FORMULATION OF POLYHERBAL VANISHING CREAM AND FACEWASH

Abstract

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.

Specification

Description:FIELD OF THE INVENTION
[0001] The present invention is related to polyherbal vanishing cream, and facewash more particularly using plant materials.

BACKGROUND
[0002] Acne vulgaris suppresses an individual's self-confidence by causing distress with regard to physical appearance, which affects a significant number of individuals during puberty and is delineated by adolescence. Several treatments have been introduced to decrease the aesthetic and psychological problems caused by acne. The topical application of therapeutic agents has been found to be more feasible than hormonal treatment and laser therapy. The ingredients in topical acne treatments, particularly herbs and naturally derived compounds, have received considerable interest as they have fewer adverse effects than synthetic agents.

[0003] Herbal creams offer several advantages over other creams. The majority of existing creams which has prepared from drugs of synthetic origin, such as acyclovir, triamcinolone, calcipotriene, mometasone, extras gives fairness to face, but it has several side effects such as itching or several allergic reactions. Herbal creams do not have any of these side effects, without side effects it gives the fairness look to skin. Method carried out to prepare herbal cream was very simple. Firstly, oil phase was prepared, the mixture of stearic acid (17%), potassium hydroxide (0.5%), sodium carbonate (0.5%) were melted at 700C. Secondly aqueous phase was prepared, mixture of alcoholic extract of crude drugs, including rhizomes of kachora plant, fruits of nagarmotha, fruits of pimpali, fruits of nutmeg, seeds of Jawas plant, rhizomes of turmeric, wheat grains and cereals of urid and harbhara (4.5%), glycerin (6%), perfume (0.5%), water (71%) heated at 70Oc. Then aqueous phase was added into the oil phase at 70Oc with continuous stirring. Now, once the transfer was completed it was allowed to come at room temperature all the while being stirred. Perfume was added at last just before the finished product was transferred to suitable container. The above prepared herbal cream was evaluated. The physical parameters such as pH, homogeneity by visual and by touch, appearance (color), rubout (spread ability, wetness), type of smear, emolliency were determined. Further studies are needed to investigate this formulation for its performance.

[0004] Mast cells participate in allergy and inflammation by secreting inflammatory mediators such as histamine and proinflammatory cytokines. Flavonoids are naturally occurring molecules with antioxidant, cytoprotective, and anti-inflammatory actions. However, effect of flavonoids on the release of histamine and proinflammatory mediator, and their comparative mechanism of action in mast cells were not well defined. Here, we compared the effect of six flavonoids (astragalin, fisetin, kaempferol, myricetin, quercetin, and rutin) on the mast cell-mediated allergic inflammation. Fisetin, kaempferol, myricetin, quercetin, and rutin inhibited IgE or phorbol-12-myristate 13-acetate and calcium ionophore A23187 (PMACI)-mediated histamine release in RBL-2H3 cells. These five flavonoids also inhibited elevation of intracellular calcium. Gene expressions and secretion of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1beta, IL-6, and IL-8 were assessed in PMACI-stimulated human mast cells (HMC-1). Fisetin, quercetin, and rutin decreased gene expression and production of all the proinflammatory cytokines after PMACI stimulation. Myricetin attenuated TNF-alpha and IL-6 but not IL-1beta and IL-8. Fisetin, myricetin, and rutin suppressed activation of NF-kappaB indicated by inhibition of nuclear translocation of NF-kappaB, NF-kappaB/DNA binding, and NF-kappaB-dependent gene reporter assay. The pharmacological actions of these flavonoids suggest their potential activity for treatment of allergic inflammatory diseases through the down-regulation of mast cell activation.

SUMMARY
[0005] 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, characterization of responsible flavonoid.

DESCRIPTION OF THE FIGURES:
[0006] Figure 1a: illustrates ixora coccinea plant.

[0007] Figure 1b: illustrates ixora coccinea leaves.

[0008] Figure 2a: illustrates kalanchoe pinnata plant.

[0009] Figure 2b: illustrates kalanchoe pinnata leaves.

[0010] Figure 3a: illustrates millettia pinnata plant.

[0011] Figure 3b: illustrates millettia pinnata leaves.

[0012] Figure 4a: illustrates vitex nigundo plant.

[0013] Figure 4b: illustrates vitex nigundo leaves.

[0014] Figure 5a: illustrates andrographis paniculata plant.

[0015] Figure 5b: illustrates andrographis paniculata leaves.

[0016] Figure 6a: illustrates punica granatum plant.

[0017] Figure 6b: illustrates punica granatum leaves.

[0018] Figure 7a: illustrates cymbopogon flexuosus plant.

[0019] Figure 7b: illustrates cymbopogon flexuosus leaves.

[0020] Figure 8a: illustrates bauhinea variegate plant.

[0021] Figure 8b: illustrates Bauhinea variegate leaves.

[0022] Table 1: illustrates Preliminary phytochemical screening of alcoholic extracts.

