Formulation and Evaluation of Herbal Ointment of Maytenus Emarginata for Burning Skin

The research aimed to evaluate the phytochemical composition of Maytenus emarginata leaves and stems, focusing on identifying bioactive compounds such as phenols and flavonoids. These compounds are known for their antioxidant and anti-inflammatory properties, which can be beneficial for wound healing. Antioxidants help neutralize free radicals, reducing oxidative stress that can impede the healing process. Anti-inflammatory agents can reduce inflammation, a common issue in burn injuries. Burn wounds are susceptible to infections by various bacterial pathogens, which can complicate the healing process. The study assessed the antibacterial activity of Maytenus emarginata extracts against several bacterial strains. The results indicated that the leaf extract better antibacterial activity, suggesting a potential role in preventing or treating infections in burn wounds. The study looks on the antibacterial qualities and phytochemical makeup of the leaves and stem of Maytenus emarginata. There are still a number of research gaps, though. First off, although the study finds phytochemicals, it doesn't separate or describe the precise bioactive substances that have antibacterial properties. Furthermore, the mode of action against bacterial strains is still unknown. Furthermore, toxicity and safety assessments—which are essential for therapeutic applications—are absent from the study. Additionally, it only concentrates on in vitro antibacterial activity, hence in vivo research is required to verify effectiveness in biological systems Furthermore, a comparative analysis with standard antibiotics is missing, which could help determine its potential as an alternative antimicrobial agent. Lastly, the study does not explore drug formulation possibilities, limiting its practical application. Addressing these gaps through advanced biochemical, pharmacological, and clinical studies would enhance the medicinal value of Maytenus emarginata for antibacterial treatments. This study help to develop the various topical formulations based on Maytenus emarginata based gels, ointments or creams for topical application on burn. Further research is needed to validate the effectiveness and safety of Maytenus extracts on burn wounds through in vivo and clinical trials.

Skin Burn

The skin, being the largest organ in the body, plays essential roles such as maintaining the body’s shape, absorbing substances, safeguarding internal organs, retaining fluids, regulating temperature, assisting with sensory functions, and defending against diseases. Burns are one of the most frequent types of skin injuries across all age groups, making skin conditions a significant global health issue. A burn occurs when thermal, electrical, chemical, or ionizing radiation causes partial or complete damage to the skin or its underlying tissues [1]. Figure 1a illustrates the structure of human skin. The thickness of the skin’s layers can vary depending on the body region. On the forearm, the outermost layer, known as the stratum corneum, is roughly 0.05 mm thick, while the epidermis measures about 0.1 mm. Beneath the epidermis lies the dermis, which is approximately 0.5 to 0.7 mm thick. The thickness of the subcutaneous fat layer can differ significantly within the same person, depending on their nutritional condition. The skin also contains sweat ducts and hair shafts, which sometimes extend into the subcutaneous fat. These structures are lined with epidermal cells. Epithelial cells from surrounding healthy tissues, including those found in sweat ducts and hair shafts, migrate to the wounded area to aid in healing. The skin’s blood supply has two main layers: one located just below the epidermis and another deeper plexus near the boundary between the dermis and subcutaneous fat. Both layers play a crucial role in providing nutrients and regulating body temperature [2]. According to the severity of the injury, which is based on the look of the skin rather than the depth of the burn, burns are frequently classified as First-degree (superficial), second-degree (partial thickness), and third-degree (full thickness) burns are the three types of burn wounds that are distinguished by their depth. Burn wounds have a complicated, multi-stage healing process. It starts with inflammation brought on by damaged blood vessels and blood component leaking. Within hours of the damage, re-epithelialization begins, and granulation tissue is formed, mostly by fibroblasts and macrophages, which aid in the restoration of the extracellular matrix. Endothelial cell migration and division are accompanied by neovascularization, which is essential for the extracellular matrix in the wound bed. Cells, the extracellular matrix, and cytokines interact to promote wound contraction [3]. First-degree burns are regarded as minor and often only damage the epidermis, the outermost layer of skin.  They often heal quickly without medical intervention, despite the fact that they can be extremely painful.  Redness and flushing of the skin are characteristics of these burns [4]. There are two forms of second-degree burns: deep partial-thickness burns and superficial partial-thickness burns. The epidermis and a portion of the dermis are affected by superficial partial-thickness burns. They are frequently distinguished by the existence of blisters that are filled with transparent liquid. Although these burns usually hurt and need to be dressed and cared for, they usually go away without leaving any scars. Conversely, deep partial-thickness burns penetrate the dermis as well as the epidermis. Because they disrupt pain receptors, they may be less painful even though they are deeper. Scarring is more likely to occur from these burns, and surgery may occasionally be necessary. Full-thickness burns, another name for third-degree burns, pierce the skin’s outermost layer. Because nerve endings are destroyed, these burns frequently don’t cause any pain. Additionally, they harm the underlying subcutaneous tissue and blood arteries. Usually, a skin transplant, antibiotics, and the surgical excision of dead tissue are all part of the comprehensive course of treatment. These burns can affect deeper structures as well as the skin and subcutaneous tissue [5].

