Homogenizer-assisted extraction of herbals
Homogenizer-assisted extraction technique was used to extract the herbals used in the development of the n-HPTF-06 and the percentage yield of the extracted herbals was found as: Aloe barbadensis (leaf) (Aq.) whole extract; Azadirachta indica (leaf) (Aq.) 16.37%; Curcuma longa (rhizome) (Aq.: Ethanol, 50:50 v/v) 11.37%; Camellia sinensis (leaf) (Aq.) 11.33%; Glycyrrhiza glabra (stem) (Aq.: Ethanol, 30:70 v/v) 7.01%; Arnica montana (Aq.: Ethanol, 50:50 v/v) 9.26%; and Calendula officinalis Aq.: Ethanol, 50:50 v/v) 15.22%.
Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR)
ATR-FTIR spectroscopy was used to characterized the prominent functional groups present in the extracted herbals which can be further used as a quality control parameter for a final form of formulation. The spectra of all herbals are depicted in Fig. 1. Characteristic ATR-FTIR spectra of Aloe barbadensis was at 3932.14 cm-1 showing sharp O-H free alcohol (stretching), 3851.00 cm-1 wide O-H (stretching), 2360.69cm-1 due to O=C= O group (stretching), 1782.60 cm-1 due to C=O anhydrous (stretching) and 1733.14cm-1 due to N-O nitro compound (stretching). In Azadirachta indica characteristic peaks were at 3744.51cm-1 showing medium O-H (stretching), 3113.13 cm-1 weak O-H carboxylic group (stretching), 2368.58 cm-1 strong C=O primary (free, stretching), 1557.07cm-1 due to N-O (stretching) and 1409.72 cm-1 medium S=O (stretching). In Curcuma longa characteristics peak were found at 3740.75cm-1 strong O-H, alcohol (stretching), 2425.99 & 2331.65 cm-1 strong C=O-H (stretching), 1688.8cm-1 strong C-H aromatic alkyl (bending) and 1549.75 cm-1 medium C=C cyclic alkenes (stretching). In Camellia sinensis the characteristic peaks were identified as 3226.44cm-1low O-H (stretching), 2967.94cm-1 C-H (stretching), 1605.53cm-1 α-β-unsaturated ketone (stretching), 1236.66cm-1 C-O alkyl aryl ether (stretching), 1144.55cm-1 C-O alcohol (stretching) and 1035.89cm-1 CO-O-CO (stretching). In Glycyrrhiza glabra characteristic peaks lies at 3743.63 cm-1 medium O-H free alcohol (stretching), 3546.22 cm-1 strong O-H intermolecular bonded alcohol (stretching), 1520.55 strong N-O nitro compound (stretching), 1416.60cm-1 O-H (bending), 1228.99cm-1 strong C-O aromatic (stretching) and 1040.50cm-1 strong CO-O-CO anhydride (stretching). In Arnica Montana the characteristics peak were found at 3326.71cm-1 medium O-H (stretching), 2977.36cm-1 strong C-H aldehyde (stretching), 1647.69cm-1 strong C=C monosubstituted alkene (stretching), 1387.42cm-1 medium C-H Gem dimethyl (bending), 1325.94 cm-1 O-H phenol (bending) and 1085.17 cm-1 strong C-H (stretching). In Calendula officinalis prominent peaks were identified at 3276.90cm-1 strong O-H (stretching), 2980.22 cm-1 weak O-H (stretching), 2133.41 cm-1 strong C=C=O ketone (stretching), 1645.58cm-1 C=O δ-lactone (stretching), 1416.82cm-1 medium O-H (bending), 1326.60cm-1 O-H phenol (bending), 1084.71cm-1 strong C-O (stretching), 1043.04cm-1 strong CO-O-CO anhydride (stretching) and 871.77cm-1 strong C-H 1,3-disubstitued (bending).
Determination of total polyphenolic content and total flavonoid content
Total polyphenolic content and total flavonoid content of the extracted herbals was quantified by using Folin-Ciocalteu Reagent (FCR) and aluminum chloride method respectively, analyzed by UV/Vis spectrophotometrically. Total polyphenolic compounds at 0.1 mg/mL of Aloe barbadensis (leaf) (Aq.); Azadirachta indica (leaf) (Aq.); Curcuma longa (rhizome) (Aq.: Ethanol, 50:50 v/v); Camellia sinensis (leaf) (Aq.); Glycyrrhiza glabra (stem) (Aq.: Ethanol, 30:70 v/v); Arnica montana (Aq.: Ethanol, 50:50 v/v); and Calendula officinalis Aq.: Ethanol, 50:50 v/v) was found as 2.43±0.04, 2.26±0.21, 2.85±0.22, 27.05±0.06, 15.44±0.15, 2.78±0.11 and 11.18±0.11 GAE of g/DWE respectively. Total flavonoid content of Aloe barbadensis (leaf) (Aq.); Azadirachta indica (leaf) (Aq.); Curcuma longa (rhizome) (Aq.: Ethanol, 50:50 v/v); Camellia sinensis (leaf) (Aq.); Glycyrrhiza glabra (stem) (Aq.: Ethanol, 30:70 v/v); Arnica montana (Aq.: Ethanol, 50:50 v/v); and Calendula officinalis Aq.: Ethanol, 50:50 v/v) was found as 6.66±0.03, 7.30±0.02, 2.83±0.01, 52.50±0.04, 46.41±0.03, 7.12±0.11 and 14.72±0.07 RTE of g/DWE respectively. The maximum content of both polyphenolic and flavonoid groups was present in Camellia sinensis and Glycyrrhiza glabra. Results were statistically analyzed by employee one-way ANOVA using Tukey’s multiple comparison test and are also depicted in Table 6.
