Formulation and Evaluation of a Herbal Gel based on Kokum and Turmeric for Acne Treatment

Acne vulgaris is a long-term inflammatory skin condition of the pilosebaceous unit, due to increase sebum production from increased androgen levels. Although it can also affect the upper arms, trunk, and back, the ailment typically presents as papules, pustules, or nodules on the face. The main lesion of acne vulgaris, known as a "comedo," is the result of several elements interacting during the disease's progression. Even though teenagers are the age group most likely to experience acne vulgaris, people of all ages can still be affected. This condition can range in severity from moderate types with few comedones to more severe forms with disfiguring inflammatory symptoms that can cause scarring, hyperpigmentation, and negative psychological effects.^{[1] [2] [3]}

ETIOLOGY

The hypersensitivity of the sebaceous glands to normal amounts of androgens in the blood leads to the development of acne. Cutibacterium acnes (C acnes), as well as other bacterial species such as Propionbacterium acne and Streptococcus aureus, and the ensuing inflammation, aggravate this process even more^. [4]

The following are suggested contributing causes to acne:

1. Using drugs such anticonvulsants, steroids, and lithium which can lead to increase in   androgen production which leads increase in sebum production.
2. Rapid multiplication of basal keratinocytes in pilosebaceous unit
3. Colonization of comedo with bacteria further worsening the acne. ^[5]
4. Use of comedogenic cosmetic product.
5. Being in too much sunshine. Wearing occlusive items like headbands, backpacks, shoulder pads, etc Face massages and makeup based on oil. Endocrine conditions, including pregnancy and conditions like polycystic ovarian syndrome.
6. The edema of the pilosebaceous duct appears to be followed by a premenstrual acne flare-up.
7. Seventy percent of patients who are female experience this. The amount of branched fatty acids in sebum is mostly determined by genetic factors; estimates of heritability range from 50% to 90%. Constant mechanical stress from washing afflicted skin with detergents and soaps. ^[6]

Severe acne is linked to psychological stress, most likely as a result of stress hormone stimulation^{. [7]}

EPIDEMIOLOGY

Teenagers and young adults are typically affected by acne vulgaris, with prevalence rates among teenagers reported to vary from 35% to over 90%. ^[8] When it comes to preadolescent acne, the illness can naturally start as early as age 7 or 12 and go away by the time a person reaches their third decade of life. Acne can, however, sometimes linger into adulthood or even appear for the first time in age. Males are more likely to be prone to experience than females to experience teenage acne. Conversely, post-adolescent acne primarily affects women. Acne vulgaris typically affects urban people more than it does rural ones. About 20% of those who are impacted experience severe acne that leaves scars. The evidence shows that the prevalence and severity of acne vulgaris may differ among specific racial and ethnic groups. Acne that is severe is more common in Asians and Africans. Although moderate acne is more. ^[9]  Neonatal babies may occasionally have acne, but most of the times it goes away on its own without the need for special care. Another kind of acne vulgaris called infantile acne can start in early childhood. Acne in youngsters between the ages of one and six is uncommon. But it might also indicate an underlying issue that needs more investigation. ^[10]

PATHOPHYSIOLOGY 

A number of host variables, such as the activation of sebaceous glands by androgens in the bloodstream, dysbiosis of the pilosebaceous follicle microbiome, and cellular immunological responses, interact in the development of acne vulgaris. Furthermore, the onset and course of the illness may also be influenced by other variables like nutrition and heredity. The original lesion, or micro comedo, is the ancestor of all clinical symptoms of acne vulgaris. It is distinguished by a tiny, hyperkeratotic plug in the lower part of the follicular infundibulum that is mainly made of corneocytes. Other acne lesions, such as closed comedones (whiteheads), open comedones (blackheads), and inflammatory papules, pustules, and nodules, progressively grow out of micro comedones. The development of different forms of acne from tiny comedones include

