Formulation and Evaluation of Herbal Gel from Ehretia Laevis Roxb Extract for Anti-Inflammatory and Anti-Arthritic Activity

Fig No. 2 Chemical Cross-linking

2. Physical Cross-linking:

In some circumstances, the transition from a solution to a gel can happen due to the creation of hydrogen bonds, the solubilization of crystalline components, concentration changes, temperature changes, or hydrophobic interactions. Dextran gels, poly (N-isopropyla crylamide) gels, cellulose gels, and others are examples of these gels.⁽⁴⁾

Fig No. 3 Physical cross-linking

3. Ionic cross-linking:

In order to create a gel, charges can be formed on polymers or other molecules (solvents) to promote cross-linking (Fig. 6). The charges on such molecules cause them to form ionic connections. In the presence of calcium ions, polysaccharide alginate, for instance, creates a gel matrix that may enclose certain components (enzymes, etc.). You may also achieve ionic gelation by changing the medium's pH. (solvent). Such mixes gel when the pH is changed; for instance, pectin gels when exposed to an acidic pH in a suitable medium.⁽⁴⁾

Fig No.4 Ionic cross-linking

Chemical Constituents

Phytochemical investigations had let to the extraction and isolation of secondary metabolites along with the primary metabolites from petroleum ether, Chloroform and Methanolic extract of its barks and leaves. These are pentacyclic Triterpenoids, Flavonoids, Alkaloids, Tannins, Phenolic components, Phenolic acids, Hydrocarbons, Aliphatic alcohols, Fatty acids, Ascorbic acids, Amino acids, Carbohydrates, Benzoquonolins, Vitamins and minerals.

a) Lupeol

Lupeol (lup-20(29)-en-3β-ol) is abundantly found I medicinal plants and has been Reported to Pomes an array of pharmacological activites, including antiangiogenic, Ant inflammatory Anticancer, and arthritis, antidiabetic, cardiovascular and Antioxidant activities Lupeol is one of the potential anticancer biomarkers.⁽²⁾

b) Flavonoids

Flavonoids are a group of natural products, which are ubiquitously present in plants (fruits, vegetables and also in certain beverages) They are associated with various therapeutic activities andare present in a variety of medicinal, nutraceutical, pharmaceutical,and cosmetic preparations. The basic structures of these compounds are often character-ized by a fifteen-carbon skeleton as a common phenyl benzopyrone linkage (C6–C3–C6) in their structures Flavonoids are a promising class of natural products sub-divided into flavonols (quercetin and kaempferol), flavones (luteolin and apigenin), flavanones (hesperetin and naringenin), flavan-3-ols (catechin and epicatechin) isoflavones (genistein), and flavanones⁽²⁾

Fig no.5 Structure of Flavonoid

c) Ursolic Acid

Ursolic acid (3 β-hydroxy-urs-12-ne-28-oic acid) is a wellknown pentacyclic terpenoid of Plant origin exhibiting a wide range of pharmacological activities, eg, antiviral, anti-ulcerosos, anti-inflammatory and anticancer activities.

d) α -Amyrin

α-Amyrin (3 β-hydroxy-urs-12-en) is the precursor of ursolic acid and predominantly Found in plant origin exhibiting an array of pharmacological activities, eg, anxiolytic, antidepressant anti-inflammatory, anti-hyperglycemic and hypolipidemic activity.⁽²⁾

e)β-Sitosterol

The physteron β -sitosterol (3β -stigmast-5-en-3-ol) is one of the important active principles of many plants. It is also used as one of the potential plant biomarkers for the treatment and Prevention of cancer.

f)Phenolic Acids and Tannins

Plant phenolic acids are a fundamental human dietary component and are well renowned for their pharmacological actions such as antioxidant, anticancer, antiallergic, antimicrobial and anti-inflammatory properties. The antioxidant potential of a particular phenolic acid depends on the number of hydroxyl groups present as well as their position on the molecule. Tannins belong to the class of polyphenols. Tannins are water soluble compounds, are present in many plants and have the ability to precipitate proteins.⁽²⁾

g) Quercetin

Quercetin (3-3-4-5-7 pentahydroxyflavanone) is a citrus polyphenolic flavonoid abundantly present in vegetables and fruits, e.g., black grapes, onion and tea. It was the first known tyrosine kinase inhibitor in the phase-I human clinical trials. Recent studies have reported for its broad spectrum of activities, including against cancer, cardiovascular diseases, inflammatory and CNS disorders. Quercetin exhibits its significant antioxidant activity by sustaining oxidative balance.