Table 6 Total Polyphenolic content and Total Flavonoid content in the homogenizer-assisted extracted herbals
Parameters
|
Aloe barbadensis (Aq.)
|
Azadirachata indica (Aq.)
|
Curcuma longa (Aq.: Ethanol, 50:50 v/v)
|
Camellia sinensis (Aq.)
|
Glycyrrhiza glabra (Aq.: Ethanol, 30:70 v/v)
|
Arnica montana (Aq.: Ethanol, 50:50 v/v)
|
Calendula officinalis Aq.: Ethanol, 50:50 v/v)
|
Total Polyphenolic content (mg GAE/g of DWE)
|
2.43±0.04$
|
2.26±0.21#$
|
2.85±0.22*#
|
27.05±0.06*#
|
15.44±0.15*#
|
2.78±0.11$
|
11.18±0.11*#
|
Total Flavonoid content (mg RTE/g of DWE)
|
6.66±0.03$
|
7.30±0.02$#
|
2.83±0.01$
|
52.50±0.04*#
|
46.41±0.03*#
|
7.12±0.11#$
|
14.72±0.07*#
|
Total polyphenolic content and total flavonoid content of extracted herbals are expressed as the mean±SEM (n=3) by standards as GAE i.e., Gallic acid equivalent and RTE i.e., Rutin trihydrate equivalent capacity of DWE i.e., Dried weight of the extract.
One-way ANOVA using Tukey’s multiple comparison test statistic is used and with p value as p<0.001(*); p<0.01(#) and p<0.05 ($).
In-vitro antioxidant potential study
Determination DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity
DPPH free radical scavenging activity of the extracted herbals was performed by using DPPH assay. Percentage inhibition of each extract was calculated and plotted against concentrations (0.5, 1.0, 2, 4, 6, 8 and 10 mg/mL) and then IC50 value of each extract was calculated from graph as represented in Table 7. IC50 value of extract namely: Aloe barbadensis (leaf) (Aq.); Azadirachta indica (leaf) (Aq.); Curcuma longa (rhizome) (Aq.: Ethanol, 50:50 v/v); Camellia sinensis (leaf) (Aq.); Glycyrrhiza glabra (stem) (Aq.: Ethanol, 30:70 v/v); Arnica montana (Aq.: Ethanol, 50:50 v/v); and Calendula officinalis Aq.: Ethanol, 50:50 v/v) was found as 1.11±0.16, 2.14±0.11, 1.72±0.17, 7.81±0.14, 2.56±0.11, 2.23±0.17 and 0.93±0.02 respectively. Results were statistically analyzed by one-way ANOVA followed by Tukey’s multiple comparison test. The order of free radical DPPH scavenging activity of the extract was Camellia sinensis>Glycyrrhiza glabra> Arnica montana>Azadirachta indica> Curcuma longa> Aloe barbadensis> Calendula officinalis.
Determination of FRAP (Ferric ion Reducing Antioxidant Power) activity
The FRAP reducing activity of the extracted herbal was performed and expressed in Fe (II) E/g of DWE as depicted in Table 7. FRAP reducing activity of Aloe barbadensis (leaf) (Aq.); Azadirachta indica (leaf) (Aq.); Curcuma longa (rhizome) (Aq.: Ethanol, 50:50 v/v); Camellia sinensis (leaf) (Aq.); Glycyrrhiza glabra (stem) (Aq.: Ethanol, 30:70 v/v); Arnica montana (Aq.: Ethanol, 50:50 v/v); and Calendula officinalis Aq.: Ethanol, 50:50 v/v) was found as 15.11± 0.28, 8.31±0.01, 17.92±0.05, 168.03±0.33, 19.47±0.16, 14.44±0.12 and 11.12±0.22 Fe (II)E/g of DWE respectively. Results were statistically analyzed byone-way ANOVA followed by Tukey’s multiple comparison test. The order of free radical FRAP activity was found as Camellia sinensis> Glycyrrhiza glabra> Curcuma longa> Aloe barbadensis> Arnica montana> Calendula officinalis>Azadirachta indica.
Determination of ABTS (2, 2’-amino-bis (3-ethylbenzothiazoline-6-sulfonic acid) free radical scavenging activity
Trolox equivalent activity of the extracted herbals was determined by using ABTS assays method. ABTS free radical scavenging activity of the extracts viz-a-viz Aloe barbadensis (leaf) (Aq.); Azadirachta indica (leaf) (Aq.); Curcuma longa (rhizome) (Aq.: Ethanol, 50:50 v/v); Camellia sinensis (leaf) (Aq.); Glycyrrhiza glabra (stem) (Aq.: Ethanol, 30:70 v/v); Arnica montana (Aq.: Ethanol, 50:50 v/v); and Calendula officinalis Aq.: Ethanol, 50:50 v/v) was found as 14.23±0.11, 1.26±0.17, 0.89±0.14, 2.75±0.016, 3.14±0.14, 2.14±0.07 and 1.18±0.11 µg TEAC/g of DWE respectively. Results were statistically analyzed by employee one-way ANOVA using Tukey’s multiple comparison test. The order of ABTS scavenging activity was found as Aloe barbadensis> Glycyrrhiza glabra> Camellia sinensis> Arnica montana>Azadirachta indica> Calendula officinalis> Curcuma longa.
