Development and Rheological Properties Evaluation of a Burn Healing Ointment Composed of Pomegranate peels and Beta-sitosterol Using Factorial Design
Maher Al-Absi1*, Abdu Faisal2, Safwan Alagbarri1, Mofeed Al- Nowihi2
1Faculty of Pharmacy, Sana’a University, Yemen.
2Biology Department, Faculty of Science, Sana’a University, Yemen.
*Corresponding Author E-mail: ph.d_hmg@hotmail.com, mahir120@gmail.com
ABSTRACT:
The present study was aimed to developed and optimized the formulation of ointment base by using factorial design includes composition for treating skin burns and wounds. The composition includes bees wax, seams seed oil, Beta-sitosterol and pomegranate peels extract. Methods: A 23 randomized full factorial design was utilized in the present study. The responses observed in this study were: Hardness, Viscosity and Spreadability (rheological properties. Pareto charts and main effects plots of each response were done to obtain a optimum proportion of ingredients. Results: The Ointment formulation of pomegranate peel extract significantly shows a good result in all the evaluation test parameters such as Hardness, viscosity and Spreadability. A multi-response prediction analysis was done to obtain the combined optimum that was used to formulate the final product with BW: VAS: SO to be 5: 1.5: 60. Therefore it was conclude that the formula run order 6 consider as a optimized formula with the appropriate rheological characteristics. Conclusion: The optimum formula for the ointment base properties were estimated using the statistical a multi-response prediction analysis, Pareto charts and main effects plots of responses. This ointment base with desired rheological properties can be used for for cosmetic and pharmaceutical usages. It can be concluded that the ointment base obtained with optimal rheological properties can be used for healing burns, scars and wounds applications.
KEYWORDS: Ointment; Beta sitosterol; Punica granatum; Pomegranate peel; Rheological properties; Factorial design.
1. INTRODUCTION:
In the last few years there has been rapid growth in the field of herbal medicine and these drugs gaining popularity both in developing and developed countries due to their natural origin and less side effects. Appropriate treatment of burns is necessary to minimize pain, avoid infection, and promote healing. Minor burns are typically treated with conventional drugs, including antiphlogistics, antiseptics or analgesics, to mitigate inflammation, prevent infection or relieve pain or discomfort of patients.
More severe burns, however, cannot adequately be treated with conventional drugs alone. Severe burns, such as deep secondary and third degree burns, invade dermal and subcutaneous tissues, resulting in destruction of most of the follicles and sebaceous glands. Obvious scars appear after healing and usually affect function and outward appearance.
Many efforts have been made to explore new agents that
can enhance healing and allow for the speedy recovery of injuries while saving
patients from amputation, similar complications and additional problems.
Medicinal plants were shown to play an important role in curing skin disorders
like cuts and burns. [1] The genus punica consists at the present time of two
species, the one under consideration and punica protopunica. The
pomegranate is one of the oldest known edible fruits.
Punicagranatum has been used for long time as a therapeutic agent for
the treatment of inflammatory diseases. The aqueous ethanolic extracts of fruit
rind, flower, and leaves of Punicagranatum have shows anti-inflammatory
activity. The pomegranate peels consider as a waste material, this peels
contained active chemical constituents such as tannins, flavonoids
β-Sitosterol responsible for anti-inflammatory activity. Herbs such as
pomegranate have been widely used in traditional systems of medicines due to
their antiseptic and anti-inflammatory properties [2] Pomegranate is one of the
important fruits stated in the Holy Qur’an. Punicagranatum belongs to
the family of Punicaceae, and is more commonly known as pomegranate, granats,
grenade and punica apple. [3] Also, of the peel, pulp and seed, pomegranate
peel had the highest antioxidant activity. [4] The ointment includes only
natural ingredients that are unadulterated or minimally processed. Topical use
of the ointment dose not Result in harmful side effects. Therefore, this study
focused on the objective to formulate an ointment base with optimal rheological
properties by the valorization of local resources as ingredients (Pomegranate
peels extract, Beeswax, Beta-sitosterol, soft paraffin and sesame oil). The
ointments and their rheological properties were studied using a full factorial
design.
