INTRODUCTION:

Hair loss

Telogen effluvium, which causes the loss of more than 200 scalp hairs per day, is the most prevalent kind of diffuse hair loss. Acute events like severe sickness, major surgery, thyroid disease, pregnancy, iron-insufficiency anaemia, malnutrition or abrupt weight loss, or vitamin D deficiency are usually the catalyst for its development.^1,2 It has to do with ageing, genetics, and variations in the testosterone hormone. Men are far more likely than women to experience inherited or pattern baldness. Baldness following a puberty might happen as any point. By the age of 70, around 80% of men exhibit symptoms of male pattern baldness. It may be brought on by hormone fluctuations, illnesses, genetics, or a typical aspect of ageing.^3,4

Greying of hair:

Greying hair is an obvious indication of ageing that happens to everyone. The pigment known as melanin, which is present in the skin, eyes, and hair, is what causes the loss of hair colour.⁵there is insufficient data on stress's ability to hasten hair greying in people, although studies on mice have raised this possibility. The lack of exact research instruments to map stress and hair colour over time is the reason behind this. Nutritional deficits, such as protein-energy malnutrition and disorders characterised by prolonged protein loss, can cause reversible hypopigmentation of the hair. A deficit in copper and iron can also result in hair greying.⁶

Pentacyclic triterpenoid ursolic acid (UA) is found naturally in a wide variety of therapeutic herbs and plants. According to recent studies, ursolic acid (UA) has a number of pharmacological actions, including anti-inflammatory, anti-tumor, and antimicrobial properties.⁷ Diosgenin, which is the sugar-free (aglycone) byproduct of this hydrolysis, is used in the manufacturing of cortisone, progesterone, pregnenolone, and other steroid derivatives for commercial purposes.⁸

MATERIALS AND METHODS:

Materials:

The R.K. University in Rajkot provided the herbs, which included guava (Psidium guajava L), Carica papaya, onion (Allium cepa L.), methi seeds (Fenugreek), and saragva (Moringa oleifera). The local market in Ahmedabad provided the coconut oil, kalonji (Nigella sativa), and rosemary seed oil (Rosmarinus officinalis L). The identification, purity, and strength of these basic components were checked. These raw ingredients underwent HPLC screening as well. Diosgenin was bought locally, and ursolic acid served as the reference standard chemical.

EXPERIMENTAL:

Formulation of herbal oil:

Accurately weighed all the herbs and oils were taken and prepare the hair oil formulations by using different concentrations and different methods. Details given in table 1.

Evaluation of oil:

The oil underwent several assessments, including microbiological tests, TLC identification of a-pinene, borneol, and b-pinene assay (β-sitosterol content by HPLC), weight per millilitre, refractive index, acid value, saponification value, and peroxide value.

Appearance:

The colour and smell of the oil were used to evaluate its appearance.

Preparation of sample solutions

Put 6gm of the oil sample into a test tube. In a water bath, add 2ml of hydrochloric acid, boil the mixture/solution for 2-3 minutes, and then extract the solution using 10ml of methanol. The methanol layer (upper) should be separated, and then evaporated until dry. Utilize the resulting solution (20µl) for HPLC analysis after dissolving the 10mg of residue in 10ml methanol.

Preparation of standard solution:

Dissolving the 10mg of Ursolic acid and Diosgenin in 10ml of methanol in 10ml of volumetric flask separately (1mg per ml methanol).

HPLC based standardization of all 8-oil samples with marker compounds:

For HPLC, eight oil samples (20µl). Fill the test tube with six grammes of the oil sample. In a water bath, add 2ml of hydrochloric acid, boil the mixture/solution for 2–3 minutes, and then extract the solution using 10ml of methanol. The methanol layer (upper) should be separated, then evaporated until dry. The residue of 10 mg should be dissolved in 10ml of methanol, and then diluted to 20µl. This solution should then be used for HPLC analysis.

RESULTS:

Chromatographic conditions:

Using an Agilent 1200 series Luna C18 (250x4.6mm, 5µ) column. Acetonitrile and methanol (70:30% v/v) were used as the mobile phase, and 0.6mL/min was the constant flow rate. The ambient temperature was maintained at 25C. Methanol was used as the diluent, and the run time was 30 minutes. The mobile phase was filtered using a 0.45µ membrane filter. At 210nm, detection was carried out using a PDA detector and an injection volume of 20µL. The strategy was selected by utilising the previously specified ideal conditions. Linearity was obtained in range of 5, 8. 10. 12, 15 and 20µg/ml of ursolic acid and diosgenin. Calibration graphs were plotted (Fig 1&2) and the coefficient of regression obtained was 0.999 of ursolic acid and diosgenin.