Figure No. 1] Illustrates the structure of human skin.

Ointment

Ointments are semi-solid compositions that, under shear stress, typically exhibit viscoelastic characteristics. Usually containing active therapeutic substances, their main purpose is topical treatment on the skin or mucous membranes. Commonly known as ointment bases, nonmedicated ointments are used to offer moisturizing or lubricating properties. These base materials can also be used to make medicinal ointments. When referring to emollient preparations with distinct properties, prescription language frequently uses phrases like creams, pastes, and cerates [6]. Ointments, creams, salves, or pastes are examples of semisolid preparations that are frequently used to apply medications topically to both undamaged and damaged skin as well as mucous membranes.  The main purpose of these compositions is to moisturize or protect the skin.  Although this function was fulfilled by traditional ointments, contemporary ointments also make it easier for drugs to enter the bloodstream [7]. Ointments and other semisolid dosage forms are frequently administered topically to the skin, eyes, nasal passages, vagina, or rectum for therapeutic, preventative, or aesthetic reasons. By enabling the medicine to permeate the surface, these formulations produce localized effects on the skin or mucous membranes at the application site. In order to treat skin disorders, ointments are made to provide drugs straight to the skin, which serves as the target organ. Usually, ointments are emulsions created by mixing water and oil, creating either water-in-oil (W/O) or oil-in-water (O/W) varieties. Small water droplets are scattered throughout a continuous oily phase in W/O emulsions, whereas tiny oil droplets are scattered in a continuous water phase in O/W emulsions.In general, ointments with both water and oil are simpler to remove with water, less greasy, and better suited for cosmetic applications[8].

Classification of ointment

1. Epidermic ointment: They act as a protective antiseptic and paraciticides.
2. Endodermic ointment: They act as a emollients, stimulants and local irritants.
3. Diadermic ointment: This ointment are intended to release the medications that has pass to the skin and produce systemic effect.

Advantages of ointment

1. They provide means of site-specific application of drug on affected area, which avoids unnecessary non-target exposure of drug thereby avoiding side effects.
2. They avoid first pass metabolism of drug.
3. Handling ointments is easier than bulky liquid dosage forms.
4. They are chemically more stable than liquid dosage form.
5. They prolong the contact time between the drug and effect area.

Disadvantages of ointment

1. Their oily semisolids preparations are staining and cosmetically less aesthetic.
2. Application with fingertips may contaminate the formulation or cause irritation when applied.
3. They are bulkier than solid dosage form.
4. When application of an exact quantity of ointment to the affected area is required, it is difficult to ascertain the same.

Figure No. 2] Herbal Ointment

Plant Profile

1] Drug (Maytenus emarginata)

Maytenus emarginata (Willd) D. Hou., a member of the Celastraceae family, is an evergreen tree characterized by resinous stems and leaves. It commonly appears as small trees, shrubs, or lianas and is known for its ability to endure various desert conditions. Locally, it is called "vickado," while in Hindi it is known as "Kankero" and in English as the "thorny staff tree." This plant is also identified by its synonyms: Celastrus emarginatus Willd., Gymnosporia emarginata (Willd) Thw., and Gymnosporia montana (Roth) Benth. Traditionally, species of Maytenus have been widely utilized to manage ailments such as gastrointestinal disorders, fever, asthma, and rheumatism [9].

