INTRODUCTION:

When a plant's seeds, berries, roots, leaves, bark, or flowers are used for therapeutic purposes, the term "herbal medicine," also known as "botanical medicine" or "phytomedicine," is used. Long before recorded history, plants were being used as medicine. Native American and African indigenous societies used herbs in their healing rituals, while Ayurveda and ancient Chinese Medicine created ancient medical systems that employed herbal remedies on a regular basis. Antibacterial activity is the capacity of a substance to prevent or eliminate bacterial cells. Numerous antibiotics and chemotherapeutic medications are utilized in the treatment of various disorders.

Herbal remedies have a wide range of therapeutic applications, including the treatment of wounds, infection-related inflammations, skin lesions, leprosy, diarrhea, scabies, venereal illnesses, snake bites, and ulcers, among others. The plants could be harmed by numerous infectious agents, including viruses, fungi, and parasites. Novel compounds that could be used to make chemotherapy medicines are abundant in traditional medicine. The first step towards achieving this goal is screening of plants used in traditional medicine. New antibacterial and antifungal agents are sought for by antimicrobial research. While the majority of these antibiotics are made from microorganisms, the chemotherapeutic drugs are made from plants. However, these antibiotics and chemotherapeutic drugs are currently produced synthetically using a variety of methods (Reiner, 1984).

Medicine can be added to ointment or not. When making medicated ointments, a medication is either suspended, dissolved, or emulsified in the base. Ointments that are used topically include keratolytics, astringents, emollients, protectants, antiseptics, and antipruritics. The suspension, solution, or dispersion used in ointment bases almost invariably contains one or more medications.

Herbalists favour using whole plants as opposed to breaking them down into their constituent parts. Whole plant extracts contain a wide variety of various elements. Together, these components have therapeutic effects and lessen the possibility of adverse consequences from any one of them.

When two or more herbs are Used in the formulation they are known as poly herbal formulations. Numerous studies have Been conducted with the extracts of Neem leaves (Azadirachta indica Family-Meliaceae) and Extract of Mango Leaves with the combination of many other herbal drugs like Pomegranate Leaves, Orange Peel.

Plants profile:

1. Neem leaves.

Fig 1: Leaves of neem tree

Azadirachta indica, sometimes referred to as neem, nim tree, or Indian lilac, is a member of the Meliaceae family of mahogany trees. Neem tree products have been utilised in Indian traditional medicine for many years, but there isn't enough clinical proof to support their potential medical advantages. In India, neem- based products have been used therapeutically for more than 2,000 years. According to reports, they have sedative, contraceptive, antibacterial, antiviral, and antifungal properties. Products made from neem are also used to selectively eradicate plant pests. Neem is utilised in ayurvedic treatment. 'Village pharmacy' is another term for neem.

Neem is used to make a variety of medications, including those for skin conditions. Neem affects the growth and behave over 500 different species of insects, mites, ticks, and nematodes, rendering them poisonous. Pests are not immediately killed by neem; instead, it slows their growth and scares them away. Because they are affordable and kind to both animals and beneficial insects, neem products are effective for controlling pests.

2. Mango leaves (Mangifera Indica)

Fig 2: Leaves of mango tree A2 The

Flowering plant species Mangifera indica, or mango, belongs to the Anacardiaceae family. When it comes to mangoes, we never look past the fruit. Because we are so enamored with the fruit, we often miss the health advantages of mango leaves. The tip of a mango leaf is sharp, shiny, and meaty. Either the powdered leaves or the combination made by boiling the leaves in water can be consumed. The leaves contain a lot of antioxidants. In South East Asia, mango leaves are cooked and consumed, proving that they are useful not only as medicines but also as ingredients in food.

Vitamins C, B, and A are abundant in mango leaves. They are also abundant in flavonoids and phenol, which give the leaves their anti-oxidant qualities. Many of the potential benefits of mango leaves can be attributed to the anti-inflammatory properties of mangoferin.

3. Pomegranate leaves:

Fig 3: Leaves of Pomegranate tree

Pomegranate skin ageing and skin inflammation, such as eczema. Pomegranate has anti-inflammatory and health-improving properties. Antioxidants aid by reducing cellular damage in your body, including the vitamin C in pomegranates. The other notable components include tannins, ellagitannins, and anthocyanins. Reliable Source Topical applications might have some benefits, even though oral and dietary consumption is the way they work best. When antioxidants were applied to the skin of mice, they partially prevented wrinkles and age spots from showing up, but they did aid to diminish their appearance. Studies on humans are currently being done. Such outcomes are assumed to be the result of increased cell regeneration, or your skin's capacity to remove dead skin cells from the surface in order to create new ones. Pomegranates are believed to have natural antimicrobials, which may help fight bacteria and fungus on your skin. Vitamin CTrusted Source. These benefits might help treat P. acnes bacteria, which might be a trigger for acne outbreaks. Pomegranate, often known as "Anar," is a plant of the Lythraceae family with the scientific name Punica grantum.

