Formulation And Evaluation of Herbal Toner Using Chrysanthemum Flower and Its Docking Study

Herbal cosmetics are formulations containing phytochemicals sourced from various botanicals, influencing skin functions and supplying essential nutrients for healthy skin and body. In cosmetic formulations, natural herbs, products, and aromatic extracts are utilized, collectively termed as herbal cosmetics. There's a prevailing belief that chemical-based cosmetics could potentially harm the skin, leading to heightened consumer awareness and increased demand for natural products and extracts in beauty care. (4) Herbal cosmetics, also referred to as natural cosmetics, are considered the safest option for skincare, protecting the skin from external environmental factors while enhancing its appearance. Cosmetics are substances applied to various parts of the body, such as the face and hands, to soothe the skin, enhance beauty, and improve appearance without altering bodily functions or structures. There's a growing trend in the use of herbal cosmetics among people, highlighting the increasing demand for these products in daily skincare routines.(9) A herbal toner is a skincare product made from natural ingredients such as herbs, flowers, and plant extracts. Typically applied after cleansing, it assists in removing remaining dirt or impurities from the skin and primes it for the application of other skincare products like moisturizers.(10) Toner is a water-based liquid containing active ingredients that cleanse the skin, maintain its pH balance, minimize pores, and provide an instant glow. Applied before washing the face, it effectively removes dirt and impurities lodged in the skin pores. When incorporated into a daily skincare regimen and used consistently, toner significantly improves skin appearance and reduces signs of aging. Additionally, its antioxidant properties help hydrate the skin. (1) Toner effectively eliminates any remaining dirt, grime, and impurities that may linger in your pores post-face wash. When integrated into your daily skincare regimen and applied consistently, it can greatly enhance the appearance and firmness of your pores, particularly as your skin ages. Additionally, toner restores the pH balance of your skin, refines rough areas, and enhances skin tone, resulting in smoother and more even skin. (3) Herbal toners offer benefits to all skin types, with particular advantages for those with sensitive or dry skin by reducing sensitivity and adding extra hydration. They serve as a refreshing option for a mid-day boost, hydrating and rejuvenating the skin. The key goals of these toners include rehydrating the skin, restoring pH balance, minimizing pores, soothing irritation, and providing germ-free protection. (10)

Fig No.1(Herbal Toner)

Effects of A Toner on Skin-

Skin toners were once commonly used as a secondary cleansing agent to eliminate residual makeup post thorough facial cleansing or to reduce excess sebum production, priming the skin for further treatments. They were categorized into alcohol-based and non-alcohol-based types to cater to diverse skin needs like oily, sensitive, or combination skin. Nowadays, skin toners have evolved into cosmeceutical solutions, serving multiple functions such as rehydrating the skin, restoring pH balance, minimizing pores, alleviating irritation, and providing antiseptic properties. (10)

Mechanism of action of the spray formulation-

Pressing the button on the spray bottle initiates a pumping action that moves air from the nozzle to the dip tube. This action reduces the pressure at the top of the tube, causing liquid to be forced up through the tube. As the liquid travels through the nozzle, it is dispensed as fine mist droplets onto the skin, penetrating the skin's surface due to the pressure applied. (3)

Advantages of herbal face toner-

Herbal face toner offers several Advantages for skin, including;

Balancing the pH of the skin ;the natural ingredients in the herbal toners can be help to balance the pH of the skin

1. Toners effectively remove lingering impurities, providing a thorough and additional cleanse for your face.
2. Consistent use of toner can reduce the visibility of pores over time.
3. Toners play a crucial role in restoring the skin's pH balance.
4. Toners are known for their ability to quickly absorb into the skin, instantly revitalizing and hydrating it.
5. Toners offer immediate refreshment to the skin upon application.
6. Serving as a protective shield, toner acts as a barrier, guarding the skin against various environmental stressors like dirt, dust, pollution, and UV radiation.
7. Toners are formulated with a variety of ingredients tailored to address different skin issues and concerns.
8. Oily or acne-prone skin benefits greatly from the use of toners, making them an invaluable addition to skincare routines. (1)

Disadvantages of skin toner-

1. Alcohol-based toners can dry out and make the skin flaky.
2. Excessive use of toner can lead to skin irritation, such as swelling and redness. (1)

Types of herbal toner-

Skin freshener or Bracers-

This toner is a gentle type containing water and glycerine, which acts as a humectant to moisturize the skin's surface. An example of this is rosewater. It's typically suitable for normal, dry, and sensitive skin types. (5)

