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Online First                                                                                                                         Indian J Pharm Drug Studies | 1  

Original Article 

Evaluation of hair growth potential of Angustifolia leaves hair gel in rats 

Manu Bharti1, Anjali Bhardwaj2, Neha Parveen3, Ashwani Gupta4 

From, 1Assistant Professor, Shri Venkateswara University Venkateswara Nagar Gajraula, Uttar Pradesh 244236, 2Professor, 

Department of Pharmaceutics, Shri Venkateswara University Venkateswara Nagar Gajraula, Uttar Pradesh 244236, 3Assistant 

Professor, Durga College of Pharmacy, Khaspur Sambal U.P., 4Associate Professor, Shri Venkateswara University Venkateswara 

Nagar Gajraula, Uttar Pradesh 244236. 

ABSTRACT  

Background: Hair loss, or alopecia, significantly affects both physical appearance and psychological well-being, particularly in cases 

such as androgenetic alopecia and alopecia areata. Current pharmacological treatments like minoxidil and finasteride have limitations, 

including side effects and decreased long-term efficacy. Objectives: An ethanolic extract of Senna angustifolia (EESA), a traditionally 

used Ayurvedic plant, was evaluated for its hair growth-promoting potential using in vivo rat models. Methodology: The research began 

with the successful extraction of phytoconstituents from Senna angustifolia leaves using Soxhlet extraction. The gel was formulated 

into two concentrations—2.5% and 5% EESA—and compared with a control and standard 2% minoxidil treatment. Hair growth studies 

were performed on Wistar rats by evaluating both qualitative (initiation and completion of hair growth) and quantitative parameters 

(hair length, follicle count, and histological observations). Results: Qualitative hair growth analysis indicated that both EESA 

concentrations accelerated hair growth, with 5% EESA (HG2) showing a significantly faster initiation and completion time (7.01 and 

32.4 days, respectively) compared to control (13.8 and 42.6 days) and even slightly outperforming minoxidil (7.07 and 33.0 days). 

Quantitative analysis supported these findings. By day 30, HG2 achieved the greatest hair length (2.8 mm), marginally higher than 

minoxidil (2.4 mm) and significantly more than the control (1.2 mm). Histopathological studies corroborated these findings with 

increased follicle density and size. Conclusion: These results suggest that Senna angustifolia extract exhibits promising hair growth-

stimulatory effects and may serve as a natural alternative or complementary treatment for alopecia with minimal irritation potential. 

Further clinical investigations are warranted to explore its therapeutic application in humans. 

Key words: Senna angustifolia, Alopecia, Ayurveda, ethanolic extract, Minoxidil alternative 

n mammals, hair aids visual and scent communication. 

Humans have less hair, mainly cosmetic. Hair loss 

(alopecia) affects appearance and self-esteem, ranging 

from patchy (alopecia areata) to complete body hair loss 

(alopecia universalis). Androgens cause alopecia, but 

treatments like finasteride and minoxidil have side effects and 

reduced long-term effectiveness [1-5]. Human hair is mostly 

fibrous α-keratin. Hair serves thermoregulation, sensing, 

protection, social cues, and camouflage. Humans have ~5 

million follicles, 80,000–150,000 on the scalp. Hair cycles 

through anagen (growth), catagen (regression), telogen (rest), 

and exogen (shedding) stages, driven by cell differentiation, 

stem cell activation, and epithelial–mesenchymal interactions 

[6-8]. Scalp hair remains in anagen for 2–5 years, catagen for 

weeks, and telogen for ~3 months. Disruptions, like 

early catagen or shortened anagen, cause hair loss, as seen 

in telogen effluvium or alopecia areata due to anagen bulb 

damage [9]. Hair loss can have a variety of causes. The most  

Access this article online 

 

Received –  14th May 2025 

Initial Review –  22nd May 2025 

Accepted – 14th August 2025 

Quick Response Code 

prevalent conditions, trichotillomania, traumas, extreme stress, 

childbirth, usage of fertility-stimulating medications, fungal 

injection, hypothyroidism, sebaceous cysts, some hereditary 

illnesses, and short anagen syndrome are among them. 

Androgenetic alopecia involves follicle shrinkage, 

shortened anagen, and prolonged telogen phases, while 

hirsutism causes excess terminal hair in females [10]. 