4 Orange Peel:

Fig 4: Peels of Orange fruit

Fibre, vitamin C, folate, vitamin B6, calcium, and other minerals can all be found in abundance in orange peels. Orange peel contains a lot of polyphenols, which shield the body from various diseases. Peels have anti-cancer properties because of the limonene molecule, which is a substance that occurs naturally. Orange fruit rind is referred to as orange peel. The orange fruit has white pith under its slightly rough outer surface. Orange peel contains a lot of the phytochemical flavonoids. Hesperidium is the name for a fruit having a thick peel, such as a citrus fruit. The inner layer of hesperidia, often known as albedo or, to non-botanists, pith.

MATERIAL AND METHOD:

Collection of plant material:

Leaves of neem, pomegranate, mango is the collect in local area and orange fruit purchase in local market.

Preparation of all Extract:

Leaves of the plant Neem, Mango, pomegranate, and orange peel were collected and washed thoroughly with distilled water and shade dried for 5 days. Dried leaves were ground into powder form. 20gm of powder was imbibed with 80ml of 90% ethanol for 3hr and transferred to the closed Container then keep it for 2days finallyfilter it. The extract was stored in the airtight Container in a cool and dark place.

Sr. No.	Ingredients	Quantity
1	Wool Fat (Lanolin)	0.5 gm
2	Hard Paraffin	0.5 gm
3	Cetosteryl Alcohol	0.5 gm
4	Yellow Soft Paraffin	8.5 gm

Formulation:

Ointment Base:

Sr. No.	Ingredients	Quantity
1	Extract	0.5gm, 1gm, 1.5gm
2	Liquid paraffin	1gm
3	Ointment base	8 gm

Preparation of herbal ointment:

The ointment base was first made by weighing precisely grated hard paraffin and placing it in an evaporating dishon a water bath. Following the melting of the hard paraffin, the remaining ingredients were added and carefully mixed to promote melting and homogenous mixing by allowing the ointment base to cool.

The herbal ointment was prepared by mixing accurately weighed extract to the ointment base by levigation method to prepare a smooth paste with 2 Or 3 times its weight of the base, gradually incorporating more base untilto form Homogeneous ointment finally transferred in a suitable container.

Preparation of Agar medium and Petri plates

Principle:

· Nutrient Agar is nutrient broth solidified by the addition of 1 – 2% agar. In addition to liquid media, solidand semisolid media are widely used for cultivation of media bacteria.

· Solid bacteria are useful for isolating bacteria or for determining the characteristics of the colonies.

· Semisolid media are prepared with agar at concentration of 0.5 or less are useful for cultivation of microscopic bacteria and for detection of bacterial motility.

Procedure:

1. Add 2.8gm agar powder in 100ml distilled water into conical flask.

2. Heat it on water bath to form clear liquid.

3. Sterilize the media at 121 degrees Celsius, 151 p.s.i. Pressure for 15 min in autoclave.

4. Allow the flask to cool up to 50 degrees Celsius and pour the medium quickly into sterile Petri plates.

5. Flame the wire loop to sterilize it.

6. Take a small amount of bacterial inoculum of E. coli, staphylococcus aureus and subtillus and add it into Petri plates A, B, C respectively.

7. Cool all the Petri plates and allow the medium to solidify.

8. After the medium solidifies place the Petri plates in an incubator at 30–35degrees Celsius for 48 hrs.

Antimicrobial activity:

a. After incubation process by using flame sterilize the cork borer.

b. Prepare 4 to 5 cups in each plate, keeping adequate distance from each other.

c. Standard and test diluted antibiotics solution is added to each cavity of plate.

d. Transfer all the plate in the refrigerator for proper diffusion of antibiotics at 4 degrees Celsius for 1– 2 hours.

e. Incubate all the plate in the incubator at 32–35 degrees Celsius for 24 – 48hrs.

f. Check the zone of inhibition.

Test for alkaloids:

Mayer’s test:

In a test tube, take 1mL of herbal ointment extract and 2-3 drops of concentrated nitric acid solution; a dark whiteprecipitate indicates the presence of alkaloids.

