Isolation, Phytochemical Investigation, Evaluation and Formulation of Antibacterial Herbal Gel Containing Grewia Tiliifolia Leaf Extract

Topical Drug Delivery - Gel:

The use of natural products for treating skin and bacterial infections shows promise due to their ease of topical application. The skin serves as a barrier of defense & is the biggest organ in the body and performing functions such as sensory perception and thermoregulation. It is typically home to low-virulence bacteria like coagulase-negative staphylococci and non-pathogenic ccorynebacteria, with the microbiota behind the waist often including Gram-negative enteric bacteria and enterococci.¹⁰ Skin infections are prevalent and present significant therapeutic challenges, particularly in light of the emergence of bacterial, viral, and fungal strains that are resistant to several drugs. The human skin is particularly vulnerable to pathogen invasion, microbial colonization, and disease transmission. Acne vulgaris, which usually develops during puberty, is mainly caused by hormonal fluctuations that affect the body's physiological processes. A severe bacterial infection leads to the release of pro-inflammatory mediators, which cause vasodilation, organ failure, and immune suppression. Staphylococcal infections, particularly when combined with streptococci, exacerbate the condition.⁹ The topical route has long been a common method for delivering medications directly to the skin. Modern techniques for creating and enhancing topical formulations require thoughtful selection of components to guarantee safety, efficacy, and patient adherence.⁵ Advancements in pharmaceutical technology have prompted scientists to explore alternative drug delivery routes, beyond oral and parenteral methods, to efficiently target specific sites. Effective drug administration ensures that therapeutics reach the intended location of the action within the required time frame. The topical drug delivery system involves applying formulations to external areas like the skin, nose, eyes, vagina for the treatment of local conditions. Topical and transdermal products are vital drug delivery systems, and their use is increasingly common in therapy. These formulations aim to deliver drugs directly to a targeted area of the skin. For skin conditions and skincare, a variety of topical products, including solids, semisolids, and liquids, are available. Among semisolids, transparent gels are becoming more popular in both cosmetic and pharmaceutical applications. ⁷ Topical antibacterial agents have key benefits over systemic treatments for skin infections, including direct delivery to the infection site, minimal systemic absorption, and a lower risk of promoting widespread antimicrobial resistance. Additionally, they can achieve higher local drug concentrations, improving effectiveness against resistant bacteria.¹⁶ "Gel" comes from "gelatine" and is associated with the Latin words "gelu" (frost) and "gelare" (to freeze or congeal). Gels are semisolid systems composed of big organic molecules or small inorganic particles combined with a liquid, according to the USP. One Approximately 99% of a gel's weight is made up of fluid, which is stabilized through surface tension among the liquid and a network of macromolecular fibers made possible through a little quantity of gelling agent.⁴ Additionally, compared to ointments and creams, gel formulations generally offer faster drug release. They are also preferred for their better patient acceptability and ease of use.¹¹

• Advantages of gel:

• Improved patient compliance and acceptance
• Simple and convenient application
• Painless and non-invasive approach
• Increased drug bioavailability
• Improved pharmacological and physiological impacts.
• Reduced systemic toxicity and limited exposure to non-target tissues/sites.⁷
• Gel formulations allow for direct drug absorption, avoiding the first-pass metabolism.¹¹

• Applications of gel:

• Used for delivering oral medications.
• Applied directly to skin, mucous membranes, or eyes for localized effects.
• Provide long-lasting drug delivery through intramuscular injections or implants.
• Act as binders, thickeners, or protective agents in tablets, suspensions, liquids, and suppositories.
• Used as lubricants for catheters.
• Serve as bases for patch testing.¹⁵
• Cosmetic gels are commonly found in products like shampoos, deodorants, toothpaste, and skin and hair care items.
• For scalp inflammations, gel-based medications with anti-inflammatory steroids are preferred over lotions and ointments, as gels are less oily and more tolerable.⁶

Grewia tiliifolia:

Herbal medicine, also known as phytomedicine, involves using plant-based remedies to treat diseases. In the early 20th century, it was the primary form of therapy due to the lack of antibiotics and analgesics. However, its use declined as allopathic medicine gained popularity, with its rapid therapeutic effects from synthetic drugs. In India, many plants are used to treat human and animal diseases, with active compounds like tannins, essential oils, and aromatic compounds offering antibacterial properties. These phytochemicals, which vary in quantity and quality across different plant parts, hold potential for developing effective drugs for human use. ^(13,14) India boasts one of the oldest, most widespread, and diverse cultural traditions surrounding the use of medicinal herbs. Around 1.42 billion people, or a quarter of the global population, rely on traditional medicine, particularly plant-based remedies, to address health issues.¹⁷ Over the past few decades, the study of medicinal plants and their traditional uses has grown globally. Traditional medicine has long utilized various plant-derived bioactive compounds to treat infectious diseases. In recent times, researchers from all over the world have become more concerned in finding natural, plant-based medicines.¹⁸ Plants have served as a source of medicine since prehistoric times, with a history spanning over 5,000 years, playing a crucial role in the healthcare systems of various societies. Due to their diverse biological and therapeutic benefits, herbal medicines are highly valued as a primary healthcare resource in both developed and developing countries.¹⁹ Researchers are increasingly exploring plant-based medicines, with Grewia Tiliifolia Vahl, a tree in the Tiliaceae family, gaining attention for its medicinal properties. Known for treating ailments such as diabetes, fever, and wound healing, various parts of the plant are used in Ayurveda.²⁰ The tree grows up to 20 meters tall, with tough, fibrous bark that peels in flakes. Its leaves are simple and alternate, and its small, round fruits are black when ripe, containing 1-2 seeds. The tree also produces small yellow flowers on thick axillary peduncles.²¹ In Ayurvedic medicine, Grewia tiliifolia, commonly referred to as Dhamani or Dhaman, is a well-known herb.  It is mostly found in parts of India, such as Mumbai. Chennai, Andhra-Pradesh, Punjab, Himachal Pradesh and Uttar Pradesh. Numerous illnesses, including fever, blood disorders, heavy menstruation, diabetes, stomach problems, hyperdipsia, rhinopathy, ulcers, skin diseases, haemorrhage, and overall weakness, are treated with this herb.  The powdered bark is prized for its aphrodisiac qualities, while the leaves, bark decoction, and infusions are used in traditional medicine to cure snakebites in cattle.  Furthermore, Grewia tiliifolia’s ripe fruits are eaten for their nutritional value.²²

       
           
       
Fig No.1: Grewia Tiliifolia

• Taxonomical classification Grewia Tiliifolia:

Table No.1: Taxonomical classification Grewia Tiliifolia

• Phytoconstituents of Grewia Tiliifolia:

Table No.2: Phytoconstituents of Grewia Tiliifolia

Traditional uses:

1. The decoction of stem bark has been traditionally utilized as a remedy for treating diabetes mellitus.²³
2. It is beneficial in conditions where kapha and pitta are imbalanced, as well as for treating burning sensations, excessive thirst, nasal disorders, ulcers, skin ailments, vomiting of blood, and general weakness.²⁴
3. It is regarded as useful in addressing various ailments, including cancer, infectious diseases like malaria, and both bacterial and viral infections.
4. Also used in treatment of Alzheimer's disease.²⁵
5. Also G. Tiliifolia used in antituberculosis treatment.²⁶

5. MATERIALS & METHODS:

• Collection and authentication of Grewia Tiliifolia plant:

       
           
       
  Fig No.2: Authentication Of Grewia Tiliifolia

The leaves of Grewia Tiliifoliawere collected from the Radhanagari Wildlife Sanctuary in January 2025, identified, and authenticated by Dr. Vinod B. Shimpale, Professor, Department of Botany, The New College, Kolhapur. The leaves were washed, dried, and ground into powder using a mixer grinder. The powdered material was then stored in airtight container at room temperature for further study.

