Formulation And Evaluation of Herbal Gel of Syzygium cumini of Antioxidant Activity

The project mainly aims in preparing pharmaceutical gel dosage form which is the “Formulation and evaluation of herbal antioxidant gel” it has antioxidant activity, which prevents free radicalsfrom the skin and helps as moisturizing and hydrating medication.

Plant profile:

Binomial nomenclature: 

• Kingdom: Plantae 
• Order: Myrtales
• Family: Myrtaceae
• Genus: Syzygium
• Species: Cumini
• Binomial name: Syzygium cumini (L) Skeels.^[1]

Fig 1: Syzygium cumini bark

Syzygium cumini (syn. Eugenia Jambolana), a member of family Myrtaceae is commonly known as Jamun or Jambul in Hindi and Black Plum or Indian Blackberry in English. S.Cumini is an evergreen tree distributed in the Indian sub-continent, south-east Asian countries and eastern Africa. S. cumini, is widely used in different countries including India for the treatment of many disorders including diabetes. Various parts of this plant have been recognized to posses several medicinal properties in the traditional system of medicine. The bark of the plant is carminative, digestive, anti-oxidant, anti-helminthic and antibacterial.^[2]

MATERIALS AND METHODS:

Materials:

The bark of Syzygium cumini was collected from local source. 

Method:

(1) Preparation of crude drug powder:

Bark of Syzygium cumini is collected from village and cleaned through washing. Then shade dried for 15 days under room temperature and grinded well. Fine powdered drug is obtained and used for extraction process.^[4]

(2) Preparation of ethanolic extract:

The powdered bark of Syzygium cumini were used for extraction. The bark of Syzygium cumini were carefully cleaned/washed and then using a soxhlet apparatus with ethanol. The extraction process was carried out for 5 hours, 7 cycle of ethanolic extract were runned repeatively. The resulting filtrate was used for phytochemical test and preparation of formulation. The product is stored in desiccator for further studies. ^[4]

Fig 2: Soxhlet apparatus

Fig 3: Ethanolic extract of Syzygium cumini

Phytochemical screening:

The preliminary phytochemical screening of Syzygium cumini bark was done by the following tests;

Test for Carbohydrates:

a) Molisch’s test: To 2-3ml aq extract, add few drops of alpha-naphthol solution in alcohol, shake add conc. H?SO? from side test tube. violet ring is formed at junction of two liquid. 

Test for Alkaloids

1. Dragendroff’s test: To 2-3 ml filtrate extract, add few drops Dragendorff’s reagent. Orange brown ppt.is formed. 
2. Hager’s test: 2-3 ml filtrate with Hager’s reagent gives yellow ppt.

Test for Tannins:

1. Ferric chloride test: Add a few magnesium turnings and concentrated hydrochloric acid to the extract. Appearance of pink or red color indicates flavonoids.
2. Potassium Dichromate: To 1-2 ml extract, add pinch of Potassium Dichromate appearance red ppt.

Test for Steroids:

1. Salkowski reaction: To 2 ml of extract, add 2ml chloroform and 2ml conc. H?SO?. Shake well chloroform layer appears red and acid layer shows greenish yellow fluorescence. 
2. Liebermann-Burchard Reaction: Mix 2 ml extract with chloroform, Add 1-2 ml aceteic anhydride and 2 drops conc. H?SO? from the side of test tube, First red, then blue and finally green colour appear. 

Test for Flavonoids:

1. Lead acetate test: to small quantity of residue, add lead acetate, yellow coloured precipitate.
2. NaOH and HCL test: Addition of increasing amount of sodium hydroxide to the residue shows colouration which decolourises after addition on acid. 

Test for Saponin:

Foam test: About 2 ml of the extract was shaken with 5ml of distilled water Foam shows the presence of Saponins^[3]

Physico-chemical Test:

1. Total ash:

The method is designed to measure the total amount of material remaining after ignition. This includes both ‘physiological ash’, which is derived from the plant tissue itself, and ‘non-physiological ash’ which is the residue of the extraneous matter (e. g. sand and soil) adhering to the plant surface. 

