INTRODUCTION:

Acne vulgaris is a disease of the pilosebaceous follicle characterized by non-inflammatory (open and closed comedones) and inflammatory lesions (papules, pustules, and nodules).

Acne vulgaris is an extremely common skin disorder that affects areas containing the largest oil glands, including the face, back, and trunk. Propionibacterium acnes (P. acnes), an anaerobic pathogen, plays an important role in the pathogenesis of acne. It is implicated in the development of inflammatory acne by its capability to activate complements and by its ability to metabolize sebaceous triglycerides into fatty acids, which chemotactically attract neutrophils. Although not a serious threat to general health, acne is one of the most socially distressing skin conditions, especially for adolescents, who must deal with a disfiguring disease that erupts just when sexual maturity makes them most sensitive about their appearance. Moreover, severe acne can lead to permanent scarring of the skin that carries the social distress throughout adulthood.

For many years, antibiotics have been used to treat acne vulgaris. However, antibiotic resistance has been increasing in prevalence within the dermatologic setting. The development of antibiotic resistance including the specific nature of the relationship of bacteria to antibiotics, how the antibacterial is used, host characteristics, and environmental factors. To overcome the problem of antibiotic resistance, medicinal plants have been extensively studied as alternative treatments for diseases. So our aim and objective to develop safe and effective polyherbal formulation for effective management of acne.

MATERIALS AND METHODS:

Plant material collection and authentication

The plant Cuscuta reflexa was collected from local area of Bhopal (M.P.) in the month of January, 2016 and seeds of Phoenix Sylvestris Roxb purchased from local market of Bhopal.

Extraction of plant material

Dried powdered whole plant material of Cuscuta reflexa and seeds of Phoenix Sylvestris Roxb has been extracted with Ethanol using Soxhlet’s apparatus for 48 hrs, filtered and dried using vaccum evaporator at 40⁰C.

Determination of percentage yield

The percentage yield of each extract was calculated by using following formula:

Weight of Extract

Percentage yield = -------------------------------------- x 100

Weight of powder drug Taken

Phytochemical Screening(Kumar et al., 2012)

The chemical tests were performed for testing different chemical groups present in extracts.

Total Phenolic content estimation: (Ali et al., 2012)

Principal: The total phenolic content of the extract was determined by the modified Folin-Ciocalteu method.

Total flavonoids content estimation (Vabkova et al., 2011)

Principal:

Determination of total flavonoids content was based on aluminium chloride method

Qualitative chromatographic Analysis

Thin Layer Chromatography

Thin layer chromatography: T.L.C. is based on the adsorption phenomenon. In this type of chromatography mobile phase containing the dissolved solutes passes over the surface of stationary phase.

Steps involved in T.L.C.

1) Preparation of plates

2) Activation of plates

3) Preparation and saturation of chamber

4) Sample application and development

5) Detection and calculation of R_f Value

TLC of extract was performed and reported.

Quantitative HPLC Study

Identification of Marker Compound (Gallic acid) by HPLC

Chromatographic conditions

The chromatographic analysis was performed at ambient temperature on a RP-C18 analytical column with a mobile phase composed of Water: Acetonitrile (80: 20%v/v) and pH is maintained at 3.00 using ortho phosphoric acid (OPA) and was isocratically eluted at a flow rate of 1 mL min-1. A small sample volume of 20 μL was used for each sample run, being injected into the HPLC system. The chromatogram was monitored with UV detection at a wavelength of 268 nm.

Determination of λmax

Figure1: Determination of λmax

Preparation of standard stock solution

10mg of gallic acid was weighed accurately and transferred to a 10ml volumetric flask, and the volume was adjusted to the mark with the methanol to give a stock solution of 1000ppm.

Preparation of working standard solution

From stock solutions of gallic acid 1 ml was taken and diluted up to 10 ml. from this solution 0.5, 1.0, 1.5, 2.0, 2.5 ml solutions were transferred to 10ml volumetric flasks and make up the volume up to 10 ml with mobile phase, gives standard drug solution of 5, 10, 15, 20, 25μg/ ml concentration.