Figure No. 3] Maytenus Emarginata

Root: Used to treat digestive issues, particularly dysentery

Stem­: The plant’s tender shoots aid in the treatment of oral ulcer. Using mustard oil, the bark is applied after being mashed into a paste to kill lice in the hair.

Leaf: Children are given crushed Maytenus emarginata leaves in milk as a vermifuge. A mouthwash made from a decoction of leafy twigs is used to treat toothaches. Using leaf ash to treat wounds and sores has a cooling effect. An ointment for wounds is made by burning the leaves and combining them with ghee. To treat jaundice, the delicate leaves are consumed uncooked [10].

Botanical classification and botanical description [11]:

Botanical classification

Kingdom: Plantae

Phylum: Magnoliophyta

Class: Magnoliopsida

Order: Celastrales

Family: Celestraceae

Genus: Maytenus

Botanical name: Maytenus emarginata (Willd.) Ding Hou

Synonyms:

Celastrus emarginatus Willd.

Gymnosporia emarginata (Willd.) Thw.

Gymnosporia Montana (Roth.) Benth.

Botanical description:

The Celastraceae family, which consists of roughly 50 genera and 850 species, often grows as trees or shrubs. The following is a list of all the Maytenus emarginata botanical descriptive characters:

Plant type: - Evergreen, Dioecious tree, very hard plant.

Growing requirements: - Light- full Sun with middy shade, Soil- sandy soil and lime soil, best growing season- monsoon, Drought tolerance- various types of stresses of desert.

1. Bark: Pale brown, smooth, cracked.
2. Leaf: Thick, coriaceous and usually longer than 40mm, Apex rounded, alternate on young branches, fasciculate on older ones.

Table No. 1]: Botanical Description of Maytenus emarginata (Willd)

 Botanical Description of Maytenus emarginata (Willd)

Phytochemical Constituents Present in Maytenus emarginat Plant

Table No. 2]: Phytochemical Constituents Present in Maytenus emarginata Plant

2]. Curcuma Longa:

General Description of Curcuma Longa:

Turmeric is a plant with a subterranean stem, or rhizome, that resembles ginger. It is a herbaceous, perennial plant that grows to a height of 60–90 cm and has tufted leaves on short stems. Its yellow flowers, measuring between 10 and 15 cm in length, bloom from late spring to mid-growing season and are arranged in dense spikes. This shrub does not produce any fruit. The turmeric rhizome has a rough, segmented outer surface. When ground, its dull orange interior transforms into a vibrant yellow color. It measures about 2.5 cm in diameter and ranges from 2.5 to 7.0 cm in length, with small branching tubers. In traditional Indian Ayurvedic medicine, turmeric was highly valued. It was prescribed in Ayurveda to treat various ailments, including skin disorders and constipation. Additionally, it was used as a digestive aid and a remedy for liver-related issues such as fever, inflammation, wounds, infections, dysentery, arthritis, injuries, jaundice, and more. In Unani medicine, turmeric is considered the top herb for treating blood-related disorders due to its ability to purify, stimulate, and strengthen the blood. Among Indians, including housewives and Himalayan hermits, turmeric is widely known as the “Kitchen Queen” and is the most essential spice in cooking. A short-term Pancha Karma treatment is said to have similar benefits to the long-term use of triphala, tulsi, and turmeric. Additionally, turmeric possesses a broad spectrum of antifungal properties [12].

Figure No. 4] Curcuma Longa

Curcuma Longa Plant Profile [12]:

Family: Zingiberaceae

Synonym: Ameshta Indian saffron Haldi Halud Haridra Ameshta Curcuma Kunyit Kunyitbasah

Chemical Constituents: curcumin, demethoxycurcumin, and bisdemethoxycurcumin

Medicinal Uses: anti-inflammatory, antioxidant, and antimicrobial properties, supporting health conditions like arthritis, diabetes, and digestive issues.

Taxonomical Classification [13]:

Class- Liliopsida

Subclass- Commelinids

Order- Zingiberales

Family- Zingiberaceae

Genus- Curcuma

Species- Curcuma longa

Microscopic Characters [12]

The outermost layer of turmeric rhizomes is made up of four to six layers of brick-shaped parenchymatous cork, which is followed by the cork cambium.  Round, thin-walled parenchymatous cells with sporadic vascular bundles make up the cortex.  The cortex contains oleo-resin cells and brownish collateral vascular bundles.  Vascular bundles are scattered throughout the pith region, creating a discontinuous ring underneath the distinct endodermis.  There are a lot of starch granules, which range in size from 5 to 15 microns [12].