2.MATERIALS AND METHODS:
Materials:
Dried peels of punicagranatum was collected from local market (Sana'a, Yemen), Beta sitosterol, AEROSIL® 200 (Silicon dioxide). The hard thickener Beeswax. The soft thickener white soft paraffin (soft paraffin). The lubricant, sesame oil were obtained from Research and development Center at Modern Pharmaceutical Company (Sana'a, Yemen)
Plant extraction:
The peels were washed with purified water, rinsed well and dried at room temperature for about 30min in open air. The dried material was properly ground into powder. This powder material was then extracted by maceration with ethanol with shaking using a shaker for three days at room temperature. The extract was concentrated at 40C of temperature to obtain semisolid extract (crude extract). The crude extract was dried by using drying agent AEROSIL® 200 (Silicon dioxide) in (3:1).
Factorial experimental design of herbal ointment:
It is desirable to develop an acceptable pharmaceutical formulation in shortest possible time using minimum number of hours and raw materials. Designing drug delivery formulations with the minimum number of trials is very crucial for pharmaceutical scientists (Hamed and Sakr, 2001). Moreover, it may be difficult to develop an ideal formulation using this classical technique (One factor at time) since the joint effects of independent variables are not considered. It is therefore very essential to understand the complexity of pharmaceutical formulations by using established statistical tools such as factorial design. In addition to the art of formulation, the technique of factorial design is an effective method of indicating the relative significance of a number of variables and their interactions (Li et al., 2005).
Selection of variables (independent, dependent) the different variables used in this study are shown in Table [1].
Factorial experimental design for formulation ointment base:
The three ingredients (factors) for ointment base formulation, which are hard thickener, Soft thickener and Lubricant, the following domain with constraint Table [2] was used. It was defined according to previous step. Base on the above implicit plan proposed by the Minitab (18) program, the following design of experiments (Full Factorial design) were made according to a full 23 factorial design for optimization with two replicates, without center points and blocks. The factors studied were the Bees wax – (BW) as hard thickener (5 and 10%), the percentage of Soft paraffin (VAS) in the ointment base – (1.5 and 3%) and the sesame oil for lubricant – (60 and 75%). All the factors studied were independent of each other. The design layout and coded value of inputs (independent) factor is shown in table [3].
Preparation of herbal ointment:
Ointments were prepared by fusion method as described by Alalor et al. [7]. The bees wax was melted at 70ºC then soft paraffin was added at 70ºC After melting the ingredients were stirred gently. Beta sitosterol (0.25%) was dissolved in half part of sesame oil then was added to previous ingredients. Pomegranate peels extract was dispersed in the remind quantity of sesame oil then was added to ingredients, the ingredients were stirred gently at 70ºC for 10 minutes and then cooled in open air.
Hardness evaluation of ointment base:
Hardness of formulations was determined by using the Brookfield Texture analyzer. It is based on the speed of displacement of probe into sample (ointment) at a given distance. The probe was moving down at a speed of 1mm/s till a 1g surface trigger was attained. At this point the probe was in full contact with the sample surface. Then the probe continued to penetrate to a depth of 5 mm at a speed of 1mm/s. At this point the probe returned to its starting position. The peak load (maximum force) was registered, and is considered a measure of firmness of the product – the bigger the force the thicker/harder is the sample. Values of peak force were expressed N (Newton).
Spreadability evaluation of ointment base:
Spreadability is a term expressed to denote the extent of area to which the ointment and gel readily spreads on application to skin or affected part. A special apparatus has been designed by Multimer [9] to study the spreadability of formulations. The spreadability is expressed in terms of times in seconds taken by two slides to slip off from ointment and placed in between the slides under the direction of certain load. Lesser the time taken for separation of two slides, resultant the better spreadability [10]. Spreadability was calculated by using the formula. (S = M.L/T). Where S= Spreadability, M= Weight tied to upper slide, L= Length of glass slides and T= Time taken to separate the slides completely from each other. In this present experiment, M = 45 gm, L= 7.5cm and. Spreadability was calculated and expressed in g.cm/s. was recorded in the Table [4]
Viscosity evaluation of ointment base:
The rheological behaviour of different formulations was done by measuring the viscosity. This viscosity expressed in centipoise (cP) was determined by Brookfield viscometer by modified method of Akanksha et al. [8]. Test sample was taken in a clean and dry 250 ml beaker, and the viscosity of the test sample was determined by standard operating procedure of Viscometer using spindle N° 5. This spindle was used for finding the viscosity of the sample at speeds of rotated at 25, 50 and 80rpm. The reading, near to 100% torque was noted. Samples were measured at 25 ± 1°C.