h) Gallic acid

Gallic acid (3,4,5-trihydroxybenzoic acid) is a naturally-occurring plant phenol ob- tained by the hydrolysis of tannins. Gallic acid is known for its diverse biological activities such as,hepatoprotective, anticancer, antimicrobial and gastroin-testinal disorders. Oxidative stress results in an accumulation and overproductionof free radicals, and is the foremost origin of several degenerative diseases such as cardio- vascular system (CVS) diseases, atherosclerosis, cancer and inflammatorydiseases. Gallic acid is a low molecular weight compound readily available in fruits, vegetables and medicinal plants. It has the ability to induce apoptosis and also acts as a strong antioxidant. It has been found in the methanolic extract of leaves of E. laevis.

i) Luteolin

Luteolin (3,4,5,7-tetrahydroxyflavone) is a flavone present in a wide variety of fruits, vegetables and in medicinal plants. Vegetables including celery, parsley, onion leaves, broccoli, peppers and carrots are rich in luteolin. Luteolin shows an array of biological properties, including antioxidant, antimicrobial, anticancer and estrogenic regulator properties. Luteolin has the ability to induce apoptosis and produce anticancer effects by causing cell cycle arrest in human oral squamous cancerous cells, human esophageal, colon, lung and liver cancers^.(2)

j) Naringenin

Naringenin [5,7-dihydroxy-2-(4-hydroxyphenyl) chroman-4-one] belongs to the flavanone series of flavonoids and is predominantly found in citrus fruits like oranges, lemons, grapes and tomatoes. It is a common polyphenolic dietary component and is derived from the hydrolysis of narirutin or naringenin-7-rutinoside. The scientific community pays considerable attention to this flavonoid because of its therapeutic potential, including its antioxidant, antidiabetic, and anti-inflammatory properties and potential against malignancies and neurodegenerative diseases. Naringenin exerts its antioxidant effects by scavenging free radical generation and enhancing several antioxidant enzyme levels such as glutathione peroxidase, catalase and superoxide dismutase.

k)Rutin

Rutin (3,4,5,7-tetrahydroxyflavone-3-rhamnoglucoside) is abundantly available as a flavonolof plant origin. The compound is abundantly present in fruit skin, buckwheat and potato skin of

this plant. It exhibits various pharmacological activities including neuroprotective, cardioprotective, antidiabetic, anticarcinogenic, anti-inflammatory, and antioxidant. It scavenges free radicals and inhibits the lipid peroxidation. It is also reported to act as a hepatoprotective agent⁽²⁾.

Phytochemical Test

                  Fig No.6 Mayer’s Test                                                                Fig No.7 Dragendroff’s Test

                       Fig No. 8  Iodide Test                                                               Fig   No.9 Froth Test

          Fig No.10 Libermann-Burchard Test                                     Fig No.11 Sodium Hydroxide Test

              Fig No.12  Ferric Chloride Test                                              Fig No.13 Lead Acetate Test

4. EXCIPIENTS

1) Alovera

Aloe barbadensis miller, commonly referred to as Aloe vera, is one of more than 400 species of Aloe belonging to family Liliaceae. Aloe vera is considered the most potent and, thereby, the most popular plant in the research field. Important component in the traditional medicine of many Countries such as China, India, the West Indies, and Japan.

Aloe vera is one of the most important medicinal plants in the world with applications in the cosmetic industry and also in the tonic or health drink product market. The main feature of the Aloe vera plant is its high water content, ranging from 99-99.5%. The remaining 0.5-1.0% solid material is reported to contain over 75 different potentially active compounds including water and fat soluble vitamins, minerals, enzymes, simple/complex polysaccharides, phenolic compounds, and organic acids⁽⁷⁾

Fig No.14  Aloe Barbadenesis Miller

Confirmation Test of Aloe-vera     

1. Borax Test

Prepare the Solution: Boil 1 gram of the aloe powder or dried juice in 100 mL of water. Let it cool, stir in 1 gram of kieselguhr, and filter it to remove impurities.

