The Development of Phyllanthus emblica Extract in Ethosomes for Hair Loss Prevention

Hair loss is a problem of concern for many people, both male and female. Even though hair loss is not a serious health problem, it can lead in some cases too low self-confidence, psychological problems, and even impaired quality of life. Genes and hormones are major causes of hair loss. Hair loss can be treated with discontinue behavior effect or using medicines such as finasteride and dutasteride which act as 5αreductase inhibitors. Although, they are effective on hair loss prevention the use of chemical products also causes many side effects as well as altered libido, erectile dysfunction, and ejaculation disorder.1


INTRODUCTION
Hair loss is a problem of concern for many people, both male and female. Even though hair loss is not a serious health problem, it can lead in some cases too low self-confidence, psychological problems, and even impaired quality of life. Genes and hormones are major causes of hair loss. Hair loss can be treated with discontinue behavior effect or using medicines such as finasteride and dutasteride which act as 5α-reductase inhibitors. Although, they are effective on hair loss prevention the use of chemical products also causes many side effects as well as altered libido, erectile dysfunction, and ejaculation disorder. 1 Literature reviews report the use of many Thai traditional herbs for hair loss prevention or treatment including Phyllanthus emblica L., Citrus hystrix DC, Acacia concinna Wall., Sapindus raruk DC, Clitorea ternatea L, Averrhoa carambola L., Carthamus tinctorius L., Zingiber officinale Roscoe., Alpinia galangal Willd., Trichosanthes cucumerina L., Lawsonia inermis Linn. 2 Phyllanthus emblica (P. emblica) has been used as an ancient Thai traditional medicine by maceration in water overnight before application to the scalp. Moreover, the literature reviews found that extracts of P. emblica have antioxidant activities [3][4][5] , stimulating proliferation of hair follicle, and inhibiting 5α-reductase activities. 2 These properties could protect from hair loss. Therefore, P. emblica may prevent hair loss but it is inconvenient to use and the exact amount required for use is not known.
Ethosomes is a novel drug delivery system that contains phospholipid, ethanol, and water. They are formed as vesicles containing P. emblica extract and can be used to increase the skin delivery to deep layers of skin, improve the systemic circulation, and enhancement of P. emblica extract effectiveness. This research aimed to develop ethosomes containing P. emblica which have good properties.

Plant material
Phyllanthus emblica fruits were collected from Na dun, Maha Sarakham, Thailand. All solvents and chemicals used were analytical grade.

Preparation of extract
The 95% ethanol of the maceration method was used as preparation P. emblica extract for 7 days at room temperature. The raw material to solvent ratio was 1:6. The extract was filtered with a Whatman No.1 filter and the filtrate was evaporated by rotary evaporator.

Total phenolic content 6
The total phenolic content was determined by Folin -Ciocalteu reagent method. 20 μl of stock solution (0.25 mg/ml) of the P. emblica extract, 100 μl of 10 % Folin -Ciocalteu reagent, and 80 μl of 1 M sodium carbonate solution were added to 96 well microplates and mixed well. The mixture was kept at room temperature for 30 min and absorbance of the color developed was recorded at 765 nm with UV Visible spectrophotometer (BMG Labtech, Germany). Total phenolic content estimated from 6 replicates was expressed in mg equivalents of gallic acid per 1 g of crude extract.
Antioxidant activity by DPPH radical scavenging assay 7 Different 2 fold-dilution of P. emblica extract (stock solution 1 mg/ ml) were prepared. 1,1-diphenyl-2-picrylhydrazyl (DPPH) solution was prepared by dissolving 6 mg of DPPH in 100 ml of 95% ethanol. Then 100 μl of P. emblica extract from each dilution was added in 100 μl of DPPH solution. The mixture was shaken vigorously and left to stand in the dark condition for 30 min. The absorbance of the solution was measured spectrophotometrically at 517 nm with 6 replicate measurements. The % radical scavenging of the extract was calculated using the following formula: % radical scavenging = [(Abs control -Abs sample )/ Abs control ] x 100 (1) Where Abs sample is the absorbance of the P. emblica extract solution and Abs control is the absorbance of the ascorbic acid which was used as standard.