Treatment aims to modify anagen duration—shortening it for 

hirsutism or lengthening it for alopecia. FDA-approved drugs 

are topical minoxidil and oral 

finasteride. Minoxidil prolongs anagen and stimulates follicle 

growth, though its effects vary by concentration [9, 11-13].  

In light of finding herbal alternatives, Senna 

(Cassia angustifolia), also called Swarna Patri in Sanskrit, an 

Ayurvedic herb, was employed in the current study. Cassia 

angustifolia belongs to the kingdom Plantae and the sub-

kingdom Tracheobionata. It is classified under the division 

Magnoliophyta and the class Magnoliopsida, within the sub-  

__________________________________________________ 

Correspondence to: Dr. Anjali Bhardwaj. Department of 

Pharmaceutics, Shri Venkateswara University Venkateswara 

Nagar Gajraula, Uttar Pradesh 244236. 

Email: anjalisharma071991@gmail.com 

I 

mailto:anjalisharma071991@gmail.com


Bharti et al.                                                                                     Hair Growth Potential of Angustifolia Leaf Gel in Rats 

Online First                                                                                                                         Indian J Pharm Drug Studies | 2  

class Rosidae. The plant falls under the order Fabales and is 

part of the family Caesalpiniaceae. Its genus is Cassia, and 

the species is angustifolia. It contains anthraquinone, a natural 

laxative approved by the World Health Organization (WHO) 

and the Food and Drug Administration (FDA). Native to Sudan 

but named in Arabia, Senna grows as a perennial after rains 

[14]. 

The genus Senna has 500 species; 26 contain anthracene 

derivatives with laxative effects. Notable ones include 

Cassia angustifolia (Alexandrine/Indian senna), Cassia fistula, 

Cassia obovata, and others recognized for laxative properties 

[14]. In the present study, we aimed to assess the quantitative 

and qualitative effects of Senna angustifolia on hair growth in 

rats.   

MATERIALS AND METHODS 

1. Identification, collection, authentication, and 

preparation of leaf extract  

The leaves of Senna angustifolia were gathered in December 

2019 at the Institute of Foreign Trade and Management (IFTM) 

University Botanical Garden in Moradabad, India. The leaves 

underwent air drying and washing. The Botanical Survey of 

India, Scientist in Charge, Allahabad (Central area), India 

performed the authentication. The department received a 

voucher specimen (Ref: BSI/CRC/Tech./2019-20/104328).  

2000g powdered leaves were Soxhlet-extracted with 

petroleum ether and 95% ethanol for 72 hours. Extracts were 

filtered, concentrated at 40–50°C, and stored at 4°C. 

2. Preliminary Phytochemical screening of the extract  

The preliminary phytochemical screening of the extract was 

carried out using standard tests to identify the presence of 

various bioactive compounds. Flavonoids were detected using 

three methods: the Shinoda Test (formation of pink, red, green, 

or blue color upon addition of magnesium and hydrogen 

chloride), the Alkaline Reagent Test (yellow coloration with 

sodium hydroxide that turns colorless with acid), and the Zinc-

hydrogen chloride Test (appearance of a red 

color). Saponins were confirmed by the Foam Test, where 

shaking the extract with water resulted in the formation of a 

stable 1 cm foam layer. Alkaloids were tested using Dragen 

dorff’s reagent, producing an orange-red precipitate, 

and Mayer’s reagent, which yielded a white or cream 

precipitate.  

For steroids and sterols, the Salkowski Test (chloroform 

and sulfuric acid) produced bluish-red or cherry coloration with 

green fluorescence, while the Libermann-Burchard 

Test showed a bluish-green color. Amino acids were detected 

by the Ninhydrin Test, resulting in a blue color upon 

heating. Carbohydrates were confirmed by Molisch’s Test, 

which showed a violet ring at the interface with concentrated 

sulfuric acid. Proteins were identified using the Biuret 

Test (pink or purple color) and Millon’s Test, which produced 

a yellow precipitate on boiling. Finally, tannins were detected 

using Ferric chloride (blue-black coloration) and Lead acetate, 

which formed a white precipitate. These tests collectively 

indicated the presence of multiple phytoconstituents in the 

extract. 

3. Thin-Layer Chromatography (TLC) 

TLC is a versatile analytical technique for detecting 

organic/inorganic compounds in micro quantities. 

Plate Preparation: Silica gel G (acts as absorbent) slurry was 

applied to plates, dried overnight or for 30 min at 80–90 °C. 