Procedure: 

1. Weigh about 2gm of powdered drug and ignite flat thin porcelain dish or a tared silica crucible.
2. Support the dish on a pipe clay triangle placed on a ring of retort stand.
3. Heat with burner, using a flame about 2 cm high and support the dish about 7cm above the flame, heat with vapour almost ceased to be evolved: then lower the dish and heat more strongly until all the carbon in burnt off.  Cool in desiccator.
4. Weigh the ash and calculate the percentage of the total ash with reference to the air- dried sample of the drug.

Total ash percentage = Pw-Fw x 100 /W

Where, Pw = Pre weight of the crucible

Fw= Final weight of the crucible 

W= Total weight of powdered plant material. ^[3]

(b) Determination of alcohol-soluble extractive value:

Alcohol- soluble extractive value is applied for the drug which contain alcohol soluble constituents such as tannins, resin, and alkaloids.

Procedure:

1. Weigh about 4 gm of coarsely powdered drug in a weighing bottle and transfer it to a dry conical flask.
2. Fill a 100 ml graduated flask to the delivery marks with the solvent ( 90% alcohol).
3. Wash out the weighing bottle and pour the washing, together with the remainder of the solvent into the conical flask.
4. Cork the flask and set aside for 24 hours, shaking frequently.
5. Filter into a 50 ml cylinder.
6. When sufficient filtrate   has collected, transfer 25 ml of the filtrate to a weighed, thin porcelain disk as used for the ash values determination.
7. Evaporate to dryness on a water bath and complete the drying in a oven at 105 degree Celsius for 6 hrs
8. Cool in a desiccator for 30 min and weigh immediately. ^[3]

(c) Determination of water-soluble extractive value:

Water soluble extractive value is applied for the drug which contain water soluble constituents such as tannins, sugars, plant acid.

Procedure:

1. About 4 gm of coarsely powdered drug in a weighing bottle and transfer it to a dry 250 ml conical flask.
2. Fill a 100ml graduated flask to a delivery mark with the water.
3. Wash out the weighing bottle and pour the washing, together with the remainder of the solvent into the conical flask.
4. Cork the flask and set aside for 24 hours, shaking frequently.
5. Filter into a 50 ml cylinder.
6. When sufficient filtrate has collected, transfer 25 ml of the filtrate to a weighed, thin porcelain dish, as used for the ash values determination.
7. Evaporate to dryness on a water bath and complete the drying in an oven at 105degree Celsius for 6 hours.
8. Cool in a desiccator for 30 min and weight immediately.
9. Calculate the percentage w/w of extractive with reference to the air dried drug. ^[3]

Formulation:

(a) Preparation of Gel formulation:

1 g of carbapol-940 was dispersed in 50 ml of distilled water kept the beaker a side to swell the carbapol-940 to form gel. Take 5ml of distilled water and required quantity of propyl paraben were dissolved by heating on water bath solution was cooled and propylene glycol 400 and CMC added. Further required quantity of extract was mixed to the above mixture and add this solution into the carbapol 940 gel with continuous stirring and add triethanolamine was added to the formulation to adjust pH required to skin.^[4]

Fig 4:Gel Formulation

Chemicals: Syzygium cumini extract, Carbopol940, Propylene Glycol, Tri-ethanolamine, Methyl paraben, Glycerine, Carboxy methyl cellulose

Apparatus: Beaker, stirrer, tripod stand, burner

Instruments: Brookfield viscometer, pH meter.

Excipient functions: 

(a)Carbopol 940: Gelling Agent

• Forms a clear, stable gel with high viscosity, enhancing gel consistency.
• Provides muco-adhesion, ensuring prolonged contact time with the skin.
• Requires neutralization with Triethanolamine to form a smooth gel.13

(b) Propylene Glycol: Penetration Enhancer & Humectant

• Increases drug penetration through the skin by disrupting stratum corneum lipids.
• Acts as a humectant, preventing drying of the gel.
• Enhances the solubility of Clitoriaternatea extract in the gel.

(c)Triethanolamine: pH Adjuster

• Neutralizes Carbopol 940, converting it into a stable gel.
• Maintains the gel’s pH within the skin-compatible range (5.5-6.5).
• Ensures better consistency and uniform dispersion of ingredients.