Development of ethosome formulation 8
Ethosomes were prepared by the cold method. In brief, the P. emblica extract was placed in a small round bottom flask and dissolved with 95% ethanol under mixing with the magnetic stirrer at 30°C. The round bottom flask was covered with aluminium foil to avoid ethanol evaporation. Soya phosphatidylcholine (Phospholipon 90G) was added and dissolved. Distilled water was added slowly with a constant rate and continuous stirring to obtain the ethosomal colloidal suspensions. The suspension of ethosomes was continuously stirred for 30 min and the resulting formulations stored at 4°C. The 9 formulations (F1-F9) of ethosomes were prepared with varied concentrations of soya phosphatidylcholine (1-3%) and ethanol (20-40%). The ethosome formulations with the highest percentage of entrapment efficiency was selected. The results are shown in Table 1.

Evaluation of the ethosome preparation
The ethosome formulation which had the highest percentage of entrapment efficiency was evaluated.

Morphology
Surface morphology examined by Scanning Electron Microscopy (SEM) (FEI, Quanta 450, USA). One drop of ethosome formulation was placed on a stub and samples were dried and coated with gold before examination.

pH measurement
The pH of the formulations was monitored by using a digital pH meter (Mettler Toledo, Switzerland) with 6 replicate measurements.

Particle size, size distribution, zeta potential
Particle size, size distribution and zeta potential were measured using Zetasizer (Malvern, UK). The size distribution was reported as the polydispersity index (PDI) with 6 replicate measurements.

Total phenolic contents
The details of total phenolic contents measurement are as mentioned above.

Entrapment efficiency 9
The percentage of entrapment efficiency (%EE) of ethosomes was determined by using the centrifugation method. 10 ml of ethosome dispersions were centrifuged using a cooling ultracentrifuge (Beckman) at 30,000 rpm. The supernatant was siphoned off carefully to divide the unentrapped P. emblica extract. 9 ml of 2% Triton-X 100 was added to the sediment to dissolves the vesicles. The percentage of entrapment efficiency was investigated in terms of % GAE in sediment measured from 6 replicate measurements. The percentage of encapsulated total phenolic content was calculated as follows:

Antioxidants
The antioxidant activity test as mentioned above.

RESULTS AND DISCUSSION
Percentage of yield P. emblica was extracted with 95% ethanol for 7 days. After evaporation to dryness, the residue was dark brown sticky extract. The percentage of yield was 12.64%.

Total phenolic contents
Total phenolic content of the P. emblica extracts was determined with the Folin-Ciocalteu method. Total phenolic contents of P. emblica extract were 406.37 ± 2.39 mg GAE/g crude extract (n=6).

Morphology
SEM photographs showed the surface morphology of ethosomes. The ethosomes were revealed to be spherical vesicles with a smooth surface as shown in Figure 1.

pH measurement
The pH of the ethosomes formulations was between in ranges of 3.83 ± 0.01 to 4.19 ± 0.02. corresponds to the results of Jantima et al. 11 who determined total phenolic contents in P. emblica from 4 sources in Thailand extracted with ethyl acetate, and found that P. emblica from Maha Sarakham had total phenolic contents 494.00 ± 19.50 mg GAE/g crude extract.

