In-vivo studies to determine Hair Growth Potential of Poly Herbal Medicated Hair Oil in Female Swiss Albino Mice

 

Pandya JK¹, Senghani MK¹*, Sukhramani PS¹, Chaudhari BG²

¹Veerayatan Institute of Pharmacy, At Post: Jakhania,

Bhuj- Mandvi Highway, Mandvi- Kutch, Gujarat-370460, India.

²Tolani Institute of Pharmacy, Adipur- Kutch, Gujarat- 370205, India.

 

ABSTRACT:

Hair loss is one of biggest problem of today’s human life throughout World. Alopecia, Baldness and hair loss are commonly found in the men as well as in female. Commonly available medicated hair oils in market for the treatment of hair disease are made up with mineral oil, which is proved to be carcinogenic in nature if applied for longer period of time. Hair plays a vital role in the personality of human and for their cure we use lots of cosmetic products. The fading (pigmentation problem), dandruff, alopecia (loss of hair) is the major problem associated with hairs. An Ayurveda is Indian traditional system of Medicine having major treatment across globe. The aim of study is to develop a poly herbal hair oil (PHO) formulation using Azadirachta indica, Rosa sinensis, Muraya koenigii, Lagenaria siceraria, Bacopa monieri, Lawsonia inermis, Eclipta alba, Embelica officinalis, coconut oil and Til oil, for better growth of hair and diminution in loss of hair (alopecia). The oil was prepared according to Ayurvedic Formulary of India and was standardized according to Protocol for Testing Ayurvedic, Siddha and Unani Medicines, Government of India. The physico-chemical evaluation was carried out for medicated hair oil. The pharmacological evaluations were performed through Pre-clinical Animal studies for qualitative, quantitative and mean- hair length studies.The statistical results of the study have demonstrated that 5% PHO possessed potent action on hair growth in the Female Swiss albino Mice animal’s model investigates as well as these provide a rationale in favor of its uses inside traditionally drug in favor of utilization in Baldness and other hair growth disorders.

 

 

 

INTRODUCTION:

Anatomy of Hair:

It is cylindrical structures or shafts made up of tightly compacted cells that grow from small sac-like organs called follicles. In man, the diameter of individual hair shafts may range from 15 to 120 pm depending upon the type of hair and the region of the body the follicle is located¹. Hair contains a family of sulfur-rich proteins called keratin (from the Greek word, keras, meaning horn)². Hair is the one of the vital part of our body and it influences the overall appearance of the person.

 

Haircare products are defined as those formulations which are used for cleansing, modifying the texture of hair, changing of the color, giving life to the stressed hair, providing nourishment to the hair and giving the healthy appearance to the hair³.

 

HAIR PROBLEMS AND DISEASE:

Hair problems ⁴:

·       Dandruff: Dandruff, scaly particles that cling to the root of the hair, can be caused by a poor diet, an infection, or even a sluggish metabolism.

·       Hair Loss: Although historically discussed as a problem among men, hair loss/ thinning hair is also common among women.  For most men, it may be male pattern balding.  Female hair loss is often caused by stress, medication, changing hormones, and even menopause.  Additionally, many hair styling products (chemicals/excessive heat) can contribute to hair loss.

·       Dry Hair:-Shampooing too often causes dry hair.  Although squeaky-clean hair is a good thing, many overdo it by washing once, sometimes twice, per day.  That will strip away all of the hair’s natural oils.

·       Spit Ends:-Over-brushing hair, excessive perming, too much heat, and lack of a good conditioner cause split ends.

 

Hair disease ⁵

Common baldness:

The most common type of hair loss is referred to as male-pattern baldness (when it occurs in women it is called female-pattern baldness).

 

Alopecia:

It is the most common cause of hair loss in men also known as hereditary baldness. Androgenetic alopecia hair follicle size is reduced and duration of anagen is diminished while an increase in the percentage of hair follicles in Telogen.

 

Alopecia areata (AA):

This is a hair loss condition where both scalp and body can be affected. It is an inflammatory and autoimmune disorder which strikes 2% population. The main characteristic of AA is hair loss in patches. When the whole scalp is affected it is called alopecia totalize, while losing hair from all body is called alopecia universalis. Pathology reveals an increase in number of Telogen and catagen folliclesincluding presence of the inflammatory lymphatic infiltrate per bulbar area.

