Assessment of Nutraceutical Potential of Herbs for Promoting Hair Growth: Formulation Considerations of Herbal Hair Oil

Herbs were acquired from Pranveer Singh Institute of Technology's medicinal garden. Herbs were collected, dried, then ground in a mortar and pestle. Grinded herbs (Murraya koenigii, Hibiscus rosa-sinensis Linn., Nigella sativa, Trigonella foenum-graecum) were combined with 60% Cocos nucifera oil, heated, cooled, and filtered. Physical appearance, viscosity, pH, sensitivity test, hair growth activity, hair weight, antimicrobial test, stability test, and other criteria were determined and are reported in this text for the created herbal hair oil.


INTRODUCTION
Hair is a protein filament that grows from dermal follicles. Mammalian hair is one of their most distinguishing features. Hair is most commonly associated with hair development, hair types, and hair maintenance, but it is also an essential biomaterial made mostly of protein, particularly alpha-keratin. Dandruff, hair loss, dry hair, split ends, frizzy hair, dull hair, heat damaged hair, colour damaged hair, grey hair, and other issues are common in cosmetics. Many products, such as hair oils, hair shampoos, hair conditioners, hair serums, hair gels, hair masks, and hair dyes, are available on the market to help with these issues. Hair oils are applied to the hair to dress it, nurture it, and give it a more healthy aspect. They also encourage hair development that is both luxurious and long. Hair tonics are made from hair oils containing herbal medications. These are essentially therapeutic plant extracts in a base of oil. Herbal hair oil hydrates the scalp while also reversing the effects of dry scalp and hair. It contains a variety of vital nutrients that help the sebaceous gland operate normally and promote natural hair growth. The current study was carried out with this objective in mind [1 -3].
Murraya koenigii (M. koenigii) is a member of the Rutaceae family and is commonly employed in the Ayurvedic system of medicine as a medicinally important herb of Indian provenance. Previous research has shown that the leaves, roots, and bark of this plant are high in carbazole alkaloids, which have powerful biological and pharmacological effects. Antioxidant, antidiabetic, anti-inflammatory, anticancer, and neuroprotective properties are among them. Murraya Koenigii has long been known for its hair-root-strengthening effects. Massage hair and scalp with a mixture of dry leaf powder and organic coconut oil to promote hair development. Premature greying of hair can be slowed down by applying a curry leaf paste to them. Moisture is 63.2 percent, protein is 8.8 percent, carbohydrate is 39.4 percent, total nitrogen is 1.15 percent, fat is 6.15 percent, total sugars are 18.92 percent, starch is 14.6 percent, and crude fibre is 6.8 percent in the plant leaves. The leaves have been found to be a good source of various vitamins, including vitamin A (B-carotene). Carbazole alkaloids, essential oils, terpenoids, and flavonoids all play important roles in the body [4,5].
Trigonella foenum-graecum, often known as fenugreek seeds, is a member of the Fabaceae family. Hair loss can be caused by a variety of factors, one of which is oestrogen insufficiency. External oestrogen administration has been linked to a disruption in the hormonal cycle and an increased risk of cancer [6]. One of the natural alternatives to oestrogen therapy can be found in a variety of plants that contain natural products, including phytoestrogens, which are molecules with mild estrogenic activity. By filling or attaching to the oestrogen receptor and providing the oestrogen action, phytoestrogen competes with oestrogen. Fenugreek seeds (Trigonella foenumgraecum L.) contain phytoestrogen, which is thought to promote hair growth [7,8].
Nigella sativa (N. sativa), often known as black-caraway and "Kalonji," is a well-known seed worldwide. Thymoquinone, thymohydroquinone, dithymoquinone, thymol, nigellicine, carvacrol, nigellimine, nigellicine, nigellidine, and alpha-hederin are some of the chemical elements found in its fixed oil. It was discovered that it affects several sections of our body and has many pharmacological actions such as antibacterial, antiviral, anti-inflammatory, and wound healing properties, as well as acne vulgaris, skin cancer, pigmentation, and many cosmeceuticals usages [9]. Telogen effluvium is a disorder in which hair thinning or shedding occurs as a result of hair entering the telogen phase too soon. Nigella sativa seed was employed in the study, which contains Thymoquinone (TQ), a major active ingredient that has anti-oxidant and antiinflammatory properties by blocking pro-inflammatory mediators including cyclooxygenase and prostaglandin D2. A total of 20 individuals with Telogen effluvium were chosen for double-blind, placebo-controlled, and randomised research. For three months, ten of these patients received daily treatment with a lotion containing 0.5 percent Nigella sativa, whereas the other ten patients received daily treatment with a placebo. Before therapy (T0), after three months of treatment (T3), and during the six-month follow-up, video dermatoscopic analysis (Trichoscan Dermoscopy Fotofinder®) and inspection by three independent dermatologists were used to assess improvement (T6). Patients treated with Nigella Sativa showed significant improvement in 70% of cases. In patients treated with Nigella sativa, videodermatoscopic research revealed a significant increase in hair density and thickness. In addition, Nigella sativa was found to alleviate inflammation in the majority of Telogen effluvium patients [10,11]. Hibiscus rosa-sinensis Linn. (Malvaceae) is a glabrous shrub that is commonly cultivated as an ornamental plant in the tropics. It comes in a variety of varieties, each with different blossom colours. The red flowered type, however, is preferred in medicine. Hair development is stimulated by the leaves and flowers, while ulcer healing is aided by the blooms. The leaves and blooms of Hibiscus rosa-sinensis are thought to offer hair growth and anti-greying effects, according to traditional texts. Furthermore, in India, herbal products for hair development on the market include extracts from various portions of Hibiscus rosa-sinensis. As a result, the current study focuses on the scientific investigation of the herb Hibiscus rosa-sinensis's hair growth potential [12].
Cocos nucifera oil, generally known as coconut oil or copra oil, is a member of the Arecaceae family. It's utilised to hydrate hair, prevent hair breakage, improve blood flow and circulation, and function as an antibacterial agent [13].
Hair oils can be made in a variety of ways, including direct boiling, paste, and cloth processes [10]. The evaluation of preparation is the next major step after preparation. The therapeutic efficacy of the product is the next and final phase. The major goal of our study is to develop and test a herbal hair oil for hair growth improvement.

