INTRODUCTION:

The skin is the largest organ on the body, accounting for about 15% of an adult's total body weight. It performs many important functions, including protecting against external physical, chemical and biological attack, as well as preventing excessive water loss of the body and playing a role in thermoregulation. Continuous skin, mucous membranes lining the surface of the body^1. Hormonal changes, particularly throughout puberty in both sexes, produce various changes in the body, in addition to balanced eating².

Acne comes from the Greek word acme, which meaning "spring of life." Acne is a skin ailment caused by irregularities in the sebaceous glands. Acne vulgaris, which simply translates "common acne," is the most prevalent type of acne. The redness is caused by the skin's inflammation in response to the infection^3,4.

Acne is caused by bacteria such as Propionibacterium acnes, Staphylococcus aureus and Staphylococcus epidermidis multiplying rapidly^5,6. A large range of antibiotics and chemotherapeutic drugs are available as topical or systemic therapy techniques in the clinical management of acne vulgaris on the global market. In mild acne, topical therapy is favoured as the first-line treatment, whereas systemic medication is required in addition to topical therapy in moderate and severe acne ^7,8.

The long-term overuse of antibiotics has led to increased resistance of acne-causing bacteria, namely as Propionibacterium acnes, Staphylococcus aureus and Staphylococcus epidermidis^9,10,11. The development of antibiotic resistance is due to many factors, including the specific nature of the relationship between bacteria and antibiotics, the manner in which antibiotics are used, host characteristics, and environmental factors. To overcome antibiotic resistance, herbal medicines have been extensively studied as an alternative treatment of various diseases^12,13. Literature survey reveals that many polyherbal gels formulated using natural plant which have antimicrobial activity^14-19.

Our current study aimed to discover medicinal plants with anti-acne properties to avoid these side effects and provide natural essence to the skin. Selection of popular herbs to know neem (Azdirachta indica)^20,21, liquorice root (Glycyrrhiza glabra)²², orange peel (Citrus aurantium var. sinensis), and palash (Butea monosperma)²³flower based on the findings that they have multiple pharmacological properties such as antibacterial, antioxidant and anti-inflammatory properties.

MATERIALS AND METHODS:

Plants Material:

Collection and drying of plant parts:

The fresh leaves of Neem (Azadirachta indica), fresh flowers of palash (Butea monosperma) were collected from the local area of Junagadh. The fresh leaves of neem and flowers of palash were dried under shade and were grinded using electronic grinder. Liquorice (Glycyrriza glabra), orange peel (Citrus aurantium var. sinensis) powder and honey were purchased from local market of Junagadh.

Preparation of herbal extracts:

a) Preparation of herbal extracts of dried drugs:

Herbal extracts of dried drugs were prepared by maceration method using rose water as a solvent (1:10). Desired quantities of herbal drugs were weighed and each herb macerated with rose water in flask separately for 3 days. After 3 days content was filtered out using simple filtration method and filtrates were collected in flask separately. The filtrates were evaporated on water bath at 60°C until the desired concentration of the extracts was obtained.

b) Preparation of palash flower extract:

Desired quantity of fresh palash flowers were weighed and boiled in rose water for 30 minutes under reflux. The content was filtered and the filtrate was evaporated on water bath at 60°C until the desired concentration of the extract was obtained and dried using vacuum evaporator^24,25.

Qualitative phytochemical analysis of plant extract:

The neem (Azdirachta indica), liquorice root (Glycyrrhiza glabra) and palash (Butea monosperma) extracts obtained was subjected to the preliminary phytochemical analysis following standard methods by kokate and khandelwal^26,27. The extract was screened to identify the presence or absence of various active principles like phenolic compounds, carbohydrates, flavanoids, glycosides, saponins, alkaloids, protein and amino acid.

Quantification of secondary metabolites:

Total phenolic content estimation:

The total phenolic content was determined using the method of folin ciocalteau reagent. A volume of 2mL of extracts or standard was mixed with 1mL of folin ciocalteau reagent (previously diluted with distilled water 1:10 v/v) and 1mL (7.5g/l) of sodium carbonate. The mixture was vortexed for 15 sec. and allowed to stand for 15 min. for colour development. The absorbance was measured at 650nm using UV-visible spectrophotometer. The total phenolic content was calculated from the standard graph of gallic acid and the results were expressed as gallic acid equivalent (mg/100 mg)²⁶.