Determination of NO (Nitric oxide) scavenging potential activity
Nitric oxide scavenging potential activity of the extracted herbals was performed by using NO method and percentage inhibition of nitric oxide free radical by given extract was expressed as capacity of inhibition as depicted in Table 7 The percentage inhibition of the NO by extracts as Aloe barbadensis (leaf) (Aq.); Azadirachta indica (leaf) (Aq.); Curcuma longa (rhizome) (Aq.: Ethanol, 50:50 v/v); Camellia sinensis (leaf) (Aq.); Glycyrrhiza glabra (stem) (Aq.: Ethanol, 30:70 v/v); Arnica montana (Aq.: Ethanol, 50:50 v/v); and Calendula officinalis Aq.: Ethanol, 50:50 v/v) was found to be 38.43%, 78.00%, 58.80%, 62.14%, 66.46%, 54.12% and 49.46% respectively. Results were statistically analyzed byone-way ANOVA followed by Tukey’s multiple comparison test. The order of percentage inhibition of NO by extracts was as Azadirachta indica> Glycyrrhiza glabra> Camellia sinensis> Curcuma longa> Arnica montana> Calendula officinalis> Aloe barbadensis.
Determination of Total Antioxidant Capacity (TAC)
Total antioxidant capacity of the extracted herbals was performed by phosphomolybdenum complex formation method. Herbal extracts viz-a-viz Aloe barbadensis (leaf) (Aq.); Azadirachta indica (leaf) (Aq.); Curcuma longa (rhizome) (Aq.: Ethanol, 50:50 v/v); Camellia sinensis (leaf) (Aq.); Glycyrrhiza glabra (stem) (Aq.: Ethanol, 30:70 v/v); Arnica montana (Aq.: Ethanol, 50:50 v/v); and Calendula officinalis Aq.: Ethanol, 50:50 v/v) showed TAC as 4.23±0.01, 8.12±0.17, 4.15±0.17, 7.14±0.16, 4.71±0.01, 1.12±0.14 and 0.77±0.01 mg AAE/g of DWE respectively. Results were statistically analyzed by one-way ANOVA using Tukey’s multiple comparison test and are also depicted in Table 7. The order of TAC activity wereAzardichata indica> Camellia sinensis> Glycyrrhiza glabra> Aloe barbdensis> Curcuma longa> Arnica montana> Calendula officinalis.
Table 7 In-vitro antioxidant potential of the homogenizer-assisted extracted herbals
Parameters
|
Aloe barbadensis (Aq.)
|
Azadirachata indica (Aq.)
|
Curcuma longa (Aq.: Ethanol, 50:50 v/v)
|
Camellia sinensis (Aq.)
|
Glycyrrhiza glabra (Aq.: Ethanol, 30:70 v/v)
|
Arnica montana (Aq.: Ethanol, 50:50 v/v)
|
Calendula officinalis Aq.: Ethanol, 50:50 v/v)
|
DPPH (IC50)
|
1.11±0.16$
|
2.14±0.11#$
|
1.72±0.17#$
|
7.81±0.14#*
|
2.56±0.11#
|
2.23±0.17#
|
0.93±0.02$,ns
|
FRAP (mg Fe (II) E/g of DWE
|
15.11±0.28#
|
8.31±0.01#
|
17.92±0.05#
|
168.03±0.33*
|
19.47±0.16#
|
14.44±0.12#
|
11.12±0.22#
|
ABTS (µg TEAC/g of DWE)
|
14.23±0.11*#
|
1.26±0.17*
|
0.89±0.14ns
|
2.75±0.16*
|
3.14±2.14*
|
2.14±0.07*
|
1.18±0.11*
|
NO (%I)
|
38.43%#
|
78.00%*
|
58.80%#
|
62.14%*
|
66.14%*
|
54.12%#
|
49.46%#
|
TAC (mg AAE/g of DWE)
|
4.23±0.01*
|
8.12±0.17*
|
4.15±0.17*
|
7.14±0.16*
|
4.71±0.01*
|
1.12±0.14*
|
0.77±0.01#
|
In-vitro antioxidant capacities of extracted herbals are expressed as the mean±SEM (n=3) evaluated by DPPH (2, 2-diphenyl-1-picrylhydrazyl); FRAP (Ferric Reducing Antioxidant Power); ABTS (2,2’-azio-bis(3-ethylbenzothiazone-6-sulfonic acid); NO (Nitric oxide) and TAC (Total Antioxidant Capacity). Where TEAC is Trolox equivalent antioxidant capacity; AAE is Ascorbic acid equivalent antioxidant capacity and DWE is Dried weight of the extract.
One-way ANOVA using Tukey’s multiple comparison test statistic is used and with p value as p<0.001(*); p<0.01(#), p<0.05 ($) and ns (non-significant).