Collection of Plant Part
Murraya koenigii, Hibiscus rosa-sinensis Linn., Nigella sativa, and Trigonella foenum-graecum were collected from the Medicinal Plant Garden of Pranveer Singh Institute of Technology, Kanpur, Uttar Pradesh, India, and were properly authenticated in the Department of Pharmacognosy for the preparation of herbal hair oil.

Formulation of Herbal Hair Oil
Precisely all the dried and fresh herbs such as Murraya koenigii, Hibiscus rosa-sinensis Linn., Nigella sativa, Trigonella foenum-graecum were weighed, grinded and mixed in 60% of Cocos nucifera oil ( Table 1). The above content was boiled for 15 min, cooled and filtered through a muslin cloth. To the filtrate 25% Cocos nucifera oil was added to make up the volume (100 mL) (Fig. 1) [14].

Physical Appearance
The general characters like colour and odour were evaluated manually.

pH Test
The pH metre was calibrated with buffer solutions of pH 4 and pH 7. The electrode was bathed in hair oil for a few minutes until the pH returned to normal [15].

Viscosity
A Brookfield viscometer (RVDV-II+PRO) was used to measure viscosity with spindle number 6. 50 mL of hair oil was poured into the beaker, and the viscosity was tested at 100 rpm [16].

Determination of Refractive Index
The refractometer's temperature was adjusted, and the oil sample was smeared over the cleaned prism before taking readings. The prism was washed with hot water when the measurements were finished. The following equation was used to rectify the readings [17].

Saponification Value
In a 250 mL conical flask, 1 mL of oil was accurately weighed, and 10 mL of ethanol:ether combination (2: 1) was added. 25 mL of 0.5 N alcoholic KOH was added to this flask. The flask was kept for 30 minutes and then cooled. Using phenolphthalein indicator, the cooled solution was titrated against 0.5 N HCl. The blank titration was carried out in the same way but without using any oil (sample). The amount of KOH used in mg was computed [15].

