INTRODUCTION:

Alopecia:

Partial or complete hair loss is one of the most common issues any patient is facing when they consult a dermatologist. Alopecia is usually linked up with psychological problems, loss of confidence, distress, and symptoms of depression. [1,2,3] The hair loss can be long lasting or even temporary.

The diagnosis of the cause of alopecia is done by laboratory testing, physical examination, checking clinical history, scalp biopsy, clinical diagnostic tests etc. which are essential to complete the process of evaluation. [4,5,6] Some common types of the non-scarring alopecia are telogen effluvium, androgenic alopecia and alopecia areata. Whereas the scarring alopecia is caused by discoid lupus erythematosus, infections, trauma or lichen planus. Other problems of the scalp include tinea capitis, trichotillomania, traction alopecia and hair shaft abnormalities. [7,8] Therefore an appropriate evaluation and management protocol is required for the suitable care of the patient and a successful treatment. Alopecia or hair loss becomes a matter of concern when a person lose more than 100 hairs/day. Although numerous medical treatments and many hair care products are available in the market, many people suffer from this dermatological disturbance globally. [9,10] Alopecia areata is a hair loss condition in the form of patches. [11] There are various causes for Alopecia such as hereditary factors, environmental triggers, exposure to certain chemicals, medicines, nutritional imbalances, oxidative stress or prolonged illness etc. [12,13]

Leech:

Using leeches has been an age-old tradition in various cultures and civilizations. Various techniques have been used involving leeches and basically using their saliva properties to cure pathological issues. Indian, Greek, Arab, Ancient Egyptian physicians have been using the medicinal leech therapy for treating basic ailments to complex systemic diseases. Disease conditions like skin disorders, nervous abnormalities, reproductive disorders, dental problems and urinary system have been potentially treated by using the leech therapy. Leeches are hematophagous animals that feed on the blood. They are recognized for their potential blood clotting properties due to their salivary gland secretions which have biologically active compounds especially anticoagulants [14]. A number of blood-sucking invertebrates exist in nature but the mechanism possessed by leeches is highly functional and effective which basically prevents blood clotting. [15]. Medicinal leech therapy has always been an attention for most of the therapeutics therefore many researchers have been conducted to identify active compounds present in its saliva and its medicinal properties. Hirudo medicinalis is the European medicinal leech species responsible for healing properties so commonly known as healing leech is preferred for medical procedures and therapies nu physicians in comparison to the American leech speeches Hirudo decora which has lesser ability to such blood [16,17,18]. Many other species of leeches are used as a medical tool like Hirudo nipponia [19,20]. Haementeria depressa [15,21] Hirudinaria manillensi [22], Hirudo verbena, Hirudo orientalis. [23] Cardiovascular Diseases are the chronic cases of abnormalities which affect veins, arteries and majorly affecting heart. [24] The ongoing rates of mortality and morbidity caused by CVDs are the main cause for increased research and alternative treatment therapies for curing CVDs with lesser side effects. [25] Saliva of leech has shown effectiveness in CVDs as they contain hirudin which was firstly isolated from H. medicinalis. [26,27,28] The saliva from leech has also shown potential inhibition of the clot-forming thrombin. [29, 30] Many other active compound shave been identified from different species of leeches like bufrudin from H. manillensis which closely resembles hirudin [22] Haemadin is a tight-binding thrombin inhibitor which is obtained from the entire body extract of Haemadipsa sylvestrisa leech species [31]. A granulin-like antithrombin was isolated from a leech species called H. nipponia [32] The human protein inhibitor Threomin (of granulocyte and monocyte) was obtained from the head extract of Theromyzon tessulatum which is a leech species and contains antithrombin activities. [33] In 1997 the Russian markets released “Piyavit” a trade name for the extract of medicinal leech saliva which contained anticoagulant and antithrombotic properties. This product was majorly prescribed as an antiplatelet and thrombolytic. Various researchers have also reported it to be an anti-inflammatory in thrombophlebitis [34] Using leeches on the affected area topically has proved with good results like lesser pain, less swelling in legs, improved normal coloration of skin on the affected area etc. [35] The leech therapy is an alternative and effective treatment for improving blood flow and treats the condition of hyperalgesia in connective tissue since ages [36]

Hibiscus:

The medicinal plant Hibiscus rosa-sinensis has been used effectively in treating many disease. It belongs to Malvaceae. It has been used in treating alopecia and androgenic alopecia [37,38,39]. The plant has been used as an anti-dandruff agent by people traditionally [40]. Hibiscus rosa sinensis has shown the presence of active biomarkers like Quercetin, β-sitosterol and linoleic acid. The flavonoid pigment found in Hibiscus rosa-sinensis is Quercetin. In flowers the- per gm level of quercitin is 7 mg [41]. The plant has been used in many supplements, beverages and foods. As Quercitin is 5α-reductase inhibitor therefore Hibiscus is used as a potential cure for prevention and treatment [42]. Hibiscus has shown increased hair growth activity as it improves the blood flow to the hair follicles by stimulating the hair follicles [43,44].