1. Seborrhoea, or increased production of sebum
2. Hyperkeratinisation of the follicles
3. acnes, formerly known as Propionibacterium acnes, is an anaerobic diphtheroid that is a normal component of the skin's microbiome.
4. Swelling

A closed comedo is created when sebum and keratinous material gradually build up within a micro comedo. When the follicular orifice is continuously distended, it eventually expands and an open comedo forms. The comedo gets its distinctive dark black colour from melanin and oxidized lipids. The inflammatory pustules and papules that result from C acnes are a result of its antagonistic cellular immune responses. Once follicles burst, proinflammatory lipids, keratin, and bacteria are released into the surrounding dermis, worsening the inflammation and causing nodule formation.  ^[11]

Pathophysiology of acne ^[12]

TYPES OF ACNE –

Fig A

Non-Inflammatory

1. Blackhead – It is mild type of acne. They are clogged with dead skin cell and oil. Black colour is due to reflection of light from obstructed hair follicles.
2. Whitehead – when top of pore closes and is clogged by sebum and dead skin cells.

Inflammatory

1. Papules – Moderate type. It is inflamed lesion without pus filled tip.
2. Pustules – Moderate type. It is inflamed lesion with white or yellow pus-filled tip.
3. Nodule – Severe type. Raised skin lesion involving deeper layer of skin.
4. Cyst – Severe type. Deep painful pus-filled lesion that causes scarring. ^[13] [14]

Assessing the severity of acne depends on several factors. Factors to consider in a comprehensive assessment of acne severity include the psychological impact of acne on the patient, the clinical type of inflammation, scarring, inflammation or sinus tract, and response to treatment tolerance. Patients with inflammatory nodular acne are diagnosed with severe acne. Similarly, people with inflammatory pustules and pustules accompanied by scarring, even without nodules, are also considered to have severe acne. [15]

The extent of skin involvement by acne can vary widely, from a few small comedones to many inflammatory nodules. Specific symptoms are influenced by patient characteristics. During puberty, comedones often appear on the forehead, nose and chin (Tzone). As acne progresses, teenagers may develop inflammatory conditions in addition to comedones. It is common for older women to experience acne on the lower face and neck, and these breakouts are associated with the menstrual cycle. [16]

TREATMENT –

Treatment of acne vulgaris aims to reduce damage and recurrence of acne. Reduce the scarring and to reduce the sebum production along with decreasing the proliferation of bacteria. Some of the synthetic agents used to treat acne are as follows- ^[17][18]

Table no 1: Topical treatment

Table no-2: Oral treatment

So, to avoid above side effects natural treatment of acne is preferred as they are gentler on skin, reduces the risk of allergic reaction, also along with treating acne they also have nourishing properties and they provide long term benefit. Some of the natural agent available are –                                            

Table no-3: Natural agents

BACKGROUND –

There has been an interest for developing polyherbal formulations for better patient acceptability, lesser side effects and traditional background. There are several treatments available that are responsible for various effects which include topical, biological, and systemic etc. Some have shown positive effects while others are shown less. Herbal formulation is tended to be a safer alternative for allopathic formulations as they have less side effects. ^[19]

A plant that contains compounds and healing properties or basically, compounds that can be used as part of semisynthetic medicine are called medicinal plants. These phytochemicals, which are not nutrients found in plants, act as plant defences against microbial infection. Acne a skin condition occurs when dead cells clog hair follicles. Acne vulgaris is defined as the development of non-painful inflammatory lesions of hair follicles and sebaceous glands affects three quarters of people aged 11 to 25. Acne can be caused by hormonal imbalances, environmental conditions or genetics which leads to the development of comedones. When bacteria enter clogged pores, they release chemicals that cause inflammation and the development of acne and pustule. Severe inflammation can cause nodules to grow into cysts, which can leave scars when they heal ^[20]. Gels play a crucial role in enhancing cutaneous and percutaneous medication delivery. Let’s explore their advantages and applications:

1. Gastrointestinal Absorption: Gels can prevent issues related to gastrointestinal absorption caused by acidic conditions. By bypassing the gastrointestinal route, they ensure effective drug delivery.
2. Interaction Prevention: Gels have the ability to prevent medication interactions with food, drink, and enzymatic activity. This makes them valuable when oral administration is not suitable.
3. First Pass Effect: Unlike oral medications, gels avoid the first pass effect. They are not inactivated by degrading enzymes in the liver.
4. Non-Invasive: Patients find gel formulations non-invasive, which is beneficial for compliance.
5. Topical Advantages: When applied directly to pathological areas, gels offer advantages in terms of direct drug release and faster absorption compared to creams and ointments.
6. Beauty Products: Single-phase gels, with uniformly dispersed organic macromolecules, are commonly used in skincare products due to their appealing appearance.

An optimal acne formulation for large hairy regions (such as the chest and back) should distribute smoothly and leave no residue or oiliness. ^[21] Carbopol® 940, known for its superior viscosity-building properties even at low concentrations, is commonly used in topical formulations. Treating acne is a promising area, especially considering the psychological impact on patients. Topical medication delivery provides a safe approach for treating skin infections. Antibiotics and anti-inflammatory drugs can be administered orally or topically as part of acne treatment. For mild to moderate acne, topical therapy is the first line of defence. Antibiotics, acids, benzoyl peroxide, retinoids, herbal agents, or a combination of these form the basis of topical treatments. While topical therapies are less risky than systemic ones, they may have limited water solubility and insufficient drug penetration through the stratum corneum. In moderate to severe cases, systemic therapy is also necessary. ^[22] [23]

The present work aimed to formulate and evaluate a polyherbal gel using active ingredients like Curcumin, Garcinol and kokum Butter for the treatment of acne vulgaris. The details of which is as follows –

1) Turmeric (Curcumin) –

Biological Source - is the dried rhizome of Curcuma longa of Curcuma Longa

Family – Zingiberaceae

Active ingredient – Curcumin

Mechanism of action - Due to safety profile of curcumin and its antibacterial and anti-inflammatory activity it has been studied extensively at high doses of clinical trials. ^[24] Therefore, it has been shown that curcumin has a potential to treat the acne. A study conducted in which a microemulsion of curcumin containing myristic acid in which curcumin is able to inhibit the bacteria (S. epidermidis), (S. Aureus), (Propionibacterium acnes) etc, which is responsible for causing acne. In another study, lauric acid-based emulsion incorporating curcumin was developed which was able to inhibit Propionibacterium acnes, the smaller size of the emulsion increased its contact and penetration into the membrane of cells. Anti-oxidant properties of curcumin showed that it results in peroxidation of lipids and he concentration of glutathione which enhances the enzyme activity of superoxidase dismutase and glutathione peroxidase in the skin ^[25] [26]

2) Kokum (Garcinol) And Kokum Butter (Stearic Acid) –

Biological Source - Kokum is the fat obtained by expression from the seeds of Garcinia indica or G. purpurea