Preparation of novel-herbosomes (n-herbosomes)
Preparation of n-herbosomes was carried out by method by thin film hydration method in different batches with ingredients prescribed in Table 1 and later on optimized by using response surface method by optimizing percentage entrapment efficiency (%EE), in-vitro drug permeation (Q) and particle size (nm).
Incorporation of n-herbosomes into Polymeric base to form novel-herbosomal loaded PEG-Poloxamer topical formulation (n-HPTF)
Optimized n-herbosomes (n-H-06) was incorporated in the PEG-Poloxamer polymeric base and n-HPTF was formulated in different batches by controlling independent variables like polymer ratio and penetration enhancer and subsequently optimized by statistical models.
Design of experiment (DoE)
Optimization of the n-herbosomes and n-HPTF was done by using response surface methodology using 32 full factorial design and optimization was done by using one-way ANOVA and expressed by surface plot, fitted surface plot, predicted and observed value and suing pareto chart for frequency of independent variables over dependent variables. The quadratics equation was found as:
%EE (CL): - 332.734 – 0.7836X – 5.9256Y + 0.006XX – 0.0056XY – 0.0157YY.
%EE (AM): - 220.1582 – 0.0224X + 3.6838Y + 0.0015XX – 0.0019XY – 0.0105YY.
PS (nm): 315.4073 + 3.0369X – 4.3125Y – 0.0056XY – 0.0088XY + 0.0196YY.
In-vitro drug permeation (CL): - 1.2232 – 0.024X + 0.2993Y + 0.0133XX – 0.0007XY –
0.0151YY.
In-vitro drug permeation (AM): 0.0797 + 0.0211X + 0.2993Y – 0.0004XX + 0.0018XY –
0.0016YY.
Spreadability (mm): - 19.3817 + 5.3888X + 1.9263Y – 0.3333XX – 0.0122XY – 0.1147YY.
Fcalculated>>>Ftabulated that confirmed that the selected independent variables had significant effect on the selected models. One-way ANOVA was employed and R2 value was obtained as: R2 (%EE of CL) = 0.9586, R2 (%EE of AM) = 0.9224, R2 (PS) = 0.8946, R2 (in-vitro drug permeation of CL) = 0.8695, R2 (in-vitro drug permeation of AM) = 0.7306, and R2 (spredability) = 0.9629 respectively. This reveals that percentage entrapment efficiency of both Curcuma longa and Arnica Montana was found to be satisfactory but particle size was little higher than usual this may be due to larger molecular chain present in the herbal extract in comparison with synthetic smaller molecular weight compounds. On the other hand,in-vitro drug permeation the actual value is less than predicted value this may be due to again larger chain present in the herbals which hinders the drug release responses from carrier and spredability and was found significant with the independent variables revealing that the selected variable fitted to the responses i.e., dependent variables. The surface responses from each run is depicted in Fig. 2. Based on which n-H-06 was optimized as n-herbosomes and n-HPTF-06 as a final formulation.
Characterization of novel herbosomes
Percentage entrapment efficiency (% EE) of n-herbosomes
Percentage entrapment efficiency of both entrapped drug Curcuma longa and Arnica montana was estimated and depicted in Table 4. The order of entrapped drug Curcuma longa was found as: n-H-06> n-H-05> n-H-07> n-H-08> n-H-09> n-H-04> n-H-03> n-H-01> n-H-02 respectively and order of entrapped drug Arnica montana was found as: n-H-06> n-H-05> n-H-08> n-H-09> n-H-07> n-H-04> n-H-03> n-H-02> n-H-01 respectively. Similar entrapment efficiency of one of the components of Curcuma longa’s that is curcumin has showed >80% entrapment efficiency when incorporated into liposomes was quoted by Ng et al., (2018).
Particle size, polydispersitivity and zeta potential of n-herbosomes
The mean vesicle size (nm), size distribution (PDI) and zeta potential (mV) are essential parameter to characterized vesiculosomes like herbosomes, liposomes, cubosomes etc. In this study all n-herbosomes was characterized for their particle size (nm) and found in the range between 214.8 nm to 280.1 nm also depicted in Table 4 and Fig. 3. Whereas polydispersitivity lies in between the range of 0.299 to 0.387 and zeta potential of all formulation was found as +26 mV revealing formation of stable herbosomes. The positive charge on zeta potential was due to stearic acid which was taken constant in all the formulation, hence measured for the optimized one i.e., n-H-06 as showed in Figure 3. To deliver drug to the deeper dermis layer the optimum particle size should be ≤300 nm [52-53] and our optimized particles lies within this range, hence herbosomes can beneficially act to the target site with more receptor binding affinity.
Characterization of optimized n-HPTF
Physical evaluation
Physical evaluation of the optimized n-HPTF-06 was done by observing colour, homogeneity, apparent phase separation upon long term storage at room temperature (250C ± 1) and cold storage condition (40C ± 1). There was no change observed attested temperature, which was further evaluated in stability chamber.
pH of optimized n-HPTF
Physiological pH is very important parameter to understand the release of drug in a unionized form. Hence, it is very important to design the formulation within range of target site of absorption of drug. pH of the optimized formulation n-HPTF-06 was evaluated by using digital pH meter and it was found as 6.5±0.11 (n=3). pH of the topicals can widely affect the permeation and absorption of the drug. Study suggested that an optimal pH of 6-7 was found good to increase the permeability through dermal layer [54-55].
