Development and Evaluation of Neem Gel Formulation using Gum Karaya as Gelling Agent
Nguyen Duc Hanh*, Le Van Thinh, Do Quang Duong
Faculty of Pharmacy, University of Medicine and Pharmacy at Ho Chi Minh City, Ho Chi Minh City, Vietnam
*Corresponding Author E-mail: duchanh@ump.edu.vn
ABSTRACT:
The aim of this study was to develop and evaluate the neem gel formulation using gum karaya as a gel forming agent. The gelling ability of gum karaya and its mixtures with the other gel-forming agents (sodium alginate, carboxymethyl starch and hydroxypropyl methylcellulose) were demonstrated by rheology properties. Gum karaya was demonstrated to be a gel-forming agent with the pseudoplastic flow. Of three gelling agents, sodium alginate was selected as the best combined excipient with gum karaya due to the better elasticity improvement of the gum karaya gel in comparison with hydroxypropyl methylcellulose (HPMC) and carboxymethyl starch (CMS). The cause-effect relations between the three independent variables (the ratios of gum karaya, calcium chloride and sodium alginate) and the two dependent variables (spreadability and viscosity of neem gel) were investigated. The optimal neem gel formulation comprised gum karaya, calcium chloride and sodium alginate with the concentrations of 1.6%, 0.0435% and 1%, respectively. The optimal neem gel was evaluated for its various properties such as pH, homogeneity, spreadability, active compound quantitation by high performance liquid chromatography (HPLC) and dynamic viscoelasticity behaviours. This study has reported for the first time the application of gum karaya in neem gel preparation. The results of the research may provide the useful data for further studies not only on the gum karaya as the gelling agent but also on the neem gel preparation for the treatment of skin diseases.
KEYWORDS: Gum Karaya; Gel; Rheology; Neem; optimization.
1. INTRODUCTION:
Neem (Azadirachta indica), a member of Meliaceace family, is the famous herbal medicine. Neem has been used safely and effectively for more than 5000 years in India to support the treatment of many common skin disorders such as acne, melasma and heal skin lesions1,2. Neem leaf extract has been reported to inhibit the growth of Propionibacterium acnes3, prevent wrinkles formation4, and inhibit the growth of fungal species such as Trichophyton rubrum, Trichophyton mentagrophytes and Microsporum nanum5.
Gum karaya is the natural polymer derived from Sterculia urens. The main components of gum karaya are D-galacturonic acid, D-galactose, L-rhamnose and D-glucuronic acid6,7.
Gum karaya is hydrophilic in nature and swells up to 60 – 100 times its dry volume when contacts with water and produces a viscous dispersion7,8. Gum karaya is chemically inert, nontoxic, less expensive, biodegradable and widely available. This gum has several applications in pharmaceutical and food industries. In pharmaceutical field, gum karaya has been applied as the controlled released excipient, stabilizer and emulsifiers due to its good emulsification efficiency, high viscosity and its stability6,9,10. However, until now little work has been reported on the application of gum karaya in the topical gel preparation.
Topical preparations such as gels, ointments, lotions and creams are the important drug delivery systems due to its convenience in delivering drug to a localized area of the skin. Gel is one of the semi-solid topical preparations providing quick onset of activity, long-term efficacy and high patient satisfaction. Various physicochemical parameters are employed to investigate the properties of gel such as pH, viscosity, rheology, drug content, in vitro and in vivo drug release11,12. The present study therefore was taken up on developing and evaluating the neem gel formulation using gum karaya as a gel forming agent and rheology as the method for excipient screening and evaluating the viscoelasticity of the optimal neem gel.
2. MATERIALS AND METHODS:
2.1. Materials:
The spray-dried powder of neem leaf extract was obtained from BV Pharma JSC (Vietnam). Sodium carboxymethyl starch (CMS) and hydroxylpropyl methyl cellulose (HPMC) were kindly provided by J. Rettenmaier and Söhne Gmbh and Co. KG (Germany). Gum karaya (29000 – 205000 Da) and calcium chloride were obtained from Sigma Chemicals (USA). Sodium alginate was a gift from FMC Biopolymer Ltd (USA). Acetonitrile and ethanol (HPLC grade) were purchased from Merck (Darmstadt, Germany).
2.2. Preparation of neem gel:
Gum karaya, glycerol and other combined gelling agents (sodium alginate or HPMC or CMS) were dispersed in distilled water. Neem leaf extract and calcium chloride were dissolved in distilled water and then mixed thoroughly with the prepared gum karaya mixture and kept for 24 h at room temperature.
