INTRODUCTION:

Polyherbal formulation known as herb-herb combination has been utilized popularly across the world due to its potent therapeutic properties. Polyherbal formulation often produces a promising effect over a single drug in the treatment of a variety of diseases. The concept of herbal blend has been recognized and practiced in westernmedicine and even in Indian traditional medicine, combined plant extracts and plant formulations are preferred instead of individual ones¹.

Topical application of plant extracts has been practiced from ancient times and it has gained a lot of importance owing to extensive application and ill-defined benefit-risk ratio. Many medicinal plants with antioxidant, and antimicrobial properties have been utilized enormously in the treatment of diseases related to the skin. Compared to ointments and creams, the topical application of gels containing these plant extracts at pathological locations offers significant advantages since the medications release more quickly and directly to the site of action².

In the current situation, scientists are trying to find out a new polyherbal therapy; gels are widely used as vehicles for drug delivery through topical routes. Polyherbal plant extracts incorporated into gels are expected to show enhanced therapeutic effects as an antioxidant agent. This is based on the fact that, the individual effect of single plants will be lesser while the use of plants with varying potency in combination theoretically produces an enhanced effect and in addition, specific actions of phytoconstituents are significant only when potentiated by other plants, but not apparent when used alone³. The goal of the current study to design polyherbal gel formulations of extracts of leaf drugs of Tamarindus indica, Nyctanthus arborstris and Memecylon malabaricum.

Tamarindus indica (Tamarind) belonging to family Leguminosae, is one of the medicinal plants containing many therapeutically valuable compounds including polyphenolic antioxidant molecules, Vitamin C and caffeic acid. Various plant parts of Tamarindus indica have been reported to possess antioxidant, lowering blood glucose, lipid level and blood pressure⁴.

Memecylon malabaricum family Melastomataceae is a smalltrees whose leaves have been used in the treatmentinflammation, bacterial infections, diabetes and skin disorders including chicken pox, herpes, psoriasis ^5,6.

Nyctanthes arbor-tristis Linn. (Night Jasmine) which belongs to the family Oleaceae is a valuable therapeutic tree with fragrant white flowers. The leaves are used as diuretic, laxative, diaphoretic, cholagogue, in the treatment of malaria, arthritis, and fungal skin infection⁷. Studies have reported that selected individual plants contained abundant quantities of polyphenolic compounds including flavonoids with antioxidant and antimicrobial activity.

It is well known that polymeric gel formulations based on cellulose and carbopol can transport water-soluble medications to the cutaneous and mucosal regions for an extended period of time⁸. Gel formulations can be designed using a variety of techniques. The traditional method involves varying one factor at a time while maintaining the same values for the other elements. However, this method is laborious and time-consuming and the interaction between factors cannot be studied.Hence to overcome these problems, in pharmaceutical formulation development application of Quality by design (QbD) for optimization is used^9-11. Central Composite Design (CCD) is one of the response surface methodologies employed in the formulation development to study the interactive effect of independent variables that influences the quality of the product. Several reports have shown that CCD results in reliable prediction of optimized formulation^12-14.

Therefore, an attempt is made to utilize the concept of herbal extract combination by formulating these extracts into polyherbal topical gel formulations and by applying the central composite design, an ideal carbopol 940 and HPMC gel formulation containing 1% each extract will be developed with the highest quality product.

MATERIAL AND METHODS:

Materials:

Carbopol 940, HPMC, triethanolamine, propylene glycol were procured from Sigma Aldrich (Germany). The solvents and chemicals used were analytical grade.

Collection and processing of plant material

Fresh leaves of Tamarindus indica, Nyctanthus arborstris, Memecylon malabarica were collected from the locality of Mangalore, authenticated and voucher specimen deposited. (Voucher No-19PY049E). To remove the dirt, leaves were washed, dried and pulverized to coarse powder.

