AU2021104419A4 - Polyherbal Gel formulation for growth of Hair Follicle - Google Patents

Abstract

The traditional system of medicine in India acclaims a number of herbal drugs for hair growth promotion newline. But herbs can also aid in your hair growth hair goals. Here we have developed a formulation consisting of horsetail and Ginseng. The silica in horsetail has been shown to encourage hair growth and hair thickness. Using this extract also impacts your collagen production in a positive way that will improve your hair health and overall look. Dermal irritancy test was conducted to evaluate the irritancy of the prepared gel formulations on intact skin of rabbits. Gel formulations of EEEA (10 %) and EELN showed no erythema and no oedema, indicating that the prepared formulations were non - irritant on the skin of rabbits. Among the various formulations the formulation containing combination of EEEA (5 %) and EELN (5 %) showed better growth initiation time and hair growth completion time and also the same formulation showed significant improvement in hair length compare to control, standard and other gel formulations containing various concentrations of horsetail and ginseng

Description

Title of Invention

Polyherbal Gel formulation for growth of Hair Follicle.

Background

The hair follicle is a cutaneous organ that remodels itself during cyclical periods of active hair growth (anagen), apoptosis-driven involution (catagen), hair shedding (exogen), and relative rest (telogen) Beside the androgenic hormones, the miniaturization of hair follicle might be explained by a shorter anagen cycle The hair follicle size and the duration of anagen phase indicate the length and the size of hair shaft, respectively The normal duration of anagen is around 2-6 years on average, and then it will turn to a short transitory period of catagen, in which the follicle will undergo apoptosis Therefore, one of the goals for treating androgenetic alopecia is to prolong the anagen

Free radicals, which are highly reactive molecules with unpaired electrons that can directly damage various cellular components, might be another factor affecting the hair loss in androgenetic alopecia. Since the oxidation process leads to progressive damage of cellular structures, the ageing phenotype of hair manifests as a decrease in hair production It has been reported that lipid peroxides on hair follicles led to the early onset of the catagen in murine hair cycles. Therefore, antioxidant compounds might be used to prolong the anagen phase and reduce the hair loss. Nowadays, the potassium channel opener minoxidil and the dihydrotestosterone synthesis inhibitor finasteride have been used for the treatment of androgenetic alopecia However, these chemicals might cause some adverse reactions. Some patients using minoxidil encounter with fast or irregular heartbeat, rapid weight gain, bloating, flushing, redness of skin, swelling of feet or lower legs, etc., whereas, finasteride can cause cold sweats, confusion, dizziness, faintness, loss in sexual ability, etc. Therefore, there is an interest in finding new compounds for the treatment of androgenetic alopecia, especially from natural sources. Deficiency of useful minerals and vitamins in body, Mental and emotional stress, Prolonged illness, Hormonal imbalance is commonly seen in hyperthyroidism, imbalance in androgen, and estrogen, Usually after childbirth due to hormonal imbalance, Certain medications such as blood thinners, vitamin A if taken in excess amount, no contraceptive pills, antidepressant drugs, and medicines used in chemotherapy for treating cancer patients, Certain infections that can promote hair loss, for example, fungal infection on scalp, Diseases such as diabetes may also be a precipitating factor in hair loss, Poor blood circulation or excess blood loss, Poor nutrition, Lack of sleep and lifestyle disorder & Hereditary factors.

Horsetail extract is taken from the horsetail plant, which is technically in the fern family. It's been around since the Paleozoic Era and has been used to stop hemorrhages and treat kidney disease in the past. The name comes from the appearance of the plant, which looks like the mane and tail of a horse. Horsetail (Equisetum arvense) is a plant that has been used as an herbal remedy for centuries.

The botanical name for horsetail is Equisetum arvense. It contains high amounts of silica, which is a compound that strengthens hair and nails. In fact, horsetail is one of the plants that contain the highest amount of silica. Equisetum Linn. is one of the most oldest living genera of vascular plant which comprises about twenty-five species .The most investigated species was Equisetum arvense L. which has been widely used in traditional medicines for the treatment of hair loss . Historically, it was used as a diuretic to increase the frequency of urination. In recent years, it has developed a reputation as a hair care and hair loss remedy.

