Formulation, Phytochemical, Physical, Biological Evaluation of Polyherbal Vanishing Cream, and Facewash

 

Vani Mamillapalli^1*, Mounika Katamaneni¹, Vamsi Meghana Tiyyagura¹, Praneetha Kanajam¹,

Aslesha Padmavathi Namagiri¹, Haripriya Thondepu¹, Bhavani Appikatla¹,

Bhavana Devangam¹, Padmalatha Khantamneni²

¹Department of Pharmacognosy and Phytochemistry, Vijaya Institute of Pharmaceutical Sciences for Women, Enikepadu-521108, Vijayawada, Krishna (Dt.), Andhra Pradesh, India.

²Department of Pharmacology, Vijaya Institute of Pharmaceutical Sciences for Women, Enikepadu-521108, Vijayawada, Krishna (Dt.), Andhra Pradesh, India.

 

ABSTRACT:

The present study aims at formulation of polyherbal vanishing cream and face wash, determination of total flavonoid content of herbs used, evaluation of formulations for various physical parameters followed by antibacterial and antifungal activities. The problems of premature ageing, damage due to irradiation, loss of pigmentation, moisture, nourishment, and acne can be solved by supplementation of health benefits provided by selected multiple herbs in the formulations. The use of polyherbal vanishing cream and face wash promotes skin health and beauty. The selected nine herbs in vanishing cream and eleven herbs in face wash formulations contain good amount of flavonoids capable of protecting skin against damage. The total flavonoid content of herbs used was determined, followed by evaluation of various physical parameters such as pH, viscosity, spreadability and compared with marketed formulations. Further, the formulations were tested against gram positive S.aureus, gram negative E.coli, and fungus C.albicans which are common associates of acne and cosmetic appliances. The results indicated that both the formulations displayed better antibacterial and antifungal activities than marketed formulations. Therefore, they can be tested further for their performance and quality control parameters.

 

 

 

INTRODUCTION:

Skin, the largest human organ is the first line of defense for external exposure to UV radiations, dust, environmental bacteria, fungi etc., which cause ageing and other infections. Skin has been reported to reflect the general inner-health status and aging [1].

UV exposure is known to negatively affect immune system functions, cause erythema, changes in skin elasticity, structure (roughness, scaling, volume, and wrinkles), trans epidermal water loss, and skin cancers. The adverse effects of ultraviolet radiation on the skin, are caused by excessive generation of reactive oxygen species [2]. Prevention is the best and most effective way to work against skin aging effects [3,4]. The best prevention strategy against the harmful action of free radicals is a balanced nutritional diet containing rich anti-oxidative flavonoids [5]. Acne vulgaris is an extremely common disorder of skin which includes follicular hyper proliferation, increased sebum secretion and colonization of organism. Acne is not infectious disease, generally caused by various factors such as environmental pollution, harmful chemicals, bacteria, fungi and treated by different mechanisms such as controlling the sebum production, use of antibiotics against bacteria and fungi that are responsible and sometimes anti-inflammatory agents [6]. Natural or synthetic sponges used in daily hygiene and in removing make-up act as reservoirs and vehicles in the transmission of bacterial species such as Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli [7]. Some strains of E. coli are pathogenic, which means that they can cause infection that leads to illnesses, such as diarrhea, urinary tract infections, respiratory illness, and pneumonia [8]. S. aureus is the most dangerous of many common staphylococcal bacteria species. These gram-positive, sphere-shaped (Coccal) bacteria often cause skin infections but can also cause pneumonia, heart valve infections, and bone infections [9]. Candida albicans, a type of yeast fungus found in moist areas of human body such as mouth, skin and genitals. Yeast infections on the skin are called cutaneous candidiasis which may result due to lack of hygiene, excessive sweating, and use of harsh facial products, rough scrubbing, and facial tissue irritation. The skin rash due to candidiasis leads to itching, ulcers, dry skin patches, burning, and pimples [10]. Facial skin is delicate, regular use of ordinary soaps can cause loss of moisture in the skin. A low fat moisturizer that disperses into the skin is called as a vanishing cream [11]. The cream acts as a moisturizer, anti-bacterial, anti-fungal agent, and fairness expert, removes ageing signs, and provides required nutrients to the skin. A face wash is a mild cleanser that does the vital job of cleansing the skin, prevents acne, anti-wrinkle, anticancer, makes germ free, smooth, fresh, moisturizes the horny layer without causing any harshness to the skin, and makes to look younger [12].