Onion:

In order to deal with the problem of baldness, there is a need to remove the effects from their roots in a gentle manner. Preference is being given nowadays to the Onion has been found beneficial in patchy baldness .The affected part should be rubbed with onion juice morning and evening till it is red. It should be rubbed with honey afterwards. Zinc present in it helps to secrete the scalp with much needed oil and avoid dandruff that may cause hair loss [45]. The main advantage of using herbal containing cosmetic is that it is pure and does not have any side effects on the human body [46]. Sulfur from onions may also help promote collagen production. Collagen results in the production of healthy skin cells and helps in hair growth. Onion juice application to the hair and scalp could increase blood supply to hair follicles, which in turn improves hair growth [47,48,49, 50]. Onion extract has the tendency to cease skin allergic reactions, proving that onion juice may induce an immunological reaction that can stimulate hair re-growth through competition of antigens. [51,52]. Another possible mechanism, is that onions belong to the genus Allium [53,54] which is rich in sulphur and phenolic compounds [55], both of which are known skin irritants. Onion may cause an irritant contact dermatitis.

Bee Hive:

Propolis is a resin-like material that honeybees use to seal small gaps in their hives. Apart from acting as a physical barrier, being rich in active compounds, it fights fungal and bacterial invasions. Honey shows antibacterial, antifungal and antioxidants activities and is highly nutritive. Crude honey shows positive potential in the management of dermatitis and dandruff. [56] Propolis contains anti-inflammatory compounds, which may help to treat balding conditions. [57] Propolis treatment stimulates migration of hair matrix keratinocytes into the hair shaft in HFs during late anagen in the depilated back skin. Propolis has been reported to have facilitated the proliferation of epidermal keratinocytes, which stimulates hair growth by inducing hair keratinocyte proliferation. After the application of propolis, the number of cells involved in hair growth get increased [58] Propolis is rich in over 200 chemical compounds, such as flavonoids, terpenoids, aldehydes, aromatic acids, aliphatic alcohols and ethers, amino acids, and sugars.[59] Propolis is also rich in number of other medicinal activities. [60,61] It also shows significant potent against dermatological disorders. Therefore, it is used widely in hair and skin products for the purpose of repairing and regenerating of the damaged tissues. [62]. A research performed over mice in 2014, after application of propolis, showed faster hair back. The propolis is responsible for the increased number of cells involved in hair growth. It stimulates hair growth by inducing hair keratinocyte proliferation as revealed by the previous studies. This indicates that in some cases, propolis may help to stimulate the growth of hair follicles that have stopped producing hair.[63] Hair loss due to deficiencies can be reduced to some extent with honey, since honey also contains a small amount of certain vitamins and minerals, like vitamin B2, vitamin B6, iron, manganese, phosphorus, and zinc.[64] Iron and its supplements are very beneficial for hair growth. Procyanidin B-2 is a compound we have identified in apple which acts as a growth-promoting factor on murine hair epithelial cells. [65, 66, 67] Botanically derived inhibitors of 5-alpha-reductase in the treatment of androgenetic alopecia. [68]. Presence of polyphenolic compounds derived from natural resources helps against alopecia. [69, 70]

MATERIALS AND METHODS:

The preparation of leech extract:

The medicinal leeches were collected from the banks of river Ganga near Ganga Barrage in Azad Nagar area of Kanpur. The leeches were kept in contact with the sodium chloride crystals or in a very concentrated salt solution. Then the bodies were washed with distilled water to remove the extra salt which may disturb the pH of the extract to be prepared. Now the washed bodies of leeches were cut in small pieces and run through the mixer for a smooth paste. The pulp was ground with sand and the extraction was done with six times the volume of distilled water to one volume of pulp. After the centrifugation the supernatant fluid was collected and filtered using a coarse paper. [71]

The preparation of hibiscus extract:

Hibiscus Rosa-Sinensis was collected from the herbal garden of Pranveer Singh Institute of Technology, Kanpur in morning of May 2018. The flowers of the plant were shaded on ground dried at a temperature of 350 - 450 Celsius for few days. The flower was then ground and powdered. The aqueous extract of the flower was prepared by boiling 20 g of the hibiscus flower powder in 1 liters of distilled water for 5 minutes. Now the extract was agitated to give a uniform extraction and it was covered until it reached room temperature. The residue of the extraction was collected by filtration (1 mm pore size) and finally the extract was concentrated in a rotary evaporator (1 hour). Now the extract was stored at -20^o Celsius for further use. [72]

The preparation of bee hive extract:

A Coconut oil from a marketed product was kept as a base oil and beehive was kept in the oil for extraction. Now the beehive was heated in the coconut oil (1 gm. hive and 100 ml coconut oil) for 1 hour on a stove. Now the extract was filtered by filtration) 2mm pore size) and the final extract was collected after getting cooled.