Statistical analysis:
Eight experiments were carried out at each experimental design point with two replicate and the mean values were stated as observed responses. Experimental runs were randomized, to minimize the effects of unexpected variability in the observed responses. Statistical analyses (p < 0.05) were performed using Minitab software (Version 18). The optimal formulations for ointment base formulation were estimated through Pareto Charts and Main Effects plots for responses of the independent variables and each dependent variable (Response).
3. RESULTS:
Optimization of formulation of ointment base:
The results for the analysis of different formulation bases aimed at choosing the best proportion of ingredients (Hard thickener (bees wax), Soft thickener (soft paraffin) and lubricant (sesame oil) are shown on the Table [4] the responses observed in the course of these analysis are: Hardness, Viscosity and Spreadability. The experimental results obtained are between, 249.5-1831 N, 1013.5-12805 cP and 13.2-23.3g.cm/s.min, for Hardness, Viscosity, and Spreadability respectively. Concerning the viscosity, the range obtained 2027 and 12800 cP is slightly comparable to result to the values obtained by Akanksha et al. [8] During their formulation, the Emulsifying wax, white soft paraffin and liquid paraffin were used as hard thickener, soft thickener and lubricant respectively. From this, our formulation ointment base from natural ingredient could be used for burn ointment. Because the viscosity governs many properties of the ointment formulation such as, spreadability, pourability of the product from the container etc. [16]. In order to have a stable and optimal formulation, it is necessary to understand the proportion and nature of each component. In addition, each formulation must be specifically designed according to the desired purpose of its use, site of application, and patient acceptability. From the above observations, the BW (bees wax) must not be high in the formulation in order to facilitate spreadability; the lubricant must not equally be too much to avoid formula from flowing off its target. Thus a multi-response prediction analysis was done to obtain the combined optimum that was used to formulate the final product with BW: VAS: SO to be 5: 1.5: 60 is given in the following figure [1] and figure [2]. Therefore it was conclude that the formula run order 6 consider as an optimized formulawith the appropriate rheological characteristics.
Effect of formulation ingredients on Hardness:
The Pareto Charts figure [3] significantly shows that the hard thickener (bees wax) has the maximum effects on hardness of ointment base while the lubricant (sesame oil) has the minimum effect, Also the binary effects of hard thickener (bees wax) and soft thickener (soft paraffin) together exert a synergistic effect on the hardness. However, the weakest effect could be of two groups of ingredient soft thickener (soft paraffin), lubricant (sesame oil). The main effects Plot for hardness figure [3] shows that the increase of percentage of hard thickener (bees wax) leads to very high hardness while the increase lubricant (sesame oil).leads to reduce the hardness.
Effect of formulation ingredients on Viscosity:
The Pareto Charts figure [4] significantly shows that hard thickener (bees wax) has the maximum effects on hardness of ointment while the binary effects soft thickener (soft paraffin) and lubricant (sesame oil) have the minimum effect.
The main effects Plot for hardness figure [4] shows that the increase of percentage of hard thickener (bees wax) leads to very high viscosity while the increase lubricant (sesame oil) leads to reduce the viscosity.
Effect of formulation ingredients on spreadability:
The Pareto Charts figure [5] significantly shows that hard thickener (bees wax) has the maximum effects on spreadability of ointment while the binary effects soft thickener hard thickener (bees wax) and soft thickener (soft paraffin) together have the minimum effect.
The main effects Plot for spreadability figure [5] shows that the increase of percentage of hard thickener (bees wax) or soft thickener (soft paraffin) lead to reduce spreadability while the increase lubricant (sesame oil) leads to very high spreadability.
3.1. Figures and Tables:
All figures and tables should be cited in the main text as Figure 1, Table 1, etc.
|
|
a |
b |
Figure 1: Optimization Plot for Minimum Hardness. (a) Multiple Response Prediction for Minimum Hardness.; (b) Optimization Plot for Minimum Hardness.
|
|
a |
b |
Figure 2: Optimization Plot Medium Spreadability (grm.cm). (a) Multiple Response Prediction for Medium Spreadability.; (b) Optimization Plot for Medium Spreadability.
|
|
a |
b |
Figure 3: Effects chartsfor Hardness. (a) Pareto Chartsfor Hardness; (b) Main Effects plots for Hardness.
|
|
a |
b |
Figure 4: Effects chartsfor Viscosity. (a) Pareto Chartsfor Viscosity; (b) Main Effects plots for Hardness.
|
|
a |
b |
Figure 5: Effects chartsfor Spreadability. (a) Pareto Chartsfor Spreadability; (b) Main Effects plots for Hardness.
Table 1: Selection Variables.