Analysis of extracts

Sample Preparation 10 mg ethanolic extract was taken in 10 ml volumetric flask and dilute upto the mark with Methanol; resultant solution was filtered through Whatmann filter paper and finally volume made up to mark with same solvent to obtain concentration of 1000 µg/ml. The resulting solution was again filtered using Whatmann filter paper no.41 and then sonicated for 10 min.

Formulation development Polyherbal GEL (Bele et al., 2010)

Method of preparation (Table-1)

Measured quantity of Methyl Paraben, Glycerin and weighed quantity of Polyethylene glycol were dissolved in about 35 ml of water in beaker and were stirred at high speed using mechanical stirrer (or sonicator). Then Carbopol 940 was added slowly to the beaker containing above liquid while stirring. Neutralized the solution by slowly adding triethanolamine solution with constant stirring until the gel is formed.

Carbopol 940 – Gelling Polymer

Triethanolamine- gelling agent, pH Adjusting agent, Neutralizer

Methyl Paraben - Preservative

Distilled Water, Glycerin and Polyethylene Glycol-solvents

Evaluation of Topical Gels

A. Appearance and consistency:

The physical appearance was visually checked for the texture of Polyherbal gel formulations and observations may be like stated in Table.

B. Washability

Formulations were applied on the skin and then ease and extent of washing with water were checked manually and observations may be like stated in table.

C. Extrudability determination of formulations

The hair 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.

D. Determination of Spreadability (Carl et al., 2001)

Principle:

An important criterion for hair gels is that it must possess good spreadability. Spreadability is a term expressed to denote the extent of area to which the gel readily spreads on application to hairs. The therapeutic efficacy of a formulation also depends on its spreading value.

A special apparatus has been designed to study the spreadability of the formulations. Spreadability is expressed in terms of time in seconds taken by two slides to slip of from formulation, placed between, under the application of a certain load. Lesser the time taken for the separation of two slides, better the spreadability.

Method:

Two glass slides of standard dimensions (6×2) were selected. The hair gel formulation whose spreadability had to be determined was placed over one of the slides. The second slide was placed over the slide in such a way that the formulation was sandwiched between them across a length of 6 cms along the slide. 100 grams of weight was placed up on the upper slide so that the hair gel formulation between the two slides was traced uniformly to form a thin layer.

The weight was removed and the excess of the hair gel formulation adhering to the slides was scrapped off. The lower slide was fixed on the board of the apparatus and one end of the upper slide was tied to a string to which 20 gram load could be applied 50with the help of a simple pulley. The time taken for the upper slide to travel the distance of 6 cms and separate away from lower slide under the direction of the weight was noted. The experiment was repeated and the average of 6 such determinations was calculated for each hair gel formulation.

Where, S=Spreadability (gcm/sec)

m = weight tied to the upper slide (20 grams)

l= length of glass slide (6cms).

t = time taken is seconds.

E. Determination of pH (Das et al., 2010).

The pH of the transdermal gels were determined by digital pH meter. One gram of gel was dissolved in 25 ml 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 times.

F. Viscosity (Goyal et al., 2011)

The measurement of viscosity of the prepared gel was done using Brookfield digital Viscometer. The viscosity was measured using spindle no. 6 at 10 rpm and 25⁰C. The sufficient quantity of gel was filled in appropriate wide mouth container. The gel was filled in the wide mouth container in such way that it should sufficiently allow to dip the spindle of the Viscometer. Samples of the gels were allowed to settle over 30 min at the constant temperature (25±/1⁰C) before the measurements.

In-vitro anti acne activity (Grace et al., 2015)

Media preparation (broth and agar media)

Composition of nutrient agar media;

Agar - 1.5 gms.

Beef extract - 0.3 gms.

Peptone - 0.5 gms.

Sodium chloride - 0.55 gms.