Total flavonoids content estimation:

The total flavonoid content was determined using the aluminium chloride colorimetric method. The stock solution of 1mg/mL crude extracts, 50µL of each extract was made up to 1mL with methanol, mixed with 4mL of distilled water and subsequently with 0.3 mL of 5% NaNO2 solution. 0.3 mL of 10% AlCl3 solution was added after 5 min. of incubation and then allowed to stand for 6 min. This was followed by the addition of 2 mL of 1M NaOH solution to the mixture and final volume of the mixture was brought to 10 mL by the addition of double distilled water. The mixture was allowed to stand for 15 min. and absorbance was measured at 510 nm. Quantification was done on the basis of the standard curve of rutin concentration ranging from 50 to 500 mg/mL (r² = 0.999). Total flavonoid content calculated from a calibration curve was expressed as mg of rutin equivalent (RU)/g of dry weight²⁶.

Total alkaloids content estimation:

The plant extract (20 mg) was dissolved in 1mL of 2N HCl and filtered. This solution was transferred to a separating funnel, 5mL of bromocresol green solution and 5mL of phosphate buffer were added. The mixture was shaken with 1, 2, 3 and 4mL chloroform by vigorous shaking and collected in a 10mL volumetric flask and diluted to the volume with chloroform. A set of reference standard solutions of atropine (40, 60, 80, 100 and 120 μg/mL) were prepared in the same manner as described earlier. The absorbance for test and standard solutions were determined against the reagent blank at 470 nm with an UV-visible spectrophotometer. The total alkaloid content was expressed as mg of AE/100 mg of extract²⁶.

c) Formulating anti acne gel:

Sufficient quantity of rose water was taken, glycerine was added to it and mixed properly. The required quantity of carbopol 934/Carboxy Vinyl Polymer 934 was added and stirred continuously, avoiding air entrapment, until a thick gel was formed. The pH was adjusted by adding triethanolamine to get a clear gel. The required quantity of each extract and honey were measured and mixed with the sufficient quantity of rose water. This mixture was added with prepared carbopol 934/Carboxy vinyl polymer gel with moderate stirring. The pH of the gel formulation was again adjusted using triethanolamine²⁶. Various batches of anti- acne gel formulated as per given in table 1. The prepared composition is filled into a suitable container.

Evaluation parameters of anti acne face gel:

Appearance and Consistency:

The physical appearance was visually checked for the texture of polyherbal gel formulations.

Grittiness:

All the formulations were evaluated microscopically for the presence of any appreciable particulate matter which preparation fulfils the requirements of freedom from particular matter and from grittiness as desired for any topical preparation.

Homogeneity:

After the gels have been set in the container, all developed gels were tested for homogeneity by visual inspection. They were tested for their appearance and presence of any aggregates.

Extrudability determination of formulations:

The polyherbal gel formulations were filled into collapsible metal tubes or aluminium collapsible tubes. The tubes were pressed to extrude the material and the extrudability of the formulation was checked^27,28. The values of each formulation are displayed in table 2.

Determination of spreadability:

The spreadability is determined by using spreadability testing apparatus. It consists of wodden block, which is provided by pulley at one end. By this method the spreadability was measured on the basic of ‘slip’ and ‘Drag’ placed on the ground slide the gel was sandwiched between the slide load of 1 kg was kept on slab. So, that gel will get spreaded without air bubbles, Excess of gel was scrapped off. Later on, 20 kg of standard weight was placed in pulley with the help of string attached to hook and time required to move till the end was noted and the length of spreaded gel is also noted The values of each formulation are displayed in table 2. Spreadability was calculated by using formula,

𝑺=𝑴∗𝑳/𝑻

Where,

S= Spreadability

L= Length moved by glass slide.

M= Weight in the pan.

T= Time taken to separate the slide completely from each other²⁹.

Determination of pH:

The pH of the anti-acne gels was determined by digital pH meter. One gram of gel was dissolved in 25mL of distilled water and the electrode was then dipped in to gel formulation until constant reading obtained and constant reading was noted. The measurements of pH of each formulation were replicated two times²⁹. The values of each formulation are displayed in table 4.

Viscosity:

The measurement of viscosity of prepared gels were carried out with brookfield viscometer The measurements was over speed setting of 100 rpm at 25 ºC using brookfield viscometer. The values of each formulation are displayed in table 4.