• Extraction of Grewia Tiliifolia:

The leaves of Grewia Tiliifolia were collected from Radhanagari, Maharashtra. The freshly collected leaves were washed, shade-dried for 4-5 days, and then chopped and ground into either a fine or coarse powder. Maceration process used to extraction of phytochemicals from G. tiliifolia leaves. These powdered leaves were soaked in ethanol for several hours, overnight, or for 2-3 days. The mixture was stirred occasionally to facilitate the release of active compounds. After soaking, the mixture was filtered, and the filtrate was collected. The filtrate was then evaporated, and the resulting extract was used for further analysis.²⁶

       
           
       
Fig No.3: Fxtraction of Grewia Tiliifolia leaves

• Pre-formulation study of Grewia Tiliifolia extract:
• Evaluation tests:

1. Test for triterpenoids and steroids:

2 ml of concentrated sulfuric acid was progressively mixed to 1 ml of the extract in a boiling tube. When terpenoids were present, a reddish – violet hue emerged, and when steroids were present, a greenish- blue hue emerged.

2. Test for flavonoids:

Five to six drops of diluted hydrochloric acid were added to a test tube that contained one milliliter of extract. Small particles of magnesium were then added. When flavonoids were present, the color turned red; when flavones were present, the color turned orange.

3. Test for phenols:

Some traces of a balanced 5 % ferric chloride solution were introduced to a test tube containing 1ml of the extract. The presence of phenolic compounds is indicated by the formation of a dark green color.²⁴

5. Quantitative estimation of extract of Grewia tiliifolia using spectroscopy:

• Determination of λ max in Methanol:

 In a 100 ml solution of methanol, 10 mg of Grewia Tiliifolia was placed in a 100 ml volumetric flask.

• Calibration Curve of G. tiliifolia in Methanol:
• Preparation of Calibration Curve for Grewia Tiliifolia:

A spectrophotometric method was employed to measure absorbance within the UV range of 200 to 400 nm in methanol for the estimation of G. tiliifolia.

• Preparation of Stock Solution:

A concentration of 100 µg/ml was achieved by combining 10 mg of Grewia Tiliifolia extract in 10 ml of methanol and then adjusting the volume to 100 ml with methanol.

• Preparation of working solutions:

Aliquots of 0.2, 0.4, 0.6, 1.0 and 1.2 ml were taken from stock solution I and diluted with methanol to a final volume of 10 ml, yielding concentrations of 5, 10, 15, 20, 25 and 30 µg/ml. Absorbance was measured at 262 nm using a UV spectrometer. The data points for absorbance were then analysed using linear regression.

• Comparative studies of FTIR Spectroscopy:

FT-IR can be employed to examine and predict potential physicochemical interactions or incompatibilities within a formulation by comparing the IR spectra peaks of individual constituents and their physical mixtures. This method helps identify compatible excipients, ensuring the selection of stable and suitable ingredients for use in both cosmetic and medicinal applications.

• Procedure of Gel formulation:

Formulation of gel base

1 g of Carbopol 934 was gradually disintegrating in 100 ml of purified water to create the gel basis, stirring constantly for an hour to clumping. After that, glycerine was added, and stirring persisted. A triethanolamine solution was subsequently incorporated gradually after preservatives such as propyl and methyl paraben were added. For 10 minutes, the mixture was agitated to create an opaque, uniform gel foundation. ²¹

Table No.3: Components List of Gel Formulation

A topical gel mixture was created utilizing an ethanol leaf extract of Grewia Tiliifolia and a gel base made from Carbopol 934, following standard drug formulation procedures. To enhance the antibacterial properties, an appropriate amount of Grewia Tiliifolia extract was incorporated into the preparation.

5. Methods of evaluation:

• Physiochemical evaluation of gel formulations:

1. Physical evaluations

Physical parameter such as colour, odour, phase separation and uniformity were checked visually.⁸

2. pH

An electronic pH meter was utilized to test the pH of a 1% water-based solution of the gel formulation that had been stored for 2 hours. The pH of gel formulation was determined for three times, and the average value along with the standard deviation was calculated.³

3. Homogeneity:

Prepared gel formulation was examined for homogeneity through visual inspection after being set in the container. In addition to being evaluated for appearance, they were examined for aggregates.⁴

4. Grittiness:

To look for any particle matter, the gel composition was inspected under a microscope. ¹¹