Morphology of the plant [14].

Root: Root initials develop from the diffuse meristem, a small cell zone located beneath the second or third node that separates the inner and outer ground tissues. This meristem extends from the primary elongating meristem. The root meristem itself arises from the diffuse meristem. At the root apex of turmeric, three sets of initials emerge from the diffuse meristem: one for the root cap, another for the plerome, and a shared zone for the dermatogens and plerome.

Figure No. 5] Roots of Curcuma Longa

Flowers: The flower is smaller than the bracts, featuring a reddish and pastel yellow border. The calyx is obtuse, three-toothed, and measures 10-15 mm in length. The flower itself is long, tubular, and semi-elliptic, with three lobes and a pale-yellow lip. Delicate and short-lived, the flowers have a thin texture and reach approximately 5 cm in length. The short, toothed, tubular calyx is split nearly halfway down one side. The corolla, tubular at the base and cup-shaped in the upper part, has three uneven lobes inserted on the cup lip. It features a hooded dorsal lobe that is transparent, thin, and pale.

Figure No. 6] Flower of Curcuma Longa

Fruit and seed: The ripe fruit resembles a small garlic bulb and has a white color. Mature seeds appear brownish-black, while immature seeds range from white to light brown. In some cases, seeds from the same fruit may exhibit embryos at different stages of development. Structurally, the embryos resemble those of cardamom and are distinctly monocotyledonous. Even in developed seeds, the nucellus remains present.

Leaves: The leaves, which grow in tufts, have long stalks or sheaths that form a pseudostem or aerial shoot. They can be alternate, obliquely erect, or subsessile. The upright leafy shoots rarely exceed one meter in height, and each sprout typically bears six to ten leaves. The slender petiole abruptly expands into the sheath. The sheaths enclosing the ligules have ciliate margins, while the ligule lobes themselves are small. The leaf blade is thin, lanceolate, and acuminate, featuring pellucid patches. It is dark green on the upper surface and pale green on the underside. While it usually reaches up to 30 cm in length and 7-8 cm in width, it can occasionally grow as long as 50 cm.

Figure No. 7] Leaves of Curcuma Longa

Nodes and Internodes: Mature mother rhizomes typically contain 7 to 12 nodes, with internodes measuring between 0.3 and 0.6 cm in length. However, the first few internodes at the proximal end are expanded, enabling the mother rhizome to reach the surface of the soil. The internodes of the primary and secondary fingers are approximately 2 cm longer than those of the mother rhizomes. Except for the first one or two nodes, every node on the mother rhizome and its fingers contains axillary buds. Secondary and tertiary branches bear only scale leaves. Branches exhibiting negative geotropic growth feature sheath or pointed scale leaves.

Aerial Shoot: The buds of the foliage leaves grow from the main finger and from the axils of the nodes of the underground bulb; the foliage leaf’s petiole is long and has a thick leaf sheath; the aerial shoot forms when the long leaf sheaths overlap.

Shoot Apex: A tunica-corpus structure is seen in the shoot’s apical meristem.  Cells in the corpus, which is situated directly beneath the tunica, divide in a variety of directions, but cells in the tunica, which is composed of two layers, divide anticlinally.  The ground meristem develops from a file of cells generated by the central rib meristem beneath the corpus layer.  The procambium, cortical region, and leaf primordium are formed by the flank meristem, which surrounds this central area.

Rhizome: Turmeric’s underground stem, known as the rhizome, usually has a single major axis.  Typically, planting material consists of a mother rhizome, sometimes referred to as the “seed rhizome,” or a whole finger.  The aerial leafy shoot develops from a single main axis produced by this seed rhizome.  The first rhizome unit, which eventually develops into the mother rhizome, is formed by the expansion of the base of the main axis.