Mix and Heat: Take 5–10 mL of the clear filtrate in a test tube and add about 0.2 to 0.5 grams of pure borax.

Heat Gently: Warm the mixture gently until the borax is fully dissolved.

Observe Fluorescence: Pour a few drops of this heated solution into a test tube almost filled with water.

Result: A distinct green-colored fluorescence will appear, confirming the presence of aloe⁽¹²⁾

Fig No.15 Borax Test

2. Clove oil

Clove is an aromatic plant rich in volatile compounds and antioxidants such as eugenol, β-caryophyllene, and α-humulene. Clove essential oil has application in the perfume, cosmetic, health, medical, flavoring, and food industries. Clove essential oil has biological activity relevant to human health, including antimicrobial, antioxidant, and insecticidal activity. The impacts of the extraction method (hydrodistillation, steam distillation, ultrasound-assisted extraction, microwave-assisted extraction, cold pressing, and supercritical fluid extraction) on the concentration of the main volatile compounds in clove essential oil and organic clove extracts are shown. Eugenol is the major compound, accounting for at least 50%. The remaining 10–40% consists of eugenyl acetate, β-caryophyllene, and α-humulene. Furthermore, the main applications in clove essential oil in the pharmaceutical preparation . It has biological applications beneficial for human health, such as anti-inflammatory, analgesic, anesthetic, antinociceptive, and anticancer activity.

It has wide role as a preserving agent and acts as strong fragrances in pharmaceutical gel formulation.⁽⁷⁾

Fig.No 16  Clove Oil

3. Glycerine

Glycerin is widely regarded as the most effective humectant. Humectants are water-absorbing substances that help keep things moist. This property of glycerin helps retain moisture in the

skin It worka by hygroscopic molecule, glycerin acts like a microscopic water magnet. Because glycerin molecules are small, they penetrate the outer layers of the skin, where they bind to water and keep the skin cells hydrated.⁽⁷⁾

Fig.No.17 Glycerine

4. Guar Gum

Guar gum is a natural, water-soluble polysaccharide extracted from the seeds of the guar bean. Highly valued for its exceptional thickening, stabilizing, and binding properties, it is widely used in gluten-free baking, cosmetics, and various industrial applications.

Guar gum powder is known for its ability to create high viscosity at low concentrations, which makes it an economical and efficient option for many applications. It is also known for its ability to form strong hydrogen bonds with water molecules, which helps it to maintain stability in various formulations.⁽⁹⁾

Fig.No 18 Guar gum

5. EXPERIMENTAL WORK

ETHANOLIC EXTRACT BY USING SOXHLET EXTRACTION PROCESS:

When the desired compound has, limited solubility and the impurity is insoluble in that solvent then the soxhlet extractor is used.

A Soxhlet extraction has three main parts

1. A percolator
2. A Thimble
3. Siphon mechanism

Fig no.19  Soxhlet apparatus

• Step 1: Collection and Cleaning of Leaves

Fresh Ehretia laevis leaves were collected.Leaves were washed properly with distilled water to remove dust and impurities.Then leaves were cleaned using absolute alcohol for sterilization.After that, leaves were rinsed three times with distilled water.

• Step 2: Drying and Powdering

Clean leaves were dried properly in shade or hot air oven. After drying, leaves were crushed and converted into fine powder.

• Step 3: Weighing of Powder

About 50 grams of dried leaf powder was weighed accurately.

• Step 4: Preparation for Extraction

The 50 g powdered leaves were placed in a thimble of the Soxhlet extractor.250 mL methanol solvent was added into the distillation flask.

• Process Step 5: Soxhlet Extraction

The solvent was heated to boil and produce vapours.Vapours moved upward through the distillation arm into the condenser. The condenser cooled the vapours and converted them back into liquid solvent. The warm solvent dropped into the chamber containing leaf powder.