5. Skin irritation test:

The skin irritation experiment was performed using human participants. After that individual underwent examination for lesions or indications of irritation.⁴

6. Viscosity:

A Brookfield viscometer (Model AMETEK) was utilized to determine the viscosity of the herbal gel at 100 rpm with spindle number.²

7. Spreadability:

Spreadability refers to how easily a gel can spread when placed on the epidermis a crippled area, It is estimated by timing the separation of two glass slide with a certain weight on the upper slide and the gel sandwiched between them. Greater spreadability is shown by a shorter separation time. The following equation is employed to determine the spreadability. 

Spreadability (S) = M × L /T,

where,

• M – is the weight attached to the top most slide
• L – is the width of the glass slide
• T – is the period taken for the slide to split up.⁶

8. In- vitro antibacterial studies:

 The antibacterial effect of the topical agent was estimated utilizing the agar well diffusion assay against the bacteria staphylococcus aureus. Various concentrations (25 µg, 50 µg, 75 µg, and 100 µg) of the topical agent were aseptically transferred from the final formulation using a sterile spatula and placed into wells created with a sterile cork-borer on Luria agar plates with a 0.7 cm diameter. After that, the plates had been incubated for 24 hours at 27?C. The plates were inspected for the existence of a zone of resistance surrounding the wells following incubation.

Stability testing:

The stability study on the selected gel formulation was conducted for three months under both room and accelerated conditions (at 40±2°C and 75±5% relative humidity) using a stability chamber. The study aimed to determine the optimal storage conditions that would preserve the physical and chemical integrity of the gel, including its viscosity, spreadability, and drug content. After three months, the stability of the gel formulation was assessed.

5. RESULT AND DISCUSSION:

The current study explores the phytochemical potential of the tender leaf part of Grewia Tiliifolia. 

Table No.4: Results of physiochemical evaluations of ethanolic extract of Grewia Tiliifolia

       
           
       
Fig. No.4: Phytochemical Analysis of Grewia Tiliifolia

• Result of λmax in Methanol:

The spectrum of Grewia Tiliifolia was recorded using a 100 ppm (100 µg/ml) solution of the plant extract in methanol, with the absorbance maximum observed at 218 nm.

       
           
       
Fig No.5: λmax of Grewia Tiliifolia

The UV absorption spectrum showed a ?max at 218 nm. The concentration verses absorbance graph for Grewia Tiliifolia exhibited an exponential pattern within the concentration range of 5-30 µg/ml.

Table No.5: Curve of Calibration Readings

       
           
       
Fig No.6: Calibration Curve of Grewia Tiliifolia Leaves Extract

• Result of comparative studies of FTIR Spectroscopy:

The FTIR spectrum of Grewia Tiliifolia shows the proximity of functional element like hydroxyl (O-H), carbonyl (C=O), and alkane (C-H), indicating the presence of organic compounds like flavonoids, terpenoids, or other plant-based phytochemicals. Key peaks include ~3300 cm?¹ (O-H/N-H), ~2920 cm?¹ & 2850 cm?¹ (C-H), and ~1720 cm?¹ (C=O). The fingerprint region (below 1000 cm?¹) is useful for identifying specific compounds.

• Results of gel formulation:

The prepared gel formulations were assessed for a variety characteristic including physical appearance, pH, homogeneity, spreadability, grittiness, and viscosity. The observations showed that the gels had a smooth texture, an elegant finish, and were transparent in appearance. Grewia Tiliifolia leaves show better antibacterial activity against staphylococcus aureus.

Table No. 6: Results of physical evaluations of gel formulation

       
           
       
Fig No.8: Gel formulation of Grewia Tiliifolia

• Results of In-vitro Antibacterial activity:

The antibacterial effect was estimated by utilizing the agar well diffusion assay with the zone of resistance (expressed in mm) recorded. The polyherbal gel exhibited dose-dependent antibacterial effects against Staphylococcus aureus, as shown in the results. Figure 10 illustrates the antibacterial activity of the formulation.

Table No. 7: Zone of Resistance

• Results of stability testing of gel: