Formulation and Evaluation Anal Inflammation (Hemorrhoids) Herbal Cream of Euphorbia Milli Leaves

HAEMORRHOIDS

Haemorrhoids is a common perianal disease, which often causes haematochezia. Besides a surgical operation or minimally invasive treatment. Haemorrhoids are swelling and inflammation of veins in the rectum and anus. The anatomical term "haemorrhoids" technically refers to "cushions of tissue filled with blood vessels at the junction of the rectum and the anus”. However, the term is popularly used to refer to varicosity of the haemorrhoid tissue. Perianal hematoma are sometimes mis diagnose and mis labelled as haemorrhoids, when in fact they have different causes and treatments. Haemorrhoids may result from straining to move stool. Other contributing factors include pregnancy, aging, chronic constipation or diarrhoea, and anal intercourse[3]. Haemorrhoids are either inside the anus internal or under the skin around the anus external. Haemorrhoids develop from 2 different places. Haemorrhoids also called piles, are swollen veins in your anus and lower rectum, similar to Herbal medicines are one type of dietary Supplement. They are sold as tablets, capsules, varicose veins. Haemorrhoids can develop inside the rectum (internal Haemorrhoids) or under the skin around the anus (external Haemorrhoids) Depending on anal origin within anal canal and relation to dentate line Haemorrhoids divided in to internal haemorrhoids, external haemorrhoids, mixed haemorrhoid^[1,2].

Fig.no.1: Haemorrhoids

 Haemorrhoids are swollen and inflamed veins located in the rectal and anal regions, often leading to discomfort and bleeding. They typically develop due to increased pressure in these veins, which can result from straining during bowel movements, obesity, or pregnancy. External haemorrhoids appear as painful or tender lumps beneath the skin around the anus. These are essentially enlarged blood vessels that become irritated, especially during difficult or strained bowel movements. Common factors contributing to haemorrhoids include: Straining during bowel movements, often due to chronic constipation or diarrhoea. Weakening of supportive tissues with age, which reduces the structural integrity of the anal and rectal veins. Pregnancy, as the growing uterus increases pressure on the pelvic veins. Obesity, which adds extra pressure to the lower body and veins. Frequent heavy lifting, which can increase abdominal pressure. Low-fiber diets, which contribute to constipation, increasing the risk of haemorrhoids^[4].

CLASSIFICATION

Depending on anal origin within anal canal and relation to dentate line haemorrhoids divided in to

1. Internal haemorrhoids.
2. External haemorrhoids.
3. Mixed haemorrhoids.

TYPES OF HEMORRHOIDS

1. External Haemorrhoids- These are under the skin around your anus. Sign & symptoms might include. Itching or irritation in your anal region. Pain or discomfort Swelling around your anus Bleeding

2. Internal Haemorrhoids- Internal haemorrhoids lie inside the rectum. You usually Can’t see or feel them, they rarely cause discomfort. Painless bleeding during bowel movements A haemorrhoid to push through the anal opening resulting in pain & irritation. If blood pools in an external haemorrhoids & forms a clot (thrombus) it can result in –

• Severe pain
• Swelling
• Inflammation
• A hard lump near your anus

CAUSES- • The vein around your anus tend to stretch under pressure of may bulge or swell. Haemorrhoids can develop from increased pressure in the lower rectum due to Straining during bowel movement • Sitting for long periods of time on the toilet. For Internal haemorrhoids

• Grade I - bleeding without prolapse
• Grade II - prolapse with spontaneous reduction
• Grade III - prolapse with manual reduction.
• Grade IV-incarcerated, irreducible prolapse^[1]

Fig.no 2: Internal Haemorrhoids grading system

CREAM:

The creams are considered an important part of the cosmetic product as a topical preparation since ancient times due to their relief on the skin and its removal. From cosmetic purposes, Medicinal creams have a variety of applications such as cleansing, beautifying, changing appearance, moisturizing etc. It also plays a vital role in treating skin conditions. These include protecting the skin from bacterial and fungal infections, and promoting the healing of minor cuts, burns, and sores. These fragile arrangements are safe for public and private use. Human skin is vulnerable to damage but has the potential to heal itself. However, a natural healing process can take time and there is also a risk of infection especially in the early stages of injury. In such cases, medicinal creams can be applied to the injured area to speed up the healing process and prevent the wound from becoming infected. This review emphasizes the use of drug delivery systems in topical formulations, specifically focusing on wound healing creams. It includes an overview of the wound healing process, formulation techniques for creams, their classification according to therapeutic action, benefits and limitations, key features, types of creams, and the various parameters used to evaluate their effectiveness^[5,6].

