FORMULATION AND EVALUATION OF HERBAL ANTIOXIDANT GEL

FORMULATION AND EVALUATION OF HERBAL ANTIOXIDANT GEL

Samruddhi R. Nale *, Varsha M. Jadhav and Vilasrao J. Kadam

Department of Quality Assurance, Bharati Vidyapeeth’s College of Pharmacy, C.B.D. Belapur, Thane, Navi Mumbai, Maharashtra, India.

ABSTRACT: Introduction: Herbal drugs have great growth potential in the global market. Natural product research continues to explore traditional medicines to develop new novel drugs. The present study aimed to investigate antioxidant potential of gel formulated with Punica granatum peel extract, Glycyrrhiza glabra root extract and Citrus sinensis peel extract. Method and Materials: Different gels were formulated by varying the concentration of the extracts and evaluation was done. The formulated gel was compared for antioxidant activity with standard marketed preparation. Results: The prepared formulations showed satisfactory rheological characteristics, release behavior, appearance, pH, and spreadability. The optimized gel formulation showed excellent antioxidant activity. The IC50 value of gel was 43.74 μg/ml, and that of marketed preparation was 51.15 μg/ml, which shows good antioxidant property of gel. Conclusion: The formulated 5% gel formulation showed good antioxidant activity when compared to the marketed preparation. Thus, the present investigation revealed that the developed gel formulation has potential antioxidant activity.

Keywords: Glycyrrhiza glabra, Punica granatum, Citrus sinensis, DPPH, Herbal Gel, Antioxidant activity

INTRODUCTION: The skin, an organ in direct touch with the environment, shields the body from environmental harm and environmental deterioration, including ultraviolet (UV) rays. However, excessive exposure to UV rays on the skin of the face accelerates skin aging. UVA and UVB make up the two categories of UV light. The epidermis absorbs UVB rays, whereas the endodermis does similarly for UVA rays. In contrast to UVA radiation, which, when absorbed, can hasten the formation of free radicals, UVB rays produce sunburn, which can be avoided with photoprotection or stated as a sun protection factor number.

Free radicals have unpaired free electrons, which makes them highly reactive substances. Antioxidants can be used to stop this. Reactive oxygen species are neutralized by antioxidants, which stop oxidative damage to cells and tissues ¹.

Punica granatum (Pomegranate) ^{2, 3}:

FIG. 1: PUNICA GRANATUM PEEL

Botanical Name: Punica granatum Linn, Family: Puniceae

Vernacular Name: Sanskrit: Dadima, English: Pomegranate, Hindi: Anar, Marathi: Dalimba.

Botanical Description: The fruit is a spherical berry measuring 5 to 12 cm and its pericarp is leathery. Each pink-red piece of pulp-like tissue that divides the interior into compartments holds a seed grain. Globose fruits with a coriaceous, woody rind and persistent callipe.

Chemical Constituents: Alkaloids, Flavonoids, PolyPhenol, Tannin, Saponins.

Pharmacological Activities: Antioxidant, Anti-inflammatory, Antiulcer, Antidiabetic, Antipyretic and Analgesic, Diuretic, CNS Effects, Snake Bite Therapy, Anticonvulsant, Antimicrobial, Immuno-modulatory, DNA Protective, Anticancer.

Part used: Peels

Glycyrrhiza glabra L. (Licorice): ^{4, 5}

FIG. 2: GLYCYRRHIZA GLABRA ROOTS

Botanical Name: Root of Glycyrrhiza glabra Linn, Family: Leguminoseae.

Vernacular Name: Sanskrit- Yashti-madhuh, English- Licorice, Hindi- Jothi-madh or Mulhatti, Marathi- Jeshtamadha.

Botanical Description: Licorice has a thick network of branching roots. The roots are long, cylindrical (round) and straight pieces of wrinkly, fibrous wood that spread out horizontally underground.

Chemical Constituents: Amino Acids, Triterpene Saponin, Flavonoids, Annins, Glycosides, Protein, Resins, Sterols, Volatile oils.

Pharmacological Activities: Antioxidant, Antiulcer, Anti-inflammatory, Anti-diabetic, antimicrobial effects. It may ease upper respiratory infections, treat ulcers, aid digestion and Skin lightening and tightening activity.

Part used: Roots

Citrus sinensis L. (Orange) ^{6, 7}:

FIG. 3: CITRUS SINENSIS PEELS

Botanical Name: Citrus sinesis (L.), Family: Rutaceae, is known botanically.

