Hair Tonic Formulation of Anti-alopecia of Angiopteris evecta Extract

 

Dolih Gozali1, Resmi Mustarichie2*

1Pharmaceutical Department, Faculty of Pharmacy, Universitas Padjadjaran, Indonesia

2Pharmaceutical Analysis and Medicinal Chemistry Department, Faculty of Pharmacy, Universitas Padjadjaran, Indonesia

*Corresponding Author E-mail: resmi.mustarichie@unpad.ac.id

 

ABSTRACT:

Synthetic cosmetic products have been used to unravel the problem of alopecia, yet synthetics are potential to give side effects such as local irritation), whilst herbal products are generally safer. Our previous research showed that water fraction of Angiopteris evecta extract had been proved as had growth hair characteristic. The aim of this study was to formulate hair tonic from water fraction of A.evecta extract which was stable, effective towards hair growth, and safe. The water fraction was into varied of 7.5%, 10.0, and 12.5%. Physical stability test performed was cycling test, storage in high temperature (40°C ± 2°C), room temperature (25°C ± 2°C), and low temperature (4°C ± 2°C). The activity of hair growth test was conducted by hair length measurements using rabbit based on Tanaka method on day 3, 6, 9, 12, 15 and 18 days.  Results showed the hair tonic was stable in storage. In addition to giving hair growth activity, the formula had greater activity equal and higher activity compared to controlled minoxidil synthetic drug. The hair tonics were safe and did not irritate skin. The best formulation was formula F3 with a water fraction of 10.0%. It is suggested to carry our hair growth test to bald volunteers in order to verify the effectiveness of this formula before it could be marketed.

 

KEYWORDS: Angiopteris evecta, hair tonic, formulation, anti-alopecia, hair growth, minoxidil.

 

 


INTRODUCTION:

Healthy, thick, long, shiny and black hair is everyone's desire, but it is not shared by everyone. One problem that is often experienced by humans is baldness or abnormal hair growth. Baldness is usually caused by hormonal disorders, drug side effects, food consumed, and stress1. Baldness or alopecia can affect all the hair that is in the body (alopecia universalis) or about all head hair (alopecia totalis). The cause of this disease is actually still unclear, but in general, alopecia can be divided based on morphological observations into two groups, namely alopecia with permanent scars and non-brush alopecia which still gives hope of hair growth. Androgenetic alopecia is one of the many non-circulate alopecias that occurs in the community2.

 

 

 

Various cosmetic products have been developed to overcome hair loss and baldness problems, both derived from synthetic ingredients and from natural ingredients. It has been proven that the synthetic ingredients used (including minoxidil) in their use have side effects such as local irritation and erythema3. Hair care is not enough to use only shampoos and conditioners, this is because hair is a living cell that needs to be nurtured, cared for and given fertilizer so that it can live healthy and beautiful. One way is to use hair tonic4. The use of hair tonic from plants has been reported by several researchers. Amin et al 5 reported the use of ethanol extract from green tea with supporting data from several other researchers6-8. 14.Sulastri et.al9, on the other hand,  used a combination of green tea and Centella herbal tea extract to test hair fertilizer.

 

In our previous research10, it has been reported that the water fraction of ethanol extract from munding (Sundanese) fern (Angiopteris evecta) root has been shown to be significantly efficacious in growing hair. This test was carried out by using the modified Tanaka method11 on animals testing Angora species. The use of water fraction from ethanol extract from A. evecta is formulated in the form of hair tonic doses. This tonic hair is chosen because they provide convenience and a comfortable application for hair skin, the absorption of trans-appendageal is large and easy to spread, not oily and leaves a residue on the skin of the hair. The resulting tonic hair was evaluated for its physicochemical and stability properties and the test of the hair growth activity of male rabbits and minoxidil as a positive control. The Tanaka method is carried out as has been done with the ethanol extract and fraction of A. Evecta

 

MATERIALS AND METHODS:

Material Plant:

The herbal materials used in this study was munding fern (A. Evecta) collected from the root of Galunggung mountain, Tasikmalaya, West Java.

 

Animal Test:

Angora male rabbits were 3-5 months old weighing 1.5-2 kg were used as an animal test. The number of test animals used is determined using the Federer formula: (n-1) (t-1) ≥ 15, where t is the number of treatments while n is the number of animals per treatment 12. Ethical approval No. 774 /UN6.C.10/PN/2017 for animal testing was obtained from the Research Ethics Committee, Universitas Padjadjaran.