Family – Guttiferae

Active ingredient – Garcinol in kokum and stearic acid in kokum butter

Mechanism of action – Garcinol generally have anti- inflammatory and anti- oxidant activity which is one of the mechanisms for the treatment of acne vulgaris. Garcinol, a potent inhibitor, affects inflammatory processes by targeting key proteins involved in inflammation. It inhibits nitric oxide synthesis in a concentration-dependent manner. Specifically, it directly inhibits the catalytic activity of two crucial enzymes: 5-lipoxygenase and microsomal prostaglandin PGE2 synthase. These enzymes are often overexpressed in various tumors. Additionally, cigarette smoke extract-induced airway inflammation may be regulated by cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) synthesis. ^[27] [28]. Some anti-oxidant studies carried in- vitro analysis Garcinol exhibits free radical scavenging activity against various radicals, including those from the hypoxanthine/xanthine oxidase system, superoxide anion, hydroxyl radical, and methyl radical. When compared by weight, the scavenging activity of emulsified garcinol is similar to that of DL-alpha–tocopherol (vitamin E) but less than that of ascorbic acid (vitamin C). Notably, garcinol more effectively suppresses the hydroxyl radical than DL-alpha–tocopherol. The proposed mechanism of action involves garcinol reacting with peroxyl radicals, followed by deprotonation of the hydroxyl group. Additionally, garcinol demonstrates a neuroprotective effect by reducing nitric oxide production and expression of inflammatory mediators (iNOS and COX-2) in rat cortical astrocyte cultures. ^{[29] [30] [31]}. Polyphenols present in garcinol play a major role in the treatment of acne by free radicle mechanism, anti-inflammatory and anti- oxidant mechanism ^[32]. Kokum butter is valued for its nutritive, demulcent, astringent, and emollient properties. It demonstrates excellent emollient characteristics and has high oxidative stability. Additionally, it can be utilized to produce stearic acid from fat, yielding approximately 45.7%. ^[33] [34].

FORMULATION CONSIDERATIONS FOR PHARMACEUTICAL GELS

Choice of carrier/solvent - Purified water is usually used as a solvent. Co-solvents can be used to improve the solubility of the therapeutic agent in the dosage form and/or to improve the penetration of the drug through the skin, e.g. alcohol, glycerol, PG, PEG 400, etc.

Inclusion of buffers - Buffers can be added to aqueous and alcohol-based gels to adjust the pH of the formulation. The solubility of buffer salts\ is reduced in water-alcohol-based vehicles. E.g. phosphate, citrate, etc.

Preservatives - Certain preservatives work in collaboration with the hydrophilic polymers used in the preparation of gels, which reduces the amount of free (antimicrobial active) preservative in the formulation. Therefore, to compensate for this, the original content of these preservatives should be improved. For example, parabens, phenolics, etc.

Antioxidants - This may be added to the formulation to improve the chemical stability of therapeutic agents susceptible to oxidative degradation. Its choice is based on the nature of the vehicle used in the preparation of the gel. Water-soluble antioxidants are usually used because most gels are water-based. E.g. sodium metabisulphite, sodium formaldehyde sulfoxylate, etc.

Perfuming agents – They are added to increase the aesthetic appeal of the formulation. E.g. Orange oil, strawberry oil, lavender oil, rose oil. ^[35]

EXPERIMENTAL WORK –

Isolation and extraction of Phytoconstituents

1) Garcinol – The fruit peels were washed with water. After 3-4 washes, the peels were ground in a blender until it breaks into a paste. sieving the chamber paste with a 20 number sieve was used to obtain a uniform, fine and uniform mass. 100 grams of smooth, fine uniform mass was added to A4 format filter paper. solvents such as methanol, ethanol, 1-propanol, hexane, chloroform are suitable for extracting garcinol. here methanol is used as a solvent. 400 mL of methanol was added to the Soxhlet. Soxhlet temperature was adjusted to 70 degrees. 0.3 Celsius degree extraction cycles were performed for 5 days. the red-violet distillate was collected in a round-bottomed flask. the distillate was transferred to a Petri dish to evaporate the methanol. for crude garcinol, the methanol was evaporated in an electric water bath the thick garcinol paste remains in the petri dish while the methanol evaporates. see (fig 1 and 2). The presence of garcinol is confirmed by TLC and chemical tests. ^[36]