 

Chemotherapy-induced alopecia:

The use of chemotherapy drugs for cytotoxic anticancer lead to the appearance of alopecia by eroding the rapidly increasing epithelium of hair follicle. It is estimated that 65% of patients suffer from hair loss due to chemotherapeutic medicament and thetreatment protocol. Anagen effluvium caused by chemotherapy is reversible in most cases, but there are certain chemotherapy agents that induce alopecia permanently.

 

Hair Oil⁶:

Hair oil is applied to the scalp for soothing and revitalizing it. In India, three types of hair oils are manufactured and marketed by small-scale industry as well as by a few units in the organized sector of the industry. The type 1 contains vegetable oil or oils as base oil and type 2 contains only mineral oil as base oil, whereas type 3 contains a mixture of vegetable oil and mineral oil. In the present study hair oil of all three types of popular brands produced in the country have been taken into consideration. But the test findings revealed that all of them belonged toonly Type-3.

 

Hair oil has no nutrition, no medicinal qualities, and no health properties. Yes, the oils do have shelf life, good fragrance, no stickiness, good color, excellent packaging the works in short everything except the oil that nourishes. All the brands of hair oil were mineral oil based which was confirmed by presence of significant quantity of un Saponification matter test.

 

Plant Profile^7-13

Medicinal plant is a major component of traditional medicine system. In India, the use of different parts of several medicinal plants to cure specific ailments has been in vague from ancient times. The indigenous system of medicine namely Ayurvedic, Siddha and Unani have been in existence for several centuries. These plants are currently in considerable significance view due to their special attributes as a large source of therapeutic phytochemicals that may lead to the formation of new drugs. Ethno-botany is the study of traditional knowledge of plants including classification, cultivation and medicinal properties in order to discover future medicines. Aimed to modern drug discovery, traditional medicinal plants have been studied which is followed by ethno-botanical lead of indigenous cures used by traditional medical systems. The demand for herbal medicines is increasing rapidly due to their lack of side effects. It is traditionally used to check hair loss, stimulate hair growth and is therefore used in hair care. Natural plant remedies have created an enormous need for information about the properties and uses of the medicinal plants.

 

List of Used Medicated Poly Herbal Plant:

·       Azadiricta indica (Neem)

·       Bacopa monieri (Brahmi)

·       Cocus nucifera (Coconut)

·       Eclipta alba (Bhringraj)

·       Emblica officinalis (Amla)

·       Hibiscus rosasinensis (Hibiscus flower)

·       Lawsonia innermis (Heena)

·       Lagenaria siceraria (Bottle Gourd)

·       Murray koenigii (Curry leaf)

 

MATERIAL AND METHODS:

Collections and Authentication for plant material:

Collection of herbal drug raw materials was from local market of Palanpur (Gandhi soap), and local area of Palanpur region, Banaskantha, Gujarat, India. Authentication of herbal plant materials was carried out by Dr. Pankaj N. Joshi, Executive Director of Sahjeevan, Bhuj -Kutch, Gujarat-India.

 

Preparation and storage of powdered materials:

All herbal raw materials were taken and properly dried in shade to remove excess of moisture. The dried raw materials Grained individually and pass sieves 10-20 #. The powder was then store at room temperature.

 

Macroscopic evaluation:

The Herbal Plant raw material were subject to macroscopical evaluation, comprise of study of organoleptics characters of the drugs. Chart inspect provide the simpler plus quicker to set up individuality, purity benefit perhaps excellence of plant material. So, the shapes, sizes, surface characteristic, textures, fracture characteristic in addition to look of cut outside, color and taste etc., were examined for identity and purity of material.

 

Formulation of polyherbal medicated hair oil:

This process is suitable for extracting water soluble or heat stable constituents. Oil/ paste and decoction prepared individually and boiled with the coconut oil with continuous stirring at a constant temperature, until the water droplets in oil stopped crackling sound and the drugs were completely extracted in the oil. The oil was then filtered through a muslin cloth and stored. It prepared by Oil: paste: decoction (1:2:4) ratio. a) Oil:-Coconut Oil (Taila) b) Paste:-The kalka of curry leaf, neem, hibiscus flower, and bottle gourd withwater. c) Decoction:-In this process the powder drug (Amla, Brahmi, and Bhringraj) isboiled with water for defined time, cooled and filtered.

 

Formulation of polyherbal medicated hair oil made by using herbal plant like E. officinalis (Amla), E. alba (Bhringraj), L. siceraria (Bottle gourd), B. monieri (Brahmi), L. inermis (Heena), M .koenigi (Curry leaf), R. sinensis (Hibiscus flower), A. indica (Neem) in various compositions like 2.5%, 5% and 7.5%.