Rheological behavior
All formulation from (n-HPTF-01 to n-HPTF-09) was used to access their rheological behavior by using ATGO digital viscometer. The rheological behavior of all formulation was found satisfactory and consistent. It was observed that formulation having high amount of polymeric ratio i.e., from n-HPTF-07 to n-HPTF-09 was slightly possesses more shear stress with respect to spinning of spindle of a given time. The rheological behavior of the all formulation is depicted in Fig.4.showing viscosity (η) with respect to shear rate (sec-1) applied.
Spreadability
Spreadabilty of the n-HPTF was performed by the method described earlier and is depicted in Table 5. It was observed that upon increasing the concentration of PEG-3350 (from 3.0 gm to 7.0 gm) and poloxamer-188 (from 2.5 gm to 10.5 gm) the spreadability of the formulation decreases and remain more consistent. Spreadability is a critical sensory parameter highly dependent upon the vehicles and humectant used in the formulation [56] and middle range of combination of PEG-3350: poloxamer-188 (5.0: 5.5 gm) was found a suitable range for ease-of-application (spreadability) i.e., 11.1±0.12 mm in 60 sec.
Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR)
The ATR-FTIR spectra of the optimized n-H-06 and n-HPTF-06 were characterized with all ingredients and their characteristic peaks and overlay spectra of the optimized formulation with ingredients are depicted in Fig. 5. The characteristic spectra of the L-α-Phosphatidylcholine showed prominent peaks at 3281.81 cm-1 broadO-H alcohol (stretching), 3010.32cm-1 medium C-H (stretching), 2922.12 cm-1 medium C-H (stretching), 1737.28 cm-1 strong C+O (stretching), 1618.99 cm-1 strong C+C (stretching), 1464.98 cm-1 medium C-H (bending), 1053.54cm-1 strong C-O (stretching) and 822.29cm-1 medium C-H (bending). Cholesterol showed their characteristics peaks at 3431.97 cm-1 broad O-H (stretching), 2929.85 cm-1 medium O-H (stretching), 2866.39cm-1 doublet C-H (stretching), 1670.7 cm-1 weak C=C (bending) and 1463.98cm-1 weak O-H (bending). And the optimized n-herbosomes (n-H-06) showed prominent peaks at 3350.91 cm-1 broad O-H (stretching), 2929.86 cm-1 medium O-H (stretching), 2978.15 cm-1 intermolecular O-H (stretching), 1643.33 cm-1 strong C=C (stretching), 1044.31 cm-1 strong CO-O-CO (stretching) and 879.13 cm-1 strong C-H (bending). Formulation base like PEG-3350 showed their characteristics peaks at 3425.32 cm-1 broad O-H (strong), 2860.61 cm-1 medium C-H (stretching), 2694.78 cm-1 medium C-H (stretching), 1340.99cm-1 medium C-H (bending) and 1097.67 cm-1 strong C-O aliphatic alcohol (stretching). Poloxamer (P-188) showed their characteristics peaks at 2969.93 cm-1 medium C-H (stretching), 2882.50 strong C-H (stretching), 1466.51cm-1 medium C-H (bending), 1100.81 cm-1 strong C-O (stretching) and 946.98 cm-1 monosubstituted C=C (bending). Garcinia indica showed prominent peak at 2953.46 cm-1 medium C-H (stretching), 2914.57cm-1 alkene C-H (stretching), 2849.33cm-1 alkane C-H (stretching), 1730.08cm-1 ester C=O (stretching) and 1174.39cm-1 medium C-O (stretching). Cetyl alcohol showed their characteristic peaks at 3271.03cm-1 broad O-H (stretching), 2916.33cm-1 strong C-H (stretching), 2848.94 cm-1 medium C-H (stretching) and 1464.81cm-1 strong C=C (stretching). Stearic acid showed their characteristics peaks at 2962.62 cm-1 medium C-H (stretching), 2914.94 cm-1 medium C-H (stretching), 1698.93 cm-1 conjugated ketone C=O (stretching), 1429.01cm-1 medium O-H (bending) and 940.62cm-1 weak C=C (bending). Xanthan gum showed their characteristics peaks at 3265.27cm-1 broad O-H (stretching), 2882.20cm-1 C-H (stretching), 1599.48cm-1 medium C=C (bending) and 1368.86cm-1 medium C-H (bending). Tocopherol acetate showed their characteristics peak at 2925.39cm-1 strong C-H (stretching), 2867.63cm-1 alkene C-H (stretching), 1758.31cm-1 strong C=O (stretching), 1460.16cm-1 medium C-H (bending) and 1204.43cm-1 C-O (stretching). Carbapol® 940 showed their characteristic peaks at 3048.70cm-1 broad O-H (stretching), 2946.83cm-1 strong C-H (stretching), 2659.46cm-1 medium C-H (stretching), 1703.76cm-1 C=O (stretching), 1414.68 cm-1 alcohol O-H (bending) and 1452.30cm-1 medium C-H (bending). PEG-200 showed their characteristics peak at 3410.49cm-1 broad O-H (stretching), 2868.24cm-1 strong C-H (stretching), 1453.92cm-1 medium C-H (bending), 1406.35cm-1 alcohol O-H (stretching) and 1099.26 cm-1 medium C-O (stretching). Tween-60 showed their characteristics peak at 3489.83cm-1 broad O-H (stretching), 2922.26cm-1 strong C-H (stretching), 2854.34cm-1 medium C-H (stretching), 1735.87cm-1 strong C=O (stretching), 1638.90 cm-1 conjugated C=O (stretching) and 