Distilled water - to make 100 ml.

pH – 7

Method of preparation

This agar medium was dissolved in distilled water and boiled in conical flask of sufficient capacity. Dry ingredients are transferred to flask containing required quantity of distilled water and heat to dissolve the medium completely.

Sterilization culture media

The flask containing medium was cotton plugged and was placed in autoclave for sterilization at 15 lbs /inch² (121^oC) for 15 minutes.

Preparation of plates

After sterilization, the molten agar in flask was immediately poured (20 ml/ plate) into sterile Petri dishes on plane surface. The poured plates were left at room temperature to solidify and incubate at 37^oC overnight to check the sterility of plates. The plates were dried at 50^oC for 30 minutes before use.

Revival of the Bacterial cultures

The bacterial cultures used in the study were obtained in lyophilized form. With the help aseptic techniques the lyophilized cultures are inoculated in sterile nutrient broth than incubated for 24 hours at 37^oC. After incubation the growth is observed in the form of turbidity. These broth cultures were further inoculated on to the nutrient agar plates with loop full of bacteria and further incubated for next 24 hours at 37^oC to obtain the pure culture and stored as stocks that are to be used in further research work.

Antimicrobial sensitivity

The antimicrobial sensitivity test is employed on to the all the microbes used under present study with ethanolic extracts obtained from plant extract of Phoenix Sylvestris and Cuscuta reflexa. For this experiment 6 mm diameter wells, stock of 100 mg/ml of extract separately applied on it. A nutrient agar plate is seeded with particular bacteria with the help of spread plate technique prior and left for 5 minutes then incubated for 24 hours at 37^oC. After incubation, plates were observed to see the sensitivity of extracts towards test bacteriums at particular concentration in the form zone of inhibition.

Antibiogram Studies (Bauer, 1966)

Broth cultures of the pure culture isolates of those test microorganisms which are sensitive towards the phytoextracts used in present study were prepared by transferring a loop of culture into sterile nutrient broth and incubated at 37^oC for 24-48hours. A loop full was taken from these broths and seeded onto sterile nutrient agar plates through sterile cotton swab to develop diffused heavy lawn culture.

The well diffusion method was used to determine the antibacterial activity of the extract prepared from the Phoenix Sylvestris and cuscuta reflexa using standard procedure (Bauer, 1966). There were 3 concentration used which are 25, 50 and 100 mg/ml for each extracted phytochemicals in antibiogram studies. It’s essential feature is the placing of wells with the antibiotics on the surfaces of agar immediately after inoculation with the organism tested. Undiluted over night broth cultures should never be used as an inoculums. The plates were incubated at 37^oC for 24 hr. and then examined for clear zones of inhibition around the wells

RESULTS AND DISCUSSION:

Determination of Percentage Yield

Yield of Extraction:

The crude extracts so obtained after the soxhlet extraction process, each extracts were further concentrated on water bath evaporation the solvents completely to obtain the actual yield of extraction. To obtain the percentage yield of extraction is very important phenomenon in phytochemical extraction to evaluate the standard extraction efficiency for a particular plant, different parts of same plant or different solvents used. The yield of extracts obtained from different samples using Pet. ether, ethanol and water as solvents are depicted in the table 7.1.

Table No.2: % Yield of Ethanolic extract

S. No.	Solvent	% Yield (w/w)
2	Phoenix Sylvestris	7.56%
3	Cuscuta reflexa	9.25%

Phytochemical screening of extracts

A small portion of the dried extracts were subjected to the phytochemical test using Kokate (1994) methods to test for alkaloids, glycosides, tannins, saponins, flavonoids and steroids separately for extracts of all samples. Small amount of each extract is suitably resuspended into the sterile distilled water to make the concentration of 1 mg per ml. The outcomes of the results are discussed separately in the table 7.2.