 

Methods:

The research method included material collection, plant determination, simplicia phytochemical screening based on modifications of the Farnsworth method13,  making Wera leaf extract based on modifications of Mustarichie et.al method14, phytochemical screening extract, extract parameter examination based on Indonesian Herbal Pharmacopeaia15 and BPOM RI16,  testing the stimulating activity of hair growth ethanol extract in rabbits based on modification of Tanaka et.al method17,  making water fraction based on Mustarichie et.al method 14, formulation of hair tonic18,19,  formula evaluation, formula hair growth activity test on rabbits, and statistical data analysis20, 21.

 

RESULTS:

Simplicia collection and determination:

Simplicia was determined as a munding fern (Angiopteris evecta (G.Forst.) Hoffm).

 

Maceration and fractionation:

The A. Evecta Wera water fraction was made by maceration and liquid-liquid fractionation and yielded water fraction of 96.54 % w/w.

 

Phytochemical screening:

The results of phytochemical screening of A. Evecta ethanol extract can be seen in Table 1.

Table 1 Phytochemical screening of A. evecta ethanol extract

Secondary metabolite

Paku munding ekstrak

Alkaloids

-

Polyphenols

+

Tannin

-

Flavonoids

+

Saponins

+

Triterpenoid & Steroid

-

Monoterpen & sesquiterpene

Quinone

+

+

Notes :  (+): detected; (-): not detected

 

Parameter examination of A. evecta root extract:

Table 2. General standard examination of Munding root extract

Parameters

Results

Ethanol soluble extractives

73.85 %

Water soluble extractives

73.12 %

Water content

4.83 %

 

Organoleptic criteria

Observation

Shape

viscous

Color

blackish green

Smell

typical

Taste

no taste

 


 

 

 

Hair Growth Stimulation Formulation:

Table 3. Formulation of anti-alopecia preparations from A. evecta roots

Components

F0

F1

F2

F3

F4     

F5

 

Concentration (% w/w)

 

Water fraction

-

-

7.5

10.0

12.5

-

Minoxidil

-

2.5

-

-

-

-

Ethanol 50%

40.0

40.0

40.0

40.0

40.0

-

Propylene glycol

2.0

2.0

2.0

2.0

2.0

-

Methylparaben

0.075

0.075

0.075

0.075

0.075

-

Propylparaben         

0.02

0.02

0.02

0.02

0.02

-

Sodium metabisulfite

0.2

0.2

0.2

0.2

0.2

-

Aquadest ad

100.0

100.0

100.0

100.0

100.0

100.0

Notes: Fo: Negative control; F1: Positive control; F2: Formula with water fraction of 7.5%; F3: Formula with water fraction of 10.0 %; F4: Formula with water fraction of 12.5%; F5: Normal control

 

 

 

Table 4. Results of Observation of Organoleptics and Homogeneity Hair Tonic preparations for one week

Formula

Test

storage time (days)

1

7

14

28

35

42

49

56

F0

Form

c

c

c

c

c

c

c

c

color

p

p

p

p

p

p

p

p

odor

tb

tb

tb

tb

tb

tb

tb

tb

homogenity

h

h

h

h

h

h

h

h

F1

Form

c

c

c

c

c

c

c

c

color

p

p

p

p

p

p

p

p

odor

tb

tb

tb

tb

tb

tb

tb

tb

homogenity

h

h

h

h

h

h

h

h

F2

Form

c

c

c

c

c

c

c

c

color

p

p

p

p

p

p

p

p

odor

bk

bk

bk

bk

bk

bk

bk

bk

homogenity

h

h

h

h

h

h

h

h

F3

Form

c

c

c

c

c

c

c

c

color

p

p

p

p

p

p

p

p

odor

bk

bk

bk

bk

bk

bk

bk

bk

homogenity

h

h

h

h

h

h

h

h

F4

Form

c

c

c

c

c

c

c

c

color

p

p

p

p

p

p

p

p

odor

bk

bk

bk

bk

bk

bk

bk

bk

homogenity

kh

kh

kh

kh

kh

kh

kh

kh

F5

Form

c

c

c

c

c

c

c

c

color

p

p

p

p

p

p

p

p

odor

tb

tb

tb

tb

tb

tb

tb

tb

homogenity

h

h

h

h

h

h

h

h

Notes: Fo Negative control; F1 Positive control; F2 Formula with water fraction of 7.5%; F3 Formula with water fraction of 10.0 %; F4 Formula with water fraction of 12.5%; F5 Normal control

p (white); tb (no smell), bk (specific odor; c (liquid); h (homogen), kh (less homogeny)

 

Table 5 Rabbit Hair Length Measurement Results in the Formula Effectiveness test

Treatment

Measurement of rabbit hair on the day - (cm)