1097.09cm-1 medium C-O (stretching). Transcutol® showed their characteristics peak at 3430.56cm-1 broad O-H (stretching), 2975cm-1 strong C-H (stretching), 2867.54cm-1 strong C-H (stretching), 1455.38cm-1 medium C-H (bending), 1287.47cm-1 medium C-O (stretching) and 1068.86cm-1 secondary alcohol C-O (stretching). Glycerol showed their characteristics peak at 3293.89cm-1 broad O-H (stretching), 2879.34cm-1 strong C-H (stretching), 2933.20cm-1 strong C-H (stretching), 1412.14cm-1 medium C-H (bending) and 1029.64 cm-1 medium C-O (stretching). Triethanolamine showed their characteristics peak at 3307.47cm-1 strong O-H (stretching), 2948.61cm-1 , 2478.52cm-1 , and 2822.85cm-1 ternary amine, 1655.19cm-1 medium C=O (stretching) and 1067.15cm-1 medium C-O (stretching). Physical mixture of the designed formulation showed characteristics peak at 3350.60cm-1 broad O-H (starching), 2980.01 cm-1 strong C-H (stretching), 2816.75cm-1 C-H (stretching), 1639.07cm-1 weak C=C (stretching), 1457.85cm-1 C-H (bending) and 1044.43cm-1 medium C-O (stretching). And finally optimized formulation n-HPTF-06 showed their characteristics peaks at 3359.71cm-1 broad O-H (starching), 2883.55 cm-1 strong C-H (stretching), 2816.75cm-1 C-H (stretching), 1638cm-1 weak C=C (stretching), 1457.85cm-1 C-H (bending) and 1094.38cm-1 medium C-O (stretching). The identical peaks in the physicochemical mixture and optimized formulation revealed no physicochemical incompatibility in this combination of drug and polymers.
In-vitro drug permeation (Q)
In-vitro drug permeation from n-HPTF-06 containing herbosomes and extracted herbals is depicted in Fig. 6. The aim of preparing herbosomes was to increase the bioavailability of Curcuma longa at the target site due to its ability to inhibit TNF-alpha factor in deep tissue injury as well as it also contains antimicrobial property along with it. Secondly, Arnica montana which is hydrophilic in nature but still selected to incorporate into herbosomes due to their potent analgesic effect which required in an immediate action at the targeted site. Other ingredients like Aloe barbadensis, Azadirachata indica, Camellia sinensis, Glycyrrhiza glabra, Calendula officinalis and Pro-Vitamin B5 showed controlled release from the polymeric matrix. Initially, a burst release pattern was seen in all ingredients due to their hydrophilic charged surface. different release pattern for all herbals suggested that it may be due to nature of solubility and affinity to entrapped by the polymer and lipid inside their core matrix as well as to the polymeric end chain, secondly this may be due molecular weight and partition efficiency to the semi permeable membrane at the diffusion site and a similar release parameter was cited by Vuddanda et al.; (2015).
Kinetic modeling
The release kinetic model’s R2 value estimated for all ingredients in optimized formulation n-HPTF-06 has shown in Table 8. Overall best fit model for optimized formulation was first order model in which amount of drug dissolved in the buffer media directly from the formulation followed by Higuchi’s model in which drug dissolution take place from the matrix of the polymeric core. The different behavior of the herbals may be due to their complex molecular chain and larger molecular size.
Table 8 Release kinetic model for the optimized formulation n-HPTF-06
Kinetic model
|
Aloe barbadensis (Aq.)
(R2)
|
Azadirachata indica (Aq.)
(R2)
|
Curcuma longa (Aq.: Ethanol, 50:50 v/v)
(R2)
|
Camellia sinensis (Aq.)
(R2)
|
Glycyrrhiza glabra (Aq.: Ethanol, 30:70 v/v)
(R2)
|
Arnica montana (Aq.: Ethanol, 50:50 v/v)
(R2)
|
Calendula officinalis Aq.: Ethanol, 50:50 v/v)
(R2)
|
Pro vitamin B5
(R2)
|
Zero order
|
0.874
|
0.874
|
0.875
|
0.969
|
0.901
|
0.974
|
0.969
|
0.971
|
First order
|
0.814
|
0.914
|
0.941
|
0.984
|
0.914
|
0.923
|
0.914
|
0.978
|
Higuchi model
|
0.819
|
0.921
|
0.874
|
0.914
|
0.901
|
0.907
|
0.907
|
0.905
|
Korsmeyer-Peppas
|
0.748
|
0.748
|
0.784
|
0.873
|
0.822
|
0.904
|
0.987
|
0.808
|
Stability study
Stability study of the final optimized formulation was studied by placing the optimized formulation n-HPTF-06 at two different temperatures as per ICH guideline for long term stability study of topical formulation i.e., 5°C±3°C and 30°C±2°C; 65%±5% RH. n-HPTF-06 was accessed for color, texture, pH, spreadability and phase separation with respect to time (in days) for sampling i.e., 0, 30, 60, 90, 120, and 180 days of storage. There was no significant change in any parameter was observed, observed data are tabulated in Table 9. Further any physicochemical incompatibility upon long term storage was accessed by using ATR-FTIR and there is no physicochemical incompatibility was seen in spectra.