Independent (Ingredient) |
Dependent (Response) |
Hard thickener (Bees wax,) |
Hardness |
Soft thickener (Soft paraffin) |
Viscosity |
Lubricant (Sesame oil) |
Spreadability |
1Selection Variables.
Table 2: Implicit Domain for Optimization Plan.
Independent (Ingredient) |
Abbreviation |
Lower limit (%) |
Upper limit (%) |
Hard thickener (Bees wax,) |
BW |
5 |
10 |
Soft thickener (Soft paraffin) |
VAS |
1.5 |
3 |
Lubricant (Sesame oil) |
SO |
60 |
75 |
2Implicit Domain for Optimization Plan.
Table 3: Full 23 factorial design layout table
Run Order |
Inputs (%) |
Outputs (Responses) |
||||
BW |
VAS |
SO |
Hardness (N) |
Viscosity (cp) |
Spreadability (gm.cm\sec.) |
|
1 |
5 |
3 |
60 |
- |
- |
- |
2 |
5 |
1.5 |
75 |
- |
- |
- |
3 |
10 |
3 |
60 |
- |
- |
- |
4 |
5 |
3 |
75 |
- |
- |
- |
5 |
10 |
3 |
75 |
- |
- |
- |
6 |
5 |
1.5 |
60 |
- |
- |
- |
7 |
10 |
1.5 |
60 |
- |
- |
- |
8 |
10 |
1.5 |
75 |
- |
- |
- |
Table 4: Full 23 factorial design for optimization with responses results
Run Order |
Inputs (%) |
Outputs (Responses) |
||||
BW |
VAS |
SO |
Hardness (N) |
Viscosity (cp) |
Spreadability (gm.cm\sec. ) |
|
1 |
5 |
3 |
60 |
352 |
3575 |
19.25 |
2 |
5 |
1.5 |
75 |
259.5 |
1013.5 |
23.3 |
3 |
10 |
3 |
60 |
1831 |
12805 |
13.2 |
4 |
5 |
3 |
75 |
379.5 |
2746.5 |
21.75 |
5 |
10 |
3 |
75 |
1203.5 |
10815 |
14.3 |
6 |
5 |
1.5 |
60 |
249.5 |
2149.5 |
20.4 |
7 |
10 |
1.5 |
60 |
1045.5 |
9348 |
17.25 |
8 |
10 |
1.5 |
75 |
516 |
5440 |
16.4 |
4. DISCUSSION:
Several studies showed that burn infection is the main cause of mortality in patients with extensive burns. Therefore, many researchers tried to achieve appropriate treatment methods to reduce the risk of wound infections and to shorten the period of treatment of patients with burn wounds. [11] Some of these treatments involve using topical antimicrobial agents which effectively reduce mortality rate of burns. [12,13] Healing of burn is still a challenge in modern medicine and there are a few drugs capable of accelerating wound healing and as an alternative plants were rich sources to survey. [12-13,15] For many years, the effect of herbal medicine on burn wound has been noted. Herbal products seem to possess moderate efficacy with no or less toxicity and are less expensive as compared with synthetic drugs. Many plants and plants-derived products have been shown to possess potent wound-healing activity. [12-14] According to these facts, burn wound, which undergoes high inflammation process and cannot be denied, should be treated with anti-inflammatory and antioxidant substances in order to prevent the probability of delayed wound healing. Generally, pomegranate has various active substances which individually can act as antibacterial, anti-inflammatory, and strong antioxidant. Some of the potential substances inside the pomegranate are tannin, flavonoid, punicic acid, and phytoestrogen. In addition, tannins can specifically act as astringent and natural wound protector to let the skin heal naturally. [16]
Ellagic acid is a simplified form of ETs, which genuinely include in tannins. Ellagic acid is an active substance which acts dominantly inside the pomegranate. Ellagic acid individually also has the antibacterial, antiinflammation, and strong antioxidant effect. [17] The addition of ellagic acid in pomegranate standardization can strengthen the function of pomegranate as a topical drug. As explained in Patwardhan et al. [18]. In previous research, pomegranate can mimic the role of ampicillin [17]. Although the antibacterial mechanism of pomegranate is not fully understood, the active substance inside pomegranate can create toxicity inside the body of bacteria through the change and interaction with enzymes and substrates [19] in previous research, the result showed of good collagen formation can be linked with an optimal healing process of the burn wound. The ointments can create an optimal process of burn wound healing. Also, it is known that the addition of 40% ellagic acid in the standardization of pomegranate extract presumably creates a synergic mechanism to support an optimal process of burn wound healing. In fact, Bess waxes are a large and complex group of lipophilic compounds that generally consist of mixtures of esters of fatty acids with long-chain monohydric alcohols [20]. Lubricant here which is sesame oil is made up of unsaturated fatty acid (90%) this will tend to reduce the hardness of the ointment base. However the binary effects of soft thickener (soft paraffin) and lubricant (sesame oil) together significantly dose show any effects.