Components
Although, the total phenolic contents extracted with ethyl acetate was more than that from 95% ethanol extraction, the ethyl acetate is toxic to the skin. Therefore, the research team used 95% ethanol extract in order to reduce toxicity so that the preparation can be used in dermal cosmetics.
As humans get older they produce more free radicals, while the endogenous defense mechanisms decrease. This imbalance leads to progressive damage to cellular structures. Thus, free radicals might lead to pattern baldness by damaging hair follicles. The researchers were interested in determining the antioxidant effect from P. emblica crude extract by the DPPH method. The IC 50 was 7.05 ± 0.17 µg/ml whereas the ascorbic acid standard solution had IC 50 at 6.42 ± 0.20 µg/ ml, corresponding with a result from Pientaweeratch et al. 4 who found the IC 50 of P. emblica crude extract from Chaopraya Abhaiphubejhr hospital, Prachin Buri, Thailand at 1.70 ± 0.07 µg/ml. The different sources of P. emblica may cause different results.
The ethosome was proper with 30,000 rpm at 4 °C for 90 min to separated sediment and supernatant parts. Then Triton X-100, a non-ionic surfactant that had no effect with total phenolic compounds analysis used as a marker in this study, was used as vesicle lysing agent for determining the percentage of entrapment efficacy. Based on the results of the study. The researchers found that increasing soya phosphatidylcholine and 95 % ethanol contents in ethosome formulations decreased the percentage of entrapment efficiency. Thus, the researchers choose F1 which had minimum soya phosphatidylcholine and 95 % ethanol contents but had the highest percentage of entrapment efficiency to apply as a hair tonic. These findings were opposite to Chen et al. 12 and Sivakranth et al. 13 Particle size, polydispersity index, zeta potential The particle size of ethosomes was in the range of 0.36 ± 0.00 to 2.34 ± 0.08 µm. The polydispersity index (PDI) was 0.30 ± 0.01 to 0.97± 0.03 and zeta potential was -10.40 ± 0.28 to 0.26 ± 0.12 mV.

Entrapment efficiency
The percentage of entrapment efficiency (%EE) of ethosomes formulations ranged from 43.34± 1.69 to 65.26± 1.80%. The F9 formulation showed minimum entrapment whereas F1 showed maximum entrapment of extract.
According to the like dissolves like rule in reference to solubility of polar and non-polar substances, 95% ethanol is a very polar substance and tends to extract a high percentage of yield. This result corresponds to the result of Kornthip et al. 10 who found for P. emblica bark that there was a greater percentage of yield in 95% ethanol (15.60%) when compared with 50% ethanol (14.18%). Moreover, a study using P. emblica from Chiang Mai produced 21.63% of crude extract when macerated with 95% ethanol. 2 The different percentage of yields may be due to different sources of P. emblica.
Total phenolics contents could have been inhibitory to a 5α-reductase enzyme which is responsible for changing androgen testosterone into the more potent androgen dihydrotestosterone (DHT). Overexpression of DHT causes androgenic alopecia in males. Therefore, we have determined average total phenolic contents of 406.37 ± 2.39 mg GAE/g crude extract which macerated in 95% ethanol for 7 days, and this   who found that increasing soya phosphatidylcholine and 95 % ethanol contents had a greater percentage of entrapment efficiency. However, if the ethanol content was more than 45%, it caused leakage of ethosome vesicles. Iizhar et al. 14 found that greater entrapment efficiency was found when the ethosomes were resized by sonication. Similarly Shirwaikar et al. 15 found that a sonication effect on ethosome vesicle arrangement which increases the stability and resized the vesicle. Also, due to the vesicle size being decreased, greater permeability and deeper penetration into the target was found. While the primary study of this research shows the percentage of entrapment efficiency was decreased when the ethosomes were resized by sonication. Base on this study, the researchers found the correlation that when the concentration of phospholipid was constant and varied the concentration of ethanol.

A B
The formulations which had more ethanol contents were larger. This correlation is opposite to that reported by Touitou et al. 16 who found when determining the concentration of phospholipid was constant and varied the concentration of ethanol. The formulations which had more ethanol contents were smaller. The polydispersity index of F1 formulation was 0.44 ± 0.03 which not more than 1. These results showed the particles of ethosomal vesicles had distributed regularly. 17 The zeta potential of F1 formulation had a more negative charge on the surface of ethosomal vesicle. F1 formulation was aggregated loosely when left overnight. However, the particles of ethosome could be quickly dispersed and suspend when shaking with a little force.
The antioxidant activity of ethosomes was greater than P. emblica extract solution. This study showed the development of P. emblica in ethosomes may improve the antioxidant activity of P. emblica. Koli and Lin 18 reported the development of ethosomes could protect active ingredients from oxidation reactions that may promote antioxidant activity.

CONCLUSION
The combination of 20% ethanol, 2% soya phosphatidylcholine, and P. emblica extract (10 mg) could be used to prepare ethosomes with good properties. The ethosomes of P. emblica extract can be used for hair loss prevention products.