The mixture of E. arvense shoot extract and ginseng has been used as a hair tonic Additionally, the superior reduction of telogen effluvium duration in patients treated with herbal drug containing E. arvense extract (seven weeks) comparing to minoxidil solution (seven weeks) has also been reported.

Horsetail extract is known to improve the circulation of your blood, which in turn leads to healthy hair follicles. It has antioxidant properties that also work as a detox for your hair and body. When your scalp gets enough blood, it increases its ability to produce more hair.

The silica in horsetail has been shown to encourage hair growth and hair thickness. Using this extract also impacts your collagen production in a positive way that will improve your hair health and overall look.

Since it has been shown to help with hair growth and stimulating the follicles, horsetail has been used in cases of hair loss. There isn't a large amount of scientific evidence to back this up, but many people swear by its effects in the way that silica interacts with keratin and improves its structure, making hair stronger.

E. arvense L. contains various chemical compounds such as silicic acid, linoleic acid, oleic acid, stearic acid, linolenic acid and traces of alkaloids (e.g., equisetin, nicotine, palustrine, and palustrinine), glucoside, flavonoids, saponosides, triterpenoids, phytosterols, calcium carbonate, potassium sulfate, potassium chloride, manganese chloride, iron, manganese, and calcium phosphate Indeed, Veit et al. isolated two styrylpyrone glucosides (3'-deoxyequisetumpyrone and 4'-O-methylequisetumpyrone) from the MeOH extract from the rhizomes ofE. arvense.

Ginseng has been shown to promote hair growth in several recent studies. However, its effects on human hair follicles and its mechanisms of action have not been sufficiently elucidated. Red ginseng extract and its ginsenosides may enhance hDPC proliferation, activate ERK and AKT signaling pathways in hDPCs, upregulate hair matrix keratinocyte proliferation, and inhibit the DHT-induced androgen receptor transcription. These results suggest that red ginseng may promote hair growth in humans.

Description of Invention

Method of Preparation

Preparation of base hair gel

• Measured quantity of methyl paraben, glycerin and weighed quantity of polyethylene glycol were dissolved in 35 ml of water in a beaker. Then it was stirred at high-speed using mechanical stirrer.

• Then Carbopol 934 or sodium CMC and PVP were added slowly to the beaker containing above liquid while stirring.

• Then triethanolamine (gelling agents) was added slowly while stirring to attain gel structure.

• The gel was finally transferred to aluminum collapsible tubes and labelled accordingly. Different composition in base gel formulation is mentioned in Table 1.

Evaluation of base hair gel Formulation Physical appearance

The physical appearance was visually checked for the colour, appearance and the feel on application of prepared hair gel formulation was noted.

pH determination

The pH of all hair gel formulations were determined by using the digital pH meter. One gram of gel was dissolved in 100 ml distilled water and stored for two hours. Electrodes were completely dipped into the hair gel formulations and pH was noted. The measurement of pH of each formulation was done in triplicate and average values were calculated.

Extrudability determination

The hair gel formulations were filled into collapsible metal tubes. The tubes were pressed to extrude the material and the extrudability of the formulations was checked. The extrudability of the formulation was determined in terms of weight in grams required to extrude a 0.5 cm ribbon of gel in 10 seconds.

Viscosity determination

The viscosity of the prepared gel formulations was measured by Brook field viscometer model LV - DV-II +. The sufficient quantity of gel was filled in wide mouth jar separately. The height of the gel filled in the wide mouth jar should sufficiently allow dipping the spindle. The RPM of the spindle was adjusted to 2.5 RPM. The viscosities of the formulations were recorded.

Spreadability

It indicates the extent of area to which gel readily spreads on application to skin or affected part. The therapeutic potency ofa formulation also depends upon its spreading value. Spreadability is expressed in terms of time in seconds taken by two slides to slip off from gel which is placed in between the slides under the direction of certain load.