Extraction of plant material:

Extracts from leaves of Tamarindus indica, Nyctanthus arborstris, Memecylon malabarica were prepared separately by conventional method (Soxhlation)

Soxhlet Extraction:

Soxhlet extraction of 20grams of leaf powder was carried out for 36 hours using 350ml ethanol as solvent at 50^oC. The solution was filtered followed by concentration to dryness using a rotary flash evaporator.

Preliminary phytochemical analysis:

Prepared extracts of Tamarindus indica, Nyctanthus arborstris, Memecylon malabarica were analyzed for the presence of important constituents by performing standard chemical tests¹⁵.

Preparation of polyherbal Hydrogel formulation:

Known concentrations of HPMC and Carbopol 940 were prepared using 100ml deionized water. The gels were swollen after 12h, after that specified volume of triethanolamine solution was added. After gel base formulation, three plant extracts (1% each) was dissolved in ethanol and added dropwise into the gel and stirred overnight at 200rpm for complete loading the extract. Finally, the total weight was adjusted to 100g with purified water¹⁶.

Optimization of polyherbal hydrogel formulation:

Central composite design with two factors-three levels were used for optimization of hydrogel formulation using Design Expert software. Independent variables selected for the study includes concentration of carbopol 940 and HPMC. The effect of these variables on spreadability (Y1) and viscosity (Y2) were analyzed by response surface methodology by keeping volume of triethanolamine constant. The optimized formulation was selected with good spreadability and viscositybased on the analysis of the data.

Characterization and evaluation of Gel formulations

Physical Appearance:

Prepared polyherbal gel formulations were visually inspected for colour, clarity and general appearance.

Determination of pH:

Digital pH meter was used for getting pH of the formulations. Gel (1gram) was added to distilled water(100ml) and pH of the mixture was recorded.

Measurement of spreadability:

Spreadability of formulations were estimated based in the slip and drag characteristics of gel using wooden block and slide apparatus¹⁷. About 2g of formulations was applied on the ground slide and on that other slide provided with hook, of same dimension was placed. A 110g of weigh was placed on the top of the slides for 5 minutes to obtain uniform film of the gel. The upper slide was given a pull of 80g. How long the top slide took to cover the distance of 7.5cm was noted.

Determination of Viscosity:

Viscosity of prepared gel formulations were determined with the help of Brookfield viscometer with spindle no 7 at 50rpm at room temperature.

Estimation of phenolic content:

Phenolic content in poly herbal gel formulation were estimated by Folin-Ciocalteau method¹⁸. The appropriate dilution of gel formulation was oxidized with Folin-Ciocalteau reagent followed by reaction with saturated sodium carbonate. The absorbance was recorded at a wavelength of 765nm using a UV-Visible spectrophotometer (Shimadzu 1700), with Gallic acid serving as the standard. The phenolic content was quantified as gallic acid equivalent per gram of the plant material.

Preparation of calibration curve:

a) Preparation of standard gallic acid solutions for calibration curve:

Gallic acid solution is prepared in phosphate buffer (pH 6.8) and treated with FC reagent (1.25ml) and 2.5ml of sodium carbonate and volume was made up with the distilled water to get a linearity concentration range of 4-24µg/ml. The absorbance was measured at 760nm using distilled water as blank.

Drug content determination:

Drug content of optimizedpolyherbal gel formulations was estimated after dissolving the gel formulation in phosphate buffer of pH 6.8. The phenolic content was estimated by using calibration equation.

Drug release study:

Franz diffusion cell with the dialysis membrane was used for determining the release of phenolic content from the optimized polyherbal gel formulation. 1g of gel formulation was placed in the donor compartment and 12ml of phosphate buffer as dissolution medium in receptor compartment. The dissolution medium was stirred at 50rpm and temperature was maintained 37±0.5˚C. Samples (1ml) were withdrawn at regular intervals of time till 8hrs and analyzed for polyphenolic content.

RESULTS AND DISCUSSION:

Response surface methodology with CCD was applied to optimize the effect of was used to optimize the effect of Carbopol 940 and HPMC concentration on spreadability and viscosity which were chosen as dependent variables. The concentration of Carbopol 940 and HPMC based on the literature were varied between 1-1.5% and 1-2% respectively. Thirteen runs were developed by employing 3²central composite design by selecting 9 combinations and 5repeated centre points^19-23.