3

6

9

12

15

18

Normal control

0.31

0.33

0.70

0.87

0.91

0.93

0.25

0.31

0.42

0.55

0.59

0.70

0.42

0.48

0.70

0.77

0.86

0.88

Mean

0.33

0.37

0.61

0.73

0.79

0.84

SD

± 0.086

± 0.093

± 0.162

± 0.164

± 0.172

± 0.121

Negative control

0.29

0.30

0.32

0.38

0.48

0.64

0.24

0.25

0.28

0.28

0.36

0.55

0.5

0.71

0.71

0.74

0.81

1.08

Mean

0.34

0.42

0.44

0.47

0.55

0.76

SD

± 0.138

± 0.252

± 0.238

± 0.242

± 0.233

± 0.284

Positive control

0.20

0.29

0.30

0.31

0.42

0.51

0.25

0.28

0.28

0.34

0.57

0.69

0.5

0.63

0.73

0.85

1.07

1.13

Mean

0.32

0.40

0.44

0.50

0.69

0.78

SD

± 0.161

± 0.199

± 0.254

± 0.303

± 0.340

± 0.319

7.5% water fraction

0.2

0.3

0.31

0.32

0.52

0.61

0.2

0.24

0.31

0.42

0.51

0.61

0.45

0.61

0.69

0.75

0.81

0.86

Mean

0.28

0.38

0.44

0.50

0.61

0.69

SD

± 0.144

± 0.199

± 0.219

± 0.225

± 0.170

± 0.144

10.0 % water fraction

0.28

0.30

0.31

0.33

0.51

0.80

0.24

0.31

0.31

0.61

0.91

0.97

0.45

0.60

0.81

0.75

0.85

0.89

Mean

0.32

0.40

0.48

0.56

0.76

0.89

SD

± 0.112

± 0.170

± 0.289

± 0.214

± 0.216

± 0.085

12,5% water fraction

0.24

0.32

0.32

0.53

0.67

0.78

0.28

0.34

0.39

0.51

0.77

0.85

0.26

0.49

0.55

0.75

0.94

0.99

Mean

0.26

0.38

0.42

0.60

0.79

0.87

SD

± 0.020

± 0.093

± 0.118

± 0.133

± 0.137

± 0.107

 

 

Fig. 1 Rabbit Hair Length Measurement on Formula Effectiveness Test

Notes: Fo: Negative control; F1: Positive control; F2: Formula with water fraction of 7.5%;

F3: Formula with water fraction of 10.0 %; F4: Formula with water fraction of 12.5%;

F5: Normal control

 

 

 


Table 6 Normality test:

One-Sample Kolmogorov-Smirnov Test

 

Respon

N

120

Normal Parametersa,b

Mean

.5193

Std. Deviation

.17354

Most Extreme Differences

Absolute

.097

Positive

.097

Negative

-.082

Kolmogorov-Smirnov Z

1.064

Asymp. Sig. (2-tailed)

.207

a. Test distribution is Normal.

b. Calculated from data

 

hair

 

Sum of Squares

Df

Mean Square

F

Sig.

Between Groups

,086

6

,014

,396

,869

Within groups

,507

14

,036

 

 

Total

,593

20

 

 

 

 

 

Table 7 Duncan’s test

                                                                                Response

Duncana,b,c

Treatment

N

subset

1

2

3

4

5

5

12

0.4275

 

 

 

 

1

18

0.4689

0.4689

 

 

 

2

18

 

0.4744

 

 

 

4

18

 

0.5000

0.5000

 

 

3

18

 

 

0.5328

0.5328

 

7

18

 

 

 

0.5689

 

6

18

 

 

 

 

0.6317

Sig.

 

0.56

0.174

0.129

0.094

1.000

 

 

 

DISCUSSION:

Leaf Collection and determination:

To ensure the identity of the plants carried out, the determination of A. evecta at the Taxonomy Laboratory, Biological Department, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Bandung. The result of the determination showed that the plants used to belong to the division of Pteridophyta, class Marattiopsida, Marattiales, and family Marattiaceae with the name Angiopteris evecta (G.Forst.) Hoffm.

 

Munding fern root processing results:

Simplified munding fern roots that had been collected and dried, sorted to separate the other undesirable parts of the plant and other impurities left behind in dried simplicia 22.  Simplicia was chopped and further reduced in size by using scissors or blenders to increase the surface area of the simplicia so as to facilitate the extraction process because the more surface area allows the solvent to reach the cell. The small particle size of simplicia expands the contact of the simplicia with the solvent, resulting in more secondary metabolites being attracted 23-25.