Vernacular Name: Marathi – Santri, Hindi – Santra

Chemical Constituents: Flavonoids, Alkaloids, Coumarins, Vitamin C and Triterpenes.

Pharmacological Activities: Antioxidant, Cardio vascular health, Antianxiety, used in treatment of depression and stress and also Menstrual disorder.

Part used: Peels

MATERIAL AND METHODS:

Plant Material: The Punica granatum peel, Glycyrrhiza glabra root and Citrus sinensis peel in powdered form was purchased from ‘Yucca Enterprises’ Wadala (e), Mumbai. Alarsin Pharmaceuticals authenticated all drugs. A/32, Street No. 3 M.I.D.C. Andheri (East). Mumbai, India. Herbal powders were stored in an airtight container. The reagents and chemicals utilized were all of LR grade.

Preparation of Extracts: Punica granatum, Glycyrrhiza glabra, and Citrus sinensis individual drugs weighing 50 g each were extracted with ethanol using the hot continuous percolation method in a Soxhlet system. The ethanolic extracts were filtered, evaporated, and dried in an electric water bath. For the creation of the gel, dried extracts of Punica granatum, Glycyrrhiza glabra and Citrus sinensis were also employed.

Development of Formulation ^{8, 9, 13}: Ingredients used for gel formulation are given in Table 1. A water-soluble gel was prepared using the dried ethanol extract of Pomegranate peels, Licorice roots and orange peels. The gel comprised carbapol-940, propylene glycol, DMSO, methyl paraben, propyl paraben, Triethanolamine, perfume and distill water.

Procedure: The amount of water needed for these formulations was split into two portions. The exact amount of extract dissolved in propylene glycol and DMSO in one portion. Carbopol 940 was uniformly mixed in a different section for 30 minutes with a mechanical stirrer set to 1200 rpm. Methyl and propyl paraben were then gradually added to this solution along with the medication solution. The pH was then corrected as necessary (6.8-7) were in the narrow range of neutral pH using Triethanolamine (TEM) and swirled gently until a clear gel was created. The volume was then increased to 100 ml by adding the remaining distilled water. A few drops of fragrance liquid were added. Formulations with different extract ratios of 1%, 3% and 5% were produced. Collapsible tubes were used to hold gel.

TABLE 1: PRELIMINARY FORMULATION DEVELOPMENT

Evaluation of Gel: The topical gel formulations from each of the twelve batches were assessed. Several characteristics, including appearance, pH, spreadability, viscosity, extrudability, drug content and in-vitro drug diffusion tests using cellulose acetate membrane, were used to choose the optimal batch. All of these factors were compared to a commercially available preparation.

Physical Observation: The formulation's colour, smell, transparency and appearance were noted.

pH Measurement: Using a digital pH metre (MK-VI, Syntronics), the pH of the formulation was determined by fully submerging the glass electrode in the gel system. Prior to usage, the pH metre was standardised using buffers with pH values of 4.0 and 7.0.

Extrudability: The formulation was filled taken into collapsible aluminium tubes and after the gels were set in the container, extrudability of formulation was determined.

Rheological Study:

Viscosity: The produced compositions' viscosities were measured using a Brookfield Synchro-electric Viscometer (Model LVT). A sufficient amount of sample was put in a cylindrical tube, and it was let five minutes to equilibrate before the dial reading at 0.3 spindle speed was measured. The dial reading on the viscometer was recorded for each speed. The dial reading that corresponded to each decrease in spindle speed was recorded. The viscosity in centipoises was obtained by directly multiplying the dial reading by the coefficients listed in the Brookfield viscometer catalogue. Utilizing spindle number 4 at a spindle speed of 0.3, the viscosity of gels was measured. To determine viscosity, the average of three observations was used.

Spreadability: 0.5g of gel was placed on a glass plate premarked with a circle of 1cm diameter, and a second plate was placed above it. Then the weight of 50g was allowed to rest on the upper glass plate for 5 min. It was observed that the diameter increased as a result of the gel spreading. Spread circle diameters were measured in centimeters and used as benchmarks for spreadability.

Assay (%Drug Content): 1 gm of gel in a 100 ml volumetric flask was used to determine the drug content of the gel formulation. A sufficient amount of methanol (for drug extraction from gel) was added to a volumetric flask, and the flask was kept in a warm water bath for drug extraction with intermittent shaking.

After cooling to room temperature, the volume was increased to 100 ml with methanol. The drug's methanolic solution was filtered through a 0.45 syringe-driven filter and 1 ml of the filtrate was diluted to 10 ml with methanol before being subjected to HPTLC analysis. The drug content was calculated using the calibration curve's linear regression equation ¹⁰.