Phytochemical Screening of Herbal Hair Oil Preparation
Using various procedures, the produced herbal oil was subjected to qualitative chemical analysis for the identification of numerous plant main ingredients such as sulphur, ascorbic acid, and saponins [18].

Ascorbic Acid Test
Added 1 drop of freshly prepared 5 percent w/v sodium nitroprusside solution and 2ml of dilute sodium hydroxide solution to 1ml of 2 percent w/v solution and 5ml of water. Drop in 0.6ml of hydrochloric acid, mix, and records found.

Assessment of Nutraceutical Potential of Herbs for Promoting Hair Growth
The Open Dermatology Journal, 2021, Volume 15 81

Sulphur Test
On the test paper, a drop of hydrogen peroxide was placed. When exposed to fumes, the paper becomes brown.

Saponin Test
Formation of stable froth was observed by shaking oil and water in a test tube.

Specific Gravity
Specific gravity bottle was taken, rinsed with distilled water, dried in the oven for 15 minutes, cooled, and then weighed (a). Herbal hair oil was filled in the same specific gravity bottle, closed, and weighed again (b). Subtracted the weight (b-a) from the weight of the sample per millilitre [15].

Anti-microbial Evaluation by Cup Plate Method
The zone of inhibition approach was used to investigate the antibacterial activity of herbal hair oil that was diffusion dependent. The plate was incubated for two days at 37°C. The inhibitory zone was measured [19].

Primary Skin Irritation Test
A basic skin irritation test was performed on shaved undamaged skin of rat with a small amount of the produced herbal hair oil. For 3 to 4 hours, the test site was monitored for erythema and edema [12].

Hair Growth Activity In Vivo
Two groups of six rats were formed. All of the rats' hair was shaved off and cleaned with surgical spirit in a 4 cm 2 area on the dorsal portion of their bodies. A 10 ml of the produced herbal oil was administered once daily to the denuded areas of group I, while a control group (Group II) got no therapy. On the 15 th , 20 th , 25 th , and 30 th days of the therapy, hair was randomly taken from the shaved area of selected rats from each group. The average length of 15 hairs was found by measuring their length. The results are expressed as a standard deviation of 15 hairs for the mean length [20].

Hair Weight Measurements
To evaluate the weight of each box, the hair was removed and weighed on day 21 and then statistically measured [3,20].

Stability Studies
The herbal hair oil was kept in the bottle at room temperature for stability study [18].

RESULTS AND DISCUSSION
The above-mentioned substances were used to make the herbal hair oil, which was then tested qualitatively.

Evaluation of Herbal Hair Oil
Developed herbal hair oil was reddish brown in colour with a transparent appearance, and when applied, it was smooth. The pH of the whole herbal hair oil was 7.3, which was suitable for hair, implying that the herbal hair oil was compatible with hair [19]. The viscosity of herbal hair oil was determined to be 30 cps ( Table 2), and Newtonian flow behaviour was observed. A Newtonian fluid is one in which the viscous stresses generated from its flow are linearly proportional to the local strain rate, or the rate at which its deformation changes over time, at every place. The viscosity of a Newtonian fluid remains constant regardless of the amount of shear applied at a constant temperature. The viscosity and shear stress of these fluids are proportional. The refractive index was used to determine the quality of herbal hair oil. Herbal hair oil was found to have a refractive index of 1.29 [20]. This demonstrates that a simple refractive index measurement in the laboratory may also be utilized as a quality control approach ( Table 2). The smaller the average length of fatty acids, the lighter the mean molecular weight of triglycerides, and vice versa, the higher the saponification value. The saponification value of herbal hair oil was discovered to be 24.32 in practice ( Table 2).