Figure No. 8] Rhizomes of Curcuma Longa

LITERATURE REVIEW

1. Shingala et al., (2024): A herbal ointment was developed and tested. To cure burns on the skin.  They examined the effectiveness of plant extracts in reducing inflammation, fighting infection, and aiding in wound healing.  The findings suggested that ointments made from herbs might be a good substitute for conventional burn care.
2. Mrabti et al., (2023):  Study investigated the use of plants in traditional medicine to cure burns on the skin.  They discovered that some plants are beneficial for skin restoration because they have anti-inflammatory, antibacterial, and wound-healing qualities.  According to the study, these plants have unique chemicals that aid in skin regeneration, which supports their use in both conventional and alternative medicine.
3. Skowro?ska and Bazylko’s et al., (2023): Study examined the potential of plants and natural items to treat sunburns and burns.  They discovered that some plants have beneficial substances that fight free radical damage, lessen inflammation, and encourage recovery.  According to the study, these plants’ extracts may help restore damaged skin, which makes them a valuable ingredient in contemporary skin care products and treatments.
4.  Maurya et al., (2023): The many kinds of ointments found in skincare products, their manufacturing processes, and their advantages.  They concentrated on key components, their combinations, and their effects on skin health.  The study also looked at recent advancements in skincare ointments with the goal of enhancing their absorption, durability, and skin-care efficacy.
5. Suryaanshi et al., (2023): In their review of turmeric covered its chemical composition, appearance, therapeutic benefits, and significance in traditional medicine.  They examined the long-standing usage of turmeric, its natural ingredients, and its health advantages, which include lowering inflammation, avoiding cancer, preventing cell damage, and eliminating bacteria.  The review backs up the notion that turmeric has a useful role in contemporary medicine.
6. Kumar et al., (2021): Examined the medicinal applications, natural components, and therapeutic qualities of turmeric.  They concentrated on curcumin, a crucial component of turmeric that possesses potent anti-inflammatory, antioxidant, and anti-germ properties.  According to the study, turmeric has long played a significant role in traditional treatment and is still utilized in contemporary medicine.  According to the experts, additional research is required to fully comprehend the advantages of turmeric and its potential use in the development of novel medications.
7. Kumar et al., (2019): Examined how Maytenus emigrata has been used traditionally in Rajasthan, India, to treat a range of illnesses, including infections, inflammation, and stomach disorders.  They discovered that the plant has beneficial substances that may have therapeutic effects.  According to the study, additional scientific investigation is required to validate its efficacy and create herbal treatments for other medical ailments.
8. Abraham et al., (2018): Examined the transmission of heat and the potential severity of skin burns.  The length of time the skin is exposed to heat, its inherent characteristics, and the way heat distributes were among the variables the researchers looked at.  In order to lower the danger of heat-related injuries, their research improved our understanding of how to avoid burns, build protective materials, and develop efficient therapies.
9. Khalandar et al., (2018): The spice turmeric, which is frequently used in cooking.  They researched its medicinal use, natural compounds, and therapeutic qualities.  Turmeric’s primary ingredient, curcumin, has been shown to have potent anti-inflammatory, antioxidant, and anti-germ properties.  More research is required to corroborate the study’s suggestion that turmeric may be utilized to treat chronic illnesses.
10.  Ranade et al., (2016): In order to find beneficial substances known as phenolics, he examined the leaves and stems of the Barleria prionitis plant.  They discovered that these substances possess antioxidant qualities that can aid in preventing cell damage.  According to the study, this plant’s natural compounds may have health benefits.  The findings indicated that further research is required and that it may be utilized to create novel medications for oxidative stress-related ailments.
11. Shelke and Mahajan et al., (2015): Reviewed ointments, discussing their types, usage in pharmacy, advantages, and manufacturing processes.  Important components such as bases, emulsifiers, and active medications were discussed, along with how they aid in the medication’s successful skin absorption.  In order to enhance the delivery of medications, the evaluation also examined recent advancements in ointment technology, possible stability problems, and testing procedures for ointments.
12. Dhawale and Ghyare et al., (2015): Using a unique method known as GC-MS, it investigated the chemical composition of the Maytenus emargin plant’s roots and fruits.  They discovered beneficial substances with potential therapeutic benefits, underscoring their significance in medicine.  The study also examined the plant’s historic uses, the range of compounds it contains, and its potential use in the creation of novel medications and therapies.
13.  Moteriya et al., (2014): The phytochemical composition and antibacterial properties of the leaves and stems of Maytenus emarginata. Previous studies have demonstrated that this plant’s active ingredients, which include flavonoids, tannins, and alkaloids, have therapeutic qualities.  It has long been utilized in traditional medicine because of its antibacterial, antioxidant, and therapeutic properties, according to other research.
14. Bahramsoltani et al., (2014): Study looked at plants and the natural substances they contain that can aid in burn treatment.  They discovered that several plants can help with burn rehabilitation because of their anti-inflammatory, antibacterial, and restorative qualities.  According to the study, plant-based components like flavonoids and tannins may aid in the healing of damaged tissue, making them potential future burn treatments.
15. Ahmed et al., (2013): The chemical makeup, toxicity, and therapeutic qualities of four Maytenus species used in Southern African traditional medicine to treat stomach disorders such diarrhea. These plants’ usage in traditional medicine is supported by the researchers’ discovery that they contain beneficial compounds and have antibacterial and antioxidant properties.
16. Sagwan et al., (2011): Maytenus emarginata as a potential cancer treatment, emphasizing its natural chemicals that can inhibit the proliferation of cancer cells.  The study discussed the traditional medicinal uses of this plant, its significance in pharmacology, and its mechanism of action.  It implied that this plant might be helpful in the treatment of cancer, but further study is required to validate this.
17. Lawrence and Bull’s et al., (1976): Study examined the factors that lead to burns, concentrating on skin exposure duration and temperature.  They discovered that these two elements have a critical role in assessing the severity of a burn.  Their work improved burn prevention and treatment, helped us understand when skin damage happens, and helped create safety regulations for different industries.