Table 1: Effect of independent factors on dependent variables containing 1% each of polyherbal extract

Runs	A: Carbopol 940 (%w/w)	B: HPMC (%w/w)	Viscosity (cps)	Spreadability (g.cm/sec)
1	1.00	1.00	3620	18.61
4	1.50	1.00	4293	16.73
3	1.00	2.00	4950	11.23
11	1.50	2.00	5262	9.70
7	0.89	1.50	4155	14.72
12	1.60	1.50	4698	11.68
2	1.25	0.79	3971	20.71
13	1.25	2.20	5061	10.98
5	1.25	1.50	4401	12.54
9	1.25	1.50	4393	12.85
6	1.25	1.50	4450	12.87
10	1.25	1.50	4381	12.99
8	1.25	1.50	4254	12.67

Values are expressed as mean±SD, n=3

The viscosityof the topical formulations should be within the desirable range sinceit affects theease of application and patient compliance. Higher viscosity will lead to difficulty in application,delayed drug release and lower viscosity will result in faster removal from application site. Increase in the concentration of carbopol 940 and HPMC resulted in increased viscosity of formulations, but effect was significant with HPMC concentration. Results shows that gels prepared under run 1 has the desired spreadability and viscosity of 3620 cps and 18.61, respectively.

Fig 1: 3D response surface plot indicating the effect of Carbopol 940 and HPMC concentration on viscosity of polyherbal gel formulation.

3D response surface plot shows the effect of Carbopol and HPMC on Viscosity was found to be linear.

The equation obtained by the design expert software for viscosity is shown below

Viscosity =+4.45+0.2191A+0.4801B

ANOVA results show that p-value was less than 0.0500 indicating that model terms are significant. Model term A and B are significant model terms.Model F-value is 57.08 indicating that model is significant.The predicted determination coefficient (R²) and adjusted R² were found to be 0.8440 and 0.9034 respectively and differences were less than 0.2 . The Predicted R² of 0.8440 is closer to the adjusted R² of 0.9034 with the differenceless than 0.2.

Fig 2: 3D response surface plot showing the effect of Carbopol 940 and HPMC concentration on the spreadability of polyherbal gel formulation

The quadratic effect of Carbopol and HPMC on spreadability was observed.Spreadability reduced with rise in carbopol and HPMC concentration.

The following equation was obtained by the design expert software for spreadability

Spreadability = +12.78-0.9637A-3.52B+0.0875AB+ 0.0942A²+1.42B²

Based on the ANOVA, model terms were found to be significant since p-value was less than 0.0500. Model terms A, B, B² were found to be significant. The model was significant since F- the value was found to be 244.01. The predicted R² of 0.9652 is in reasonable agreement with the Adjusted R² of 0.9902; i.e. the difference is less than 0.2.

All the polyherbal gel formulations were green in colour, translucent and had good homogeneity free of lumps. The pH of the formulations were in normal skin pH range of 6.8-6.9. Results of the evaluation show that variation in the combination of Carbopol and HPMC effects the physical properties of gel formulations. The drug content of optimized formulation was found to be 97.05%.

For the optimized formulation, the experiment values and predicted values of viscosity was found to be 3620cps and 3754cps respectively which produces a percentage bias of 3.56. Similarly, the predicted and experiment values for spreadability was found to be 18.867 and 18.61gcm/sec with a percentage bias of 1.36. Finally based on the results obtained by DOE, formulation with run 1 containing HPMC and carbopol 1:1 w/w was chosen as the optimized formulation which has desired viscosity and high spreadability.

Drug release study:

Optimized Gel formulation showed 88.02% percentage release of phenolic content after 8 hours of the study as shown in the figure 3. This showed the optimized formulation shows the faster release of drug.