Phytochemical Screening
Herbal hair oil contains ascorbic acid, sulphur, and saponins, according to phytochemical analysis ( Table 3). Because of its powerful antioxidant action and nutritional importance as vitamin C, ascorbic acid is one of the most commonly used natural antioxidants. It has therapeutic properties in addition to antioxidant potential. It is a watersoluble vitamin that is easily absorbed but not stored in the body. Ascorbic acid is necessary for the preservation of collagen, which makes up around one-third of the total protein in the body. The ideal amount of ascorbic acid is used to prevent oxidative degradation of the oils. For a good reason, sulphur is commonly referred to as one of the building blocks of hair. Our hair is made up of keratin, a long-lasting protein with a high sulphur concentration. Sulfur is needed for proteins (like keratin) to keep their structure, which helps maintain hair's overall health, strength, and suppleness [21]. Sulfur has been shown to promote hair development in research. Sulfur has been shown to increase the length of your hair's growing phase. Longer hair is associated with a longer growing phase (before resting and shedding) [18,19]. Finally, sulphur has been associated with the treatment, alleviation, and prevention of psoriasis, dandruff, eczema, and folliculitis. Saponins are the most common phytochemicals that act as natural surfactants. Natural saponins provide body and gloss to hair, making it feel fuller, silkier, and smoother. Herbal hair oil has a specific gravity of 0.83 [21].

Antimicrobial Study
The cup plate method was used to test the antimicrobial activity of Cocos nucifera oil and herbal hair oil using the Candida albicans organism, and the findings are shown in Table 2 [15].

Primary Skin Irritation Test
The irritation caused by herbal hair oil was evaluated using a primary skin irritation test on rabbits' shaved undamaged skin. The absence of erythema and/or edema in the prepared herbal hair oil suggested that it was non-irritant to rabbit skin ( Table 2) [20].

Hair Growth Activity and Hair Weight Tests
At the end of the second week, hair began to grow back from the denuded area, and the length of the hair began to increase until the treatment cycle was completed ( Table 2). During the fourth week, when compared to the control group, it was discovered that the herbal oil treatment group had a substantial effect. This could be attributed to the gentle rubbing of shaved skin while applying extracts and placebo (liquid paraffin), which improves blood circulation in the local area and may have an influence on hair growth [22]. When compared to the control group, the herbal oil treatment group had a larger effect on hair length, with 8.89 mm at the conclusion of the course compared to 2.91 mm in the control group ( Table 2). This could be caused to follicles moving from the telogen to anagen phase of the hair growth cycle too soon. Hair weight was found to be 219.49 and 150.12 mg for group I and group II, respectively. In Table 2, it can be seen that herbal hair oil corresponded to a higher weight in the third week [23].

Stability Study
The prepared hair oil was maintained at room temperature following the chemical analysis in a glass container. Following the time period, the same oil was tested again. Physical testing revealed a reddish-brown colour, a specific gravity of 0.91, and a viscosity of 30 cps in 30 days stored prepared oil.

CONCLUSION
One of the most well-known hair treatments is herbal hair oil. Herbal hair oil not only hydrates the scalp, but also helps to heal dry scalp and hair. It contains various vital nutrients that support regular sebaceous gland activity and encourage natural hair growth. In this study, dried portions of Murraya koenigii, Hibiscus rosa-sinensis Linn., Nigella sativa, and Trigonella foenum-graecum were made into herbal hair oil and evaluated for their potential as an effective topical formulation for hair growth-promoting action. The results showed that herbal hair oil exhibited good pH, acceptable viscosity, and was stable at room temperature. Furthermore, the animal investigation revealed that the herbal hair oil created has a promising effect on hair growth stimulation with no negative side effects. As a result, it is clear that the herbal plant may be a preferable option for future formulations.

ETHICS APPROVAL AND CONSENT TO PARTI-CIPATE
This study was approved by Animal Ethics Committee of Pranveer Singh Institute of Technology, Kanpur, India, under the registration number: 1273/PO/Re/S/09/CPCSEA.

HUMAN AND ANIMAL RIGHTS
The institutional and international guide for the care and use of laboratory animals was followed.

CONSENT FOR PUBLICATION
Not applicable.

FUNDING
None.

AVAILABILITY OF DATA AND MATERIALS
The data that support the findings of this study are available from the corresponding author [R.T], upon reasonable request.