2) Turmeric - The rhizome of Curcuma longa was dried and powdered to get the turmeric powder. This turmeric powder was used to extract curcumin. There are various methods to extract curcumin from Turmeric we have used the Soxhlet extraction process. There are various solvents used for extraction of curcumin like methanol, chloroform, acetone. We have used methanol here out of all these solvents. For extraction of curcumin from turmeric we have weighed 20 g of turmeric powder wrapped up in filter paper and kept in Soxhlet apparatus after that add 400 ml of methanol and finish setting up of the Soxhlet apparatus. The temperature was set to 50°C. 4 cycles of extraction was done in 8 days. The yellowish orange extract of curcumin with methanol was obtained in RBF. This was transferred into China dish and kept on water bath for heating to evaporate excess methanol and concentrate the extract. (See Fig 3 and 4) For confirmation of curcumin TLC and other chemical test were done. ^[37] [38]

Fig 1                                                                        Fig 2

    Fig 3                                                  Fig 4

Preformulation Studies –

FT – IR  - FTIR study of individual API that is curcumin from turmeric, garcinol from kokum, stearic acid from kokum butter was done, FTIR study of individual excipient that is carbapol 940 was done. FTIR study of combined API of curcumin, garcinol and stearic was done to check the interactions, FTIR study of all API and Excipient was done to check the interaction between excipient and API

TLC of garcinol –                                                                                    

The extract was reconstituted in methanol and used for TLC.                    

Stationary phase – Silica Gel G plate

Fig 5

Mobile phase used: Toluene: Ethyl acetate: Formic acid (4: 1: 0.5 v/v/v) Chamber saturation: 30 mins.

Procedure – Standard Spot value of Garcinol was compared from reference and the extracted garcinol on Stationary phase. Take the above mobile phase and add to TLC chamber wait for the chamber to saturate then add the spotted TLC plate. Wait for the spots to run. Remove the TLC plate from chamber and dry the eluted plate and visualise. Rf value for std and reference was calculated. The Rf value of garcinol extract was found to be 0.691 which resembles the standard value of garcinol (see fig 5) ^[39]

                                                 Fig 6                                                              Fig 7

TLC for curcumin –

To check the presence of curcumin, 1mg of Curcumin was dissolved in 1ml methanol

Stationary phase – Silica Gel G plate

Mobile phase – Chloroform: Ethanol: Glacial acetic acid – 94:5:1

Procedure - Apply the spot of std curcumin and the extracted curcumin on Stationary phase. Take the above mobile phase and add to TLC chamber wait for the chamber to saturate then add the spotted TLC plate. Wait for the spots to run. Remove the TLC plate from chamber and dry the eluted plate and visualise. Rf value for std and reference was calculated. Three distinct spots of curcumin extract were seen out of which Rf value of 3^rd spot matched with the standard. The Rf value of third spot and curcumin std was found to be 0.65. This confirms the presence of curcumin in the extracted extract (see fig 8)

                                                            Fig 8                                             Fig  9

UV studies for curcumin –

Procedure -

Procedure -

Formulation  Table –   A total number of three batches were prepared for the analysis of gel characteristics  and properties. 

Table no 7: Formulation Table

Batch B2 was found to be most optimized in terms of proper gel and was used for antimicrobial activity and evaluation.

Table no 8: Formulation Table for Batch B2

EVALUATION OF GEL -

1. Organoleptic parameter

This includes evaluation of colour, odour, texture, homogeneity and phase separation of polyherbal gel.

2. pH

The pH of gel formulations was measured using a digital pH meter. A precise amount of 0.25 grams of gel was dispersed in 25 mL of distilled water and left to settle for 2 hours. pH measurements for each formulation were taken three times, and the average values are presented in the table. (See fig 10)

Fig 10

3. Viscosity and rheological study

The viscosity and rheological properties of the gels were analyzed under room temperature conditions using the LV model. Testing was conducted at 12 rpm with a spindle number of 64, ensuring the entire spindle was immersed in the beaker.

4. Spreadability - Spreadability was assessed by placing a polyherbal formulation gel between two glass slides of standard dimensions, sandwiching them together, and applying a weight of 20 grams on the upper slide. Excess gel adhering to the surface of the glass slides was removed, and then the spreadability was determined. The formula used for calculation is (d^2 * π) / 4. (See fig 11)

Fig 11

5. Washability

For washability assessment, formulations were applied to the skin, and the ease and thoroughness of washing with water were manually evaluated.