Fig 3: In vitro drug release study of optimized polyherbal formulation

Optimized Gel formulation showed 88.02% percentage release of phenolic content after 8 hours of the study. This showed the optimized formulation shows faster release of drug.

CONCLUSION:

The optimized polyherbal gel formulation containing Carbopol and HPMC polymers was fabricated using an experimental design method. Polyherbal gel formulation prepared using Carbopol and HPMC in the ratio 1:1 produced a gel of satisfactory physicochemical parameters with the release of polyphenolic content of 88.02% after 8 hours. Hence this gel formulation containing the polyherbal extract can used for faster release of maximum polyphenolic constituents which will be useful for higher antioxidant activity. Further, this formulation can be evaluated for in -vitro and in-vivo antioxidant activity

ACKNOWLEDGMENT:

Authors are thankful to Nitte (deemed to be) University for providing fund with grant numberN/RG/NUSR2/ NGSMIPS/2021/3and NGSMIPS for giving the facilities for work.

CONFLICT OF INTEREST:

None.

REFERENCES:

1. Kurian A, George S, Thomas B. A review on phytochemical characterization of kwatha–ayurvedic polyherbal formulation. J of Nat Remed. 2021; 17; 21(2): 87-98.

2. Bhinge SD, Bhutkar MA, Randive DS, Wadkar GH, Kamble SY, Kalel PD, Kadam SS. Formulation and evaluation of polyherbal gel containing extracts of Azadirachta indica, Adhatoda vasica, Piper betle, Ocimum tenuiflorum and Pongamia pinnata. J Res Pharm. 2019; 23 (1): 44-54.

3. Risberg K, Fodstad O, Andersson Y. Synergistic anticancer effects of the 9.2.27PE immunotoxin and ABT-737 in melanoma. PLoS One. 2011; 6(9): e24012.

4. Kuddus, S.A., Bhuiyan, M.I., Subhan, N. et al. Antioxidant-rich Tamarindus indica L. leaf extract reduced high-fat diet-induced obesity in rat through modulation of gene expression. Clin Phytosci. 2020; 6(68): 1-13.

5. Ramaiah M, Ganga RB, Chakravarthi G. Antidiabetic activity of methanolic extract of Memecylon malabaricum cogn (melastomataceae) leaves. Int J Pharm Bio Sci. 2013; 4: 822-8

6. Dhanabal SP, Muruganantham N, Basavaraj KH, Wadhwani A, Shamasundar NM. Antipsoriatic activity of extracts and fractions obtained from Memecylon malabaricum leaves. J of Pharmand Pharmacol. 2012; 64(10): 1501-9.

7. Peddinti R. Phytochemical, antibacterial and in vitro antioxidant analysis of Nyctanthes arbortristis leaves. Int J PharmSci Invention. 2020; 9(10): 6-9

8. Najafabadi AR, Moslemi P, Tajerzadeh H. Intranasal bioavailability of insulin from carbopol-based gel spray in rabbits. Drug Deliv. 2004; 11: 295-300.

9. Jyothi D, Priya S, James JP. Development and optimization of polymeric nanoparticles of glycyrrhizin: physicochemical characterization and antioxidant activity. Int J App Pharm. 2024; 16(1): 166-71.

10. Sebastian G, Priya S, James JP, Sannidhi MA, Prabhu VK. Computational tools assisted formulation optimization of nebivolol hydrochloride loaded PLGA nanoparticles by 32 factorial designs. Int J App Pharm. 2022; 14(4): 251-8.

11. Jyothi S, Cheriyan SP, Ahmed SRR, Priya S, James JP. Microwave assisted green synthesis of silver nanoparticles using Coleus amboinicus leaf extract. Int J App Pharm. 2020; 12(3): 56-61.

12. Peng X., Fang X. Development and optimization of solid lipid nanoparticle formulation for ophthalmic delivery of chloramphenicol using a Box-Behnken design. Int. J. Nanomed. 2011; 6: 683–692.

