3.56 X12 -0.7654 X22 - 2.39 X32
Eq. (3)
% Yield = 61.52 + 8.99 X1 + 1.32 X2 + 1.56
X3 + 1.42 X1X2 + 0.47 X1X3 - 0.4025 X2X3 - 4.29 X12 -1.10 X22
- 2.39 X32 Eq. (4)
Negative and positive sign before
coefficients of factors X1, X2 and X3 indicated antagonistic and synergistic
effects of factors on % EE and
% yield of TFG-PH. The p-value for main effect (X1, X2 and X3) and quadratic
effect (X12, X22 and X32) was less than 0.05 (p
< 0.05) which indicated significant main and quadratic effect of factors on
response variables [14,18,19]. %
EE and % yield rapidly increased with increase in
Phosphatidylcholine (X1) from lowest
level (–1.68) to highest level (+1.68) as indicated
through response surface graphs (Figure 2 and 3) [29-32].
3.2.
Optimized Formulation
of TFG-PH as Per Design-expert®
11.1.2.0 software
The desirability function
was investigated by Design-Expert software (Trial Version 11.1.2.0, Stat-Ease Inc., MN) to reach optimized TFG-PH on set premise
of maximizing entrapment
efficiency (% w/v) and yield (% w/v). The composition and processing
conditions for optimized TFG-PH were Phosphatidylcholine (X1 = 1.31 % w/v), cholesterol (X2 = 6.218 % w/v) and rotation speed (X3 = 94.5 rpm). Desirability function for
optimized TFG-PH was found 0.918 which will produce entrapment efficiency and yield of 72.18 % w/v and 64.89 % w/v, respectively (Figure 4).
3.3.
Physical Characteristics
of Topical Phytosomes-Based Cream of Optimized TFG-PH:
Topical phytosomes-based cream of optimized TFG-PH was
pink coloured, transparent, homogeneous with smooth texture and without any
phase separation. Immediate skin feel of cream was moisturizing, non-greasy, no
grittiness having spreadability (spreading diameter after 1 minute) 48 mm, pH
6.41 and viscosity 32000 cps which are ideal values for any topical cream. No phase
separation was visible after thermodynamic stability study via heating-cooling
cycle and centrifugation test which illustrated stability of topical
phytosomes-based cream [24,27].
4.
CONCLUSION:
Various strategies for treatment of rheumatoid
arthritis produce enduring risks which requires continuous monitoring.
Therefore, herbal supplements are better and safer alternative to manage
rheumatoid arthritis. Therefore,
Trigonella foenum-graecum ether extracts phytosomes were fabricated by thin-film hydration technique using L-α-Phosphatidylcholine and cholesterol. Central composite design was employed to explore the optimized TFG-PH having phosphatidylcholine (X1 = 1.31 % w/v), cholesterol (X2
= 6.218 % w/v) and rotation speed (X3 = 94.5rpm). Desirability function for optimized TFG-PH was found
0.918 which will produce % EE and % yield of 72.18 % w/v and 64.89 % w/v, respectively. Optimized TFG-PH was further incorporated into transparent, homogeneous, moisturizing, non-greasy,
spreadable and stable phytosomes-based
cream which could generate
superior therapeutic effect owing to enhanced permeability and skin retention
potential of phytosomes without
producing any adverse effect.
5. CONFLICT OF INTEREST:
The authors declare that there is no conflict of
interest.
6.
ACKNOWLEDGMENTS:
The authors express gratitude to Chitkara College of
Pharmacy, Chitkara University, Punjab, India for infrastructural support to
carry out this work. The authors thanks to Stat-Ease, Inc. for providing access
to Design-Expert software (trial version 11.1.2.0, Stat-Ease Inc., MN).
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