Lesser the time taken for the separation of two slides, better the spreadability. It is calculated by using the formula

S = M. L / T where,

M= Weight tied to upper slide L= Length of glass slide T= Time taken to separate the slides

Stability studies

The stability studies were carried out for all the prepared gel formulations by freeze - thaw cycling. Here, by subjecting the formulations to a temperature of 40 C for one month, then at 250 C for one month and then 400 C for one month and syneresis was observed. After this, the gel is exposed to ambient room temperature and liquid exudate separating is noted.

Homogeneity

After the gel formulations have been set in the container, all developed gels were tested for homogeneity by visual inspection. They were tested for their appearance and presence of any lumps, flocculates or aggregates.

Grittiness

All prepared gel formulations were evaluated microscopically for the presence of any appreciable particulate matter which was seen under light microscope. Hence obviously the gel preparation fulfils the requirement of freedom from particular matter and from grittiness as desired for any topical preparation (Kaur et al., 2010).

Results

Optimization and selection of gel formulation

One of the main ingredients of the gel formulation is the gelling agent. The concentration of viscosity enhancer or gel former is of immense value as a less concentration will lead to simple solution or lotion with very low consistency, while high

concentration may lead to formation of gels with high viscosity leading to non - uniform distribution of drug and problem with handling of gel formulation. Two different gel formers with various concentrations were tried in order to select the best gelling agent. Gels containing sodium CMC showed phase separation and are poor in consistency as indicated by spreadability and extrudability values, hence these gel formulations were rejected. Gels containing 0.5 % and 1 % of Carbopol 934 form a very thin gel that liquefies within 4 and 5 hours of preparation respectively. With 1.5 % Carbopol 934 gelling agent to some extent better gel was obtained but the problem of liquefaction after 28 hours was observed. Gel formulation containing 2 % of Carbopol 934 formed uniform and smooth gel that does not liquefy upon keeping long time.

The pH of the formulation was determined in order to be sure that the formulation can be used without the risk of irritancy to the skin. The pH was found to be 7.56 for G4 gel formulation which was very nearer to the neutral skin pH, thus the formulation G4 can be used without the risk of irritancy to the skin. This also indicated that the selected ingredients of the formulation did not alter the pH of the formulation

The spreadability of formulations was found to decrease with increasing the concentration of gelling agent. The value of spreadability for optimized gel was found to be 10.2 cm indicating that the gel is easily spreadable by small amount of shear. The results indicated that the formulation can be applied easily without being runoff. This promises that the formulation maintain a good wet contact time when applied to the site of application. According to the present study, 2 % of Carbopol 934 was selected as the optimized concentration of gelling agent and this gel formulation is used for further herbal hair gel preparations.

Preparation of herbal hair gel formulation

Herbal hair gel formulation was prepared with Ethyl acetate extract in ethanol 5

% (HG1) and 10 % (HG2), 5 % (HG3), 10 % (HG4) and combination of these HG1 & HG2 (5 %) and (5 %) (HG5) and pharmaceutical parameters were evaluated. Measured quantity of methyl paraben, glycerin and weighed quantity of polyethylene glycol were dissolved in about 35 ml of water in beaker. Then it was stirred at high-speed using mechanical stirrer. Then carbopol 934 and PVP were added slowly to the beaker containing above liquid while stirring. Crushed menthol was incorporated slowly in above dispersion after smooth dispersion is obtained. Then triethanolamine (gelling agents) was added slowly while stirring to attain gel structure. The ethyl acetate soluble fraction of ethanolic extract of horse tail and Ginseng was levigated using stainless steel spatula and porcelain slab.

Different composition in preparing herbal hair gel is mentioned in Table 2

Evaluation of herbal hair gel formulations

The physical appearance was visually checked for the colour, appearance and the feel on application of prepared herbal hair gel formulation were noted. And also other evaluation parameters like pH, viscosity, extrudability, spreadability, homogeneity and grittiness, feel of application, stability and consistency were evaluated as per the methods mentioned earlier in evaluation for base gel formulations. The results for the evaluation parameters of herbal hair gel are tabulated in Table 2.