• Step 6: Dissolution of Active Compounds

The warm solvent dissolved the required phytochemicals from the leaf powder.

• Step 7: Siphoning Process

When the chamber became full, the liquid automatically siphoned back into the flask. This carried the dissolved extract into the flask.

• Step 8: Repetition of Cycle

The same process was repeated many times for several hours. This helped in complete extraction of active compounds.

• Step 9: Recovery of Extract

After completion, the methanol solvent containing extract was collected.Solvent was removed using a rotary evaporator.

• Step 10: Final Extract

Thick concentrated methanolic extract of Ehretia laevis leaves was obtained.The remaining insoluble material in the thimble was discarded.⁽³⁾

6. Phytochemical Screening Test

For performing these first we have to make acidic alcoholic extract. Preparation of acidified alcohol

1. Take 100 ml ethanol
2. Add 1-2ml conc.HCL

Now , we have to weigh 10g crude drug .Then add sufficient acidic alcoholic solvent about 50-100ml.Then shake and heat gently on water bath 30-60min.Then filter by using muslin cloth.

6. FORMULATION

1. 1. Material:

All reagents used were alovera, glycerin, sodium hydroxide (NaOH), Clove oil, Distilled water, ethanol 80%, ehretia laevis roxb leaves extract. Instrumentation: Hot air oven , blender, pH meter, Viscometer (Brookfield viscometer), water bath.

1. 2. Preparation of Gelling Agent

Required amount of alovera are dispersed in distilled water. This mixture is often kept aside to allow the polymer to swell completely ( For keep 1-2 hour).

1. 3. Preparation of Ethanolic Extract Solvent
1. Take 100 ml ethanol
2. Add 1-2ml conc. HCL

Now , we have to weigh 10g crude drug .Then add sufficient acidic alcoholic solvent about 50-100ml.Then shake and heat gently on water bath 30-60min.Then filter by using muslin cloth.⁽¹⁴⁾

1. 4. Encorporation of Ingredients

Other components like glycerin (humectant), clove oil (preservative, penetration enhancer) are dissolved and added to polymer base.

1. 5. Neutralizer (Ph adjustment)

To achieve the desired consistency and the pH (close to pH 7 skin friendly) a neutralizing agent such as sodium hydroxide (NaOH) are added. It also form clear gel.

1. 6. Blending and Homogenization

Plant extract is slowly mixed into gel base under constant gently stirring until a uniform and transparent gel is formed and also after this process add thickening agent such as guar gum⁽⁴⁾

Table No.2: Four Different quantity of batches of Ehretia Laevis Extract Gel Formulation

Formula = 𝑨𝑷𝑰 𝒊𝒏 % 𝒙 𝐭𝐨𝐭𝐚𝐥 𝐠𝐞𝐥 𝐢𝐧 𝐠𝐦

                                   𝟏𝟎𝟎

Fig. No 20 BEhretia Laevis Extract Gel

7. EVALUATION PARAMETER OF TOPICAL GEL

• Appearance and Homogenecity
• pH of Gel
• Viscosity
• Spreadability
• Extradurability Study
• Stability Study
• Grittiness
  1. Appearance and Homogenecity

Physical appearance and homogeneity were evaluated by visual inspection.All developed gels were tested for homogeneity by visual inspection after the gels have been set in the container.They were tested for their appearance and presence of any aggregates. The gel was smooth, good consistency. The gel appeared to be in light greenish colour.⁽⁶⁾

1. 2. pH of Gel

the reading at room temperature. The The pH was measured in each gel, using a pH meter, which was calibrated before each use with standard buffer solutions at pH 4, 7, 9. The electrode was inserted in to the sample 10 min priors to taking pH of the gel was close to skin pH near to 6 to 7. ⁽⁴⁾

Fig.No 21 Digital PH Meter

1. 3. Viscosity

The measurement of viscosity of the prepared gel was done with a Brookfield viscometer. The gels were ranging between 790 to 820 centipoise using spindle number 64.At each speed, the corresponding dial reading was noted. ⁽⁶⁾

Fig.No 22 Brookfield Viscometer

1. 4. Spreadability

It displays the extent of the area to which the gel quickly spreads upon application to the skin or affected area. The usefulness of a detail also depends on how well-known it is. Spreadability is expressed in terms of the number of seconds it takes two slides to separate from gel that is sandwiched between them while being subjected to a particular load.