AIM AND OBJECTIVES

AIM: Formulation and evaluation of herbal ointment of Maytenus emarginata for burning skin.

OBJECTIVES:

1. Develop a herbal ointment that effectively soothes and heals burning skin, reducing pain, redness and inflammation.
2. Ensure optimal extraction and integration of bioactive compounds such as flavonoids, tannins, and phenolic compounds known for their anti-inflammatory, antioxidant and wound-healing properties.
3. Create a stable ointment base that maintains the potency, texture and efficacy of the herbal ingredients throughout its shelf life.
4. Incorporate hydrating agents to reduce dryness, prevent moisture loss and improve skin elasticity in affected areas.

METHODOLOGY AND MATERIALS

Ingredients

Table No. 3] Ingredients

Collection of plant

The leaves of Maytenus emarginata were collected during August 2024 from areas of Saygata forest, Bramhapuri, Chandrapur district, Maharashtra. The leaves were washed throughly under running tap water 2-3 times to remove dirt and it burnt into oven and grind to fine powder with the help of mechanical grinder and store in airtight container.

Preparation of Herbal Extract

Leaves of the plant were collected and washed thoroughly with distilled water and burn the leave and grind in fine powder. 100gm powder was imbibed with 350ml of 90% ethanol for 3hrs. and transferred to conical flask with addition of 150ml of 90% ethanol for maceration for 7 days with occasional stirring. Finally, ethanolic extract was collected and concentrated to get blackish green residue. The extract was stored in the airtight container in cool and dark place.

Figure No. 9] Maceration Process

Phytochemical evaluation for Maytenus emarginata [15]

Test for tannins:

About 2 ml of filtered extract was taken in a test tube and 2 ml of ferric chloride was added. The presence of blue-black coloured precipitate indicated the presence of tannins.

Test for saponins:

To 0.5 ml of extract, 5ml of distilled water was added in a test tube. The solution was shaken vigorously and observed for stable persistent froth indicating presence of saponins.

Test for cardiac glycosides:

To 2 ml of extract 1 ml of glacial acetic acid containing one drop of ferric chloride solution was added. This was underlayed with 1ml of sulphuric acid. A brown ring at the interface indicated the presence of a deoxysugar characteristic of cardenolides. A violet ring may appear below the brown ring, while in the acetic acid layer a grennish ring may from just above the brown ring and gradually spread throughout this layer.

Test for Flavonoids:

Dilute ammonia about 5ml was added to the extract. Concentrated sulphuric acid 1ml was added to this reaction mixture. A yellow colouration that disappears on standing indicates the presence of flavonoids.