From the physical evaluation the color of prepared herbal hair gels was pale green in color and appearance of gel was translucent and it was smooth on application. So it shows significant physical evaluation parameters. The subjective properties such as consistency were good and texture of prepared herbal hair gel was found to be smooth. The pH value of the prepared gel formulation was observed at room temperature and valued range at 7.2 to 7.6. As we go from epidermis to dermis, pH of the skin increases and attained the neutral value. So gel formulation having pH range 7.2 to 7.6 are desirable to skin since they do not interfere with the physiology ofskin.

The prepared herbal gel formulation were subjected to accelerated stability testing. The prepared herbal gel was stored at 40 C, 250 C and 450 C in refrigeration, room temperature and oven for a period of 30 days to study effect of temperature and at different humidity condition. The physical parameters were evaluated during study period. The result of the study indicates that the preparation is physically stable at all temperatures tested.

Evaluation of EEEA and EELN gel formulation against dermal irritancy (OECD Guideline 404)

Skin irritation

The substances that induce inflammation are known as irritants. Inflammation may be caused by use either immediately or through prolonged use or on repeated use. Irritants may be classified as primary irritants and secondary irritants. Primary irritants cause inflammation on the first contact (the duration of contact may be of several hours). Secondary irritants are harmless on first contact but cause inflammation on repeated contact and inflammation becomes more severe with progressive contacts. Contact urticaria means local oedema (swelling) and erythema (increased blood flow) at the site of application of a substance. Contact urticaria may be allergic or nonallergic. Some substances cause ill defined response when applied to the skin. This response is generally termed as stinging. The other terms which refer to this response are itch, tingle, burn or sore. This response starts within minutes of application of substance, intensifies within next 5 - 10 minutes and then vanishes. This phenomenon is different from irritation and does not lead to inflammatory change.

Test Procedure Application of the test substance Half a gram of the herbal gel formulation was applied to a small area (approximately 6 cm 2 ) of skin and covered with a gauze patch, which is held in place with non-irritating tape. In cases in which direct application is not possible (e.g., liquids or some pastes), the test substance was first be applied to the gauze patch, which is then applied to the skin. The patch was loosely held in contact with the skin by means of a suitable semi - occlusive dressing for 4 hours duration of the exposure period and then removed. If the test substance is applied to the patch, it was attached to the skin in such a manner that there is good contact and uniform distribution of the substance on the skin. Access by the animal to the patch and ingestion or inhalation of the test substance was prevented. At the end of the exposure period, which was normally 4 hours, residual test substance was removed using water or an appropriate solvent without altering the existing response or the integrity of the epidermis.

Initial test (In vivo dermal irritation/corrosion test using one animal) It was strongly recommended that the in vivo test be performed initially using one animal, especially when the substance was suspected to have corrosion potential. This was in accordance with the sequential testing strategy. When a substance has been judged to be corrosive on the basis of a weight-of theevidence analysis, no further animal testing is needed. For most substances suspected of being corrosive, further in vivo testing is normally not necessary. However, in those cases where additional data are felt warranted because of insufficient evidence, limited animal testing may be carried out using the following approach: Up to three test patches were applied sequentially to the animal. The first patch was removed after three minutes. If no serious skin reaction was observed, a second patch was applied at a different site and removed after one hour. If the observations at this stage indicate that exposure can humanely be allowed to extend to four hours, a third patch was applied and removed after four hours, and the response was graded. If a corrosive effect was observed after any of the three sequential exposures, the test was immediately terminated. If a corrosive effect was not observed after the last patch was removed, the animal was observed for 14 days, unless corrosion develops at an earlier time point. In those cases in which the test substance was not expected to produce corrosion but may be irritating, a single patch was applied to one animal for four hours.