 

Frequently researched antioxidants such as flavonoids, have been referred as agents capable of promoting skin health and beauty The use of flavonoids in cosmetic formulation provides both medicinal and cosmetic benefits [13].  They have been reported to possess potent anti-oxidant properties and have been widely used in the skin care industry either as topically applied agents or oral supplements in an attempt to prolong youthful skin appearance. Several thousand molecules having a polyphenol or flavonoid structure have been identified in plants being generally involved in defense against UV radiation, UV-induced skin inflammation, oxidative stress, DNA damage, and risk of skin cancers or aggression by pathogens [14]. These   polyphenolic   compounds or flavonoids   have   proven  to improve  skin  microcirculation, protection of the blood vessels, [15] inhibit  the  synthesis  of  melanin [16],  inhibit  the  release  of inflammatory  mediator - histamine [17], reduce erythema, inhibit  the  platelet  aggregation [18] and thus produce beautifying effects on the skin surface. The contribution of the traditional preparations, which are normally polyherbal, is increasing day by day because of the general impression that these products are safe with multiple applications; while the single-molecule based modern drugs used in allopathic system can cause severe adverse effects [19]. Topical polyherbal formulations, containing flavonoids are used against acne, skin tan, and other skin problems due to their anti-microbial, anti-inflammatory and anti-oxidant activities. Skin, the organ of beauty has been an interesting research fields but also a common field of interest for humans throughout the years, from ancient times to nowadays. With this view the medicinal plants for the study were selected based on literature review. The current research has been planned to carryout in vitro flavonoid content of the selected plant species, formulate, and evaluate the polyherbal vanishing cream and face wash for pharmaceutical parameters followed by antibacterial and antifungal activities.

 

MATERIALS AND METHODS:

The plant materials were collected collected in the month of December during afternoon from local grounds of Prasadampadu and Enikepadu, Vijaya Institute of Pharmaceutical Sciences for Women, Enikepadu, coordinates 16°32′45″N 80°34′12″E of Vijayawada rural region, Krishna district, Andhra Pradesh, India. Dr. P. Satya Narayana Raju, Plant Taxonomist, Department of Botany and Microbiology, Acharya Nagarjuna University, Guntur, Andhra Pradesh, India, identified and authenticated the plant specimens namely jungle geranium Ixora coccinea, negundo Vitex nigundo, kalmegh Andrographis paniculata, pomegranate Punica granatum, bryophyllum Millettia pinnata, pongam Kalanchoe pinnata, lemon grass Cymbopogon flexuosus and kachnar Bauhinea variegate (Figure 1a-8b). The plant materials sandal wood, honey, seeds of mustard, green gram, fruits of myrobalan, amla, and lemon, were purchased from the local market. The liquorice root powder, camphor oil, lemon grass oil, and xanthan gum were collected from the department of Pharmacognosy, Vijaya Institute of Pharmaceutical Sciences for Women, Enikepadu, Vijayawada. Marketed formulations used were Himalaya kesar, alfalfa face cream and Patanjali neem, tulsi facewash. The collected plant materials used in the formulation of vanishing cream are jungle geranium (Figure 1a and 1b),  kalanchoe (Figure 2a and 2b), and bryophyllum (Figure 3a and 3b).The collected plant materials used in the formulation of face wash are nirgundi (Figure 4a and 4b), kalmegh (Figure 5a and 5b), pomegranate (Figure 6a and 6b), lemon grass (Figure 7a and 7b), and kachnar (Figure 8a and 8b).