2.3. Gelling ability of gum karaya:
Three samples (S1-S3) with different gum karaya concentrations were prepared (Table 1). Gum karaya and glycerol were dispersed in distilled water and kept for 24 h at room temperature. Neem leaf extract was dissolved in distilled water and then mixed thoroughly with the prepared gum karaya mixture. The viscosity and spreadability of each sample were evaluated.
Table 1 - Formulations for evaluating the gelling ability of gum karaya
|
S1 |
S2 |
S3 |
|
|
Neem leaf extract (%, w/w) |
1 |
1 |
1 |
|
Gum karaya (%, w/w) |
1 |
2 |
3 |
|
Glycerol (%, w/w) |
10 |
10 |
10 |
|
Distilled water (q.s.) |
100 |
100 |
100 |
Nine samples (S4-S12) containing binary mixtures of gum karaya and different concentrations of combined gelling agents (sodium alginate or HPMC or CMS) were prepared (Table 2). The viscosity, spreadability, elastic modulus G’ and viscous modulus G” of each sample were investigated.
Spreadability was measured by placing 1g of gel within 1 cm diameter circle on a glass plate. The second glass plate (280g) was then placed over the gel for 1 min. The diameter of the spreading gel circle (d) was measured11. The area of the spreading gel (S) was calculated by the following equation.
S = (πd2)/4
The HAAKE viscotester iQ viscometer (Thermo Fisher Scientific, Germany) with parallel plate geometry P35Ti (50 mm diameter, 1 mm gap) was used to measure the viscosity (in Pa.s) of neem gels. The controlled rate mode and the temperature were set at 1 s-1 and 25 ± 1oC, respectively. The results were calculated using Rheowin Data manager 4.6.3.0 (Thermo Fisher Scientific, Germany).
Rheological measurements were conducted by a Haake Viscotester iQ rheometer (Thermo Scientific, Germany) equipped with two-parallel plate geometry P35Ti (50 mm diameter, 1 mm gap). The temperature was controlled using a Peltier system. The results were calculated using Rheowin Data manager 4.6.3.0 (Thermo Fisher Scientific, Germany).
Steady-state shear measurements:
Evaluation of the steady shear behaviour of neem gels was performed at 25 oC and in the shear rate (γ) range of 0.01 - 100 s-1.
Dynamic shear measurements:
Dynamic viscoelasticity properties of neem gel were investigated in a controlled stress mode. Frequency sweep measurements at a constant shear stress of 1 Pa were performed at 25 oC in the range of 5 to 20 Hz for investigation of the viscoelastic nature of neem gel. Temperature dependent viscoelasticity of neem gel was investigated with the temperature sweep test in the range of 25 - 37oC and the shear stress was set at 1 Pa.
Table 2 - Formulations for evaluating gelling ability of mixtures containing gum karaya and combined gelling agents
|
S4 |
S5 |
S6 |
S7 |
S8 |
S9 |
S10 |
S11 |
S12 |
|
|
Neem leaf extract |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
|
Gum karaya |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
1 |
|
Sodium alginate |
0.5 |
1.0 |
1.5 |
- |
- |
- |
- |
- |
- |
|
Calcium chloride |
0.058 |
0.058 |
0.058 |
- |
- |
- |
- |
- |
- |
|
HPMC |
- |
- |
- |
0.5 |
1.0 |
1.5 |
- |
- |
- |
|
CMS |
- |
- |
- |
- |
- |
- |
0.5 |
1.0 |
1.5 |
|
Glycerol |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
10 |
|
Distilled water (q.s.) |
100 |
||||||||
Fourteen formulations (F1 – F14) were generated by Design Expert software (version 6.0.6, Stat–Ease Inc., Minneapolis, USA) using the D-optimal design to study the effects of independent variables on dependent variables. Percentages of gum karaya (X1), % calcium chloride (X2) and % sodium alginate (X3) were selected as three independent variables whereas spreadability (Y1) and viscosity (Y2) were chosen as dependent variables. Percentage of gum karaya (X1) and % sodium alginate (X3) were studied at three levels and % calcium chloride (X2) was studied at two levels. The selection ranges of independent variables were based on the results of initial trials.
All experiments were repeated in triplicate. The data were analyzed and the best model was selected by using the BCPharSoft OPT software (Vietnam). The optimal formulation was repeated in triplicate. The observed response data of the optimal formulation were then compared with the predicted data obtained by the BCPharSoft OPT software.
It is observed from Fig. 1 that at 1% (w/w) of gum karaya, sample S1 was identified as the liquid solution with a low viscosity of 1.93 Pa.s. When increasing the concentration of gum karaya to 2% (S2) and 3% (S3), the viscosity of the samples was increased. S2 and S3 formed the gel systems with the viscosity of 12.57 Pa.s and 35.3 Pa.s, respectively. The viscosity and spreadability of S3 were found to be significantly increased when compared with S2 (p <0.05). However, since the gel created by gum karaya at the concentration of 3% (w/w) could not simultaneously meet the requirements of viscosity and spreadability of the desired gel system, the combined excipients (CMS, HPMC and sodium alginate) were added to gum karaya solution to improve the properties of neem gel.