Test for steroid:

To 1ml extract 1ml of chloroform was added; then 1ml of concentrated sulphuric acid was added carefully to form coloured layer. Upper layer turns red. Sulphuric acid layer forms yellow with green fluoresence, indicates thr presence of steroid.

Test for triterpenes:

To 1ml of extract 1nl of chloroform was dded, then 1ml of concentrated sulphuric acid was added. Apperane of yellow colour in the lower layer of the solution in standing indicates the presence of triterpens.

Test for alkaloid:

To 1ml of extract, few drops of Mayer’s Reagent (1.36g of mercuric chloride and 5g of potassium iodide in 100 ml of distilled water) were added. The presence of creamy yellow colourd precipitate indicated the presence of alkaloids.

Mayer’s test: Take 1ml of extract in the test tube and add 2-3 drops of mayre’s reagent, a dull white precipitate indicate the presence of alkaloids.

Dragendoff’s test: Take 1ml of extract in the test tube and add 2-3 drops of Dragendroff’s reagent, orange precipitate give positive results.

Wagner’s test: Take 1ml of extract in the test tube and add 2-3 drops of Wagner’s reagent, presence of brown precipitate indicates the presence of alkaloids.

Phytochemical evaluation for curcuma longa [16]

Test for Alkaloid

Dragendroff's reagent was used to test this, and Wagner's reagent was used to confirm it. In a test tube, 3ml of crude ethanolic extract was placed, and 1ml strong HCl acid was slowly added. The mixture was warmed for 20 minutes, then cooled before being filtered through Whatman No 1 filter paper (125mm diameter.) After that, the filtrate was put through the Dragendroff and Wagner tests.

1. Dragendroff's test: In 1 mL of filtrate, two drops of Dragendroff's reagent were added. There was a creamy precipitate, indicating the presence of alkaloids. The Wagner's test was used to confirm this.
2. Wagner's test: Wagner's reagent was combined with 1ml of the filtrate. The presence of alkaloids was revealed by the formation of a brownish precipitate.

Test for Tannins

3mL plant extract was combined with an equivalent amount of newly produced FeCl3. The presence of tannin was revealed by a green coloration.

 Test for Coumarin

1.5 mL NaOH (0.25 M, 10%) was combined with 2 mL Turmeric extract. The presence of Coumarin was demonstrated by the production of a yellow colour.

Test For Saponin

In 5ml distilled water 40mg extract was dissolved with vigorous shaking till a stable persist froth was obtained. In froth 2-3 olive oil drops were added with vigorous shaking.

Test for Diterpenes

The copper acetate test was used to accomplish this test. A dropper was used to add 10 drops of copper acetate solution to 1ml of turmeric extract and an equal amount of water. A rich green coloration was found, indicating the presence of diterpenes.

 Test For Pholbatannis

1 mL of the plant extract was combined with 1 percent HCl acid (dilute) and gently heated. The presence of phlobatannins was shown by the formation of a reddish-brown precipitate.

Test for Glycosides

The Legal's test was used to look for cardiac glycosides, which involved mixing 1 mL pyridine with 3 mL ethanolic Turmeric extract. A dropper was used to add 5 drops of freshly made 2 percent sodium nitroprusside solution and 5 drops of 20 percent NaOH solution. A pinkish-red coloration was detected, which faded into brownish-yellow after standing for a while, indicating the presence of cardiac glycosides.

 Test for Phenol

In a test tube, 1ml of the extract was combined with 4 drops of freshly made alcoholic FeCl3 solution. The plant extract has a bluish-black coloration, indicating that phenol is present.

Preparation of herbal ointment (Fusion method):

Ointment containing beeswax, wool alcohol and coconut oil are prepared by melting ingredients in a porcelain dish over a water bath. In this process higher melting point substance should be melted first and add then other ingredients of the bases in order of their melting point. After melting all bases ingredients the herbal extract add drop wise in the melted base with continuous stirring. Remove from waterbath and allow to cool down at room temperature with continuous stir.