Fig No-2

Skin Tonics-

These toners are slightly more potent as they contain a small amount of alcohol (up to 20%), along with water and a humectant. (2)

Fig No-3

Acid Toners –

Acid toners are potent formulations containing alpha hydroxy acid and/or beta hydroxy acid. (3)

Fig No-4

Use of natural ingredients in herbal toner preparation-

Aloe Vera-

Aloe vera is rich in amino acids, including leucine and isoleucine, as well as vitamins A, C, E, B, choline, B12, and folic acid, which have cleaning properties and antioxidant properties. (11) Aloe vera harbors a rich array of potentially active components, numbering at 75, which include a diverse range of vitamins, enzymes, minerals, sugars, lignin, saponins, salicylic acids, and amino acids. (6) Aloe vera's anti-inflammatory characteristics aid in diminishing redness associated with acne, burns, and wounds. Its antioxidant attributes shield skin cells from damage, while its moisturizing properties alleviate dryness and mitigate skin irritation. (13)

Fig No-5 (Aloe Vera)

2. Turmeric-

1. Turmeric is a remarkable skincare ingredient with a long history of resolving various skin concerns like acne, darkening, pigmentation, and rashes. Its efficacy in promoting skin health and radiance has been acknowledged for generations.

2. Turmeric's skin-brightening and lightening properties are exceptional, thanks to its antioxidant compound curcumin, which offers a multitude of skincare benefits. It effectively combats acne, acne scars, and reduces skin pigmentation.

3. Furthermore, Curcuma longa, the botanical name for turmeric, exhibits a range of pharmacological activities beneficial to the skin, including antioxidant, antimicrobial, anti-inflammatory,     anti-carcinogenic, anticoagulant, antidiabetic,          and immunological properties. (13)

Fig No-6 (Turmeric)

Rose Water-

The active constituents of this substance include phenyl ethanol, linalool, citronellol, nerol, and geraniol.

It serves various purposes:

• Enhancing flavor and providing a mild astringent effect on pores to prevent their buildup of dirt on the skin.
• Exhibiting anti-inflammatory properties.
• Acting as a moisturizing and anti-aging agent.
• Being utilized in cosmetics and skin protection applications.
• Providing an astringent effect on skin pores.
• Serving as a skin whitening agent.(6)

Fig No-7- (Rose Water)

Peppermint Oil-

Mentha piperita L. is a fragrant perennial herb characterized by its creeping stolons and classification within the Lamiaceae family. (5)

1. Active Constituents-Menthol
2. Use-Astringent, antimicrobial property (4)

Fig No-8 (Peppermint Oil)

Lemon Leaves -

Common Name: Lemon leaves

Botanical Name: Citrus limon (L.) Osbeck

Family: Rutaceae

Antioxidant Activity

1. Due to flavonoids and phenolic compounds.
2. Protects cells from oxidative damage.

Uses

1. Infusions for calming nerves and promoting sleep.
2. Folk remedy for colds, cough, and headaches.
3. Used in aromatherapy and herbal teas.

Fig No-9 (Lemon Leaves)

Tulsi-

In India, Tulsi, known as Holy Basil, holds significant cultural importance in Hindu tradition. It is utilized for treating a range of disorders and is valued for its antiviral properties. (9)

Fig No-10 (Tulsi)

Need of investigation-

Synthetic chemicals can have adverse effects on the skin, prompting a rising preference for herbal cosmetics among consumers seeking natural alternatives for personal care. This shift towards herbal products is driven by their ability to provide essential nutrients, promote health, and offer satisfaction by avoiding artificial chemicals. Herbal cosmetics represent the latest, safest, and most popular trend in beauty and fashion.

Drug Profile

Chrysanthemum-

Fig no -11 (Chrysanthemum)

Scientific Name: - Dendranthema grandiflora. Family: - Asteraceae.

Order: - Asterales.

Chemical Structure: -

Plant Part: - Petal.

Use: - anti-inflammatory, antioxidant effects and to protect the skin from Uv radiation.

Biological Activities of Chrysanthemum Flower (12)

Table no-1(Material) (10)

Ingredients And Their Roles

Procedure

Extraction Process of Tulsi and lemon leaves -

Take Tulsi leaves transfer it into 100 ml of water. Boil it for 15 mi. Then take lemon leaves into another beaker and add 20 ml water and boil it gently. Collect the juice of aloe vera into the beaker. Filter the all-liquid extracts. (10)

In Laboratory Extraction –

Formulation Process:

22ml of Tulsi extract and 14 ml Chrysanthemum extract take in a beaker and stir it. Add the 6.6 ml rose water in above mixture. Then add the 2 ml of lemongrass and the 1ml aloe vera extract. Makeup the volume with distilled water and stir the mixture (10).