Table 9 Stability study of the optimized n-HPTF by using ICH guidelines Q1 A (R2) (Long term study)
Characteristics of Optimized formulation (n-HPTF-06)
|
Temperature
(°C) and %Relative Humidity (%RH)
|
Time (in days)
|
Initial observations
|
30 days
|
60 days
|
90 days
|
120 days
|
180 days
|
Color
|
5°C±3°C
30°C±2°C; 65%±5% RH
|
Pale yellow
Pale yellow
|
Pale yellow
Light brown
|
Pale yellow
Light brown
|
Pale yellow
Light brown
|
Pale yellow
Light brown
|
Pale yellow
Light brown
|
Texture
|
5°C±3°C
30°C±2°C; 65%±5% RH
|
Smooth & no grittiness
Smooth & no grittiness
|
Smooth & no grittiness
Smooth & no grittiness
|
Smooth & no grittiness
Smooth & no grittiness
|
Smooth & no grittiness
Smooth & no grittiness
|
Smooth & no grittiness
Smooth & no grittiness
|
Smooth & no grittiness
Smooth & no grittiness
|
pH
|
5°C±3°C
30°C±2°C; 65%±5% RH
|
6.5±0.11
6.5±0.17
|
6.5±0.10
6.5±0.11
|
6.5±0.12
6.5±0.11
|
6.5±0.10
6.4±0.13
|
6.4±0.14
6.4±0.12
|
6.4±0.11
6.4±0.12
|
Spredability
(Spreading in 1 min at the force of 25 gm)
|
5°C±3°C
30°C±2°C; 65%±5% RH
|
11.02 mm2
11.11 mm2
|
11.00 mm2
11.02 mm2
|
11.01 mm2
11.13 mm2
|
11.10 mm2
11.15 mm2
|
11.17 mm2
11.14 mm2
|
15.14 mm2
15.19 mm2
|
Phase separation
|
5°C±3°C
30°C±2°C; 65%±5% RH
|
No phase separation
No phase separation
|
No phase separation
No phase separation
|
No phase separation
No phase separation
|
No phase separation
No phase separation
|
No phase separation
No phase separation
|
No phase separation
No phase separation
|
Skin irritancy study of optimized n-HPTF
The optimized n-HPTF-06 was used for the skin irritancy study on female Sprague Dawley rats (n=6) and observed under prescribed protocol of (OECD 402). The formulation was applied over dorsal area of the rat’s skin by dividing the skin surface into two sections on the same rat to see any immediate contact irritation on the leftover skin (if any). Subsequently, rat skin was observed initially for 1h and 6 h and observations were recorded and further the skin was restrained by using medical adhesive so that formulation remained in contact with the skin for a long time. On the next day, adhesive was removed and wiped out and the skin was gently sanitized by using 70 % ethanol and observation was recorded and further observations were made till 72 h and primary dermal irritation index (PDII) was scored. No erythema or edema was observed on skin of the rats at any point of study. Hence, the optimized formulation was considered as a safe formulation possessing no-irritancy upon application on rat’s skin is depicted in Table 10.
Table 10 Primary dermal irritation scores in female Sprague Dawley rats (n=6) after exposure to optimized n-HPTF
Time (h)
|
Incident of dermal irritation
Erythema Edema
|
Total PDI*
|
PDII**
|
1
|
0
|
0
|
0
|
0
|
6
|
0
|
0
|
0
|
0
|
24
|
0
|
0
|
0
|
0
|
48
|
0
|
0
|
0
|
0
|
72
|
0
|
0
|
0
|
0
|
PDI* (Primary Dermal Irritation = average erythema + average edema)
PDII** (Primary Dermal Irritation Index)
Experimental design and dose optimization for bio-efficacy study of n-HPTF
Sprague Dawley rats used for this study were grouped into three groups: Group (I, II, and III) as mentioned above and frostbite was induced comparative study of the frostbite condition was monitored in formulation treated groups as well as in untreated condition. Visual observation was done and changes recorded and further the condition was analyzed by histological study. No treatment complications or death of animals were observed during the course of this study.
n-HPTF ameliorates cold injury, promotes skin frostbitten wound healing and maintains skin integrity in-vivo
A sequential analysis was required before reaching the final conclusion vis-a-vis the optimized n-HPTF formulation. In cold-injury, tissue undergoes multifarious pathological alterations which may lead to amputation of exposed extremities if the sequences of changes/alteration(s) are not controlled. When the skin comes in contact with chilled wind or in sub-zero temperature, it impairs the normal physiological condition and causes desensitization of the non-selective cationic channel, vasoconstrictions, tissue hypoxia, alteration in flow of electrolyte concentration inside the cells, ice-crystal formation, generation of reactive oxygen species, tissue necrosis. Cumulative and prolonged exposure persistence of these conditions accentuates thrombus formation, and thus leading to amputation/loss of extremities. Thus, in this study animals were grouped in three major groups viz; Group I (untreated), Group II (standard formulation twice a day; 0.5 gm application), Group III (treatment was followed by applying n-HPTF twice a day; 0.5 gm/each application) and observation was recorded on 3, 7, 14, 21, and 28 days till the complete healing of frostbitten rat skin.