 

 

Fig. 1a: Ixora coccinea plant

 

  

Fig. 1b: Ixora coccinea leaves

 

Fig. 2a: Kalanchoe pinnata plant

 

 

Fig. 2b: Kalanchoe pinnata leaves

 

 

Fig. 3a: Millettia pinnata plant

 

Fig. 3b: Millettia pinnata leaves

 

 

Fig. 4a: Vitex nigundo plant

 

 

Fig. 4b: Vitex nigundo leaves

 

 

Fig. 5a: Andrographis paniculata plant

 

 

Fig. 5b: Andrographis paniculata leaves

 

 

Fig. 6a: Punica granatum plant

 

 

Fig. 6b: Punica granatum leaves

 

 

Fig. 7a: Cymbopogon flexuosus plant

 

 

Fig. 7b: Cymbopogon flexuosus leaves

 

 

Fig. 8a: Bauhinea variegate plant

 

Fig. 8b: Bauhinea variegate leaves

 

Preparation of extracts and preliminary phytochemical screening:

The leaves, seeds and fruits were cleaned of debris and other vegetative parts, washed properly with water, dried and powdered using an electric mixer, sieved and stored in an air tight container for further use. All the plant materials 5g each were soaked in alcohol separately for seven days, filtered, filtrates were concentrated to get residues, dried and kept in refrigerator.  Further, all the selected plant materials were subjected to preliminary phytochemical screening using standard methods (Table 1) [20,21,22].

 

Table 1: Preliminary phytochemical screening of alcoholic extracts

Al-Alkaloid; Ta-tannin; Fl-Flavonoid;

Sa-Saponin; St-Steroid;Tr-Triterpene

 

Quantative determination of flavonoids:

The extract (1.5ml) was added to 1.5ml of 2% methanolic AlCl₃ (Finar). The mixture was vigorously shaken on centrifuge (Lab India) for 5 minutes at 200 rpm and the absorbance was read at 367nm after 10 minutes of incubation  (Bio-tech). Quercetin (Sigma-Aldrich) was used as standard for the calibration curve (Figure 9, Table 2). The assay was carried out in triplicate [23].

 

C = c.V/m

 

C –   Total phenolic compounds mg/g of plant extract

c –    The concentration of standard established from the calibration curve mg/ml

V –   The volume of extract in ml

m -   The weight of pure plant extract

 

Table 2: Total flavonoid content of ethanolic extracts

Values represent mean ± S.D.of three parallel measurements

.

 

Fig. 9: Standard calibration curve of quercetin for flavonoids

 

Formulation of poly herbal vanishing cream:

The alcoholic extract was prepared by adding 100ml ethanol to combined selected powdered crude drugs (Table 3) 5g each, in to a conical flask, capped with aluminum foil followed by maceration for 5 days. The oil phase was prepared by adding stearic acid (17%), potassium hydroxide (0.5%), sodium carbonate (0.5%) were taken into a porcelain dish and melted at 70ºC. The oil phase was prepared by adding alcoholic extract of crude drugs (4.5%), glycerin (6%), and water (71%) into another porcelain dish and heated at 70ºC. The aqueous phase was added to the oil phase by constant stirring at 70ºC. It was allowed to cool to room temperature, by constant stirring. Perfume (0.5%) was added at last just before the finished product was transferred to suitable container [24] (Table 3, Figure 10a).

 

Evaluation of vanishing cream for physical parameters:

The vanishing cream was evaluated for various parameters as per official guide lines [25,26,27] and compared with standard marketed formulation. The appearance of the cream was judged by its color, pearlescence, and roughness [24]. The pH of the suspension was determined by using digital pH meter (India Mart) at 27⁰C by dispersing 5g of the sample in 45 ml of water [19]. The formulation was tested for the homogeneity by visual appearance and touch [24]. Spreadability may be expressed by the extent of the area to which the topical application spreads when applied to the affected parts on the skin. The spreadability value also determines therapeutic efficiency of the formulation. Lesser the time taken for separation of two slides, better the spreadability [28]. Hence, it was found necessary to determine the spreadability of the formulation. The most widely used method for determining the spreadability of semisolid preparations is parallel plate method. A modified laboratory apparatus was used to evaluate spreadability. The setup consists of two glass slides placed on a tripod stand.  Excess of cream (3g) was placed in between glass slides. The upper slide is movable and the lower slide was made immovable, firmly fixed to the stand. 100g weight was placed on the slides for a period of 5 min to compress the cream to uniform thickness. The excess cream was scrapped off. Then 50g weight was added to one side of the slide and the slide was pulled till it covers a distance of 10cm. The time required to separate two glass slides by a distance of 10cm was taken as a measure of spreadability and was noted in seconds. A shorter interval indicates better spreadability. Spreadability was calculated by using the formula given below. The determinations were carried out in triplicate and the average of three readings was recorded. The results were given in the table 4, and figure 10a [29].