Preparation of Onion Extract:

The onion was collected from the herbal garden of PSIT and identified by the pharmacologist of Department Pharmacy, PSIT, Kanpur. The cold water extract of fresh onion bulbs was produced by rinsing the onion bulbs with distilled water. Not the moisture on onion was removed by drying then inside an oven in the lab at a constant temperature of 600 Celsius. [73]Now the dried onion was ground using a mortar and pestle. An Approximately 12gm weight of powder was dissolved in 100 ml of distilled water. Now the extract was kept for 2 days for soaking and then filtration was done using 1mm filter paper.

Preparation of Oil:

All extracts were mixed in equal quantity in an appropriate quantity of coconut oil. Emulsifier was added in approximate quality to emulsify the entire preparation. Now using a homogenizer the entire preparation was mixed and a uniform solution of oil with herbal extracts was formed and oil was prepared as shown in Figure 1.

Figure 1: The figure shows the method for preparation of oil.

Standardization of Oil:

The prepared oil was evaluated for its various parameters such as organoleptic and physical standards in order to get assure about its efficacy, stability, quality as well as purity.

Physicochemical analysis:

a. Determination of color and odor:

Organoleptic characteristics for various sensory characters like color and odor etc. was carefully noted down. Color and odor of the oil sample were found to be typical of their constituents. [74,75,76]

b. Determination of specific gravity:

Specific gravity bottle by was cleaned and shaken with acetone and then with ether. Bottle was dried and weight was noted down. The specific gravity bottle was filled with the oil and weight was noted. The procedure was repeated using distilled water in place of oil. In case of oil, it may also change with thermal effects. The specific gravity of the oil was calculated from the following relationship. Specific Gravity at 30°C = A-B / C-B Where, A = weight of specific gravity bottle with oil at 30°C (g); B = weight of specific gravity bottle at 30°C (g); C = weight of specific gravity bottle with water at 30°C (g). [77]

c. pH Determination:

The digital pH meter was used for the pH determination.

d. Determination of acid value:

10g of oil was taken in a conical flask. To it was added 50 ml of acid free alcohol-ether mixture (25 + 25ml) previously neutralized by the addition of 1 ml of Phenolphthalein solution and titrated against 0.1N potassium hydroxide solution. End point is figured out with the appearance of pale pink color which persists for 15 seconds. The acidity increases with oxidation because triglycerides are converted in to fatty acids and glycerol. So, there is direct relationship of acid value with rancidity. In present work, acid value of oil was below 2 which indicated the better quality.

Acid value = 5.61V N / W Where, V = Volume of standard sodium hydroxide used (ml); N = Normality of the sodium hydroxide solution; W = Weight of the sample (g). [78, 79]

e. Determination of peroxide value:

Peroxide value = 10 (a-b) / w Where, a = ml of NaOH required to neutralize the substance, b = ml of NaOH required for blank, w = weight of sample in (g). [80, 81, 82]

f. Determination of saponification value:

About 2 g of the oil was weighed in 250 ml round bottom flask. 25 ml of the alcoholic solution of KOH was added and a reflux condenser was attached. It was kept for boiling on water bath for 1hr, the contents of the flask were rotated frequently. The flask was cooled and 1ml phenolphthalein solution was added and excess of alkali was titrated with 0.5N HCl. The number of ml required was noted. The long chain fatty acids found in fats have a low Saponification value because they have a relatively fewer number of carboxylic functional groups per unit mass of the fat as compared to short chain fatty acids. Saponification value = 28.05 (B-S) W Where, S = ml of KOH required to neutralize the substance; B= ml of KOH required for blank; and; W = Weight of the sample taken for the test (g). [83]

Alopecia Induction:

Animals:

Eighteen Wistar rats weighing about 150-250 g all male were caged at 28±2 °C in three separate cages (six animals in each cage). The humidity was controlled to (50-55%). Now the animals were fed with standard pellet diet and water ad libitum in the CPCSEA approved animal house of PSIT, Kanpur (Institute Animal Ethics Committee number is 1273/PO/Re/S/09/CPCSEA for Research for Education Purpose on small animals) and the experimental design is as shown in Table 1

Experiment:

Table 1: Experimental Design

S. No.	Group Number	Group Name	Dosing	Number of Animals
1.	Group I	Control Group	Application of Parachute- Coconut Oil	6
2.	Group II	Standard Group	Application of 2% minoxidil ethanolic solution	6
3.	Group III	Treatment Group	Application of Herbal oil prepared in the laboratory	6

The screening of the hair growth potential was evaluated for Wistar rats using the method stated by Saraf et al. [84]Hairs from the dorsal portion of the Wistar rats were removed by clipping 2-4 cm length if hair and applying hair removal cream (Veet - a marketed hair removal product) on them. The length of hair was measured on 15th, 21st, 30th and 40th days in all groups. Now the length of hair was compared in both groups after the end of therapy for 40 days.

RESULTS:

Standardization of Herbal Oil:

The prepared oil was reddish brown in color due to addition of hibiscus, which was also the reason behind the pleasant odor of the formulation. The characteristic odor of other constituents were masked by the flowery fragrance of hibiscus. pH was found to be neutral to suit all the different types of scalp as well as hair. If Saponification value is more than normal range it indicates lower molecular saturated fatty acids. If acid value is more, then chances of photo-oxidation and rancidity are more. The obtained values of these tests were found within normal limits in prepared formulation which indicate good quality of product. In addition, no rancidity was found in finished product. The specific gravity of the sample was 0.851, which was closer to plain coconut oil, for which it was 0.9, showing that the sample was not too dense. The acid value was 1.5 w/v, indicating the amount of free fatty acid present in the oil. The saponification value was found to be 95 w/v. It gave an idea of the molecular weight of an oil/fat, and the oil contained a long chain of fatty acids. All the evaluation data observed for the herbal oil is mentioned in Table 2.

Table 2: The parameters observed and evaluated by standardization of the oil are as follows:

S. No.	Parameters	Result
1.	Color	Reddish brown
2.	Odor	Pleasant
3.	pH	6.7
4.	Specific gravity	0.72
5.	Acid value	1.2
6.	Peroxide value	1.5
7.	Saponification value	90

Evaluation of Effectiveness of Herbal Oil:

The length of hair before and after application of oil was recorded and compared in Group I, Group II and Group III on 15th, 21st, 30th and 40th days. The parameters evaluated for concluding the results of the experiment were the initiation of hair growth after shaving on the dorsal are of Wistar rat, recording and comparing hair length in all groups on dates 15, 21, 30 and 40 after shaving of hair as shown in Table 3, 4 and Figure 2, 3.

Table 3: The mean hair length was recorded on 15, 21, 30 and 40 days in rats of Group I, group II and Group III

S. No.	Days	Group I (Mean Hair length)	Group II (Mean Hair length)	Group III (Mean Hair length)
1.	15th day	1cm	1.1 cm	1.5 cm
2.	21st day	1.4cm	1.3 cm	2 cm
3.	30th day	2 cm	2.1 cm	2.4 cm
4.	40th day	2.2 cm	2.4 cm	3 cm

Figure 2: A comparative graph of hair length on days 15, 21, 30 and 40 after hair removal

X Axis: Number of days on which hair length was recorded

Y Axis: The length of hair grown on dorsal area of wistar rat in centimeters.

Table 4: The table shows initiation of hair growth in days (mean) in all groups

S. No.	Time to initiate hair growth in days
Group I	8
Group II	7
Group III	5

Figure 3: A comparative representation of time taken for initiation of hair growth in Group I, II and III in days (mean)

X Axis: Shows the different Groups on basis of dosing & treatment I, II and III

Y Axis: Shows the time taken to initiate hair growth in days

CONCLUSION:

The results obtained from the standardization of prepared formulation confirm the ingredients present in it. Due to the presence of natural ingredients, this formulation proves to be a boon to fight the trending problem of hair fall due to various reasons. The results were tabulated, graphed and compared to draw the conclusion of the effectiveness of the prepared herbal oil. Minoxidil was used as a standard to evaluate the effectiveness of the herbal oil prepared. The herbal oil prepared was found to be more effective than the standard preparation of Minoxidil for stimulating hair growth in cases of treating Alopecia.

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Received on 10.10.2018 Modified on 05.11.2018

Research J. Pharm. and Tech 2019; 12(2):757-763.

DOI: 10.5958/0974-360X.2019.00134.3

S. No.	Group Number	Group Name	Telogen	Anagen	A/T
1.	Group I	Control Group	53.3 ±0.4520 %	44.2 ±0.4251%	0.829
2.	Group II	Standard Group	32.5 ±0.4332***%	65.3 ±0.5230***%	2.009
3.	Group III	Treatment Group	30.4 ±0.7934***%	68.1 ±0.5475***%	2.24