Table No. 4] Formulation of bases

Different formulations of ointment

Table No 5] Different formulations of ointment

1. Physical determination:

The Prepared ointment formulations were inspected visually by

a) Colour

b) Homogeneity

c) Consistency

2 Viscosity

The viscosity of the prepared gel formulations was determined using BrookField DV-III ULTRA programmable rheometer model RV, helipath spindle set (Brookfield Engineering laboratories, USA) using 4-bar spindle. The viscosity was measured in centipoises (cps) at 6 rpm for 1 minute and temperature 25ºC using 20-gram sample. This experiment was performed for medicated formulations.

3 Spreadability:

The spreadability is expressed in terms of time in seconds taken by two slides to slip off from ointment, placed in between two slides under the direction of certain load. Lesser the time taken for separation of two slides, the better the spreadability of ointment. The spreadability was calculated by using the following formula.

S=M.L/T

 Where, M = weight tied to upper slide

              L = length of glass slides

             T = time taken to separate the slides

Figure No. 10] Spreadability

4 Determination of pH:

2.5gm Ointment sample was taken in 100 ml dry beaker, 50 ml water was added to it. Beaker was heated on water bath maintained at about 60ºC to 70ºC for 10 minutes, cooled to room temperature. The pH of water extract was measured by using pH meter. The pH measurements were done by using a digital type pH meter by dipping the glass electrode into the ointment formulation.

Figure No. 11] Determination of pH

5. Skin irritation studies:

Ointment formulation was evaluated for the non-irritancy test. Apply the ointment on skin and Observation of the sites was done for 30mins.

6. Washability

Formulation was applied on the skin and then easy extend of washing with water was checked.

 In-vitro diffusion study for anti-bacterial activity:

 In Vitro diffusion study: Antibacterial activity: The antibacterial screening of herbal gels was done by disc diffusion method.  The gels were tested against bacterial agents namely E. coli.  A loopful of the pure bacterial culture was suspended in nutrient broth and incubated for 24 hours.  Nutrient agar media was sterilized and poured into petri plates.  After solidification, 0.1ml of the inoculum was spread over the agar evenly using a rod.  6mm diameter cavity was prepared and formulated gel is placed in the cavity.  A standard antibiotic was used as the control.  The inoculated plates are incubated for 24 hours.  Later, the zone of inhibition around the disc was measured and recorded.

Figure No. 12]: Zone of inhibition F2

Figure No. 13] Zone of inhibition F3

Phytochemical Constituents Present In Maytenus emarginata Plant

Table No. 6]: Phytochemical Constituents Present In Maytenus emarginata Plant

Phytochemical Constituents Present In curcuma longa Plant

Table No. 7] Phytochemical Constituents Present In curcuma longa Plant

Physical evaluation

Table No. 8] Physical evaluation

PH

Table No. 9] PH

The herbal ointment containing Maytenus emarginata or curcuma longa extract has pH range of 6.33 to 6.53, which is slightly acidic to neutral.                                   

Viscosity

Table No. 10] Viscosity

The viscosity is illustrated according to the change in type of fatty alcohol and concentration and the molecular weight of the ointment bases. found that increasing the concentration of the emulsifying agent in the ointment’s formulation led to increased viscosity of the formulation.              

Spreadability

Table No. 11] Spreadability

Pharmaceutical semisolid preparations of ointment. Their common property is the ability to cling to the application surface for a reasonable period before they are washed off or worn off. They usually serve as vehicles for topically applied drugs, as emollients, or as protective.

Skin Irritation Test: Minor or No irritation observe

The herbal ointment with wax bases that contains extracts of Maytenus emarginata or Curcuma longa is probably going to be mild on the skin, reducing the possibility of discomfort.  The anti-inflammatory and antioxidant qualities of Maytenus emarginata and Curcuma longa are well-known for their ability to calm and shield the skin.  The wax base functions as a barrier to prevent irritation and promote the effectiveness of the herbal extracts.  Sensitive skin or skin disorders that call for mild, non-irritating treatments might benefit from this formulation.

Washability: Cannot remove with water

Herbal ointment contain wax bases are typically anhydrous or water-free formulation. Due to ther hydrophobic property these ointment cant not removed with water.

Anti-bacterial activity:  the anti-bacterial test of ointment from Maytenus emarginata leaves extaract show promising result the study has demonstrated that the extract exhibits significant antibacterial activity using E. coli bacterial culture the test of anti-bacterial activity are positive.

Table No. 12] Zone of inhibition