The viscosity of nine binary mixtures (S4-S9) of gum karaya and different combined gelling agents are shown in Fig. 2. Among the three combined gelling agents, sodium alginate (S4, S5, S6) could significantly improve the viscosity of binary mixtures of gum karaya when compared with HPMC (S7, S8, S9) and CMS (S10, S11, S12) at all concentrations (p< 0.05).
In the semisolid system, elastic modulus G' represents the elasticity of the solid while viscous modulus G” represents the viscosity of the liquid. The G’ and G’’ values of four samples containing 1% of gum karaya (S1, S6, S9 and S12) and 1.5% of combined gelling agents (S6, S9 and S12) are shown in Fig. 3. It was found that the G” modulus of four samples containing 1% of gum karaya (S1, S6, S9, and S12) were not significantly different in the frequency range of 5 - 20 Hz.
The elastic modulus G' of all three binary mixtures of gum karaya and 1.5% of combined gelling agents (S6, S9 and S12) were found to be higher than elastic modulus G' of the sample without the combined gelling agents (S1). Moreover, the G’ modulus of the sample employing sodium alginate as the combined gelling agent was found to be higher than the other two. Therefore, sodium alginate could improve in the gelling properties of the binary mixture better than CMS and HPMC. As a result, sodium alginate was chosen as the combined excipient for neem gel using gum karaya as gelling agent.
The levels of three independent variables and the constraints of the two dependent variables are shown in Table 3.
Table 3 - Variables in experimental design
|
|
Level |
||
|
Independent variables |
Low |
Medium |
High |
|
X1: % Gum karaya (w/w) |
1 |
1.5 |
2 |
|
X2: % Calcium chloride (w/w) |
0.029 |
0.058 |
- |
|
X3: % Sodium alginate (w/w) |
0.5 |
1 |
1.5 |
|
Dependent variables |
Constraints |
||
|
Y1: Spreadability (cm2) |
Maximum |
||
|
Y2: Viscosity (Pa.s) |
Maximum |
||
Table 4 shows 14 formulations generated by Design Expert software. The ranges of spreadability (Y1) and viscosity (Y2) were found to be 32.2 – 100.2 cm2 and 6.97 – 80.11 Pa.s, respectively.
Table 4 - The independent variables of 14 formulations (F1 - F14) and their responses
|
Formula-tion |
Independent variables |
Dependent variables |
|||
|
X1 |
X2 |
X3 |
Y1 |
Y2 |
|
|
F1 |
1 |
0.029 |
1 |
59.4 ± 2.1 |
22.45 ± 1.51 |
|
F2 |
1.5 |
0.029 |
1.5 |
38.5 ± 0.9 |
69.89 ± 2.54 |
|
F3 |
1.5 |
0.058 |
1.5 |
37.4 ± 1.6 |
73.52 ± 3.57 |
|
F4 |
2 |
0.029 |
1.5 |
38.5 ± 0.6 |
80.11 ± 1.86 |
|
F5 |
2 |
0.058 |
1.5 |
32.2 ± 1.6 |
78.70 ± 2.03 |
|
F6 |
1.5 |
0.058 |
0.5 |
62.2 ± 1.4 |
10.69 ± 0.32 |
|
F7 |
1 |
0.029 |
1.5 |
38.5 ± 1.3 |
70.77 ± 1.17 |
|
F8 |
1 |
0.029 |
0.5 |
100.2 ± 1.0 |
8.02 ± 0.47 |
|
F9 |
1.5 |
0.029 |
1 |
49.0 ± 2.2 |
35.79 ± 2.61 |
|
F10 |
1 |
0.058 |
0.5 |
84.9 ± 1.6 |
6.97 ± 0.15 |
|
F11 |
1.5 |
0.029 |
0.5 |
93.3 ± 1.0 |
12.94 ± 0.27 |
|
F12 |
1 |
0.058 |
1 |
55.4 ± 1.5 |
29.63 ± 1.26 |
|
F13 |
2 |
0.058 |
1 |
65.0 ± 3.5 |
41.86 ± 0.34 |
|
F14 |
2 |
0.058 |
0.5 |
45.3 ± 0.7 |
17.32 ± 0.64 |
The data in Table 4 were used as inputs for BCPharSoft OPT to study on the cause - effect relations and optimize the neem gel formulation.
Training parameters were set at:
· Test groups: Y1(3, 11), Y2(5, 8)
· Transfer function: Back Propagation Learning.