 

Evaluation Parameters of Herbal Hair Oil and Herbal Plant Materials:

 

Physical parameters of medicated poly herbal hair oil^14-19:

PHO was evaluated by determination of Acid Value, Saponification Value, pH, Viscosity, Specific gravity, Refractive index and organoleptic property.

 

In-vivo studies to determine Hair Growth Potential of Poly Herbal Medicated Hair Oil formulation in Female Swiss Albino Mice:

The objective of this study was to evaluate the hair growth potential of Poly Herbal Oil (PHO) in female Swiss albino mice. It was believed that the poly herbal oil at different concentrations, have potential toenhance the hair growth in human and hence a proof of concept study in normal mice was planned to evaluate the hair growth potential of PHO in female Swissalbino mice. The test facility is certified by the Committee for the Purpose of Control and Supervision of Experiment on Animals (CPCSEA) for breeding andexperimentation, certificationnumber1780/PO/RcBi/S/14/CPCSEA.

 

Animal study:

The study design included 5 groups containing 4 animals each, each animal was anesthetized and the dorsal side was carefully cleaned using a disinfectant, followed by shaving a patch measuring 2x2 cm by an electronic razor. The patch was then cleaned with a swab soaked in spirit, finally 400 µL of formulation was applied and mice were kept back in respective cage with water and feed ad libitum. The animals were monitored daily for growth and dosage was applied directly on to the skin. The feed and water were replenished daily and bedding along with the cage was changed on every alternate day. Body weight of the animal was recorded daily as well as examined for any abnormalities in the animals. Animals were photographed for visible hair growth monitoring on day 0,7 and 14, whereas hair samples were plucked on day 7 and 14 for hair growth measurement.

 

RESULTS AND DISCUSSION:

Physical parameters for medicated polyherbal hair oil:

 

Table 1: Physical parameters for medicated poly herbal hair oil

Sr. No.	Parameters	Concentration (%)
		2.5	5	7.5
1.	pH	4.1	4.2	4.3
2.	Specific gravity	27.42	27.4	27.45
3.	Acid value	3.9	2.7	2.9
4.	Saponification value	60.2	50.4	55.3
5.	Viscosity	0.326	0.435	0.495

 

In physical parameters present in prepared formulation (hair oil) are in above table. Like pH, specific gravity, acid value, saponification value, viscosity etc.

 

General characteristics for poly herbal formulation of hair oil

Table2: General characteristicsof poly herbal formulation of hair oil.

Sr.no.	Concentration (%)	Color	Odor
1.	2.5	Greenish black	Characteristic
2.	5.0	Greenish black	Characteristic
3.	7.5	Greenish black	Characteristic

 

In general characteristics of prepared formulation (hair oil) different concentration color, odour was observed for various concentrations of PHO give greenish black color and characteristic odor.

 

Photography of skin patch post PHO application:

To monitor visible growth of hair in shaved skin patches, the animals were photographed on day 7 and 14 of the study.

 

Figure 1: Visible monitoring of hair growth post PHO application.

 

 

The visual monitoring during entire study showed that growth occurred slowest in case of vehicle control; the hair growth observed in case of 2.5% and 7.5.0% PHOwere similar or comparable to that of Minoxidil group (Figure-1). Highest growth was found in case of 5.0% PHO followed by Minoxidil (positive control), however in week 2 of the study visually the growth of hair in 5.0% PHO and Minoxidilwere comparable and almost equal. But upon hair strand length measurement significant difference could be observed n 5.0% PHO compared to that of Minoxidil, 2.5% and 7.5.0% PHO.

 

Measurement of hair length:

10-12 strands plucked from each animal of each group was measured in length. The data of 30 independent recordings were averaged and compared. Highest length was observedin case of 5% PHO solution, 4.6±1.074 mm in week 1 which grew to 6.3±1.251 mm in week 2 (table1 and figure 1). The trend of hair length was again in order of: vc<pc ≤ 2.5%PHO ≤ 7.5%PHO< 5 % PHO.

 

Table3: Average length of hair strands

Average length of hair strands
Dosage	Week 1	Week 2
Vehicle control	2.90±0.10	2.60±0.18
Positive control (Minoxidil)	2.80±0.16	2.10±0.13
2.5% PHO	2.70±0.15	2.90±0.10
5.0%PHO	4.60±0.34	6.30±0.40
7.5%PHO	2.60±0.16	2.70±0.33

 

Figure 2: Summary of average length of hair strands.