Table No.3: Result of Phytochemical Screening of Ethanolic extract

Chemical Tests	*Phoenix Sylvestris*	*Cuscuta reflexa*
Alkaloids
Mayer’s reagent	-	-
Hager’s reagent	-	-
Wagner’s reagent	-	-
Dragendorff’s reagent	-	-
Glycosides (+Ve)
Baljet test	-	-
Legal’s test	-	-
Keller-Kiliani	-	-
Phenols/Tannins
Ferric chloride	+	+
Gelatin Solution	-	-
Lead acetate test	-	-
Flavonoids
FeCl3 test	-	+
Alkaline reagent test	+	-
Shinoda test	+	+
Saponins
Foam test	+	-
Hemolytic test	+	-
Lead acetate	-	-
Fixed oil/Fats
Spot	-	-
Saponification	-	-
Gums and Mucilage
Water	-	-
Carbohydrates
Molish test	-	-
Fehling’s solution test	-	-
Benedict’s test	-	-
Amino acids
Ninhydrin Test	-	-
Millons Test	-	-
Xantoprotein Test	-	-
Terpenoids
Lieberman Burchard Test	+	+
Salkowski test	+	+
*Steroids*
Lieberman Test	-	+
*Protein*
Biuret test	-	-

Results of Comparative Thin Layer Chromatography of ethanolic extracts

From the Rf value it was confirmed the presence of gallic acid as phenolic compound in the extract

Table No.4: Calculation of R_f.Value

S. No.

Compound

Extract

R_fValue

(Std)

Gallic acid

Toluene: Ethyl acetate: Formic acid

(7:5:1)

0.65

Figure 2: Photograph of T.L.C.

Spot -1 Gallic acid

Spot -1 Ethanolic extract of Phoenix Sylvestris

Spot -1 Ethanolic extract of Cuscuta reflexa

Results of Estimation of Total Phenolic and Flavonoids Contents

Total tannin content estimation (TFC)

The content of total phenolic compounds (TPC) and total tannin content was expressed as mg/100mg of gallic acid equivalent of dry extract sample using the equation obtained from the calibration curve: Y = 0.002X- 0.025, R²= 0.980, where X is the absorbance and Y is the tannic acid equivalent (GAE).

Calibration Curve of Gallic acid

Table No.5: Preparation of calibration curve of Gallic acid

S. No.	Concentration	Absorbance
0	0	0
1	25	0.049
2	50	0.093
3	75	0.155
4	100	0.255
5	125	0.315
6	150	0.421

Figure 3: Graph of Estimation of Total Phenolic content

Total flavonoids content estimation (TFC)

Total flavonoids content was calculated as quercetin equivalent (mg/g) using the equation based on the calibration curve: Y=0.004 X - 0.001, R²=0.996, where X is the absorbance and Y is the quercetin equivalent (QE).

Table No.6: Preparation of calibration curve of Quercetin

S.No.	Concentration	Absorbance
0	0	0
1	25	0.119
2	50	0.195
3	75	0.297
4	100	0.387
5	125	0.517
6	150	0.626

Figure 4: Graph of Estimation of Total Phenolic content

Results of Estimation of Total phenolic and Flavonoids Content

Table No.7: Estimation of Total phenolics and Total flavonoids content

S. No

Extracts

Total phenolic content

(mg/100gm of dried powder)

Total flavonoids

Equvalent to Quercetin mg/ 100 mg of dried extract

Phoenix Sylvestris

0.045

0.98

Cuscuta reflexa

0.052

1.25

The total phenolic and flavonoids content of the ethanolic extract were also determined. In extract was found more Flavanoidal content in comparison to Phenolic contents.

Identification of Marker Compound (Gallic acid) by HPLC

A reverse phase C-18 column equilibrated with mobile phase methanol: acetonitrile (50:50, v/v) was used. Mobile phase was filtered through Whatmann filter paper and degassed. Mobile phase flow rate was maintained at 1 ml/min and effluents were monitored at 256 nm. The sample was injected using a 20 μl fixed loop, and the total run time was 10 min. The sample solution was chromatographed and a concentration of gallic acid in Extract sample was found out using regression equation.