Table 1. Various Batches for Preparation of Formulation

Batches	F1	F2	F3	F4	F5	F6	F7	F8
Neem extract	1 gm	1 gm	1 gm	1 gm	1 gm	1 gm	1 gm	1 gm
Liquorice root extract	1 gm	1 gm	1 gm	1 gm	1 gm	1 gm	1 gm	1 gm
Orange peel extract	1 gm	1 gm	1 gm	1 gm	1 gm	1 gm	1 gm	1 gm
Palash flower extract	1 gm	1 gm	1 gm	1 gm	1 gm	1 gm	1 gm	1 gm
Carbopol 934	0.5 gm	1 gm	1.5 gm	2 gm	0.5 gm	1 gm	1.5 gm	2 gm
Triethanolamine	0.5 mL	0.5 mL	0.5 mL	0.5 mL	1 mL	1 mL	1 mL	1 mL
Glycerine	1 mL	1 mL	1 mL	1 mL	1 mL	1 mL	1 mL	1 mL
Honey	1 mL	1 mL	1 mL	1 mL	1 mL	1 mL	1 mL	1 mL
Rose water	1 mL	1 mL	1 mL	1 mL	1 mL	1 mL	1 mL	1 mL

Stability Study:

The stability study was performed as per ICH guidelines. The formulated gels were filled in the collapsible tubes and stored at fixed temperature and humidity condition. Stability study of the two formulations were carried out at 40ºC± 2ºC/75%±5% RH temperature and relative humidity for the period of 3 months. The different parameters which were recorded for the change during this period were as follows:

a. Changes in appearance

b. Changes in pH

c. Changes in viscosity

d. Changes in Spreadability³⁰

The values of formulations are displayed in table 5.

RESULT AND DISCUSSION:

Phytochemical analysis of hydroalcoholic extracts of plants showed the presence of flavonoid, Phenol, alkaloids, carbohydrate and saponins while, protein, glycosides and oils and fats were reported to be absent Table 2. Quantitative phytochemical assay was performed by calculating total phenolic content, total flavonoid content and total alkaloid content Table 3. The various characteristics studies of formulation like extrudability, spredability and pH displayed in Table 4.

Table 2. Result of phytochemical screening of extracts

Sr. No	Constituents	*Azadirachta Indica*	*Glycyrriza Glabra*	*Butea Monosperma*
1.	Alkaloids	+ve	+ve	+ve
2.	Glycosides	-ve	+ve	-ve
3.	Flavonoids	+ve	+ve	+ve
4.	Diterpenes	-ve	+ve	-ve
5.	Phenol	+ve	+ve	+ve
6.	Amino Acids	-ve	-ve	-ve
7.	Carbohydrate	-ve	+ve	+ve
8.	Proteins	-ve	-ve	-ve
9.	Saponins	+ve	+ve	-ve
10.	Oils and fats	-ve	-ve	-ve

Table 3. Estimation of total phenolic, flavonoids and alkaloid content

Sr. No.	Estimation Test	Extracts
Sr. No.	Estimation Test	*Azadirachta Indica*	*Glycyrriza Glabra*	*Butea Monosperma*
1	Total alkaloid	0.062	0.246	0.458
2	Total Phenolic	0.672	0.481	0.456
3	Total flavanoids	0.387	0.387	0.213

Table 4: Results of various parameters

Formulation	Extrudability	Spreadability (gm-cm/sec)	pH	Viscosity (cps)
F1	Average	14.23±0.13	6.32± 0.11	3250±10
F2	Average	15.65±0.16	6.02±0.15	3156±15
F3	Average	13.15±0.25	6.25±0.12	3265±18
F4	Average	14.65±0.45	7.75±0.14	3358±20
F5	Average	14.12±0.15	7.95±0.16	3454±25
F6	Average	13.35±0.33	8.15±0.13	3562±22
F7	Good	13.25±0.22	8.00±14	3589±24
F8	Good	12.20±012	7.00±0.10	3685±20

Table 5. Stability study of gels at 40ºC± 2ºC / 75% ± 5% for 3 months

Formulation

Color

Appearance

Spreadability (gm-cm/ sec)

Viscosity

(cps)

Yellowish

Homogenous

14.65±0.45

7.6

3300

Yellowish

Homogenous

12.20±012

6.9

3600

CONCLUSIONS:

The research study, an attempt was made to formulate poly herbal anti acne face wash gel using different natural ingredients like neem, liquorice, orange peel, honey and to evaluate the prepared formulations for the desired parameters. Prepared formulation was evaluated for physical parameters like colour, odour, grittiness, greasiness, pH, consistency, spreadability, foam ability and wash ability. Based on the results we conclude that the prepared formulation can be effectively used for facial care.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENT:

The authors express their sincere thanks to Principal, Teaching and non-teaching Dr. Subhash University, Junagadh, Gujarat, India for providing all facilities to conduct this work.

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Received on 03.06.2022 Modified on 31.10.2022

Research J. Pharm. and Tech 2023; 16(12):5691-5695.

DOI: 10.52711/0974-360X.2023.00920