Grading of skin reactions - Erythema and Eschar Formation

N o ery them a ........................................................................................................... 0

Very slight erythema (barely perceptible) .............................................................. 1

W ell defined erythem a ..... ............................................................................. . 2

M oderate to severe erythem a .......................................................................... . 3

Severe erythema to eschar formation preventing grading of erythema............... 4

Maximum possible: 4

Oedema Formation

N o o ed em a ..................................................... ......................................................... 0

V ery slight oedem a (barely perceptible) ................................................................... 1

Slight oedema (edges of area well defined by definite raising) ........................... 2

Moderate oedema (raised approximately 1 mm) .................................................. 3

Severe oedema (raised more than 1 mm and extending beyond area of exposure)... 4

Maximum possible: 4

Both the control and the test animals were observed every day for any occurrence of skin irritation or toxic reactions such as oedema or erythema. Per observation of skin,

A value between 0 and 4 was recorded where 0 meant no skin erythema and eschar formation and 1, 2, 3 and 4 stood for very slight, well defined, moderate and severe erythema to eschar formation, respectively. It also scored from 0 - 4, where 0 stood for no oedema and 4 stood for severe oedema

Evaluation of EEEA and EELN for hair growth promoting activity

Qualitative hair growth study Qualitative hair growth analysis was undertaken by visual

observation of two parameters: hair growth initiation time (i.e. minimum time to initiate hair

growth on denuded skin region) and hair growth completion time (i.e. minimum time taken to complete cover the denuded skin region with new hair)

Table 1 : Different composition of base gel formulation

Methyl Sodium Carbopol PVP paraben Glycerine PEG Triethanol Formulation CMC 934 (g) (mg) sodium (ml) (ml) amine

(g) (mg) (ml)

GI - 0.5 5 75 3 6.25 0.6

G2 - 1.0 5 75 3 6.25 0.9

G3 - 1.5 5 75 3 6.25 1.2

G4 - 2.0 5 75 3 6.25 1.5

G5 0.5 - 5 75 3 6.25 0.6

G6 1.0 - 5 75 3 6.25 0.9

G7 1.5 - 5 75 3 6.25 1.2

G8 2.0 - 5 75 3 6.25 1.5

Table 2. Formulae of herbal hair gel

Formulation HG1 HG2 HG3 HG4 HG5

5%of 10%of 5%of 10%of 5%ofEEEA+ Herbal Extract (g) EEEA EEEA EELN EELN 5 % of EELN

Carbopol934(g) 2 2 2 2 2

PVP (mg) 5 5 5 5 5

Methyl paraben sodium (mg) 75 75 75 75 75

Glycerine (ml) 3 3 3 3 3

PEG (ml) 6.25 6.25 6.25 6.25 6.25

Triethanolamine (ml) 1.5 1.5 1.5 1.5 1.5

Table 3. Evaluation of base hair gel formulation

Formulations

Parameters Gi G2 G3 G4 G5 G6 G7 G8

Feel of Smooth Smooth Smooth Smooth Smooth Smooth Smooth Smooth application

Spreadability 12.7 10.9 10.2 6.3 5.5 4.8 4.2 (g.cm/sec) 13.5

Excellent Consistency Poor Good Good uniform Poor Poor Fairly Good

gel good

pH 6.69 6.91 7.12 7.56 6.91 7.02 7.35 7.41

Viscosity 85,065 1,00,080 1,52,013 50,075 62,000 81,072 92,070 (cps) 82,000

Extrudability Good Good Excellent Excellent Poor Poor Good Good

Table 4. Evaluation parameters of herbal hair gel formulation

Formulations

Parameters HGI HG2 HG3 HG4 HG5

Colour Pale green Pale green Pale green Pale green Pale green colour colour colour colour colour

Appearance Translucent Translucent Translucent Translucent Translucent

Odour Pleasant Pleasant Pleasant Pleasant Pleasant odour odour odour odour odour

Spreadability 10.7 10.6 10.2 10.7 10.9 (g cm/sec)

Homogeneity Good Good Good Good Good

Feel of Smooth Smooth Smooth Smooth Smooth application

Consistency Good Good Good Good Good

pH 7.6 7.2 7.5 7.4 7.2

Viscosity 1,52,000 1,51,065 1,52,070 1,52,013 1,52,075 (cps)

Extrudability Excellent Excellent Excellent Excellent Excellent

Stability Stable Stable Stable Stable Stable

Table 5

Effect of EEEA and EELN gel formulation on hair growth initiation and completion time

Hair growth in days Per cent

Treatments reduction in hair Initiation time Completion time growth completion time*

Negative control - simple gel 11.8 0.94 42.6 1.50

Positive control - 2 % minoxidil 6.07 0.42 33.0 0.71 23.55***

5 % EEEA (HG1) 10.4 0.86 35.0 0.41 15.51

10 % EEEA (HG2) 8.01 0.64 33.4 0.49 23.96***

5 % EELN (HG3) 11.3 0.57 40.2 0.99 7.69.