 

Maceration:

Munding fern root extraction process was carried out by maceration method using 96% ethanol. Ethanol was chosen as a solvent in the extraction process because it had high solubility, most of the secondary metabolites were insoluble, non-toxic, and inert so they did not interfere with other components. The use of ethanol as a solvent could prevent the growth of fungi and bacteria in the extract, so as to minimize the occurrence of contamination in the extract. The low ethanol boiling point facilitated the evaporation process in the extraction process by using less heat 26,27.

 

Phytochemical screening:

Seen in Table 1, that alkaloids,  tannin, triterpenoid as well as steroids were not detected in our A. evecta ethanol extract. Mismawati et.al 28 and Molla et.al 29 reported the presence in their respectively, East Borneo and Bangladesh samples. Saleride et.al 30, however, did not found alkaloids content in their study on A. evecta (Forst) Hoffm. collected from the Kodaikanal Botanical garden, India. These differences most likely due to the origin of the plant being examined.

 

Parameter examination of A. evecta root extract:

Examination of A. evecta root extract parameters performed based on Indonesian Herbal Pharmacopeaia 15 and BPOM RI 16. This included observation on organoleptic criteria, ethanol soluble and water-soluble extractives, and water content as shown in Table 2. These results consistent with our previous report 31.

 

Fractionation result:

The method of liquid-liquid extraction (LLC) was applied in order to get the water fraction of A.evecta ethanol extract. The method used was a modification of the method of Mustarichie et.al 16. The LLC method was a simple, fast, economical, and commonly used as a method of fractionation 32. It was obtained for 1.20% n-hexane fraction, 2.26% ethyl acetate fraction and 96.54% for the water fraction. This water fraction was then used to formulate make hair tonics.

 

Hair tonic formulation results:

Hair tonics are formulated as shown in Table 3. This formulation was made in reference to POUCHER’s Perfumes, Cosmetics and soaps Handbook 18 and Excipients Handbook of Pharmaceuticals19.

 

Formula evaluation results:

The formula was evaluated its general characterization, the physical and chemical evaluation including pH, organoleptic, stability, pH, and viscosity.

 

Organoleptic Test:

Organoleptic examination was done by using the five senses to observe changes inhomogeneity, color, odor, and form from the hair tonic preparation during storage time 33. The organoleptic examination was carried out multiples of 7 days. The results of organoleptic observations are shown in Table 4. It was revealed all of the prepared formulations were no changes occurred during 2 months of storage.

 

 

 

Freeze-thaw test (stability test):

The freeze-thaw test was carried out by storing the preparation at 4oC for 24 hours then at room temperature for 24 hours and then transferred to 40°C for 24 hours (1 cycle). After that, it continued for up to six cycles. Every single cycle was completed, look at whether there was a phase separation.  It revealed that all the formulas made were not separated. Our method was a modification of the micro-laboratory method 33 which stated that one cycle was -10°C, room temperature, and 45°C.

 

pH test:

pH measurement could not be done with a pH meter because the sample contained organic ethanol solvents which would damage the electrode. Measurements were then made using universal indicator paper. pH testing was carried out at the same time as other tests. There were no changes in pH during monitoring time.

 

Viscosity test:

Every formula of hair tonic was tested for viscosity using a Brookfield Viscometer with suitable spindle and speed (rpm). The viscosity value (cPs) found in the Brookfield Viscometer is the value of the preparation viscosity 34. Measurements for each formula were carried out on days -1, 3, 7, 14, 28, 30. There was no significant change in viscosity on the formula tested. Formula F2, F3 and F4 had stable viscosity at 0.230, 0.2450, and 0.2471 mPa.s

 

Pharmacological Test:

Research on the efficacy of formula in rabbit hair growth activity was carried out by the modification of Tanaka et.al  method 17. A total of 3 male rabbits were used. Before the treatment was carried out, the test animals were acclimatized first for 7 days in order to get used to living in the environment and new treatments. After that, one day before testing, the animal was shaved clean and smeared with 70% alcohol as an antiseptic and rested for 24 hours.