Wagner’s test:

1mL of herbal ointment extract is put to a test tube with 2 to 3 drops of Nitric Acid solvent and sulphuric acid, areddish brown precipitate appears, indicating the presence of alkaloids.

Test for carbohydrates:

Molish test:

In a test tube, add 1mL of herbal ointment extract with 1 mL of α-naphthol solution and a few drops of strongsulphuric acid to produce a purple or reddish violet colour indicates a positive result.

Fehling’s test:

Take 1ml of Herbal ointment extract in the test tube and to this added the equal quantities of Fehling’s solution A and B, it gives a brick precipitate indicates the presence of carbohydrate.

Test for steroids and sterol Salkowski test:

Take 1ml of Herbal ointment extract and dissolved in the chloroform, the Same amount of concentratedsulphuric acid is added. Cherry red color is observed in the chloroform layer.

Test for glycosides:

Baljet test:

In a test tube, add 1mL of herbal ointment extract and add sodium picrate solution; a yellow to orange colourshows the presence of glycosides.

Keller - Kiliani test:

The Herbal ointment extract was dissolved in acetic acid containing a Trace of ferric chloride and transferred to the surface of concentrated sulphuric acid. Reddish Brown color is formed which gradually becomes blue indicating the presence of glycosides.

Test for saponins:

Foam test:

1mL herbal ointment extract + 1mL distilled water. The foam was seen when the sample was shaking.

Test for flavonoids:

Shinoda test:

In a test tube, put 1ml of the herbal ointment extract, ferric chloride, and strong hydrochloric acid. The presence of flavonoids is indicated by a red colour.

Test for tannins:

Ferric chloride test:

Take 1ml Herbal ointment extract, ferric chloride was added. Dark Blue or greenish black color indicates thepresence of tannins.

Potassium dichromate test:

In the Herbal ointment extract, potassium dichromate solution was added. A precipitate indicates the presence oftannins.

Test for proteins and amino acids:

Add 1ml of 40% sodium hydroxide and 2 drops of 1 % copper sulphate to the herbal ointment. A violet colorindicates the presence of proteins.

Evaluation of herbal ointment:

Organoleptic evaluation (color, odor) was done by sensory and visual inspection and compared to the marketedHerbal ointment. The evaluations were carried out on the Herbal Ointment by using the following parameters;

· Color and odor

· Color and odor of prepared ointment were checked by visual examination.

· pH

· A digital pH meter was used to determine the ointment’s pH. The pH of 1 g of ointment dissolved in

· 50ml ofdistilled water was determined.

· Stability study

· The stability study was done for the herbal ointment at the room temperature for 15 days

· Skin irritation test

· The skin irritation test was performed on human skin. The ointment was applied for 42 minutes on human skin and irritation effect was observed.

Phytochemical analysis of leaves

Sr. No	Sample	Solvent extraction Methanol
Sr. No	Sample	F1	F2	F3
	Phytochemical analysis test	Phytochemical analysis test result	Phytochemical analysis test	Phytochemical analysis test
1	Test for carbohydrate	+	+	+
2	Test for alkaloids	+	+	+
3	Test for steroid and sterol	+	+	+
4	Test for glycoside	+	+	+
5	Test for saponins	-	-	-
6	Test for flavonoids	-	-	-
7	Test for tannins	+	+	+
8	Test for proteins and amino acid	+	+	+

CONCLUSION:

The purpose of the study was to prepare antimicrobial herbal ointment using locally available plants. On the basis of antimicrobial efficacy, four different local plants were taken and their ethanolic extracts were incorporated in the most effective ratio in appropriate base. The final product readily spread on skin surface, showed no irritant effect, diffused well and was stable at different temperatures. Different extract of whole plant powder was subjected to phytochemical evaluation. The ethanolic extract showed the presence of various phyto constituents such as Steroids, Glucoside, tannins, alkaloids and carbohydrates. All the extract was tested for anti-microbial activity. Ethanol extract showed good activity even at low concentration and also shows maximum zone of inhibition.

REFERENCES:

1. Rajasree PH, Vishwanath V, Cherian M, Eldhose J. Singh R. Formulation and evaluation of antiseptic polyherbalointment. International Journal of Pharmacy and Life Sciences. 2021; 3(10): 2021-31.