Preparation of the Calibration Curve of the Gallic acid

Each of the standard drug solutions were injected 3 times and the mean peak area of drug was calculated and plotted against the concentration of the drug. The regression equation was found out by using this curve.

Table No.8: Preparation of Calibration curve

S. No.	Conc.	Mean AUC
1.	0	0
2.	5	215.256
3.	10	425.569
4.	15	643.534
5.	20	865.755
6.	25	1080.256

Figure 5: Calibration Curve of the Gallic acid

Table 9: Characteristics of the analytical method derived from the standard calibration curve

Compound

Linearity range

μg/ml

Correlation co-efficient

Slope

Intercept

Quercetin

5-25

0.999

43.26

-2.372

Chromatogram of standard gallic acid

Figure 6: Chromatogram of standard gallic acid

Chromatogram of ethanolic extract of Phoenix Sylvestris

Figure 7: Chromatogram of aqueous extract

Chromatogram of ethanolic extract of Cuscuta reflexa

Figure 8: Chromatogram of ethanolic extract

Result of Assay of extract

Table No 10: Result of Assay of Extract formulation

S. No.	Ethanolic Extract	Assay (%)
1	*Phoenix Sylvestris*	0.365
2	*Cuscuta reflexa*	0.458

Results of High performance liquid chromatography showed the presence of gallic acid in both the extract. The percentage estimation of gallic acid in Phoenix Sylvestris and Cuscuta reflexa herbal extract found to be 0.365% and 0.458 % respectively.

Results of Formulation Development

Evaluation of gel formulation

Table 11: Results of Psychorheological Characteristics

Formulation	Colour	Clogging	Homogeneity	Texture
HG1	Brown	Absent	Good	Smooth
HG2	Brown	Absent	Good	Smooth
HG3	Brown	Absent	Good	Smooth
HG4	Brown	Absent	Good	Smooth
HG5	Brown	Absent	Good	Smooth
HG6	Brown	Absent	Good	Smooth

Results: In the above formulations of gels, it has been noted that all of them has clear colour, No clogging, good homogeneity and smooth texture.

Results of washability and Extrudability

Table 12: Results of washability and Extrudability

Formulation	Washability	Extrudability
EG1	Good	Average
EG 2	Good	Average
EG 3	Good	Average
EG 4	Good	Average
EG 5	Good	Average
EG 6	Good	Average

Results: In the above formulations of gels, they have good washability as well as extrudability.

Results of spreadability

Table 13: Results of spreadability

Formulation	Spreadability (gcm/sec)
EG1	13.33±0.32
EG 2	13.06±0.64
EG 3	13.06±0.54
EG 4	13.05±0.21
EG 5	12.00±0.11
EG 6	12.54±0.12

Results: In all above formulations of gel the spreadibility of EG-5 is good.

Determination of pH

Table 14: Determination of pH

Formulation	pH
EG1	7.1± 0.11
EG 2	7.2±0.15
EG 3	7.2±0.11
EG 4	7.1±0.14
EG 5	7.3±0.12
EG 6	7.0±0.13

Results: The above formulation of topical gels has different Ph value for different formulation

Results of Viscosity

Table 15: Results of Viscosity

Formulation	Viscosity (cps)
EG1	2500
EG 2	2700
EG 3	3000
EG 4	3200
EG 5	3500
EG 6	4500

Results: In the above formulations the viscosity of different sample of gel were determined and found that there is increase in viscocity. The formulation HG-5 has good viscocity.

Results of Drug Content

Table 16: Results of Drug Content

Formulation	% Drug Content
EG1	87.97±0.12
EG 2	88.25±0.25
EG 3	90.23±0.14
EG 4	95.56±0.23
EG 5	98.89±0.45
EG 6	89.98±0.35

Results: In the above formulation of different gels the percentage of drug content was found that HG-5 HAS maximum percentage of drug content.