Hard thickener contributes to increase viscosity of the ointment base. This is due to the lipophilic compounds that generally consist of mixtures of esters of fatty acids with long-chain monohydric alcohols which constitute the wax constituting the hard thickener (bees wax). In fact, waxes are recognized as a potential excipient or enhancing the aesthetic and maximizing the therapeutic benefits of topical formulations by increasing the viscosity [20]. In addition, Pattanayak et al. [21] had shown by comparative study that when the viscosity of formulation increases, the spreadability decreases and vice versa.
5. CONCLUSIONS:
This study aimed to formulate an ointment base with optimal rheological properties by the available of local resources as ingredients (Pomegranate peels extract, Beeswax, Beta-sitosterol, soft paraffin and sesame oil). Using the full factorial design experiment, it was found that the mixtures of Beeswax, soft paraffin and sesame oil not only improve spreadability of ointment base but also modify its viscosity and hardness. The optimum formula for the ointment base properties, were located, using the statistical Pareto charts and main effects plots of responses. This ointment base with desired rheological properties can be used for further work as matrix for incorporation of extracts for cosmetic and pharmaceutical usages. It can be concluded that the ointment base obtained with optimal rheological properties can be used for cosmetic and pharmacological applications.
6. SUPPLEMENTARY MATERIALS:
The following are available online at www.mdpi.com/xxx/s1, Figure S1: title, Table S1: title, Video S1: title.
7. AUTHOR CONTRIBUTIONS:
For research articles with several authors, a short paragraph specifying their individual contributions must be provided. The following statements should be used “Conceptualization, X.X. and Y.Y.; methodology, X.X.; software, X.X.; validation, X.X., Y.Y. and Z.Z.; formal analysis, X.X.; investigation, X.X.; resources, X.X.; data curation, X.X.; writing-original draft preparation, X.X.; writing-review and editing, X.X.; visualization, X.X.; supervision, X.X.; project administration, X.X.; funding acquisition, Y.Y. All authors have read and agreed to the published version of the manuscript.”, please turn to the CRediT taxonomy for the term explanation. Authorship must be limited to those who have contributed substantially to the work reported.
8. FUNDING:
Please add: “This research received no external funding” or “This research was funded by NAME OF FUNDER, grant number XXX” and “The APC was funded by XXX”. Check carefully that the details given are accurate and use the standard spelling of funding agency names at https://search.crossref.org/funding, any errors may affect your future funding.
9. ACKNOWLEDGMENTS:
The authers are grateful to Dr. Safwan adbsul Warith who help us to do this work. Also thankful to the authority of the Modern Pharma Pharmaceutical Industry, Sana'a, Yemen.
10. CONFLICTS OF INTEREST:
The authors declare no conflicts of interest regarding the publication of this paper.
11. REFERENCES:
1. Kokane, D.D., More, R.Y., Kale, M.B., Nehete, M.N., Mehendale, P.C., Gadgoli, C.H., 2009. Evaluation of wound healing activity of root of Mimosa pudica. J. Ethnopharmacol. 124 (2), 311-315
2. Li, Y., Guo, C., Yang, J., Wei, J., Xu, J., Cheng, S., 2006. Evaluation of antioxidant properties of pomegranate peel extract in comparison with pomegranate pulp extract. Food Chem. 96 (2), 254-260
3. Voravuthikunchai, S.P., Sririrak, T., Limsuwan, S., Supawita, T., Iida, T., Honda, T., 2005. Inhibitory effects of active compounds from Punica granatum pericarp on verocytotoxin production by enterohemorrhagic Escherichia coli O157: H7. J. Health Sci. 51 (5), 590-596.
4. Guo, C., Yang, J., Wei, J., Li, Y., Xu, J., Jiang, Y., 2003. Antioxidant activities of peel, pulp and seed fractions of common fruits as determined by FRAP assay. Nutr. Res. 23 (12), 1719-1726
5. “The Ayurvedic Pharmacopoeia of India” first edition part-I volume-II page no; 31 to 33.