13. Savic Gajic I., Savic I., Boskov I., Žerajić S., Markovic I., Gajic D. Optimization of Ultrasound-Assisted Extraction of Phenolic Compounds from Black Locust (Robiniae Pseudoacaciae) Flowers and Comparison with Conventional Methods. Antioxidants. 2019; 8: 248.

14. Gajic I.S., Savic I., Gajic D., Dosic A. Ultrasound-Assisted Extraction of Carotenoids from Orange Peel Using Olive Oil and Its Encapsulation in Ca-Alginate Beads. Biomolecules. 2021; 11: 225-29.

15. Kokate CK, Purohit AP, Gokhale SB. Pharmacognosy. 10th ed. New Delhi: Nirali Prakashan Pvt Ltd; 1998; 92-4.

16. Aiyalu R, Govindarjan A, Ramasamy A. Formulation and evaluation of topical herbal gel for the treatment of arthritis in animal model. Braz J Pharm Sci. 2016;52:493-507

17. Gaikwad L, Yadav V, Dhavale P, Choudhari B, Jadhav S. Effect of carbopol 934 and 940 on fluconazole release from topical gel formulation: a factorial approach. Int J Curr Pharm Res. 2012; 2: 487-493.

18. Bukhari SB, Bhanger MI, Memon S. Antioxidative activity of extracts from fenugreek seeds (Trigonellafoenum-graecum). Pak J Anal Environ Chem. 2008;9(2):78-83.

19. Nagy, M.; Otremba, P.; Krüger, C.; Bergner-Greiner, S.; Anders, P.; Henske, B.; Prinz, M.; Roewer, L. Optimization and validation of a fully automated silica-coated magnetic beads purification technology in forensics. Forensic Sci. Int. 2005; 152(1): 13-22.

20. Jainey P. James, Sneh Priya, Divya Jyothi. Effect of PLGA polymer on Antimicrobial Activity and the Release Studies of Nanoparticle Hydrogel Containing Mimosa pudica Extract. Research J. Pharm. and Tech. 2018; 11(7): 2876-2880. doi: 10.5958/0974-360X.2018.00530.9

21. Sneh Priya, Divya Jyothi, Jainey P James, Amala Maxwell. Formulation and Optimization of Ethosomes loaded with Ropinirole Hydrochloride: Application of quality by Design Approach. Research J. Pharm. and Tech. 2020; 13(9): 4339-4345. doi: 10.5958/0974-360X.2020.00767.2

22. Avinash, Ullas Prakash D’souza, Divya Jyothi, Partha Bhowmik. Evaluation of Antidepressant Activity of Leaf Extract of Curculigo orchioides Gaertn in mice. Research J. Pharm. and Tech. 2023; 16(11): 5178-2. doi: 10.52711/0974-360X.2023.00839

23. Puneeth Deepak Ail, Divya Jyothi, Rokeya Sultana, Lenisha Crasta. Synthesis and Optimization of Silver Nanoparticles using Leaf extract of Tamarindus indica by Box-Behnken Design for Enhanced Antimicrobial Activity. Research J. Pharm. and Tech. 2023; 16(10): 4583-0. doi: 10.52711/0974-360X.2023.00746

24. Vinod Kumar, Prashant Kumar, Saurabh Sharma. Application of Box-Behnken Experimental Design in Process Parameter Optimization for Production of Berberine HCl loaded Chitosan Coated Sodium Alginate Nanoparticles. Research Journal of Pharmacy and Technology. 2023; 16(3): 1139-6. doi: 10.52711/0974-360X.2023.00190

25. Sneh Priya, Poornima, Cynthia Lizzie Lobo. Formulation and Optimization of Rivastigmine-Loaded PLGA and Chitosan Nanoparticles for Transdermal Delivery. Research Journal of Pharmacy and Technology. 2023; 16(7): 3175-2. doi: 10.52711/0974-360X.2023.00522

Received on 02.04.2024 Revised on 29.07.2024

Accepted on 01.10.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):5702-5706.

DOI: 10.52711/0974-360X.2024.00868