TYPES OF CREAM

They are divided into two types:

Oil-in-Water (O/W) creams are emulsions where small oil droplets are evenly distributed within a continuous water phase. In this type of formulation, oil acts as the internal phase while water serves as the external medium, making it an oil-based emulsion suitable for non-greasy and easily washable applications.

In contrast, Water-in-Oil (W/O) creams consist of water droplets dispersed within a continuous oil phase. Here, water forms the internal phase and oil acts as the external medium, resulting in a water-based emulsion. These formulations tend to be more occlusive and are ideal for dry or dehydrated skin due to their ability to retain moisture^[5,6,7].

METHODOLOGY

6.1 PLANT PROFILE

EUPHORBIA MILII –

Euphorbia milii, the crown-of-thorns, Christ plant or Christ's thorn, is a species of flowering plant in the spurge family, Euphorbiaceae, native to Madagascar. The specifies commemorates Baron Milius, once Governor of Réunion, who introduced the species to France in 1821 ^[9,10].

Fig.no 3: Euphorbia milii plant

• Botanical Name : Euphorbia milii Des Moul.
• Synonyms: Euphorbia splendens subsp. Bojeri (Hook.) Denis
• Common Name : Crown of Thorns,                          
• English: Christ-plant,Christ-thorn                                     
• Spanish: corona de Cristo, espina santa, martirio              
• French : épine du Christ                                                   
• Chinese: tie hai tang
• Scientific name: Euphorbia milii
• Family: Euphorbiaceae
• Plant Form : Shrub
• Genus: Euphorbia
• Kingdom: Plantae
• Order: Malpighiales                                                                

About Euphorbia milii Plant:

• Habit: A small, armed shrub with stem fleshy, with long spines and bluish-white colour and milky latex.
• Leaves: Alternate, oblong, acute or obovate, mucronate, spines on the branches in pairs, in between each pair of spines, is a reduced branch which bears pairs of small spines. In between each pair of spines a leaf is developed, therefore, the spines are modified stipules.
• Inflorescence: Cyathium
• Flowers:

• Cyathia grouped dichotomously, stalked from the axils of upper leaves.
• The peduncle is divided into two successively three times and the cyathia are seated on the ultimate pedicles.
• Each cyathium is supported by two, petaloid, crimson or scarlet, orbicular mucronate bracts which have fused to the involucre in their lower part.
• The involucre bears at the top margin, 5 broad, rounded glands, alternate, with the glands is a reddish scale or bracteole. Inside the involucre, there are many male flowers, consisting of a stalked stamen with two-anther lobes.
• Ovary 3-locur, 3-lobed, with one ovule in each cell, styles 3, connate for half their length, each divided into a pair, short stigmas.

• Flowering and Fruiting Time: Throughout the year.
• Significance: It is very popular in all gardens.

Leaves:

Euphorbia milii, commonly known as Crown of Thorns, has fleshy, bright green, obovate leaves that are typically found on new stem growth, and are known to drop off as the stems mature, leaving a spiny appearance ^[8,9,10].

Morphology Features:

The "songosongo" plant, also known as Euphorbia Milii or crown of thorns, is indigenous to Madagascar. It is a woody succulent prickly shrub that reaches a height of 1.5 to 2 meters (5-7 feet). The spines become straight and narrow when they reach a length of 3 cm. The oblong, acute-shaped, fleshy green leaves can reach lengths of 3-3.5 cm and widths of 1.5 cm. These tiny, cup-shaped flowers, which range in hue from red to pink to white, are hung in two petal-like brats and measure up to 12 mm. Since euphorbia thrives in warmer temperatures, it produces flowers throughout the year but especially in the spring and summer^[10,11,12].

Chemical Constituents:

Euphorbia milii contains a variety of phytochemicals, including terpenoids, flavonoids, alkaloids, phenolic compounds, and tannins. These compounds are believed to contribute to the plant's various medicinal properties. Specific examples of phytochemicals found in E. milii include beta-amyrin acetate, beta-sitosterol, cycloartenol, lupeol, euphol, and flavonoids. The medicinal values of the plants depend on the presence of certain chemical substances (secondary metabolites) that are involved in production of different kinds of effects on human body^[13,14,15].

Pharmacological Activity:

Fig.no 4: Pharmacological activity

6.2 DETERMINATION OF TOTAL ASH:

PROCEDURE

1. 1. 1. About 2 g of the powdered drug was weighed accurately in a tared silica crucible.
      2. Crucible with powdered drug was incinerated in muffle furnace by gradually increasing heat (temperature) at 500-800°c until freed from carbon.
      3. After complete incineration, furnace was off and crucible was remained in it till temperature fall down.
      4. The crucible was removed from the furnace and cooled after the incineration about 1 hour in a desiccator.
      5. The ash was weighed and percentage of total ash with reference to the air dried sample was calculated.