10 % EELN (HG4) 13.4 0.61 36.2 0.58 18.39

5 % EEEA + 5 % EELN (HG5) 6.03 0.81 28.2 0.76 34.75*** * (Mean of control - Mean of treatment) / Mean of control X 100 ***P < 0.05 significant when compared to control (ANOVA followed by Dunnett's test; Values are mean ±SEM

Summary

Hair loss is a dermatological disorder, and the surge for discovering natural products with hair growth promoting potential is continuous. Hair loss or alopecia is a common patient complaint and a source of significant psychological and physical distress. Androgens are considered to be one of the most important causes for alopecia apart from a variety of other factors. Minoxidil, a drug of scientific origin was scientifically proved for the treatment of alopecia. Though the side effect associated with this drug has limited its pharmacological benefits hence the drug of plant origin is necessary to replace the synthetic one. Thus it is very important to develop new therapeutic materials to stop hair loss and to enhance hair growth. Alternative medicine is one interesting area, which is getting more popular. Although it has not yet been incorporated into the mainstream of medical care because of limited scientific evidences and lack of mechanistic understanding, alternative medicine is becoming an increasingly attractive approach all over the world. Natural products in the form of herbal formulations are available on the market and are used as hair tonic, hair growth promoter, hair conditioner, hair-cleansing agent, antidandruff agents, as well as for the treatment of alopecia and lice infection. A number of herbal products have been acclaimed with hair growth promoting activity. The traditional system of medicine in India acclaims a number of herbal drugs for hair growth promotion but lack of sound scientific backing and information limits their use. The determination of physicochemical constants such as moisture content, ash values, extractive values and elemental composition of aerial parts of Horsetail and Ginseng were carried out to confirm the identity of herb and to ascertain the quality and purity of the drug. These physical parameters are useful in establishing quality profile of drug and constitute an important component of qualitative evaluation. Plants with possible antimicrobial activity should be tested against an appropriate microbial model to confirm the activity and to ascertain the parameters associated with it.

Horsetail and Ginseng are important plants among many traditional herbs in the treatment of

many diseases, which are usually free from side effects, are economical and also easily accessible to humans. On the basis of our results of the present study, it is concluded that the ethyl acetate fraction of ethanolic extract of Horsetail and Ginseng and the isolated compounds EA - 1 and LN - 1 have significant antibacterial and antifungal activity. So these two plants can be considered for preparation of nutraceuticals with potent antimicrobial activity suitable for the prevention of human disease. Therefore these extracts can be considered as a potential source of natural antioxidant agent. gel formulations were prepared with EEEA 5 %, EEEA 10 %, EELN 5 % and EELN 10 % and physical parameters were evaluated. Hence this herbal gel formulation was utilized for further pharmacological studies. Dermal irritancy test was conducted to evaluate the irritancy of the prepared gel formulations on intact skin of rabbits. Gel formulations of EEEA (10 %) and EELN showed no erythema and no oedema, indicating that the prepared formulations were non - irritant on the skin of rabbits. Among the various formulations the formulation containing combination of EEEA (5 %) and EELN (5 %) showed better growth initiation time and hair growth completion time and also the same formulation showed significant improvement in hair length compare to control, standard and other gel formulations containing various concentrations of horsetail and ginseng.

Claims (5)

Claims:

1) Herein we claim a Polyherbal Gel formulation for growth of Hair Follicle, which helps in hair growth.

2) The gel formulation developed in1 is developed with various concentrations of horsetail and ginseng.

3) The gel formulation developed in 1 is non - irritant on the skin.

4) The gel formulation developed in 1 is applied topically as cosmetics but contain ingredients that influence the skins biological function.

5) We also claim the method of preparation and evaluation of formulation claimed in 1.