6. Dr. K. R. Khandelwal and Dr. Vrunda Sethi “Practical Pharmacognosy Techniques and Experiments” Nirali Prakashan Printed by Yogiraj Printers Page no; 23.1-23.11
7. C. A. Alalor, I. I. Cecilia, P. A. Chukwuemeka, Evaluation of the Antibacterial activity of Herbal ointments formulated with Methanolic extract of Cassia alata. Asian Journal of Biomedical and pharmaceutical Sciences. 2 (2012) 15-19.
8. D. Akanksha, G. Vikas, K. J. Neetesh, S. Shalendra, B. Neelam, K. J. Dinesh Formulation and Evaluation of Neomycin Sulphate Ointment containing Natural Wound Healing Agent Curcuma longa, International Journal of Pharmaceutical Sciences and Drug Research. 1 (2009) 105-118.
9. Mutimer MN, Riffikin C, Hill JA, Marry E, Cyr NG, Glickman. Synthesis of Methylsilyl Derivates of Procaine and Their Diffusion. J. Am. Pharm. Assoc. Sci, Ed, 45, 1956, 212.
10. Ehrlich HP, Hunt TK. Effect of Cortisone and Vitamin A on Wound healing, Ann. Surg. 167, 1968, 324.
11. Manafi A, Kohanteb J, Mehrabani D, Japoni A, Amini M, Naghmachi M, Zaghi AH, Khalili N. Active immunization using exotoxin A confers protection against Pseudomonas aeruginosa infection in a mouse burn model. BMC Microbiol 2009; 9:23.
12. Hazrati M, Mehrabani D, Japoni A, Montasery H, Azarpira N, Hamidian-shirazi AR, Tanideh N. Effect of Honey on Healing of Pseudomonas aeruginosa Infected Burn Wounds in Rat. J Appl Anim Res 2010;37: 161-165.
13. Hosseini SV, Tanideh N, Kohanteb J, Ghodrati Z, Mehrabani D, Yarmohammadi H. Comparison between Alpha and silver sulfadiazine ointments in treatment of Pseudomonas infections in 3rd degree burns. Int J Surg 2007; 5: 23-6.
14. Upadhyay NK, Kumar R, Siddiqui MS, Gupta A. Mechanism of Wound-Healing Activity of Hippophae rhamnoides L. Leaf Extract in Experimental Burns. Evid Based Complement Alternat Med 2011;2011: 659705.
15. Kahkeshani N, Farahanikia B, Mahdaviani P, Abdolghaffari A, Hasanzadeh Gh, Abdollahi M, Khanavi M. Antioxidant and burn healing potential of Galium odoratum extracts. Res Pharm Sci 2013;8: 197-203.
16. Jesus NZT, Falcao HS, Gomes I, Leite TJA, Lima GRM, Barbosa-Filho JM, et al. Tannins, peptic ulcers and related mechanisms. Int J Mol Sci 2012; 13:3203–28;https://doi.org/10.3390/ijms13033203
17. Jurenka J. Therapeutic applications of pomegranate (Punica granatum L.). Altern Med Rev 2008; 13(2):128-44
18. Patwardhan CB, Warude D, Pushpangadan P, Bhatt N. Ayurveda and traditional chinese medicine: a comparative overview. Evid Based Complement Altern Med 2005; 2(4):465-73; https://doi.org/ 10.1093/ecam/neh140
19. Ghudaib KK, Hanna ER, Jawad AH. Effect of ellagic acid on some types of pathogenic bacteria. J Al-Nahrain Univ 2010; 13(2):79-85; https://doi.org/10.22401/JNUS.13.2.09
20. I. Biljana, T. Loftsson, M. A. Hjalmarsdottir and G. M. Asgrimsdottir, Topical Formulation Comprising Fatty Acid Extract from Cod Liver Oil: Development, Evaluation and Stability Studies. Mar. Drugs. 14 (2016) 105-116
21. S. Pattanayak, S. S. Nayack, S. C. Dinda, D. Panda and K.P. Naval, Evaluation of herbal ointments formulated with methanolic extract of Cajanus scarabaeides, J. Pharm. Allied health Sci. 1 (2) (2011) 47-59
Received on 26.07.2020 Modified on 04.09.2020
Accepted on 29.09.2020 ©Asian Pharma Press All Right Reserved
Asian J. Pharm. Tech. 2021; 11(1):59-65.
DOI: 10.5958/2231-5713.2021.00010.6