6. Stability study of polyherbal gel

A stability study was conducted on the gel at 40°C and for a duration of 30 days by keeping it in a preset stability chamber. The evaluation included assessing colour, odour, appearance, texture, and the presence of phase separation to ensure stability.

As a part of study Ph and viscosity of gel was carried out on day 1,30

7. Microbial study of polyherbal gel

Weigh 2% of nutrient agar in a conical flask and dissolve it with distilled water, keep it for cooling and cover the flask with cotton or aluminium. Take four Petri plates, two pipettes and keep it for autoclaving at 120° C for 30 min. For maintaining the sterilized condition keep two burners and do the microbial work in between. Take Whatman   filter paper and prepare disk by sterilizing them. Prepare standard solutions of clindamycin, sample solution (our gel) and a solvent for testing the efficacy of the drug at various concentrations 1μg/ml, 5μg/ml and 7μg/ml. Pour the agar in a sterilized petri plate and keep it un disturbed for 2 minutes. After agar gets solidify take a sterilized inoculating loop and dip into prepared bacterial culture medium, streak the bacterial culture completely over the plate. After streaking place, the disc containing drug solution are kept in the agar medium and kept for a day in a controlled temperature environment. Observe the results on next day and note down the readings. ^[41]

RESULTS AND DISCUSSION –

1. Physicochemical characteristics

2. Chemical tests

Chemical test for Garcinol

Chemical test for curcumin  ^[40]

3. Spectroscopic analysis

Calculation of Dilution factor - DF= 10*10*10*10= 10000

CALCULATION

Concentration of unknown is calculated by substituting value of y i.e. absorbance in the above equation it was found to be 0.443318 microgram Multiplying it by the dilution factor4433.18 microgram of curcumin was present in 0.1 g turmeric powder, as 20 g of turmeric powder was used for extraction 0.886 g of curcumin is present in 20 g turmeric powder

UV Spectra of curcumin                                                                            

0.886 g of curcumin is present in 20 g turmeric powder

4. FTIR study –

Structure of curcumin

IUPAC - (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl) hepta-1,6-diene-3,5-dione

FT IR interpretation for garcinol

IUPAC - (1S,3E,5R,7R)-3-[(3,4-dihydroxyphenyl)-hydroxymethylidene]-6,6-dimethyl-5,7-bis(3-methylbut-2-enyl)-1-[(2S)-5-methyl-2-prop-1-en-2-ylhex-4-enyl]bicyclo[3.3.1]nonane-2,4,9-trione

FT IR interpretation for stearic acid

IUPAC - octadecanoic acid

FT-IR interpretation for Carbapol 940

IUPAC - prop-2-enoic acid

FT-IR interpretation for all API + Carbopol

5. Preformulation

TLC of curcumin -Distance travelled by solvent – 6 cm, Distance travelled by solute – 3.9 cm, Rf value – 0.65

TLC of garcinol- Distance travelled by solvent – 4.2 cm, Distance travelled by solute – 2.5 cm, Rf value – 0.614

6. EVALUATION OF GEL

Discussions - Physicochemical evaluation showed that the formulated gel had skin-compatible properties with a pH of 6.97, viscosity of 12345 cps, and a spreading coefficient of 6.15 cm2

7. Stability study

Discussions - Batch 2 is optimal match because of consistency and texture.

8. Microbial Study

Image of Zone of Microbial inhibition – (See fig 12)

Discussions - Batch 2 i.e. optimal batch show 30 mm2 zone of inhibition on staphylococcus aureus. Kokum butter is suitable for oily and acne-prone skin because of non-comedogenic rating. The polyherbal gel formulation demonstrated antibacterial activity indicating a synergistic effect.

                                    Fig 12  A                                                                      Fig 12 B