 

 

Average length of hair strands. Here each data point is average of 30 independentreading. The adjacent values indicate sem values determined for each data set.

 

Histopathology:

At the terminal sacrifice of animals, 1.0 cm x 1.0cm skin sample from the dorsal region of mice from all the groups. Mouse from each group was euthanized at 2 weeks of the experiment. Skin biopsies were taken from the shaved area and fixed using 10 % neutral formalin solution and formatted with paraffin. The paraffin-formatted skin tissue wassliced to a thickness of 5 µm for hematoxylin and eosin (H&E) staining. The stained tissuewas observed by optical microscopy.

 

Figure 3: Histopathological slides prepared for skin sections of mice from different groups.

 

 

The transverse sections of the skin showed evident increase in depth and size of skin follicles insamples treated with PHO as compared to vehicle control. The blue arrows indicate hair follicles in the skin.

 

Statistical analysis:

All the relevant statistical analysis was performed for individual group data against vehicle control group data. The statistical analysis i.e. Paired t test was done using graph pad server.

 

SUMMARY AND CONCLUSION:

The aim of this study was to formulate and evaluate poly herbal medicated hair oil. Hair loss is one of biggest problem of today’s human life throughout World, Alopecia, Baldness and hair loss are commonly found in the men as well as infemale. The present work was to formulate an effective hair tonic and brine tonic medicatedherbal hair growth oil with their evaluation parameter including pre-clinical- animal study.

 

Collection of herbal raw materials was carried out from local market of Palanpur (Gandhi Soap), and local area of Palanpur region, Banaskantha, Gujarat, India. Authentication of plant materials was carried out by Dr. Pankaj N.Joshi, Executive Director of Sahjeevan, Bhuj - Kutch, Gujarat- India. Herbal raw materials were evaluated through various parameters like phytochemical screening, physical parameters, analytical methods, Ash value, extractive value etc. The results of which indicated the authenticity of crude drugs used informulation of oil. There were three different compositions containing formulations prepared during present research work. The pre-clinical study was carried out on female Swiss albino mice for evaluation of Hair growth potential of prepared different formulation of PHO. The aim of animal study was to examine the effect of PHO on hair growth. Animals weighing 20±5 gm were divided into 5 groups of 4 animals each. The 2x2 cm patch was shaved using an electronic razor, 400µL of various compositions containing PHO or relevant controls were applied daily. The animals were maintained at ambient conditions; feed and water were recorded and replenished daily. The animals were monitored daily for growth of hair on the treated patch; the animals were photographed on day 0, 7 and 14 for visual monitoring of hair growth (Figure 1). The visual monitoring of hair patch showed that hair grew fast in case of PHO treated samples compared to vehicle control group. Minoxidil, treated group also showed increase in hair growth compared to control group however it was less compared to 5% PHO group animals. Minoxidil treated group showed comparable growth compared to vehicle control in first week, however appreciable growth was recorded in all minoxidil treated group animals in week 2. It is important to point out that in week 2, the growth in case of 5% PHO was much better as compared to vehicle control and other PHO.

 

To gain a better understanding of the effect of PHO on hair growth, length of hair from the treated patch was measured. Hairs were plucked from the patch of each animal and the length of hair was measured on day 7 and 14, it was not possible to pluck hairs on day0. The data presented here is an average of 30 individual measurements for each group. The hair length at week 1 clearly showed that the 5% PHO showed maximum growth as compared to other PHO and controls, the observation made in week 2 was in corroboration with that of week 1, where again 5% - PHO showed maximum growth (Figure 2). The overall trend of hair length was found as: VC <PC ≤ 2.5% PHO ≤ 7% PHO < 5% PHO H & E-stained skin tissues at 2 weeks after depilation were used to observe the status ofthe hair follicles and hair growth by optical microscopy.

 

In order to investigate the effectof PHO on the skin and hair follicles, transverse section of skin samples were examined. The Histopathological data clearly showed that an evident increase in depth and size ofhair follicles in PHO treated animals versus vehicle control group animals (Figure 3). At 2weeks after depilation, many fully grown hair follicles were observed in the PHO treated mice compared to vehicle control groups. Other observations made was that though the follicles were densely placed in skin treated with 2.5% -PHO, the depth and size/ number of follicles in 5% PHO treated skin was highest explaining the increased length of hair in the group as compared to other samples. The 7% PHO and Minoxidil had comparable hair growth observed. In conclusion the 5% PHO gave best results both in length, size, depth and density of hair follicles.