Antibacterial Activity of Phytochemical Extracts

Bacterial Cultures

For the studies of antimicrobial effect of phytochemicals obtained from cuscuta reflexa extract a medicinally important plant, microbial successfully procured from Microbial Culture collection, National Centre forcell science, Pune, Maharashtra, India The lyophilized cultures of bacterial strains upon culturing in nutrient broth for 24-48 hours at 37^oC in an incubator resulted into turbid suspension of activated live bacterial cell ready to be used for microbiological study. The list of bacterial species with suitable codes used in the antimicrobial studies is given in the table. From the broth of respective revived cultures of bacteria loop full of inoculum is taken and streaked on to the nutrient agar medium and incubated again at same culture conditions and duration that yielded the pure culture colonies on to the surface of the agar culture that are successfully stored in refrigerated conditions at 4^oC as stock culture to be used for further experimentation.

Antimicrobial Studies

The lawn cultures were prepare with all the microbes used under present study and sensitivity of bacteria towards the various phytochemicals extracts obtained from the Phoenix Sylvestris and Cuscuta reflexa were studied at the concentration of 100 mg/ml using well diffusion method.

Antibiogram studies

The present investigation in this research work, the antimicrobial activity of extract obtained from the cuscuta reflexa was evaluated against bacterial and fungal pathogens used under present study. The fresh pure 100% extracts obtained from plant used to suitably dilute upto the concentrations of 100, 50 and 25 mg per ml and applied on to the test organism using well diffusion method. Results of the experiment are being concluded in the Table, which clearly shows the anti-microbial activity of extracts of Phoenix Sylvestris and cuscuta reflexa.

CONCLUSION:

A herb is a plant or part of a plant valued for its medicinal, aromatic, or savoury qualities. Herbs can be viewed as biosynthetic chemical laboratories, producing a number of chemical compounds. Herbal remedies or medicines consist of portions of plants or unpurified plant extracts containing several constituents, which often work together synergistically. Herbal medicine or herbalism is the use of herbs or herbal products for their therapeutic or medicinal value. They may come from any part of the plant but are most commonly made from leaves, roots, bark seeds, and flowers. They are eaten, swallowed, drunk, inhaled, or applied topically to the skin Herbal products often contain a variety of naturally occuring biochemicals from plants, many of which contribute to the plant’s medicinal benefits.

Acne vulgaris is a disease of the pilosebaceous follicle characterized by non-inflammatory (open and closed comedones) and inflammatory lesions (papules, pustules, and nodules). Its pathogenesis is multifactorial - the interplay of hormonal, bacterial, and immunological (inflammatory) factors results in the formation of acne lesions. Although acne is not a life -threatening condition, it can have detrimental effects on the quality of life of affected individuals.

Natural remedies are more acceptable in the belief that they are safer with fewer side effects than the synthetic ones. Herbal formulations have growing demand in the world market. It is a very good attempt to establish the herbal gel containing ethanolic extract of Phoenix Sylvestris seeds. and whole plant Cuscuta reflexa. This study revealed that the developed polyherbal formulation F6 consisting both extract was comparatively better than other formulation.

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Received on 11.06.2017 Accepted on 28.06.2017

Research J. Topical and Cosmetic Sci. 8(1): Jan.-June 2017 page 01-11

DOI: 10.5958/2321-5844.2017.00001.2

Ingredients (%)	HG1	HG2	HG3	HG4	HG5	HG6
*Phoenix Sylvestris* Extract	1	1	1	1	1	1
*Cuscuta reflexa* Extract	1	1	1	1	1	1
Carbopol 940	0.25	0.30	0.35	0.40	0.45	0.5
Polyethylene Glycol	2	2	2	2	2	2
Methyl Paraben	0.08	0.08	0.08	0.08	0.08	0.08
Triethanolamine	1.2	1.2	1.2	1.2	1.2	1.2
Distilled Water (q.s)	100	100	100	100	100	100