Sample Application: 1, 2, and 5 μL of MESM were spotted 2–

2.5 cm from the bottom using a syringe/micropipette. 

Solvent System: Non-reactive solvents were used. A 2:1:0.5 

mixture of n-Hexane:Acetone was used for Senna angustifolia 

extract. 

Development Chamber: A glass ascending chamber was 

used. 

Spot Detection: Plates were placed in an iodine chamber to 

visualize spots. 

Retardation factor (Rf) calculation: 

Rf = Distance travelled by the spots from solvent front 

        Distance travelled by solvent from the origin front 

4. Preparation of formulation  

Three herbal gels were made using Carbopol base 

with methylparaben, glycerine, polyethylene glycol, 

polyvinylpyrrolidone, and triethanolamine. Carbopol 934 and 

extracts were dissolved in water, stirred (800 rpm, 1 

hr), glycerin added, then triethanolamine dropwise until 

neutral. Stirring continued until a clear gel formed. 

Senna angustifolia extract amounts varied (Table 1). 

 Table 1: Preparation of formulations  

Formulation Hair gel 

(HG) 1 

Hair gel 

(HG) 2 

Control 

Herbal Extract [g] 2. 5% of 

EESA 

5% of 

EESA 

--- 

Carbopol 934 [g] 2 2 2 

Polyvinylpyrrolidone 

[mg] 

5 5 5 

Methyl paraben sodium 

[mg] 

75 75 75 

Glycerine [ml] 3 3 3 

Polyethylene glycol [ml] 6.25 6.25 6.25 

Triethanolamine [ml] 1.5 1.5 1.5 

5. Hair growth study preparation   

For the hair growth study, 150–250 g male or female rats were 

used from the Animal House, IFTM University. The 

experimental protocol was approved by the Institutional 



Bharti et al.                                                                                     Hair Growth Potential of Angustifolia Leaf Gel in Rats 

Online First                                                                                                                         Indian J Pharm Drug Studies | 3  

Animal Ethics Committee (Approval No. 

2019/837ac/MPH/03). Rats were kept in standard cages with 

proper lighting, ventilation, free access to water, and a regular 

diet. A 7-day acclimatisation period was observed before the 

study began. 

5.1.1 Primary Dermal Irritation Study 

Using the Organisation for Economic Co-operation and 

Development (OECD) 404 guidelines, Western rats were tested 

for skin irritation with EESA gel (2.5% and 5%). Fur was 

shaved from a 6 cm² dorsal area 24 hours prior. Half a gram of 

gel was applied, covered with gauze and tape. Erythema 

and edema were scored at 60 minutes, 24, 48, and 72 hours after 

patch removal. Primary Dermal Irritation Index (PDII) was 

calculated as the average irritation score over 12, 24, 48, and 72 

hours: <0.5 = non-irritating; 0.5–2.0 = slightly irritating; 2.1–

5.0 = moderately irritating; 5.0 = severely irritating. 

6. Treatment for hair growth activity in vivo 

Twenty-four rats were divided into four groups (n=6). Hair was 

removed from a 3 cm² dorsal area using electric shavers and a 

commercial remover. Group 1 (negative control) received plain 

gel; Group 2 (positive control) received 2% minoxidil; Groups 

3 and 4 received 2.5% and 5% EESA gel, respectively. All 

treatments were applied topically once daily. The treatment was 

continued for 30 days, and the hair growth pattern was observed 

and tabulated. 

Figure 1: Treatment for Hair Growth Activity 

7. Qualitative Hair Growth Study 

Two parameters—hair growth initiation time (i.e., minimum 

time to initiate hair growth on the denuded skin region) and hair 

growth completion time (i.e., minimum time taken to cover the 

denuded skin region with new hair)—were visually observed to 

conduct a qualitative analysis of hair growth.  

8. Quantitative Hair Growth Study  

8.1 Hair length determination  

Rats’ shaved dorsal areas were randomly selected for hair 

collection on days 10, 20, and 30 of treatment using sterile 

forceps. After measuring the length of each hair, the findings 

were expressed as the mean length ± standard error of 25 hairs.  

8.2 Hair follicle counting  

At a constant magnification of 100x, digital photomicrographs 

were captured from representative sections of the slides. 

Following a predetermined area cropping of 1500 μm in width, 

each image's deep subcutis hair follicles were manually 

counted.  