At day 0, purplish skin was observed after 2 Hrs of induction of frostbite in all the rats and the area was marked for further morphometric analysis.
At day 3, the major changes in the skin were noticed in all the three groups: [Group I: the skin became more injured and appeared more wounded in comparison to Groups II and Groups III animals which received topical formulations]. The severity of cold injury among the groups was evident from visual inspection of the dead epidermis and more inflamed and flared skin.
At day 7, Group I animals of the skin became oozier and developed hard crust over skin as well as pus formation was also observed. In Group II and Group III the hard crust was also observed but in case of Group III the skin around the crust was normal and no sign of inflammation was observed in comparison to Group I and II.
At day 14, the hard crust from the animal skin was crust out in almost all groups but in case of untreated groups the condition of skin worsened and was a significant sign of ice-crystal formation, which upon nucleation damaged the deep skin layers in both Group I and II with very slight observable differences whereas the skin of Group III animal which were treated with n-HPTF was remain in contact which reveals that n-HPTF increases the ionic interaction and thus maintains integrity of the skin.
At day 21, Group III showed accelerated healing rate followed by contraction and narrowing the wound area, suggesting an earlier re-epithelialization than the others groups of animal.
At day 28, Group III showed complete healing and no scars over skin was witnessed by necked eyes over the skin of animals.
In conclusion, based on the ingredients incorporated in n-HPTF which was priory selected with the function of their targeted mode of action over the entire squeal helped in alleviation of the frostbite symptoms and promoted rapid tissue healing as compared to untreated group and standard drug treated group and the comparative digital representation of all the three groups with respect to days 0, 3, 7, 14, 21 and 28 are also depicted in Fig. 7.
Morphometric analysis of frostbitten wound area
Animals groups followed by their prescribed dosage form was observed for their healing rate and measured by using Vernier-caliper at day 3, 7, 14, 21 and 28 respectively and percentage frostbitten-wound contraction was calculated by formula mentioned above. The results was analyzed by using student t-test and found to be significant at p- value < 0.05. n-HPTF showed ≈98.96% recovery than the standard treatment (65.65 %) at day 28 suggesting that an improved healing rate and the supporting morphometric measurement in graphical form is also depicted in Fig. 8.
Channelized recovery of frostbitten skin by n-HPTF evaluated histopathologically
The histological study was designed to observe the detailed changes occurring during the course of induction of frostbite in cold injury induced model. During the study protocol, detailed histology of skin layers, demarcation and thickness of skin layers, morphology of underlying papillary layer as well as dermis layer with abundant blood capillaries and connective tissue cells was studied.
Day 3: Animals of all three groups represented similar changes symptomatically to frostbite condition. Few differences was noticed in the animal Group I and II, in which the skin layers was less inflamed (Fig. 9 (B1 and C1) as compared to untreated groups as marked by red arrows in Group I (Fig. 9 (A1).
Day 7: In Group I and II (Frostbitten and frostbitten + standard drug treated) underlying papillary layer was completely delocalized due to progression of frostbitten wound (Fig. 9 (A2 and B2) but in Group III papillary layer just below the epidermis was found intact with their position which is due to the antioxidant properties possess by extracted herbals incorporated to the n-HPTF are able to maintain papillary which is widely known for the regulation of vascular temperature hence inhibit the ice-crystal formation as depicted in Fig. 9 (C2), similar observation was evaluated by Auerbach et al., 2014.
Day 14: Group I (untreated) exhibited complete disruption of all layers of the skin and losses of skin integrity, disruption of blood vessels and connective tissue and histopathologically depicted in Fig. 9 (A3) whereas, in Group II, the demarcation was observed and skin gaps between epithelial junctions could be clearly observed. However, in case of Group III a clear morphology, with defined skin integrity was observed at day 14 suggesting that n-HPTF is effective in controlling the subsequent ischemic condition inside the cells those results due to cold induced stress. The herbal ingredients in n-HPTF possess significant amount of antioxidant capacity as reveals in the in-vitro antioxidant capacity of the homogenizer assisted extracted herbals which are showing promising therapeutic for the cold injury condition.
Day 21: Group I, showed formation of larger vacuoles during the phase of natural healing attaining by dermis and epidermis layer of mammalian skin (Fig. 9 (A4)) similar results were seen in Group II with less number of larger gap filling vacuoles (Fig. 9 (B4)), but in case of Group III, the skin layer exhibited a well-defined structure and a clear delineation between epidermis and dermis layer of skin suggesting that the herbal ingredients of n-HPTF are able to maintain ionic interaction between cells and thus accelerate the production of collagen as well as connective tissue which can be clearly observed in Fig 9 (C4) as well 40X resolution image is depicted in Fig 9 (E) showing dense collagen fiber suggesting recovered healing.