In-vitro Drug Diffusion Studies: Based on the antifungal study and gel evaluation, selected batches of formulation were tested for drug diffusion using a Franz diffusion cell with a surface area of 2.09 cm² and a receptor compartment having capacity of 20 ml. The cell is divided into two chambers: the donor and the receptor. As a receptor medium, a combination of pH 6.8 phosphate buffer and methanol (70:30) was used. The addition of 30% methanol to phosphate buffer pH 6.8 was done to ensure sink conditions.

The temperature of the diffusion medium was thermostatically controlled at 37^o C 1o C by surrounding water in the jacket, and the medium was stirred at 500 rpm by a magnetic stirrer. One gramme of gel sample was placed on a 0.45µ cellulose acetate membrane and then fixed in the diffusion cell's donor and receptor compartments. For 30 minutes before applying formulations, the membrane was allowed to equilibrate with the receptor medium. To prevent vehicle evaporation, the donor compartment was covered with aluminium foil. At predetermined intervals, 2 mL samples were withdrawn and replaced with an equal volume of fresh fluid. The withdrawn samples were spectrophoto-metrically estimated at 250 and 265 nm wavelengths using the UV simultaneous method ¹¹.

In-vitro Antioxidant Activity:

Materials and Methods:

Measurement of Antioxidant Activity: The antioxidant activity of the ethanolic extracts was determined by their ability to scavenge the free radical 1,1-diphenyl-2-picryl hydrazyl (DPPH). DPPH is a stable free radical with an odd electron in its structure commonly used in chemical analysis to detect radical scavenging activity. 1ml of each extract solution at different concentrations (20, 40, 60, 80 and 100 g/ml) was added to 3ml of 0.5mM ethanolic DPPH free radical solution.

After 30 minutes, the absorbance of the preparations was measured at 517 nm using a UV spectrophotometer and compared to the absorbance of standard ascorbic acid concentrations (20, 40, 60, 80, and 100 g/ml). The method described was used to measure the absorbance with some modifications. Then the % inhibition was calculated by the following equation:

% 𝑟𝑎𝑑𝑖𝑐𝑎𝑙𝑠𝑐𝑎𝑣𝑒𝑛𝑔𝑖𝑛𝑔 𝑎𝑐𝑡𝑖𝑣𝑖𝑡𝑦 = [(𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑏𝑙𝑎𝑛𝑘 – 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒) / 𝑎𝑏𝑠𝑜𝑟𝑏𝑎𝑛𝑐𝑒 𝑜𝑓 𝑏𝑙𝑎𝑛𝑘] × 100

From calibration curves, obtained from different concentrations of the extracts, the IC₅₀ (Inhibitory concentration 50%) was determined. IC₅₀ value denotes the concentration of sample required to scavenge 50% of the DPPH free radicals ¹².

RESULT AND DISCUSSION:

Formulation Development and Evaluation of Gel: Precipitation and turbidity occurred in batches of the HPMC gel-containing drug, possibly due to system incompatibility; thus, this batch was discarded. Sodium CMC batches formed an overly stiff gel system with an unappealing appearance. Batches containing carbopol 940 (G7-G15) formed a proper gel, so these nine batches were chosen for further investigation.

Physical Appearance of Gel Formulations: Gel formulations were examined visually for clarity, colour, homogeneity, consistency and particle presence. A microscope was used to examine homogeneity. To investigate the consistency of the formulations, a small amount of gel was pressed between the thumb and index fingers and the gel's consistency was noted. All gel formulation batches (G7-G15) were translucent with a smooth and homogeneous texture.

TABLE 2: EVALUATION OF BATCHES

*_Average of Three Determinations

In-vitro Drug Release:

FIG. 4: IN-VITRO DRUG RELEASE OF ELLAGIC ACID, GLYCYRRHIZIC ACID AND HESPERIDIN

In-vitro Antioxidant Test:

DPPH Free Radical Scavenging Activity (In-vitro Antioxidant Activity): The present findings exhibited a concentration-dependent free radical scavenging (antioxidant) activity by the extracts throughout the selected concentration ranges with reference to ascorbic acid. The IC₅₀ values of Punica granatum peel extract were found to be 50.47μg/ml, for Glycyrrhiza glabra root extract 52.38μg/ml and Citrus sinensis peel extract was found to be 54.08 μg/ml. The IC₅₀ value for ascorbic acid was found to be 61.9μg/ml which Indicates that the extract shows better antioxidant activity than standard ascorbic acid.