8.3 Histological observation  

According to the histology observation, each treatment group 

will have five to seven hair follicles per millimeter of skin. 

However, there will be a noticeable difference between the 

treated and control groups in the various cycle phases 

(Anagen/Telogen). In all groups, around 40% of hair follicles 

will be in the anagen phase by the tenth day.   

9. Statistical analysis  

One-way ANOVA was used to analyze the data statistically for 

the test and control groups, and Dunnett's test was the next step. 

Data differences were deemed extremely significant when P < 

0.05. GraphPad Instate version 3.06 was the computer program 

that was utilized. The information is presented as mean ± SEM. 

RESULTS 

The percentage (%) Yield of Extract from 

Senna angustifolia leaves powder was Percentage yield = 3.57 

% weighing 735gm. The TLC profile is described in Table 2. 

 

 

 

 

 

 

 

 

 

 

Figure 2: TLC Profile of Senna angustifolia extract 

Table 2: TLC: Senna angustifolia with Rf value  

Sr. 

No. 

Solvent System Ratio Spot No Colour Rf 

Value 

1. n-hexane and 

acetone 

2:0.5 1 Light 

yellow 

0.95 

2. n-hexane and 

acetone 

2:0.5 2 Yellow 0.88 

3. n-hexane and 

acetone 

2:0.5 3 Light 

Brown 

0.55 

4. n-hexane and 

acetone 

2:0.5 4 Pale Brown 0.45 

5. n-hexane and 

acetone 

2:0.5 5 Light 

Brown 

0.38 



Bharti et al.                                                                                     Hair Growth Potential of Angustifolia Leaf Gel in Rats 

Online First                                                                                                                         Indian J Pharm Drug Studies | 4  

Moving on to the primary dermal irritation study, using OECD 

scoring, no erythema or edema was observed in rats after 72 

hours of herbal gel application. The PDII was zero, classifying 

the gel as non-irritating per OECD standards. 

EESA gel was tested on telogenic rats. Anagen onset was 

indicated by darkening of pink skin. By day 10, HG1 (2.5%) 

and HG2 (5%) showed more black patches than minoxidil and 

the control. By day 30, full hair regrowth occurred in HG1 and 

HG2, while the control showed 60% regrowth. Results suggest 

HG2 promotes early telogen-to-anagen transition. 

As for the qualitative hair growth study, over 30 days, hair 

growth initiation and completion were monitored with a 

magnifying lens. EESA 5% (HG2) treated animals showed 

significantly faster hair growth start and finish than the control 

and minoxidil groups. Control animals began hair growth at 

13.8 days (Table 3). 

Table 3: Effect of EESA gel formulation on hair growth initiation, completion time, and growth of hair length   

*p < 0.05 significant when compared to control (ANOVA followed by Dunnett’s test values are mean ± SEM 

Figure 3: Hair growth on 0, 10, 20 and 30th day using  HG 2

Further, hair follicles, skin thickness, and color were assessed. 

EESA 2.5% (HG1) showed visible changes from day 20, while 

EESA 5% (HG2) showed early effects by day 10. HG2 

significantly enhanced hair regrowth, follicle density, 

and subcutis thickness, indicating telogen-to-anagen transition. 

H&E staining confirmed higher anagenic follicle percentages: 

HG2 (71%), HG1 (65.1%), minoxidil (69.4%), vs. control 

(47%), with significant values obtained (p<0.05) for HG2 and 

the positive control group. Increased follicle size and density 

confirmed HG2’s superior hair growth effect.  

H&E staining showed increased follicle size and number, 

indicating telogen-to-anagen transition. The control group had 

47% anagenic follicles, while HG2 (69%) and minoxidil 

(69.4%) showed higher anagen rates and follicle density, 

confirming enhanced hair growth. 