 

S=m.l/t

 

Where,

S=Spreadability, m=Weight tied to upper glass slide, l=Length of glass slide, t= Time taken to separate them.

 

Wetness was determined by applying cream on skin surface of human volunteer. Type of smear was determined by applying the cream on the skin surface of human volunteer. After application of cream, the type of film or smear formed on the skin was checked [30]. Emolliency, and amount of residue left after the application of fixed amounts of cream along with slipperiness was checked. The viscosity determinations were carried out at 25ºC using a Brookfield Viscometer (DV II+ Pro model), spindle number S-64 at 20rpm speed. The determinations were carried out in triplicate and the average of three readings was recorded [19]. The type of emulsion of the prepared vanishing cream was determined by dilution test and dye solubility test. In the dilution test the emulsion (cream) was diluted with water to determine the type of emulsion. The emulsion on dilution with water, was stable as water is the dispersion medium indicating o/w type of emulsion. In the dye solubility test the emulsion (cream) was mixed with a water soluble dye (Amaranth) and observed under the microscope. The continuous phase appeared red, indicating o/w type emulsion as water is in the external phase and the dye will dissolve in it to give color [19] (Table 4).

 

Formulation of poly herbal face wash:

Desired quantities (5g) of herbal drugs (Table 5) were weighed and each herb is separately macerated with rose water (100ml) in conical flask with moderate shaking for 3 days [28]. After 3 days filtration of the extracts was carried out two times. The extracts were evaporated on water bath at 60ºC temperature to a desired consistency. The required quantity of gelling agent i.e. xanthan gum (Table 5) was weighed accurately and dissolved in hot rose water (not more than 60ºC; 50% weight of the batch size) with moderate stirring. Air entrapment was avoided and allowed to soak overnight. Required quantity of lemon juice was dissolved in desired amount of honey by gentle stirring. The required amount of concentrated herbal extracts were added to the remaining amount of rose water and mixed with above honey mixture by gentle stirring. This was finally mixed with previously soaked xanthan gum formulation. Prepared formulation was filled in a suitable container and labeled accordingly (Table 5, and  Figure 10b).

 

Table 3. Formulation of vanishing cream

 

Table 4. Evaluation parameters of herbal vanishing cream

 

 

Table 5. Formulation of  face wash

Qty-Quantity

 

Evaluation of face wash for physical parameters:

The prepared face wash was evaluated for various parameters as per official guide lines [25,26, and 27] and compared with standard marketed face wash [53]. Physical parameters such as colour, appearance and consistency were checked visually. Formulations were applied on the skin then washed with water and visually checked for its washability nature [54]. pH of 1 % aqueous solution of the formulation was measured by using a calibrated digital pH meter at constant temperature [55]. The viscosity of face wash was determined by using Brookfield Viscometer. The Values Obtained for sample is noted and compared with marketed formulation. Spreadability was determined by parallel plate method [28,29]. The face wash was applied on left hand dorsal surface of 1 sq. cm and observed in time interval 1 to 2 h for irritant reactions if any [56] (Table 6).

 

Table 6. Evaluation parameters of face wash

 

 

 

 

 

Evaluation of anti-bacterial activity of prepared formulations:

The micro-organisms gram-positive bacteria Staphylococcus aureus (NCIM 2794) and gram-negative bacteria Escherichia coli (NCIM 2256) were selected to test the anti-bacterial activity i.e. ability to inhibit the growth of microbes in the formulated vanishing cream (Table 7 and Figure 11) and face wash (Table 8 and Figure 12). Prior to testing, each of the bacterial strain was cultured in nutrient agar plates and incubated for 18 to 24 hrs at 37ºC to obtain colonies. After overnight incubation, colonies were selected with a sterile disposable inoculating loop and transferred to a glass tube of sterile physiological saline and mixed thoroughly. Each bacterial suspension turbidity was compared with 0.5 Mc Farland standard solution which produces turbidity equivalent to 1.5×10⁸ CFU/ml (Colony forming units). Antimicrobial susceptibility was tested by using well-diffusion method (National Committee for Clinical Laboratory Standards). The formulations were tested on Mueller Hinton plates to detect the presence of bacterial growth. All plates were inoculated with the test bacteria which have been previously adjusted to the 0.5 Mc Farland standard solution turbidity by the pour plate method.