 

The results of the study have demonstrated that 5% PHO possessed potent action on hair growth in the Female Swiss albino Mice animal‟s model investigates as well as these provide a rationale in favor of its uses inside traditionally drug in favor of utilization in Baldness and other hair growth disorders. Further study is to establish the exact bio-molecular level mechanism of action of PHO.

 

REFERENCES:

1.     Harkey M R, Anatomy and Physiology of Hair.1993; 63 (1-3): 9-18. doi: 10.1016/0379-0738(93)90255-9.

2.     KS, WA, AKV et al. Physiology and Anatomy of Hair in drug abusing cases. International Journal of Medical Toxicology and Forensic Medicine,2012;2(4):153-159, doi.org/10.22037/ijmtfm.v2i4(Autumn).3765.

3.     Saraf S et al. Herbal Hair Cosmetics: Advancements and recent findings. World Journal of Pharmaceutical Research, 2014; 3(2): 3278-3294, ISSN 2277 – 7105.

4.     Gupta S, KR. Hair Loss- An analysis and Updated Treatment. IOSR Journal of Dental and Medical Sciences (IOSR-JDMS), 2015; 14: 77-81, doi:10.9790/0853-14727781.

5.     Cafardi J. The Manual of Dermatology, 2012: 15-17, ISBN-13:978-1461409373.

6.     Anonymous, Comparative test, the tress caressed-Hair oil massage consumer,2011, PMID: 12715094

7.     Subhashiset al. A Review on Role of Medicinal plants in Immune system.Asian Journal of Pharmacy and Technology, 202; 10 (04):273-277, doi: 10.5958/2231-5713.2020.00045.8.

8.     Mohammad et al.Ethnobotanical study on Medicinal Plants used by Traditional HealthPractitioners and Villagers of Garhphulghar Gram, Chhattisgarh, India, Asian Journal of Pharmaceutical Research, 2017; 7(2):98-105, doi: 10.5958/2231-5691.2017.00017.X

9.     Indian Medicinal Plants -A compendium of 500 species. Orient Longman Publications, Part 3, 1997: 256-263.

10.  Meena et al. A Review on Herbal Plants used in Skin and Hair Treatment. Research Journal of Topical and Cosmetic Science, 2010; 1(1): 13-17, doi: Not Available.

11.  Pratibha et al. Physicochemical Investigation of Some Marketed Herbal Hair Oil, Research Journal of Topical and Cosmetic Science, 2016; 7(2): 70-72, doi: 10.5958/2321-5844.2016.00011.X.

12.  Chaudhari et al. Antimicrobial Studies on Selected MedicinalPlants in Khandesh Region, Maharashtra, India, Research Journal Pharmacognosy and Phytochemistry,2010; 2(5): 386-390.

13.  Anonymous. The Ayurvedic Pharmacopeia of India, Part I-VII,Government of India, PDH.81.Pt.I.Vol.IX2000-2015-(DSK-II)

14.  M.Pande, S.Ingale, S.Gupta. The Pharmacognostic and phytochemical studies on the leaves of Murrayakoenigii (L) Spreng”, Indian Journal of Science and Technology, 2009; 2(3), ISSN: 0974- 6846, doi: 10.17485/ijst/2009/v2i3.9.

15.  Conway D. The Magic of Herbs. Readers Union publishedJonathon Cape Ltd, 1975, ISBN 10: 0583124275.

16.  Schauenberg P, Paris F. Guide to Medicinal Plants. Lutterworth, 1990, ISBN0718822617.

17.  Afzal A, Rahamatullah A, Sapna R, Rajeev D, et al. Formulation and evaluation of polyherbal hair care Powders. Ancient Science of life, 1996: 15-20, PMCID: PMC3331134.

18.  K.Sudheer Kumar, S.Gomathi, S.SeetarmSwamy et al.Formulation and evaluation of polyherbal hair poly herbal hair oil-an economical cosmetic. International Journal of Advanced Research in Medical and Pharmaceutical Sciences,2016; 1(2), doi:10.22413/ijarmps/orcid:0000-0003-4711-3822.

19.  Pooja SB, Megha S, Rajesh KN et al. Preparation evaluation and hair growth stimulating activity of herbal hair oil. Journal of Chemical and Pharmaceutical Research, 2009, 1(1): 261-267, ISSN: 0975-7384.

 

 

 

 

 

Accepted on 28.11.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(3):1409-1414.