(d) Methylparaben: Preservative

• Prevents microbial contamination, extending shelf life.
• Effective against bacteria and fungi, ensuring product stability.
• Widely used in pharmaceutical and cosmetic formulations due to its safety and effectiveness at low concentrations.18

(e)Glycerine: Moisturising agent

• Glycerin attracts water from the air and binds it to the skin, helping to retain moisture and prevent dryness. 
• It's a natural humectant, meaning it helps the skin hold onto moisture. 
• It can help soften and heal dry, cracked, or irritated skin. 

(f)Carboxy methyl cellulose: Thickening agent 

• CMC is used as a thickening and stabilizing agent in food products like salad dressings, sauces, and dairy products. 
• It can improve the texture and consistency of foods, especially in low-fat or diet products. 
• It helps prevent ingredient separation and settling.

Formulation table:

Table no.1: Formulation table

(3) Evaluation parameters of gel formulation:

1. Visual inspection: The gel were examined for their physical properties by visual inspection of colour, clarity, homogenesity, odour etc. ^[4]
2. Appearance: All the formulation of antioxidant gel was pale yellow/yellow in colour. ^[4]
3. Consistency: The consistency was checked by applying on the skin. ^[4]
4. Greasiness: The greasiness was assisted by the application on the skin and the slide ^[4]
5. Irritancy test (patch test): Skin irritation test was performed for the selected gels on human volunteers to find out any irritation problems which could make it unsuitable for topical use. Skin irritation test was performed, for each gel on three volunteers. Approximately 1gm of gel was topically applied to the hand near the wrist over 2 square inch area and observed for any lesions, irritation, allergy or edema etc. ^[4]
6. Washability: Gel Formulation was applied on hand and was observed continuously under running water^[4]
7. Determination of pH: The pH of gel was determined using digital pH meter by dipping the glass electrode completely into the gel and by using pH paper ^[4]

Fig 6: pH test of different batches

8. Determination of spreadability: Spreadability was measured on the basis of “slip” and “drag” characteristics of the gels  ground glass slide was fixed on wooden block.an weight is provided by pulley ground slide was fixed on this block. 2gm of gel placed on the slide make sandwich weight of 100 gm. placed on the top of the two slides for 5 minutes excess of gel (about 2 gm.) was scrapped out from edges top plate subjected to pull of 20 gm. weight with help of stirring attached hook. Time in seconds required by the top slide cover a distance 7.5was noted. Spreadibility was determined using following formula, S=M.L/T         where,  S is the spreadability in grams.cm/sec, M is the mass in grams, T is time in seconds^[4]
1. Determination of viscosity: Viscosities of the formulated gels was determined using viscometer (speed 60 rpm. At 250 c. was used for gels, corresponding dial reading on the meter was noted. ^[4]

Fig 7: Viscosity test of different batches

10. Clarity: The prepared gel formulation was evaluated in glass container and observed under the glass. ^[4]

Result and discussion:

As we performed all evaluation parameters tests, the results are also found for every single tests of the gel formulation. Evaluation of herbal gel has been performed with parameters given for the evaluation purpose of the gel. It is evaluated by all factors like physicochemical as well as phytochemical factors.

Table no.2: Preliminary Characteristics of Gel

Table no.3: Test for carbohydrates, Alkaloids, flavonoids, tannins, steroids and saponin

Table no.4: Physico-chemical test results

Calculation:

(a) Ash value:

Weight of empty dish (x) = 22.50g

Weight of powder (y) = 2 g

Weight of dish with powder = 24.50 g

After incineration weight of dish (z) = 22.34 g

Weight of ash (z-x) = 22.34- 22.50 

                                = 0.16 

Total Ash = (100 ×Weight of Ash) / Weight of Powder 

                 = (100 ×0.16) / 2

                 = 8 % w/w

Fig 9: Ash value

2. Alcohol-soluble extractive value:

Weight of porcelain with dried extract = 66.62 g

Weight of dried extract = Weight of porcelain with dried extract - Weight of porcelain                                                                = 66.62 - 66.42