Punica granatum Peel Extract:

FIG. 5: GRAPH SHOWING FREE RADICAL SCAVENGING ACTIVITY OF PUNICA GRANATUM EXTRACT

TABLE 3: ANTIOXIDANT ACTIVITY OF PUNICA GRANATUM EXTRACT

Glycyrrhiza glabra Root Extract:

FIG. 6: GRAPH SHOWING FREE RADICAL SCAVENGING ACTIVITY OF GLYCYRRHIZA GLABRA EXTRACT

TABLE 4: ANTIOXIDANT ACTIVITY OF GLYCYRRHIZA GLABRA EXTRACT

Citrus sinensis Peel Extract:

FIG. 7: GRAPH SHOWING FREE RADICAL SCAVENGING ACTIVITY OF CITRUS SINENSIS EXTRACT

TABLE 5: ANTIOXIDANT ACTIVITY OF CITRUS SINENSIS EXTRACT

The antioxidant activity of prepared gel was then compared with marketed formulation which is Patanjali Soundarya gel.

FIG. 8: GRAPH SHOWING FREE RADICAL SCAVENGING ACTIVITY OF GEL FORMULATION AND MARKETED GEL EXTRACT

TABLE 6: ANTIOXIDANT ACTIVITY OF GEL AND MARKETED GEL FORMULATION

IC₅₀ value of prepared gel was found to be 43.74 μg/ml and that of marketed gel was found to be 51.15 μg/ml, which show that the prepared gel has good antioxidant property.

CONCLUSION: In the present work, an attempt has been made to formulate and evaluate the combination of herbal drugs and incorporation in gel formulation. Gel containing crude extracts of Punica granatum peel, Glycyrrhiza glabra root, and Citrus sinensis peel extract with different concentrations was prepared.

Which is having good antioxidant. It can be summarized that the prepared formulations showed satisfactory rheological characteristics, release behaviour, appearance, pH, spreadability and antioxidant activity.

ACKNOWLEDGMENT: Authors would like to thank Bharati Vidyapeeth’s College of Pharmacy, c.b.d. Belapur, Navi Mumbai for providing all the research work facilities.

CONFLICTS OF INTEREST: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

REFERENCES:

1. Arif Budiman, Arni Praditasari DR and Aulifa DL: Formulation of Antioxidant Gel from Black Mulberry Fruit Extract 2019; 216–22.
2. Bagade VB, Jadhav VM and Kadam VJ: HPTLC Method Development and Validation for Simultaneous Estimation of 2013; 3(9).
3. Eghbali S, Askari SF, Avan R and Sahebkar A: Therapeutic Effects of Punica granatum (Pomegranate): An Updated Review of Clinical Trials 2021; 2021.
4. Linn GG: International Pharmacognostical, Physicochemical and Phytocemical Study of 2020.
5. Qureshi J, Memon Z, Mirza KM and Motiani V: Anti-hyperglycemic and anti-dyslipidemic activities of Glycyrrhiza glabra root extract in diabetic rats anti-hyperglycemic and anti-dyslipidemic activities of Glycyrrhiza glabra root extract in diabetic rats 2020; (6).
6. Park JH, Lee M and Park E: Antioxidant activity of orange flesh and peel extracted with various solvents. Prev Nutr Food Sci 2014; 19(4): 291–8.
7. Arora M and Kaur P: Antimicrobial & Antioxidant Activity of Orange Pulp and Peel 2013; 2(1): 412–5.
8. Gaikwad VL, Yadav VD, Dhavale RP and Choudhari PB: Current Pharma Research 2012; 2(2): 487–93.
9. Guleri KT and Preet KL: Research Article 2013; 3(6): 51–3.
10. Helal DA, El-rhman DABD, Abdel-halim SA and El-nabarawi MA: Formulation and Evaluation of Fluconazole Topical Gel 2012; 4: 12–4.
11. Haque T: Chemical Enhancer : A Simplistic Way to Modulate Barrier Function of the Stratum Corneum 2018; 8(2): 169–79.
12. Kedare SB, Singh RP. Genesis and development of DPPH method of antioxidant assay. J Food Sci Technol 2011; 48(4): 412–22.
13. Syed Ayaz Ali and Moizul Hasan: A review on different formulations of Punica granatum. International Journal of Research and Analytical Review 2019.
    

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    How to cite this article:

    Nale SR, Jadhav VM and Kadam VJ: Formulation and evaluation of herbal antioxidant gel. Int J Pharm Sci & Res 2023; 14(6): 3076-83. doi: 10.13040/IJPSR.0975-8232.14(6).3076-83.

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