2. Pandey A Jagtap JV, Patil AA, Joshi RN, Kuchekar BS. Formulation and evaluation of antibacterial and antifungal activity of herbal ointment containing Aloe vera, Azadirachtaindica, and Curcuma longa. Journal of Chemical and Pharmaceutical Research. 2010:2(3):182-86.

3. Yamini K. Onesimus T. Preparation and Evaluation of Herbal Antiacne Gel. International Journal of Pharma and Biosciences. 2013; 4(2).

4. Kokate C. K., Gokhale S.B., Purohit A.P. A Textbook of Pharmacognosy, Nirali Prakashan 34" Edi. Sept 2013; 9: 117.

5. Valarmathy, K., Krishna, G., Kausar, A., Salma, M., and Kusum, D. P. A study of antimicrobial activity of ethanolic extracts of various plant leaves against selected microbial species. International Journal of Pharma Sciences and Research. 2010; 1: 293–295.

6. Toghyani, M., Toghyani, M., Gheisari, A., Ghalamkari, G., and Eghbalsaied, S. Evaluation of cinnamon and garlic as antibiotic growth promoter substitutions on performance, immune responses, serum biochemical and haematological parameters in broiler chicks. Livestock Science. 2011; 138: 167–173. 10.1016/j.livsci.2010.12.018

7. Yadav, S., and Jha, R. Strategies to modulate the intestinal microbiota and their effects on nutrient utilization, performance, and health of poultry. Journal of Animal Science and Biotechnology. 2019; 10: 2. 10.1186/s40104-018-0310-9.

8. Hannan A, Asghar S, Naeem T, Ikram Ullah M, Ahmed I, Aneela S, Hussain S. Antibacterial effect of mango (Mangifera indica Linn.) leaf extract against antibiotic sensitive and multi-drug resistant Salmonella typhi. Pak J Pharm Sci. 2013 Jul; 26(4): 715-9. PMID: 23811447.

9. Mirza B, Croley CR, Ahmad M, Pumarol J, Das N, Sethi G, Bishayee A. Mango (Mangifera indica L.): a magnificent plant with cancer preventive and anticancer therapeutic potential. Crit Rev Food Sci Nutr. 2021; 61(13): 2125-2151. doi: 10.1080/10408398.2020.1771678. Epub 2020 Jun 8. PMID: 32506936.

10. Medina D, Ulloa G, Camere R, et al. Antibacterial activity of Bixa orellana L. (achiote) against Streptococcus mutans and Streptococcus sanguinis. Asian Pac J Trop Biomed. 2016; 6(5):400–403. DOI: 10.1016/j.apjtb.2016.03.005.

11. Camere R, Ulloa G, Medina D, et al. Antibacterial activity of Myrciaria dubia (Camu camu) against Streptococcus mutans and Streptococcus sanguinis. Asian Pac J Trop Biomed. 2016; 6(9): 740–744. DOI: 10.1016/j.apjtb.2016.07.008.

12. Mayta-Tovalino F, Gamboa E, Sanchez R, et al. Development and formulation of the experimental dentifrice based on Passiflora mollisima (Tumbo) with and without anion Fluor: antibacterial activity on seven antimicrobial strains. Int J Dent. 2019; 2019: 9056590. DOI: 10.1155/2019/9056590.

13. Ameen F, Srinivasan P, Selvankumar T, et al. Phytosynthesis of silver nanoparticles using Mangifera indica flower extract as bioreductant and their broad-spectrum antibacterial activity. Bioorg Chem. 2019; 88: 102970. DOI: 10.1016/j.bioorg.2019.102970.

14. Miguel M.G., Neves N.A., Antunes M.D. Pomegranate (Punica granatum L.): A medicinal plant with myriad biological properties—A short review. J. Med. Plants Res. 2010;4:2836–2847.

15. Orak H.H., Demirci A.S., Gümüş T. Antibacterial and antifungal activity of pomegranate (Punica granatum L.cv.) peel. Electron. J. Environ. Agric. Food Chem. 2011; 10: 1958–1969.

16. Hotwani K, Baliga S, Sharma K. Phytodentistry: use of medicinal plants. J Complement Integr Med. 2014; 11: 233–51.

17. Akdemir Evrendilek G. Empirical prediction and validation of antibacterial inhibitory effects of various plant essential oils on common pathogenic bacteria. Int J Food Microbiol. 2015; 202: 35–41.

Received on 10.12.2023 Modified on 09.03.2024

Research J. Topical and Cosmetic Sci. 2024; 15(1):33-37.

DOI: 10.52711/2321-5844.2024.00006

Shraddha Parjane, Someshwar Mankar