 

Table 7: Antibacterial and antifungal activity of vanishing cream

Mktd- Marketed

 

The plates were allowed to dry for 3-5 min to remove the excess moisture in laminar air flow chamber. 50ul aliquots of each test and standard marketed cream and facewash formulations with different concentrations (0.1 mg/10ml, 0.2mg/10ml, 0.3mg/10ml) were dispensed into each well after the inoculation of the plates with bacteria. The plates were sealed, labelled, and placed in an incubator set at 37ºC. After 24h of incubation, each plate was examined for inhibition zones. Inhibition zone reader was used to measure the inhibition zones in millimeters [57] (Figure 11 and Figure 12).

 

Evaluation of anti-fungal activity of prepared formulations:

The fungal organism Candida albicans (ACTM 2091) was selected for the study of anti-fungal activity of prepared vanishing cream and face wash formulations. The fungal organism was cultured in subouraud dextrose agar (SDA) medium at  22ºC. Colonies were observed after 5 days, and were diluted to 0.5 Mc Farland which is equal to 1.5×10⁸ CFU/ml. All plates were inoculated with the test fungi which have been previously adjusted to the 0.5 Mc Farland standard solution by pour plate method in SDA medium. Anti-fungal susceptibility was tested by using well-diffusion method (National Committee for Clinical Laboratory Standards). 50µl aliquots of each test and standard marketed cream and facewash formulations with different concentrations (0.1 mg/10ml, 0.2mg/10ml, and 0.3mg/10ml) were dispensed into each well after the inoculation of the plates with fungi. The plates were sealed, labelled, and placed in an incubator set at 22°C. After 5 days of incubation, each plate was examined for inhibition zones. Inhibition zone reader was used to measure the inhibition zones in millimeters [57] (Figure 11 and figure 12).

 

Table 8: Antibacterial and antifungal activity of face wash

Mktd- Marketed

 

RESULTS AND DISCUSSION:

The results of the preliminary phytochemical screening study indicate that tannins and flavonoids were found to be present in all the selected plant extracts (Table 1). The ethanolic extract of karanj was found to contain highest amount of total flavonoids 36mg/g followed by lemon grass 35mg/g equivalent to quercetin among the plant extracts. The plant extracts of jungle geranium (Ixora), green gram, nirgundi contain total flavonoids 34mg/g equivalent to quercetin. The plant extracts of bryophyllum, liquorice, kalmegh, kachnar contain total flavonoids 33mg/g equivalent to quercetin. The plant extracts mustard, amla, sandalwood contain total flavonoids 32mg/g equivalent to quercetin. Pomegranate and myrobalan were found to contain less amount of flavonoids 31mg/g equivalent to quercetin when compared to other plant extracts (Table 2).

 

Flavonoids are indispensable components in a variety of pharmaceutical, medicinal, neutraceutical, and cosmetic applications. Studies have indicated that quercetin helps to to inhibit the production of superoxide radicals [58, 59], suppress lipid peroxidation, [60], a process  responsible for inflammation, along with ageing [61,62]. Serum flavonoid content of flavonoids improves capillary blood flow to the dermis. Dermal microcirculation causes increased cutaneous blood flow which may contribute to enhanced delivery of oxygen and nutrients to the skin, a proposed impact of flavonoids on skin health [63]. This effect leads to improved skin structure, texture, and water homeostasis. Flavonoids reduce the UV-induced DNA damage, by promoting DNA repair. Topical application of flavonoids may protect skin by absorbing UV rays before they can interact with and damage cellular components, thereby providing a sunscreen effect [64]. The prepared formulations comply with parameters as per official guidelines [25,26,27] (Table 4 and table 6). The formulations were compared with marketed formulations (Himalaya kesar, alfalfa face cream). The appearance of formulated and marketed vanishing creams was smooth (Table 4 and figure 10a). The formulated cream was in creamish white, the marketed cream was in pinkish white colour. The odour of both the creams was found to be pleasant. The pH value of formulated cream was 6.3 whereas of the standard cream was 6.1. Both the formulations were elegant by visual and touch examination. The spreadability values were 8 sec and 6 sec for formulated and marketed creams. They were found to be non-greasy and emollient. The viscosity measurements were 27025cps and 25623cps (centipoise). They were both o/w type formulations.