The total ash value for given sample Euphorbia milli was found to be 12 % W/W.

Formula:

Total ash = (Weight of ash/Weight of sample) x100

Fig.no 5: Determination of total ash

6.3 DETERMINATION OF ACID-INSOLUBLE ASH VALUE:

Procedure:

1. Using 25 ml of dilute HCl, wash the ash from the dish used for total ash into a 100 ml beaker.
2. Place a mere guaze over a Bunsen burner and boil for five minutes.
3. Filter through an 'ashless' filter paper, wash the residue twice with hot water.
4. Ignite a crucible in the flame, cool and weigh.
5. Put the filter paper and residue together into the crucible heat gently until vapours cease to be evolved and then more strongly until all carbon has been removed.
6. Cool in a desiccator.
7. Weigh the residue and calculate acid-insoluble ash of the crude drug with refer to the air-dried sample of the crude drug.

Formula:

Acid -insoluble ash value = (Weight of acid -insoluble ash/Weight of sample) x 100

Fig no.6: Acid insoluble ash value

6.4 DETERMINATION OF WATER-SOLUBLE ASH:

Procedure:

1. Using 25 ml of water, wash the ash from the dish used for total ash into a 100 ml beaker.
2. Place a mere guaze over a Bunsen burner and boil for five minutes.
3. 3.Filter through an 'ashless' filter paper, wash the residue twice with hot water.
4. 4 Ignite a crucible in the flame, cool and weigh.
5. Put the filter paper and residue together into the crucible heat gently until vapours cease to be evolved and then more strongly until all carbon has been removed.
6. Cool in a desiccator.
7. Weigh the residue and calculate water-insoluble ash of the crude drug with refer to the air-dried sample of the crude drug.

Formula:

Water-insoluble ash value = (Weight of water-insoluble ash/Weight of sample) x100

Fig.no:7: Water -insoluble ash

6.5 DETREMINATION OF LOSS ON DRYING (LOD)

Procedure:

1. Weigh accurately 2g of the powdered drug into a weighed flat and thin porcelain dish.
2. Dry in the oven at 100°C or 105°C, until two consecutive weight do not differ by more than 0.5 mg.
3. Cool in a desiccator and weigh. The loss in weight is usually recorded as moisture.

Formula:

Loss on Drying = (Initial weight -Final weight after drying/Initial weight) x100 ^{[16,17,18,19]}.

Fig no.8: Loss on Drying

6.6 METHOD OF EXTRACTION:

? Infusion:

• It consist of pouring water over the drugs and
• Then allowing it to keep in contact with water for the stated period, usually 30 minutes, with occasional stirring and Processes used for extraction
• finally filtering off the liquid.

Fig.no. 9: Infusion

Maceration:

In this extraction method, coarsely powdered plant material—such as leaves, stem bark, or root bark—is placed in a container. A suitable solvent (menstruum) is then added until the material is fully submerged. The container is sealed and allowed to stand for a minimum of three days. During this period, the mixture is stirred or, if kept in a bottle, shaken occasionally to facilitate thorough extraction. After the extraction period, the liquid portion (micelle) is separated from the solid residue (marc) using filtration or decantation. The micelle is then subjected to evaporation, either in an oven or over a water bath, to remove the solvent. This method is especially appropriate for extracting compounds from thermolabile (heat-sensitive) plant materials.

Fig.no10: Maceration

Decoction:

This method involves continuous hot extraction using a measured amount of water as the solvent. The plant material, which is first dried, ground, and finely powdered, is placed in a clean container. Water is added and the mixture is stirred thoroughly. Heat is applied throughout the procedure to accelerate the extraction of active constituents. The entire process typically takes around 15 minutes. This technique is ideal for extracting compounds that are both water-soluble and stable under heat.

Fig no.11: Decoction

Percolation:

\This extraction method uses an apparatus known as a percolator, which is a narrow, cone-shaped glass vessel with openings at both ends. The plant material, which is dried, ground, and finely powdered, is first moistened with a suitable solvent in a clean container. After moistening, an additional amount of solvent is added, and the mixture is allowed to stand for about 4 hours. Following this, the material is transferred into the percolator with the lower opening closed and left undisturbed for 24 hours to allow saturation. Then, more solvent is poured from the top until the plant material is fully saturated. The bottom outlet is then opened, and the extract is allowed to drip slowly. During the process, more solvent is added gradually, enabling gravity to carry it through the plant material, facilitating extraction. The solvent addition is stopped once approximately 75% of the total intended volume has been used. The extract is collected, and any remaining liquid is separated through filtration and decantation. The residual plant material (marc) is pressed to recover remaining extract, and finally, enough solvent is added to make up the required volume of the preparation. ^[20,21].