It is calculated by using the formula:

S = M. L / T

where,

M = wt. tied to upper slide

L = length of glass slides

T = time taken to separate the slides.

The spreadability of the formulated Herbal gel was found to be 7.5cm.⁽⁶⁾

Fig.No 23 Spreadability Test

1. 5. Extradurability Study

To assess extradurability a sealed collapsible tube holding gel was pressed immovably at the folded end. At the point when the top was emptied, gel discharged till the weight dispersed. Weight in grams necessary to evacuate a 0.5 cm ribbon of the gel in 10 sec was resolved. The usual expulsion pressure in g was recorded.⁽⁶⁾

1. 6. Stability Study

The stability study of the gel is to be done as per ICH recommendations the gel was store at 30⁰ C± 2⁰ C/ 60% ± 5% RH and 40⁰ C± 2⁰ C/ 75% ± 5% RH. The formulation were examined in the change in physical appearance, pH, spread ability and Viscosity.⁽⁶⁾

1. 7. Grittiness

On the off chance that no apparent particulate matter was seen with a light magnifying lens, the four definitions were evaluated infinitesimally for the existence of particles. The gel arrangement so obviously meets the need of independence from particular matter and from coarseness as desired for any efficient preparation.⁽⁶⁾

Table no 3 : Spread-ability

Table no 4 : pH

Table no 5: Homogeneity

Table no 6 : Grittiness

8. RESULT AND DISCUSSION

Drug Identification by ultraviolet absorption spectroscopy.

A solution of 5 μg/ml concentration containing ehretia laevis roxb in pH 6.8 phosphate buffer and was scanned between 200 to 400nm for getting the absorbance at 278 nm λmax.

Calibration curve of Ehretia Laevis roxb:

Calibration curve of ehretia laevis roxb was carried out in Phosphate buffer of pH 6.8 and absorbance was taken by using UV spectrophotometer.

Preparation of Phosphate buffer of pH 6.8:

Take about 50ml of potassium dihydrogen phosphate in a 200ml volumetric flask and add 22.4ml of 0.2M Sodium hydroxide and made up to 200ml with distilled water. Check the pH of resulting solution and adjust to pH 6.8 by using 0.2M sodium hydroxide solution.

Calibration curve of Ehretia Laevis Roxb in pH 6.8:

50 mg of drug Ehretia Laevis Roxb was dissolved in pH 6.8 Phosphate buffer and volume was make up to 100ml to make stock solution of concentration 500μg/ml. Then 1 ml of stock solution was taken and diluted upto 100ml with the buffer of pH 6.8 and to get concentration of 5μg/ml and in similar way dilution were made as 5, 10, 15, 20 and 25 μg/ml respectively and absorbance measured at 278nm by UV visible spectrophotometer. The absorbance values were plotted against concentration (μg/ml) to obtain the standard calibration curve.

Calibration of Ehretia Laevis Roxb in pH 6.8 Phosphate buffer

Graph 1:  Calibration of Ehretia Laevis Roxb

Table no 7: Calibration of Ehretia Laevis Roxb

Cumulative % drug release of gel

The release rate of ehretia laevis roxb extract gel was determined using franz diffusion cell. The % Cumulative Drug  Release  was performed  using 49ml of pH 6.8 Phosphate buffer, at 37± 0.5 ℃ at 75 rpm. Aliquot volume of the solution was withdrawn from the dissolution apparatus predetermined intervals (20min) and the samples were replaced with fresh dissolution medium to avoid sink condition. Absorbances of these solutions were measured at 278nm. Cumulative % Drug release was calculated using an equation obtained from a standard curve. % Drug release mention in below table.

Table No 8: Cumulative % drug release of gel

Graph 2: %   Culumative Drug Release