Formulation Pics:

Picture of formulation on the day of manufacturing

Fig no -12 Formulation

Characterisation

• • Organoleptic Properties-

The organoleptic characteristics of herbal face toner, such as colour, Odour, and appearance, were investigated.

• • Homogeneity-

Visual inspection was used to assess homogeneity for the presence of any clogs and their appearance.

• • Surface Tension –

By using a stalagmometer, the researchers collected the formulation of the toner and measured its surface tension. This information helps in predicting how the toner will spread on the skin, ensuring even distribution and a consistent user experience. (10)

• • Skin Irritation-

Small amount of the toner was sprayed on hand dorsal skin and kept for some time result was found Non-irritant on the skin. (4)

• • PH measurement -

In herbal toners, pH testing is employed to ascertain their acidity or alkalinity levels, crucial for maintaining optimal skin health and effectiveness. To conduct the test, a 25ml formulation was placed in a beaker with graduations, and a calibrated pH meter was immersed in the formulation for a duration to obtain the measurement.

• • Light Exposure Testing -

To check for product discoloration, the product is left in its actual packaging in a light chamber or under direct sunlight for 48 hours.

• • Perfume Stability-

The formulation herbal face toner was applied after 30 days, to record fragrance.(10)

• • Skin Conditioning-

The appearance of the skin after application of the mist was seen to be smooth, hydrated and supple

• • Temperature Variation-

The formulation was exposed to different temperatures at 45o C and -10 o C for 3 months to check the stability

Result And Discussion-

• • Organoleptic Properties-

Colour, Odour, Appearance Was Conformed in laboratory Scale.

• • Determination of pH

pH of the four formulation F1,F2,F3 was found to be closer to be closer to skin pH , including that they can be safely used on skin pH of all three formulation

Fig no-13 Ph Test of Herbal Toner

Table No-5   Determination of pH

• • Skin Irritation Test-

It is carried out by applying product on the skin for 10 min.

Table No-6 Skin Irritation Test

Anti-Microbial Activity-

1.Introduction:

The antibacterial activity is estimated by comparing the inhibition of growth of sensitive micro- organisms produced by known concentration of the isolated substance or extract or synthetic compound to be examined against a reference substance.

2 Method of Analysis:

Two general method usually employed; One is the disc -plate method [Agar disc diffusionmethod)-The agar cup plate method depends upon diffusion of the antibiotic from a vertical agar [disc ] Cylinder through a solidified agar layer on a Petri dish. Sterile Agar isinoculated by suspension of the microbial inoculum. Then a disc with diameter of 6 to 8 mm is punched aseptically , and then of the antimicrobial solution at desired concentration is introduced into the disc . Then, agar plates are incubated under suitable conditions depending upon the test microorganism. The antimicrobial agent diffuses in the agar medium and inhibits the growth of the microbial strain entirely in a zone around the cylinder containing a solution of the substance to be tested.

Product Details:

Product Details:

1. Description Of Equipment/Instruments:

Name of Equipment/Instrument

1. Media And Reagents Preparation:

Antibiotic Assay Medium No. 19 (pH is 6.1 ± 0.2):

Ingredients:

Prepared of medium Antibiotic Assay Medium No. 19 in 600 ml of purified water, heat boiling to dissolve the medium completely check the pH of media. If required add sufficient 1 M sodium hydroxide or I M hydrochloric acid, distributed in 200 ml flasks as required so that after sterilization as quantity as per required for analysis., sterilized by autoclaving it at15 lbs pressure (121°C) for 15 min.

Preparation of the sample solution

Use Directly 100 µl and 200µl To Inoculate.

Preparation of the STD solution

Weigh 1 mg of each sample and dissolve/dilute with 1 ml DMSO in volumetric flask. Vortex for 1-2 min to effect the dissolution use directly 100 µl to inoculate.