DISCUSSION 

Senna (Cassia angusifolia) is commercially obtained from 

South India and some from the north part of India. The senna 

leaves contain rhein, chrysophanol, emodin, aloe emodin, mono 

Treatment Hair Growth in days 

  

Percent reduction in hair 

growth completion time 

Mean length of hair in mm 

  Initiation time Completion time 10th Day 10th Day 20th Day 30th Day 

Negative 

Control- Simple 

Gel (HG3) 

13.8 ± 0.42 42.6 ± 1.50 0.0 ± 0.00 0.0 ± 0.00 0.3 ± 0.0 1.2 ± 0.20 

Positive Control- 

Standard (2% 

minoxidil) 

7.07 ± 0.42 33.0 ± 0.71 0.6 ± 0.21 0.6 ± 0.21 1.4 ± 0.24 2.4 ± 0.20* 

2.5 EESA (HG1) 11.4 ± 0.66 36.0 ± 0.45 0.1 ± 0.0 0.1 ± 0.0 1.4 ± 0.24 1.6 ± 0.24 

5% EESA (HG2) 7.01 ± 0.64 32.4 ± 0.51 0.6 ± 0.20 0.6 ± 0.20 1.6 ± 0.24 2.8 ± 0.20* 



Bharti et al.                                                                                     Hair Growth Potential of Angustifolia Leaf Gel in Rats 

Online First                                                                                                                         Indian J Pharm Drug Studies | 5  

and diglucosides, kaemferol, palmidin, myricyl alcohol and 

mucilage. The leaves are commercially employed as a hair 

black dye [15]. The leaves have anti-dandruff, nourishing, 

conditioning, and strength building properties that can help to 

grow your hair [16].  

Gasmi A et al., in 2023, described natural compounds 

obtained from Urtica dioica, Humulus lupulus, Serenoa repens, 

Vitis vinifera, Pygeum africanum, and Cucurbita pepo for 

reducing hair loss [17]. In another study, it was deduced that 

hair growth-promoting properties of phytochemicals used in 

various herbal compounds are associated with the tumour 

growth factor-β (TGF-β) pathway, β-catenin, Janus kinase-

signal transducer and activator of transcription 3 J (AK/STAT3) 

pathway, Extracellular Signal-Regulated Kinase pathway 

(ERK) signaling pathway, and 5α-reductase inhibitory 

property. Some studies proposed that phytochemicals are more 

effective than conventional hair loss regimens such as 

minoxidil and finasteride [18].  

Owing to the presence of the phytochemical properties in 

Senna leaf extract, ESSA was employed in the current study 

exploring the hair growth-promoting potential, formulated into 

a topical gel, and evaluated in vivo using rat models. The study 

demonstrated that ESSA, prepared via Soxhlet extraction 

(3.57% yield), contains bioactive compounds like flavonoids, 

tannins, glycosides, amino acids, and proteins that support hair 

follicle stimulation. The extract was formulated into gels (2.5% 

and 5%) and tested against a control and 2% minoxidil. The 5% 

EESA gel showed superior hair growth activity, initiating and 

completing growth faster than both control and minoxidil, and 

producing the longest hair length (2.8 mm vs. 2.4 mm with 

minoxidil and 1.2 mm in control). Histological analysis 

confirmed its efficacy, with 71% anagenic follicles, increased 

follicle density, and thicker skin, indicating that Senna 

angustifolia effectively promotes hair regeneration by 

stimulating the telogen-to-anagen transition. 

CONCLUSION 

The ethanolic extract of Senna angustifolia (EESA), when 

formulated as a topical gel, significantly enhanced hair growth 

in rats, as demonstrated by: faster initiation and completion of 

hair regrowth, greater hair length over time, and higher density 

and proportion of anagenic hair follicles, along with absence of 

dermal irritation or adverse effects. These results suggest that 

EESA—particularly at 5% concentration—is a promising, safe, 

and effective natural alternative for hair growth promotion. Its 

efficacy was found to be comparable to minoxidil, a standard 

FDA-approved treatment, thus supporting its potential 

application in the development of herbal therapies for alopecia 

and other hair growth disorders. Further studies, 

including mechanistic investigations, clinical trials in humans, 

and long-term safety evaluations, are recommended to validate 

these findings and facilitate their translation into commercial 

use. 

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Natural Herbal. 2024. 

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How to cite this article: Bharti M, Bhardwaj A, Parveen N, 

Gupta A. Evaluation of hair growth potential of Angustifolia 

leaves hair gel in rats. Indian J Pharm Drug Studies. 2025; 

Online First. 

Funding: None;                 Conflicts of Interest: None Stated 

http://dx.doi.org/10.1016/S0975-3575(10)80110-5
https://naturalherbal.info/author/natural-herbal/
http://dx.doi.org/10.2174/1381612829666230505100147

	16. Senna Leaves Benefits for Hair: A Comprehensive Guide. Natural Herbal. 2024.