Table 5 - Model statistics from BCPharSoft OPT outputs
|
Dependent variables |
R2 training |
R2 test |
|
Y1 |
1.00 |
0.98 |
|
Y2 |
0.99 |
0.98 |
All R2 values were found to be more than 0.9 indicated the very good reliability of the models and these models (Table 5). Therefore, these models could be used for multivariate optimization.
The three-dimensional (3D) response surface plots were employed to study the cause-effect relations between the independent and dependent variables.
Fig. 4a shows that when % gum karaya (X1) increased, the spreadability of gel (Y1) decreased. When % gum karaya (X1) increased, viscosity (Y2) increased (Fig. 5a). These could be explained by the swollen ability of gum karaya in water and the cohesion property of gum karaya. The gum karaya subunits tended to stick together making the gel difficult to be spread. Similar results were also reported by Balwinder Singh13.
When calcium chloride (X2) content increased, the spreadability of gel (Y1) decreased (Fig. 4b) and the viscosity increased (Fig. 5b). These may be explained by the presence of α-L-guluronic acid molecules (G block) in the structure of sodium alginate. One calcium ion may replace sodium ions of two α-L-guluronic acid units (G blocks) of two alginate molecules and created the 3D structure of gel and increased the viscosity and decreased the spreadability of neem gel. Similar results were reported by Martinsen14, F. Lardy15.
When % sodium alginate (X3) increased, spreadability of gel (Y1) decreased (Fig. 4b) and viscosity increased (Fig. 5b). The sodium alginate molecule contains many hydroxyl radicals and polar carboxyl groups. Therefore, sodium alginate is soluble in water. The hydrogen bonds between the carboxyl and hydroxyl groups helped to link the polysaccharide molecule together.
As a result, the viscosity of the sodium alginate solution could be increased. The viscosity of the neem gel is proportional to the sodium alginate concentration, the calcium ion concentration16. When the concentration of sodium alginate solution (X3) increased, the viscosity, the firmness of the gel and its ability to adhere to the skin and mucous membrane increased17,18. Sodium alginate and gum karaya could give the synergistic effect when simultaneously forming a gel system with significant improvement in viscosity. Gum karaya is not soluble but swelled in water. In contrast, sodium alginate is water soluble. These two types of gel-forming adjuvants could produce a better matrix12. This result was found to be consistent with the study of Leroux MA19.
The optimized neem gel formulation, achieved by BCPharSoft OPT software, contained gum karaya, calcium chloride and sodium alginate at the percentages of 1.6%, 0.0435 % and 1 %, respectively. Three replicated batches of the optimized neem gel formulation were prepared and statistically compared with the predicted data using one sample T-test. The spreadability (Y1) and viscosity (Y2) of the optimized neem gel were found to be at 54.7 ± 0.8 (cm2) and 36.14 ± 1.72 (Pa.s), respectively. It was demonstrated that all predicted values were in good agreement with the observed values (p>0.05).
The optimal neem gel was characterised by dynamic viscoelastic analysis. The optimal neem gel was demonstrated as a non-Newtonian fluid. The flow curve conformed to pseudoplastic flow or shear thinning behaviour. This property made the gel easier to apply on skin. This rheology property could be considered in the quality control of homogeneity, ensuring the therapeutic effect of gel products between batches14. The dynamic viscoelastic property of the optimal neem gel was investigated by the elastic modulus G’ and the viscous modulus G”. It was found that the elastic moduli G’ of the optimal neem gel were higher than the viscous moduli G” at all the frequencies analysed (5-20 Hz) revealing suitable gel stability. According to the Dahlquist standard, the optimal neem gel with the elastic modulus G’ in the range of 100 - 700 Pa which was less than 105 would possess the good adhesion to the skin and mucosa20. The optimal neem gel showed the gel behaviour with the investigated temperature range of 25 to 37°C. It was interesting that the optimal neem gel after spreading in the human skin tended to increase its temperature and increase its viscosity and may let the neem gel stay longer on the skin surface and hence, improve its therapeautic efficacy.
This study successfully developed and evaluated the neem gel formulation. Gum karaya was demonstrated to be a gel-forming agent with the pseudoplastic flow. Sodium alginate significantly improved the elasticity of the gel-forming binary mixture containing gum karaya. All three independent variables (% gum karaya, % calcium chloride and % sodium alginate) were found to significantly affect the two dependent variables (spreadability and viscosity of neem gel). The properties of the optimal neem gel were confirmed by its dynamic viscoelastic analysis.
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.
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Received on 07.08.2019 Modified on 04.10.2019
Accepted on 09.11.2019 © RJPT All right reserved
Research J. Pharm. and Tech. 2020; 13(4): 1859-1864.
DOI: 10.5958/0974-360X.2020.00335.2