[0023] Table 2: illustrates total flavonoid content of ethanolic extracts.

[0024] Figure 9: illustrates Standard calibration curve of quercetin for flavonoids.

[0025] Table 3: illustrates formulation of vanishing cream.

[0026] Table 4: illustrates evaluation parameters of herbal vanishing cream.

[0027] Table 5: illustrates Formulation of face wash.

[0028] Table 6: illustrates Evaluation parameters of face wash.

[0029] Fig. 10a: illustrates Prepared vanishing cream.

[0030] Fig. 10b: illustrates Prepared face wash.

[0031] Table 7: illustrates Antibacterial and antifungal activity of vanishing cream.

[0032] Table 8: illustrates Antibacterial and antifungal activity of face wash.

[0033] Figure 11: illustrates Evaluation of Antibacterial and antifungal activity of vanishing cream.

[0034] Fig. 12: illustrates Antibacterial and antifungal activity of face wash.

DETAILED DESCRIPTION OF THE INVENTION:
[0035] 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 & 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).

[0036] 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 5 g 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].

[0037] Quantative determination of flavonoid: The extract (1.5 ml) was added to 1.5 ml 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 367 nm 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

[0038] Formulation of poly herbal vanishing cream: The alcoholic extract was prepared by adding 100 ml ethanol to combined selected powdered crude drugs (Table 3) 5 g 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).

[0039] 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 5 g 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 (3 g) was placed in between glass slides. The upper slide is movable and the lower slide was made immovable, firmly fixed to the stand. 100 g 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 50 g weight was added to one side of the slide and the slide was pulled till it covers a distance of 10 cm. The time required to separate two glass slides by a distance of 10 cm 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 20 rpm 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.

[0040] Formulation of poly herbal face wash: Desired quantities (5 g) of herbal drugs (Table 5) were weighed and each herb is separately macerated with rose water (100 ml) 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).

[0041] 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 & 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).

[0042] Evaluation of anti-bacterial activity of prepared formulation: 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 37 oC 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. The plates were allowed to dry for 3-5 min to remove the excess moisture in laminar air flow chamber. 50 ul aliquots of each test and standard marketed cream and facewash formulations with different concentrations (0.1 mg/10 ml, 0.2 mg/10 ml, 0.3 mg/10 ml) 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 37 oC. After 24 h 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).

[0043] 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). 50 µl aliquots of each test and standard marketed cream and facewash formulations with different concentrations (0.1 mg/10 ml, 0.2 mg/10 ml, and 0.3 mg/10 ml) 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).

[0044] 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 36 mg/g followed by lemon grass 35 mg/g equivalent to quercetin among the plant extracts. The plant extracts of jungle geranium (Ixora), green gram, nirgundi contain total flavonoids 34 mg/g equivalent to quercetin. The plant extracts of bryophyllum, liquorice, kalmegh, kachnar contain total flavonoids 33 mg/g equivalent to quercetin. The plant extracts mustard, amla, sandalwood contain total flavonoids 32 mg/g equivalent to quercetin. Pomegranate and myrobalan were found to contain less amount of flavonoids 31 mg/g equivalent to quercetin when compared to other plant extracts (Table 2).

[0045] 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].

[0046] 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).

[0047] The polyherbal face wash (50 ml) 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. 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).

[0048] 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 7and 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 0.3 mg/10ml concentration. The prepared cream exhibited better antibacterial activity against gram negative E. coli (11 mm) than marketed formulation (9 mm). It also exhibited better antifungal activity (12 mm) than marketed formulation (10 mm). 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).

[0049] 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 0.3 mg/10 ml. The formulation exhibited better antibacterial and antifungal activity than marketed formulation at 0.3 mg/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.
, Claims:CLAIMS:
I/WE CLAIM:
1.A method of evaluating polyherbal vanishing cream, and face wash, comprising:
a sandal wood;
a honey;
a seeds of mustard;
a green gram;
a fruits of myrobalan;
an amla;
a lemon,
a liquorice root powder;
a camphor oil;
a lemon grass oil;
a xanthan gum
a himalaya kesar;
an alfalfa face cream;
a patanjali neem;
a tulsi facewash;
a kalanchoe;
bryophyllum;
a nirgundi;
a kalmegh;
a pomegranate;
a lemon grass;
a kachnar (
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;
all the plant materials of 5 g each were soaked in alcohol separately for seven days, filtered, filtrates
were concentrated to get residues, dried and kept in refrigerator;
whereby the extract (1.5 ml) was added to 1.5 ml of 2% methanolic AlCl3 (Finar);
the mixture was vigorously shaken on centrifuge for 5 minutes at 200 rpm and the absorbance was
read at 367 nm after 10 minutes of incubation;
the alcoholic extract was prepared by adding 100 ml ethanol;
whereby combined selected powdered crude drugs 5 g each, into 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%) was 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; and perfume (0.5 %) was added at last just before the finished product was transferred to suitable container.

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