INTRODUCTION:

Wrightia tinctoria (W.tinctoria) known to possess a broad range of biological Drug-related behaviors such as anti-bacterial¹, anti-tubercular, anti- oxidant, and anti-hypertensive properties. W.tinctoria is named after the Scottish doctor and botanist William.Wright².

The whole plant or its particular component (leaf, bark, seed and root) is considered to have a deep past use in indigenous communities in India. Entomedically the plant's bark is used to relieve abdominal pain³, skin ailments and wounds also as anti-oxidant⁴, anti-bacterial and anti-fungal agent⁵, as Antidote for snake poisoning, and anti-pyretic⁶. Its leaves used to possess anti-psoriatic⁷, anti-diabetic⁸, anti-microbial⁹, anti- ulcer activity¹⁰, anti-helminthic activity¹¹, cytotoxic activity¹², anti-inflammatory activity¹³, anti-cancer, and anti-fungal activity¹⁴. Alopecia often referred to as alopecia or baldness usually refers to hair loss from the head or part of the body¹⁵. In this study, we used the W.tinctoria leaf extract. W.tinctoria R. Br., (Apocynaceae) (W. tinctoria), Small Dark Gray deciduous with a light gray color, scaly, smooth bark with green, fragrant flowers scattered all over the world and it does happen tremendously in India¹⁶. The leaves are opposite, lanceolate, length of 8-15cm. It has a smooth, light-gray, scaly bark. The fruits are pendulum, and long paired follicles are joined to their ends. The hairy seeds have been released into the fruits as dehisces. This tree's leaves produce a blue dye, called Pala Indigo Leaves are plain, contradictory, and distichous. In the Indian Medicines System, various parts of W.tinctoria R.Br have been widely used, such as Ayurveda, Unani, and Siddha¹⁷. In Siddha medicine the plant is widely utilized to treat psoriasis and other skin diseases. Oil 777 made from fresh coconut oil plant leaves has been reported to be analgesic, anti- inflammatory, and antipyretic, to be an efficient entreating burning sensation, stomach disorder, and dropsy. W.tinctoria was recommended for chest treatment (asthma), colic, and diuretic infections. Most of the health-promoting and disease curing potential of plants and their plant products are associated with their phytoconstituents¹⁸. Accumulating evidence showed that bioactive phytoconstituents are found in W.tinctoria. Preliminary phytochemical review of the leaves showed Alkaloids, cardiac glycosides, flavonoids, tannins, steroids, and terpenoids.

Plant Extract Fresh Leaves and bark of twigs of W.tinctoria was gathered from the Annavaram forest. The plant was authorized by the ANU Botany Department, Guntur. Minoxidil and aminexil topical solution purchased from the local market manufactured by Glen mark pharmaceuticals. Composition-minoxidil IP5%w/v, aminexil1.5% w/v. Coconut Oil: Pure coconut oil is purchased from the local market. Veet Purchased from local market Formalin solution (10%) Weigh 0.386 gm. Sodium dihydrogen phosphate (NaH2PO4) dissolve in 200ml double distilled water. Weigh 1.022 gm. Of Sodium dihydrogen phosphates (NaH2PO4) and dissolve separately in 200ml double distilled water and dissolve separately Mix over both solutions and make up a limit of 500 ml by adding 100 ml of distilled water. To this 500 ml solution add 100 ml of 40% formalin to fill up the volume till 600ml. To the 600 ml solution add 400 ml twice distilled water to compose to 1000 ml solution. Thus, obtained solution is 10% formalin. Animals: - 30 Albino Wistar rats, weighing 200 – 250 g, were obtained from purchased from Sainath Animal Agencies, Hyderabad. Livestock is held in cages at 25 ± 3 ° C room temperature, with 12h dark/12h light cycles. The rats were fed with rat pellets and the ad libitum bath. The experiments were certified by the Ethical Committee on Institutional Animals. The experiment was being recognized by the Ethical Committee on Institutional Animals (KVSRSCOPS-29-03- 2019-004).19

Extraction procedure:

Pure coconut oil is used as the carrier oil from the tree cut fresh twigs, leaves and barks into plastic food grade tubes and immediately immersed them in the oil. As the latex oozes out inthe process, dissolve quickly the components of plant in oil. The oil ratio is approximately 2:1 or until the parts of the plant are dipped. The contents of tubes are stirred and turned over 1-2 hours between 30-40 hours depending on the environment in the shade. After that it was exposed to sun so that the contents that were maintained in sun remove moisture. Stir and turn for 2-5 days, so that the mixture develops a shade of purple violet. The material is filtered and exposed in the sun for a few hours. This extract is used in the treatment, as an external application for thirty days.²⁰

Phytochemical analysis:

Most of the health-promoting and disease curing potential of plants and their plant products are associated with theirphytoconstituents. The presence of bioactive plant constituents was shown in accumulating records in leaf, bark, root, and seeds. Preliminary phytochemical examination of the leaves endorses the presence of alkaloids, glycosides, flavonoids, tannins, and terpenoids.²¹

Procedure for in-vivo studies:

The chemical components were evaluated for alopecia activity on albino Wistar rats as an external application. Animal models were used to aid in the In- vivo study of hair growth for 30 days. 30 animals are distributed into 5 groups; each group consists of 6 animalsas shown in (Table1).

Table 1: Experimental Animal Groups

Group	Treatment with
1	Control
2	Minoxidil
3	Coconut oil
4	Extract (for 5minutes)
5	Extract (for 24 hours)

The procedure for doing alopecia activity:

Commercially available hair remover (Veet) has been applied to the dorsal area, removing hair from 1 sq. area in cm. The surgical spirit was used to clean the shaved area. The first group is maintained as a control, Washing was carried out daily, even though no solution was applied. For standard group 1ml of minoxidil solution is applied and washed off after 5 minutes. An appropriate quantity of coconut oil is added to the third group for 5 minutes and washed off. For fourth group leaf extract is rubbed for 5 minutes, put on 5minutes, then washed away. Fifth group the steps taken in group four were repeated, except that the solution was left overnight for 24 hours before being washed off ³⁹. For all groups, care was taken to make assured the application time each day was approximately the same, to ensure the time lapse between each application is consistent.²²

RESULTS AND DISCUSSIONS:

Swiss ADME studies:

The compounds were evaluated by the software’s-DOCKING, Swiss ADME. The compound showing more soluble in aqueous solubility with elevated partition coefficient and high permeability represented in (Fig. 1 and Fig. 2) it obeys Lipinski rule. Swiss, ADME of Indirubin. The Compound that is more soluble in aqueous solubility within elevation partition coefficient and high permeability reference in (Fig. 2) it obeys Lipinski rule. The Swiss ADME Showing the increased Number of Bonds on Hydrogen and reducing the partition coefficient from the aqueous process to the permeable lipid bilayer membrane of passive diffusion which is helpful to predict molecule general properties, bioactive scores, Toxicity and a resemblance to the products. The Swiss ADME concept of BOILED egg representing Brain or intestinal Permeation process estimated method represented that the compounds can penetrate the blood-brain barrier and not the substrates of PgP-carriers mention in (Fig. 3).²³

Characterization of W.tinctoria:

The Chemical components of W.tinctoria were characterized by IR, NMR, MASSspectroscopic methods.IR data: 3413 (N-H), 2984 (Ar-H), 2874 (C-H), 1598 (C=O), 1584 (C=N), 1468 (C=C) represented in Supplemental data. IR data: 3413(N-H), 2984(Ar-H), 2874(C-H), 1598(C=O), 1584(C=N), 1468 (C=C) represented in Supplementary data. A BRUKER NMR AVANCE spectrometer, operating at 320 MHz for 1H and 80 MHz for 13C, 1 dimensional 1H and 13C NMR spectra Dissolved as a solvent may be dimethyl sulfoxide6 (DMSOd6). An inner reference volume of 20 μL of Tetramethylsilane (TMS) has been added.13C NMR spectra are reported in a 2J modulated sequence and can distinguish between peer protons. (Quaternary and –CH2) from the odd (CH3; –CH–) even down. NMR was represented in Supplementary data. Spectra revealed the content of Aromatic Proton and the presence of C=C at 6.5-8 ppm.²⁴

Fig. 1: Swiss ADME of Quercetin, LIPO - Lipohilicity, FLEX - Flexibility, POLAR – Polarity, INSOLU – Insolubility, INSATU–Saturation it obeys Lipinski rule.

Fig. 2: Swiss ADME of Indirubin. LIPO -Lipohilicity, FLEX-Flexibility, POLAR-Polarity, INSOLU- Insolubility, INSAT-Saturation it obeys Lipinski rule.

Fig. 3: Brain or Intestinal estimation permeation method (BOILED EGG for all compounds)

Docking Studies:

Molecular docking was done by Auto Dock, Constituents 1 and 6 shown better docking values than standard, i.e., minoxidil (Table 2). Target Sulfotransferase is an enzyme present inside scalp hair follicle. It plays a major role in treating androgenic alopecia. Sulfotransferase converts minoxidil to concerning its active form to give better results in curing alopecia. Rising in the amount of Sulfotransferase represented in (Fig. 4) and (Fig. 5) can effectively cure androgenic alopecia. Compounds Quercetin and Indirubin shows more docking values than the other constituents shown in (Table 2).²⁵

Table 2: Molecular Docking values for all the compounds

S. No	Compound	Docking Values
1	Quercetin	-7.375
2	Squalene	-5.123
3	Beta sitosterol	-4.709
4	campesterol	-5.182
5	Stigmasterol	-4.503
6	Inirubin	-6.701
7	Lauric acid	-0.386
8	Standard minoxidil	-6.672

Fig. 4: Molecular docking study for Quercetin

Fig. 5: Molecular docking study for Indirubin

In- vivo studies:

For determination of hair length, hair was randomly plucked from the dorsal shaved region by means of sterile forceps from a rasped dorsal region of rats for seven, fourteen, and twenty-eight days.²⁶

Hair Growth 30^thday of treatment:

Hair Growth of each group of rats was observed on 30^thday of treatment. The result shows Minoxidil and 24 hrs. Extract treated rat shown significant results when compared with other group of rats (Fig. 6).²⁷

Histopathological studies:

Each group of rats was anesthetized on 15^th and 30^th day of treatment. Skin biopsies from the shaved part were obtained and 10% of formalin were retained. Sections of the tissues have been implanted on glass slides. Cut tissues have been stained with eosin and hematoxylin, and hair follicular phases were found out under a microscope were represented in (Fig. 7 and Fig.8).²⁸

(A) Control

(B) Minoxidil

(D) 5Min Extract

(E) 24 Hrs. Ectract

Fig. 6: Rat hair growth on 30^th day of treatment showing Minoxidil and 24 hrs. Extract have a significant result.

(A) Control rat skin

(B) Minoxidil rat skin

(D) 5 Min Ectract rat skin (E) 24hours Ectract rat skin

Fig. 7: Effect of W.tinctoria on skin cells on 15^thday. (A) Control, (B) Minoxidil, (C) Coconut oil, (D) 5min W.tinctoria extract (E) 24 hours W.tinctoria extract.

(A) Control rat skin

(B) Minosidil rat skin

(D) 5min Ectract rat skin

(E) 24 hours Ectract rat skin

Fig. 8: Effect of W.tinctoria on skin cells on 30^thday. (A) Control, (B) Minoxidil, (C) Coconut oil, (D) 5min W.tinctoria extract (E) 24 hours W.tinctoria extract.

Statistical analysis:

The results have been expressed as Mean± SEM. One-way ANOVA followed by Tukey's post- hoc test. Differences with *P<0.05, **P<0.01, and ***P<0.001 were considered as Statistically Important. Statistical analysis was conceded out by means of Graph Pad Prism was shown in (Table 3). Values are Mean±SEM (n=6).²⁹ One ANOVA Way followed by Tukey's post-hoc test. *p<0.05, **p<0.01 and ***p<0.0001 were considered as statistically significant. a- Compared to the control group and B- Relative to the minoxidil group (Table 3 and Fig. 9), the number of days taken to initiate and complete the development of the rasped dorsal hair region was represented.³⁰In comparison to the standard (Minoxidil), the time it took for the initiation of hair growth (A) was obtained to be 7 days for 24 hours’ extract, 5-minute extract, coconut oil and the complete hair growth (B) were found to be in 24 days for 24 hours’ extract followed by 5-minute extract (26 days), and coconut oil (27days).³¹ The number of days required for the hair development completion was obtained to be less in all the groups as compared to control represented in (Table 4 and Fig. 10). Values are Mean ± SEM (n=6). One ANOVA Way followed by Tukey's post-hockey test. *p<0.05, **p<0.01 and ***p<0.0001 were considered as statistically significant. a- compared to the control group and b-comparison to the minoxidil group was represented in (Table 5 and Fig. 10).³² The increase in the length of hair from the denuded area at the final of 1^stweek and the further observations in length in the due course of the treatment were done on the 14^thand 28^thday represented in (Fig.10).³³ On comparing with the control, all the extract-treated groups and the standard were fully covered with hair on the 4th week of the treatment. At the end of treatment, the maximum length of hair was observed in the group treated with the extract (24 hours) is nearer to the standard group.³⁴ The growth of hair was sparse for 5-minute extract and coconut oil and there was no substantial difference in the texture of hair. The difference in treatment and control groups in many cyclic phases of hair follicles, such as the Anagen and Telogen phases was investigated and the results reported.³⁵ Nearly 45-50 percent of follicles were in anagen process on the 15^th day of treatment. At the closing stages of the treatment, 24 hours treated extract of W.tinctoria exhibited the maximum quantity of hair follicles at the anagen phase.³⁶ The extract and coconut oil treated for 5 minutes shown a higher percentage of anagen phase follicles than the control, but less than the extract-treated for 24 hours.³⁷ In the pictographs, on the 15^th an 30^th day of the analysis the skin of animals in each category was depicted.³⁸ The amount of hair follicles in the Anagen cycle was found to be higher in treatment groups relative tocontrol. 24 hour extract of W.tinctoria had shown a better activity on the 30th day as that ofthe standard minoxidil group(Fig. 11).^39-40 The overall process of this present investigation represented in Graphical Abstract (Fig. 12).

Table 3: Qualitative analysis of hair growth

Treatment group	Number of days taken to initiate hair growth	Number of days taken to complete hair growth
Control	8.0±0.63	30±0.77
Minoxidil (standard)	6.0±0.63	22±0.73a***
Coconut oil	7.0±0.63	27±0.44b***
Extract (5 minutes)	7.0±0.63	26±0.77b**
Extract (24 hours)	7.0±0.57	24±0.57

Fig.9: Extraction of W.tinctoria with coconut oil was done (A) and (B), Qualitative analysis of hair growth of 24 hrs. Extract was compared with standard minoxidil, the values are stated in Mean ± SEM (n=6).

Table 4: Effect of various compounds on hair length of albino rats

Treatment group	Length of hair(mm)
Treatment group	Day 7	Day 14	Day 28
Control	2.15±0.15	4.63±0.13	14.78±0.36
Minoxidil (standard)	3.36± 0.17a***	8.42±0.22a***	17.7±0.37a***
Coconut oil	2.58±0.18b*	5.78±0.21b***	15.60±0.20b*
Extract (5minutes)	2.93±0.16	6.6±0.34b***	16.05±0.25
Extract (24 hours)	3.45±0.17	8.2±0.18	16.33±0.21

Fig. 10: Effect of various compounds on hair length of albino rats of 24 hrs. extract was compared with standard minoxidil, the values was mentioned in Mean ±SEM (n=6).

Table 5: Hair Growth activity of extract of W.tinctoria on anagen/ telogen phases

Treatment Group	Day15		Day30
Treatment Group	Anagen	Telogen	Anagen	Telogen
Control	45	55	52	48
Minoxidil [standard]	56	44	71	29
Coconut oil	46	54	59	41
Extract (5 minutes)	48	52	62	38
Extract (24 hours)	57	43	73	27

Fig. 11: Effect of various components on Hair Growth activity of W.tinctoria on (A) anagen and (B) telogen phases was mentioned in Mean ± SEM (n=6).

CONCLUSION:

The present work involves the evaluation of In-vivo action to hair development of W.tinctoria extracts on albino Wistar rats. It also involves histopathological studies which include the hair development activities at anagen and telogen phases on the 15^thand 30^thday. The findings of this study reveal that the chemical constituents of W.tinctoria which include Quercetin and Indirubin play an essential role in hair promotingactivity.

CONFLICTS OF INTEREST:

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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Received on 03.11.2020 Modified on 27.02.2021

Research J. Pharm.and Tech 2022; 15(2):643-649.

DOI: 10.52711/0974-360X.2022.00106