= 0.2g

25 ml of alcoholic extract gives = 0.2 g of dried extract

100 ml of alcoholic extract gives = 0.2/25 x 100

                                                      = 0.8g 

4 g of air-dried drug gives  = 0.8g of alcoholic soluble extractives

100g of air-dried drug gives = 0.8/4 x 100

                                              = 20 g of alcoholic soluble extractives

Fig 10:Alcohol-soluble extractive value

3. Water-soluble extractive value:

Weight of porcelain = 65.14g

Weight of porcelain with dried extract = 65.25g

Weight of dried extract = Weight of porcelain with dried extract - Weight of porcelain

                                      = 65.25 – 65.14                             

                                      = 0.09 g

25 ml of aqueous extract gives = 0.09 g of dried extract

100 ml of aqueous extract gives = 0.09/25 x 100

                                                  = 0.36 g 

4 g of air -dried drug gives   = 0.36 g of water -soluble extractives

100 g of air -dried drug gives = 0.36/ 4x 100

                                             = 9 g of water- soluble extractives

Fig 11: Water-soluble extractive value

Table no.5: Some basic parameters like; pH, spreadability, viscosity, homogenesity and clarity is well measured as per IP

Result of activity determination:

In-vitro Antioxidant activity test:

Superoxide Dismutase (SOD):

Procedure:

1. Prepare the assay solutions and add to tubes in the order shown in Table 1. The following recipe is for one sample (20 tubes of the assay system).
2. Turn on the visible light wavelengths on the spectrophotometer. For measuring total SOD activity, prepare a blank solution by adding 200 μl of PB to 800 μl of total assay solution. Place the solution in a 1 ml disposable cuvette and blank the machine at 560 nm using the appropriate blank.
3. The XO concentration of the assay system should be adjusted to an absorbance/min result between 0.02-0.025. For the total SOD assay, add 100 μl of PB, 800 μl of total SOD assay solution, and 100 μl of total SOD assay XO solution. Measure the absorbance of the sample immediately. If the reading is < 0.02, adjust with XO stock. If the reading is > 0.025, adjust with DETAPAC. Repeat until the proper absorbance is measured.
4. Using a stock solution of 1,000 ng/μl SOD, prepare three dilutions as stated below (Table 2). Measure the absorbance of the dilutions at 560 nm and prepare a standard curve. Calculate the Km of SOD. If the assay is working properly, the Km should be near 7-15 ng. This standard is utilized only to ensure that the assay is working correctly.
5. To prepare and run the samples, label tubes (10 × 75 or 12 × 75 mm glass tubes) for the total SOD assay. The tubes required are (13 tubes total): blank, control 1, control 2, sample dilution 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. The blank is the background subtraction, and controls are XO added only. XO sample results represent NBT-associated reduction only, where the XO control is equal to 100% NBT reduction.
6. Using the original sample lysate, prepare 500 μl of a 1 μg/μl sample. Using the diluted sample, prepare total SOD assay tubes to contain 2, 5, 10, 15, 25, 50, and 100 μg of protein. Next, using the original lysate, prepare total SOD assay tubes containing 200, 300, and 500 μg protein. Add the necessary amount of PB to each tube for a final volume of 100 μl. Add 200 μl of PB to the blank tube and 100 μl of PB to the control XO tubes. vii. Add 800 μl of the appropriate assay solution to the tubes. Mix by tapping the racks gently on the laboratory bench. Incubate the total SOD assay tubes for 10 min at room temperature.
7. Transfer the solutions to 1 ml disposable cuvettes. Use the total assay blank tube to calibrate the spectrophotometer. If using a spectrophotometer with a 6-well chamber, arrange the samples as Part A: XO control, dilution 1, 2, 3, 4, 5. Add 100 μl of the total SOD XO solution to the cuvettes and immediately read the samples. Run the kinetics assay for 5 min. Record the rates for each dilution. Repeat with the second dilution series, Part B: XO control, dilution 6, 7, 8, 9, 10.
8. To calculate SOD activity, determine the % inhibition using the following formula:

a) Critical Step: Add BSA first to prevent BCS precipitation

Table no.6: Solutions prepared for Superoxide Dismutase System

b) Preparation of three dilutions

Table no.6: Dilution Preparation

Calculation:

Table no.7: Results observed for F3 batch