Fig no.12: Percolator

SOXHLET EXTRATION:

Soxhlet extractor was invented in 1879 by Franz von Soxhlet, a German chemist. The original use of this apparatus was the extraction of a lipid from a solid material. It is an apparatus used in the laboratory for extracting a compound with a limited solubility in a solvent and where the impurity is insoluble in that solvent.  This is one of the conventional solid/liquid extraction method which consists in extracting directly from the raw material using a solvent and an extractor.

Chemical:

The chemical solvents and other products that have been used in the experiments and methods are the following:

• • Distilled water
  • Whatman filter
  • Ethanol (C₂H₆O) was supplied by Molar Chemicals Kft, purity: 99.5%.
  • Diethyl Ether (C₂H₅) was supplied by molar chemical Kft, purity: 98%%.

Method of extraction:

For this operation, an approximate amount of 50 g was measured into the paper thimble placed inside the chamber. Also 240 ml of solvent was introduced in the solvent flask. three solvents with different polarities were applied: ether, chloroform and 70% ethanol. The Soxhlet apparatus was connected to a cooler and the round bottom flask containing the solvent was immersed in silicon oil. First the cooling water of condenser was turned on, then the heating element of silicon bath was switched on for the solvent is heated, and the extraction can start. The extraction run until the solvent in the Soxhlet apparatus became transparent again, which showed the end of extraction. It took around 3 days (18 hours more or less) in the case of raw material with diethyl ether, and one day extract with chloroform and 4 days (24 hours) for raw material with 96% ethanol extraction. After the Soxhlet extraction was done, the evaporation of the liquid is necessary in order to get the extract without solvent. For this, each flask had to be evaporated in the rotary evaporator using a vacuum pump and a control valve. After this step only the extract obtained as a product remains in the flask, which is collected in a sample bottle. The extracted material was dried and discarded. The solvent evaporated and condensed is collected in another flask and returned to a bottle for reuse^[22].

Fig no.13: Soxhlet Extraction

Estimation of total tannin content:

In this method, 100 mg of extract was accurately weighed and was dissolved in 100 ml of methanol to make the stock solution. This was further diluted 5 times and 1 ml of this resultant solution was transferred to a test tube. To this 7 ml. of methanol was added followed by addition of 0.5 mL. of Folin-Ciocalteau reagent and 1.5 mL. of 20% Na?CO? solution. The final volume was made up to 10 mL with methanol. Tannic acid was used as a reference standard for this estimation. The final solution was shaken vigorously to achieve a proper distribution and was kept for 15 min in dark. Further, absorbance was recorded at 775 nm against blank using UV spectrometer and the total tannin contents was expressed in terms of mg/g tannic acid equivalent^[23].

Graph Calculation

Fig no.14: Total tannic content

6.11 Thin Layer Chromatography:

Steps to Prepare TLC Plate:

1. Prepare the slurry:

• Mix 1 part silica gel G with 2–3 parts distilled water in a beaker.
• Stir quickly to get a uniform slurry.

2. Coat the glass plate:

• Pour the slurry onto one end of the glass plate.
• Spread it evenly using a spreader or TLC applicator.
• Aim for a uniform thickness of ~0.25 mm.

3. Air dry:

Let the coated plate stand vertically for about 10–15 minutes to remove surface moisture.

4. Activate the plate:

Place the plate in an oven at 110–120°C for 30 minutes to remove all moisture and activate the adsorbent.

5. Cool and store:

Allow the plates to cool

6. Sample Preparation:

Dissolve tannic acid in a suitable solvent like methanol or ethanol to make a dilute solution (~1–2 mg/mL).

7. Spotting the Plate:

• Using a capillary tube, apply a small spot of the tannic acid solution about 1 cm from the bottom edge of the TLC plate.
• Allow the spot to dry completely. You may repeat the spotting 2–3 times to concentrate it.

8. Prepare the Mobile Phase:

• Mix solvents: n-Butanol: Acetic acid: Water (4:1:5).
• Pour into a TLC chamber (jar or beaker with lid).
• Place a piece of filter paper in the chamber to saturate the atmosphere (improves separation).