Preparation of Test organism and suspension:

• Test organisms
• Propinobacterium acne ATCC no.11827
• Staphylococcus aureus Slant ATCC no.6538
• Stock culture: Staphylococcus aureus ATCC no.6538.
• Streak a loopful of Staphylococcus aureus ATCC no.6538 on, two slants of pre incubated Nutrient agar. Incubate the slants at 30-35°C for 24 hours in an incubator
• Stock culture: Propinobacterium acne ATCC no.11827

Streak a loopful of suspension ATCC. 11827 on two slants of pre incubated Nutrient agar. Incubate the slants at 30-35°C for 24 hours in an incubator After incubation pick up the growth from incubated slant and inoculate in 3 ml of saline solution and vortex to prepare the uniform suspension. Adjust the O.D. of culture to approx. 60-70 % OD at 530 nmusing sterile saline and calorimeter. After adjusting O.D. store the test organism in refrigeration at 2-80C Note: Approximately viable count is 10° to 10 cfu/ml against 60-70 %OD at 530 nm.

Plate Preparation for analysis:

After the suspension is prepared, use each 2 ml of culture suspension of Propionibacterium acne is to inoculate separately in 200 ml of sterile molten and cooled medium at 40°C - 45°C Antibiotic Assay Medium No. 19. 15-20 ml of Sterilized agar medium is poured into a sterile Petri plate with the help of sterile measuring cylinder give a depth of 3 to 4 mm. Allow to cool at room temperature by placing the dishes or plates on a level surface. Keep plates in refrigerator for 15 to 20 minute for hardening. Ensure that the layers of mediumare uniform in thickness. Make 4-5 agar cups on each plate using 8-10 mm SS borer. Label the plates for sample, standard and negative control samples and analysis details.

Analysis:

The volume of solution added to each cylinder or cavity must be uniform and sufficientalmost to fill the holes when these are used. Add 100 µl 1mg/ml Solution A to agar cup labeled as STD. Add 100 µl Img/ml = Solution B to agar cup labeled for each compound ID labeled on plate.Add 100 µl DMSO to agar cup labeled as N (Negative). Leave the dishes or plates standing for 15-20 min. at 2-8°C or as appropriate, as a period of pre- incubation diffusion to minimize the effects of variation in time between the applications of the different solutions. Incubate them for about 24-48 hours at the temperature 30-35°C for bacteria and 20-25°C for yeast and mould. After completion of incubation accurately measure the diameters or areas of the circular inhibition zones and record the results.

Observations:

Dose of compound: 100µl     

Control: DMSO

200µl STD: 1mg/ml

During the study it has been found that sample N16 showed good inhibiting the growth of micro-organisms against Propionibacterium acne as compared to marketed preparation. During the study it has been found that sample N16 showed moderate inhibiting the growth of micro- organisms against s. aureus as compared to standard.

Stability Study-

Docking Study:

Introduction:

Molecular Docking Studies:

Molecular docking is in-silico method which predicts the placement of small molecules or Ligands within the active site of their target protein (receptor). It is mainly used to accurate Estimation of most favorable binding modes and bio-affinities of ligands with their receptor, Presently it has been broadly applied to virtual screening for the optimization of the lead Compounds. Molecular docking methodology comprises mainly three goals which are interconnected to each other like: prediction of binding pose, bio affinity and virtual screening. In the molecular Docking method, the basic tools are searching algorithm and scoring functions for creating and analyzing conformations of the ligand. Molecular docking involves the identification of pertinent peptide-protein complexes, the determination of the optimal shape for small molecules, and the exploration of how various compounds interact to develop highly potent substance. It proves valuable for investigating the interaction between proteins and pharmacological ligands or receptors. Interaction energy is gauged using a docking value. To rapidly quantify the strength of interactions among different compounds post-docking, a scoring function is employed in mathematical analysis. Most scoring functions stem from quantum chemical force fields grounded in physics, predicting energies at low (negative) levels. This implies a heightened likelihood of binding interactions and system stability. Docking options include rigid docking, leading to accurate positioning of the peptide within the receptor environment, and flexible docking, wherein the ligand's torsional angles and the side chains of intermediates at the catalytic site come into play.

The docking process comprises two fundamental steps:

1. Forecasting the conformation of the ligand within specific sites of orientation and positioning
2. Evaluating the binding affinity (14) (15)

Basic Principles of Molecular Docking:

Molecular docking is a valuable tool for comprehending fundamental biochemical mechanisms, deciphering the actions of small molecules at target protein binding sites, and simulating the atomic- level interaction between a protein and a small compound. The process of docking entails two fundamental stages:

1. The conformation and positioning of the ligand within these designated sites.
2. Evaluating the binding affinity between the ligand and the protein (14)

Antioxidant Activity Docking Results-

Fig No-16 Ligand-Receptor Complex

3D Structure