 

Then on the back of the sheared test animals, 6 boxes were made with 2x2 cm area for each box (test place). With each box the following test materials are used:

·      Box 1: normal control (without any treatment, F5)

·      Box 2: negative control (Matrix, F0)

·      Box 3: positive control (2.5% minoxidil, F1)

·      Box 4: Test material 1 (Formula with a water fraction of 7.5%, F2)

·      Box 5: test material 2 (Formula with 10.0% water fraction, F3)

·      Box 6: Test material 3 (Formula with 12.5% ​​water fraction, F4)

 

 

 

Determination of the application area was done randomly and it was expected that hair growth activities of all regions with different representatives could be represented. The application of the test material was done twice a day, namely morning and evening as much as 0.5 mL. The research parameter in the hair stimulating activity test was the length of the hair of the test animal in each section. Observations were made for 18 days, assuming polishing on the first day as day 0. Observations were carried out every 3 days, calculated on the 3rd, 6th, 9th, 12th, 15th, and 18th days by taking the three longest strands of hair in each section. The hair of the test animal that had been taken was then stored in a flat plane, and affixed to the tape then measured using a caliper. Results of this measurement can be seen in Table 5 and Fig. 1. In the 18th day, growth was best demonstrated by the Formula F3 and had better growth activity compared with positive control hair growth (Minoxidil). To verify all the data statistical analysis was then carried out.

 

Statistical analysis:

The statistical method used to see the difference in hair growth data between groups was ANOVA (Analysis Of Variance). Statistical analysis was performed with the help of IBM SPSS Statistic 24.0 software 20,21,39. After obtaining rabbit hair growth data with a range of three days, then the data were averaged and then obtained statistically using the ANOVA method. To find out that the data we have described the real situation, the data needs to be analyzed using statistical analysis. In this study, the activity of stimulating hair growth between the formulas F2, F3, and F4 was done by looking at the data normality test. Normality testing is the most basic statistical analysis before further statistical analysis was carried out. The normality test here serves to see whether the data we get follows or approaches the normal distribution or not. In this study, the data normality test was carried out using the Kolmogorov Smirnov test. Based on Kolmogorov Smirnov test rabbit hair length data is normally distributed with a significance value> 0.05 (see Table 6). Then the homogeneity of the data was tested with the Levene test. Based on the Levene test, rabbit hair length measurement data was said to be homogeneous with a significance value for response> 0.05. With the fulfillment of the normality and homogeneity of this data, to determine the presence or absence of significant differences between treatment groups, one-way analysis of variance analysis was carried out.

 

Based on the Anova Test results obtained with a significance value of the treatment of 0.00 <0.05, which means that there are differences in the effect of some treatments on hair growth stimulant activity, then continued with Duncan's Test. On Duncan's test that there were 5 different subsets of test results. Table 7 showed that there was a difference in the activity of hair fertilizers from each faction although it was not real. Of the 5 subsets, the Formula with 10% water fraction provides the best results for hair growth compared to other treatments.

 

It worth to mention that study of the use of plants extract for anti-baldness has been reported by researchers but none they ever mentioned to formulate A. evecta water fraction. Purwal et.al36, for example, studied hair formulation of Emblica officinalis, Bacopa monnieri, Trigonella foenumgraecum, Murraya koenigii in various concentrations in the form of herbal oil for their hair growth activity. The use of green tea was reported by Amin et.al 5, Sulastri et.al9 used combination of green tea and centella herbal tea. Formulation and evaluation of hair tonic from Ziziphus jujuba leaf extract was the article of Sekar et.al37. Jojoba, tulsi, and coconut oil were formulated and reported by Badhe et.al38. Hair growth gel formulation containing extracts of Hibiscus rosa sinensis flower 1%, Eclipta alba whole plant 1% and Solanum nigrum plant berries 0.5% reported by Chakraborty et.al39. Plants used in traditional phytotherapy for hair care by tribals in Sabarkantha district, Gujarat, India was articled by Punjani and Kumar 40.

 

CONCLUSION:

From this work, it could be concluded  the following:

a.    Hair tonic containing A. evecta water fraction  7.5%, 10.0%, and 12.5% organoleptically (form, odor, color, and homogeneity) stable during 2 months storage

b.    The formulated formula showed in good and stable for 30 days of freeze-thaw, pH, and viscosity tests.

c.    All of the formulae showed activity towards hair growth. Moreover, it showed greater activity than minoxidil 2.5%, whereas formula 3 (10.0% water fraction) gave the best activity.

 

It is advisable to carry out hair growth test to bald volunteers in order to verify the effectiveness of the formula before it could be marketed. Further research on the formulation of A. evecta  extract using current technology would be worth to develop.

 

ACKNOWLEDGMENT:

We thank PDUPT-RISTEK DIKTI to support and finance this project.

 

CONFLICT OF INTEREST:

There is no conflict of interest among authors

 

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Received on 29.10.2018         Modified on 24.11.2018

Accepted on 21.12.2018      © RJPT All right reserved

Research J. Pharm. and Tech. 2019; 12(3): 1079-1085.

DOI: 10.5958/0974-360X.2019.00177.X