9. Develop the Plate:

• Place the TLC plate vertically in the chamber. The solvent level must be below the sample spot.
• Close the chamber and allow the solvent to rise until it’s about 3/4th of the plate height.
• Remove the plate and mark the solvent front with a pencil immediately.

10. Dry the Plate:

Air-dry or gently warm to remove solvent vapors.

11. Visualization:

• Spray with FeCl? solution (1% ferric chloride in ethanol or water).
• Heat gently if required. Tannic acid spots appear blue-black or greenish.

12. Calcuation of Rf value:

Rf = Distance travelled by the solute/Distance travelled by the solvent front^[24,25].

Fig no.15: TLC plate

6.12 PREPARATION OF CREAM

PROCEDURE:

1. All ingredients were weighed accurately.
2. Accurately weigh beeswax and liquid paraffin and heat them together in a beaker at 70–75°C until completely melted. This mixture forms the oil phase (cream base).
3. In a separate beaker, dissolve borax, methyl paraben, and aluminium sulphate in distilled water and heat to the same temperature as the oil phase (around 70–75°C). This is the aqueous phase and acts as the preservative vehicle.
4. Gradually add the hot aqueous phase to the oil phase with continuous stirring to form a uniform cream base. Continue stirring until a homogenous emulsion forms.
5. Allow the emulsion to cool down to below 40°C while stirring slowly.
6. Add mentha , Euphorbia milli extract, and prednisolone while stirring gently to ensure even distribution.
7. Add starch to act as a thickener and stabilizer.
8. Incorporate tartrazine (colouring agent) and vanilla (flavouring agent) in quantities sufficient for desired appearance and fragrance.
9. Mix thoroughly until the cream is smooth and homogenous.
10. Transfer the prepared cream into clean, dry containers.
11. Label properly and store in a cool, dry place ^[27,28].

Fig.no. 16: Formulated Cream

FORMULATION COMPONENT:

Table no. 1: Formulation of cream

6.13 Evaluation of cream –

1) Evaluation of cream pH of the cream:

Using normal buffer solutions, the pH meter was performed calibration. Approximately 0.5 g of the cream was balanced and dissolved in 50.0 ml of distilled water, calculating its pH. In a 100ml beaker, 5 ± 0.01g of the cream was correctly measured. 45ml of water was applied and the cream was spread therein. Using the pH meter, the pH of the suspension was estimated at 27°C. The cream pH was found to be in the range of 4.5 to 6.5 which is good for the PH of the skin. All the formulations showed pH closer to the appropriate skin.

2) Organoleptic evaluation:

The cream thus acquired was evaluated for its organoleptic properties, such as color, odor, and condition. The appearance of the cream was measured and graded by its roughness and Colour.

3) Homogeneity:

The formulations were tested for the homogeneity by visual appearance and by touch.

4) Spreadability studies:

Spreadability is expressed in terms of your time in seconds taken by two slides to slide far- away from the formulation, placed between, under the appliance of a particular load. Two glass slides of ordinary dimensions were selected. The formulation whose spreadability had to be determined was placed over one of the slides and the other slide was placed on top of the formulations was crammed between the two slides across the length of 5 cm alongside the slide. 100 g weight was placed upon the upper slide so as that the formulation between the two slides was pressed uniformly to form a thin layer. The load was removed and thus the quite formulation adhering to the slides was scrapped off and one of the slides was fixed on which the formulation was placed. The second movable slide was placed over it, with one end tied to a string to which load could be applied with the help of a simple pulley and a pan. A 30g weight was placed on the pan and thus the time taken for the upper slide to travel the space of 5.0cm and separate distant from the lower slide under the direction of the load was noted.

The Spreadability (S) are often determined using the formula.

Spreadability = m x l /t

m = weight tied to the upper slide (25g)

1=length of glass slide (5cm)

T = time taken in seconds. ^[29,30]

6) Antimicrobial test:

Protocol- The Nutrient agar media was used. Staphylococcus aureus microorganism culture was used. Incubation time was setup for 24hrs.

Method-Agar bore well diffusion method.

Procedure- Staphylococcus aureus (Gram +ve bacteria) suspension was introduced in each plates & 40ml of sterile nutrient agar media was poured into each sterilized plates. The plates were left on the flat solid surface & allow to harden. In each plate 1cup, 10mm in diameter was bored in the medium with cork borer. The disks of agar were removed by sterilized dissecting needle while being careful not to damage the cups. In each plate equal amount of cream formulation having same strength was placed in the cup & the plates were incubated at37°C± 2°C for 24hrs.in incubator. The entire operation was carried out under aseptic condition & zone of inhibition was calculated. The zone of inhibition obtained for prepared formulation was showed in figure no.17^[31,32].