 

The results indicate that the formulated cream passes the physical parameter tests as per official guidelines [25, 26,27] and further it was compared with marketed formulation (Table 4). The polyherbal face wash (50ml) was formulated (Table 5 and figure 10b) as per the prescribed procedure and evaluated for various physical parameters according to official guidelines [25, 26, 27]. The formulated polyherbal face wash was evaluated for physical parameters (Table 6) and compared with marketed formulation (Patanjali neem and tulsi face wash). The results indicate that the formulated face wash was in greenish brown colour. The marketed formulation was in green colour. The formulations exhibited good consistency and washability. The pH values measured were 5.2 for formulated face wash and 4.7 for marketed face wash.

 

 

 

Fig. 11 Evaluation of Antibacterial and antifungal activity of vanishing cream

 

Fig. 12: Antibacterial and antifungal activity of face wash

 

 

The viscosity values measured were 1520 cps and 1690 cps  for  formulated and  marketed  formulations.  Both the formulations exhibited good spreadability and no irritation. The formulated face wash obeys the physical parameter tests as per Pharmaceutical guidelines  prescribed, the results were compared to marketed formulation (Table 6). The formulated polyherbal vanishing cream was evaluated for antibacterial activity against gram positive S. aureus, and gram negative E. coli bacteria. The antifungal activity was tested against C. albicans. The results were compared with marketed formulation (Table 7 and Figure 11). The results indicate that prepared formulation of polyherbal vanishing cream showed better antibacterial and antifungal activities than marketed formulation. It showed maximum growth of inhibition at 0.3mg/10ml concentration. The prepared cream exhibited better antibacterial activity against gram negative E. coli (11mm) than marketed formulation (9 mm). It also exhibited better antifungal activity (12mm) than marketed formulation (10mm). The results of antibacterial and antifungal activities of the prepared cream formulation are in line with the findings of Eichie et al., 2011 [65]. Therefore, it can be quoted that the prepared formulation acts as an effective antibacterial, antifungal vanishing cream (Table 5; Figure 6). The formulated polyherbal face wash was evaluated for antibacterial activity against gram positive S. aureus, and gram negative E.coli bacteria. The antifungal activity was tested against C. albicans. The results were compared with marketed formulation (Table 8 and figure 12). The prepared formulation showed maximum antibacterial and antifungal activities at 0.3mg/10ml. The formulation exhibited better antibacterial and antifungal activity than marketed formulation at 0.3mg/10ml. The formulation showed better results towards gram positive S.aureus. It exhibited activity equivalent to marketed formulation against E.coli. Therefore, the results form an evidence to quote that the formulation acted as an effective antibacterial, antifungal facewash compared to marketed face wash. The results of antibacterial [66] and antifungal activities [65] of prepared polyherbal facewash formulation are comparable to the studies of Amgad et al., 2015 and Eichie et al., 2011 [66,65]. The exhibited antibacterial and antifungal activities  may be attributed to the presence of flavonoids in the prepared formulations.

 

CONCLUSION:

In the current study polyherbal vanishing cream and face wash were formulated, evaluated for total flavonoid content, various physical parameters, antibacterial and antifungal activities. The results indicated that the formulations passed the tests for pharmaceutical physical parameters and exhibited better antimicrobial activities. The prepared polyherbal formulations nourish, moisturize, protect the skin against premature ageing, irradiation, and acne may be due to the presence of flavonoids. The prepared formulations  may be further studied their performance, quality control tests followed by  isolation, characterisation of responsible flavonoid,

 

ACKNOWLEDGEMENT:

The   authors are thankful to Vijaya Institute of Pharmaceutical Sciences for Women for providing infrastructural facilities, encouragement and support.

 

CONFLICT OF INTEREST:

The authors declared no conflict of interest.

 

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Received on 17.04.2020          Modified on 08.05.2020

Accepted on 25.05.2020     ©AandV Publications All right reserved