Table- 1: Formulation of hair oil

Herbs used	Direct boiling method F1(10)	Maturation method F2 (10)	Water extraction method F3(10)	Ksheerapaka method F4(10)	Direct boiling method F1(20)	Maturation method F2(20)	Water extraction method F3(20)	Ksharapaka method F4(20)
Methi seeds	10gm	10gm	10gm	10gm	20gm	20gm	20gm	20gm
PapayaLeaves	10gm	10gm	10gm	10gm	20gm	20gm	20gm	20gm
Onion bulb	10gm	10gm	10gm	10gm	20gm	20gm	20gm	20gm
Moringa Leaves	10gm	10gm	10gm	10gm	20gm	20gm	20gm	20gm
GuavaLeaves	10gm	10gm	10gm	10gm	20gm	20gm	20gm	20gm
Coconutoil(or)CM	70ml	70ml	70ml	CM(QS)	70ml	70ml	70ml	CM(QS)
Kalonji oil	30ml	30ml	30ml	30ml	30ml	30ml	30ml	30ml
Rosemary oil in drops	4 - 5	4 - 5	4 - 5	4 - 5	4 - 5	4 - 5	4 - 5	4 - 5

Figure 1: Calibration curve of Ursolic Acid

Figure 2: Calibration curve of Diosgenin

Table 2: Recovery data of Ursolic acid and Diosgenin

S. No.	Sample Name	CONC. of Ursolic Acid (µg/µl)	Conc. of Diosgenin (µg/µl)
1	F1/10	3.39	4.97
2	F2/10	3.96	3.83
3	F3/10	0.86	1.21
4	F4/10	7.32	3.84
5	F1/20	6.39	3.82
6	F2/20	6.39	3.95
7	F3/20	2.13	3.83
8	F4/20	8.80	3.52
9	Rosemary	8.46
10	Methi		5.19

Figgure 3: Concentaraton of Ursolic acid and Diosgenin in different formulations

Figure 4: Standard chromatogram of Ursolic Acid and Gallic Acid

DISCUSSION:

The literature review demonstrated the accuracy and simplicity of the HPLC approach for the simultaneous analysis of formulations including several herbs. Hair and scalp health are enhanced by ursolic acid. Similar to how they produce the waxy covering of fruits, ursolic acid and its derivatives build oil-resistant barriers on the skin and hair. Following the oil's evaluation through morphological and pharmacological studies, HPLC, and microbiological studies, we discovered that two formulations produced good results when compared with market compounds using HPLC Analytical methods. The major chemical compounds, such as ursolic acid diosgenin, improve hair growth and reduce grey hair. According to the literature review, the HPLC technique offers a straightforward and precise way to analyse polyherbal mixtures simultaneously. The condition of the scalp and hair is enhanced by ursolic acid. Similar to how ursolic acid and its derivatives make the waxy coating of fruits, they also build oil-resistant barriers on the skin and hair. The oil was evaluated by morphological and pharmacological studies, HPLC, and microbiological studies. The major chemical compounds, such as ursolic acid diosgenin, improve hair growth and reduce grey hair. Using HPLC analytical methods, we found that two formulations produced good results compared with market compounds. Because of its high sample throughput at a low operating cost and analytical assurance provided by multi-level calibration, which produced precise findings in the measurement of the quantity of analysis, HPLC is a commonly utilised technology for herbal medications. When compared to other HPLC chemicals, only the F4 20gm formulation produced satisfactory results in the RP-HPLC examination of the created varied formulations. Other than HPLC, High-Pressure Thin Layered Chromatography (HPTLC) is also a beneficial technique to which serves as quality control tools in various formulations. The present work will be useful for quality control of herbal formulations having Ursolic acid and Diosgenin.

CONCLUSION:

The current study clearly shows that various formulations with phytoconstituents as the main ingredient can employ the suggested analytical RP-HPLC procedures. The current study measured the amounts of diosgenin and ursolic acid in manufactured polyhedral hair oil both qualitatively and quantitatively.

ACKNOWLEDGEMENT:

The instruments required to accomplish this work were provided by RK University, Rajkot, India, for which the authors are very grateful.

CONFLICT OF INTEREST:

There are no conflicts of interest, according to the writers.

REFERENCES:

1. Singh HK, Dhawan BN. Neuropsychopharmacological effects of the Ayurvedic nootropic Bacopa monnieri Linn. Indian Journal of Pharmacology. 1920; 29(2): 359–365.

2. Murthy PBS, Raju VR, Ramakrisana T. Estimation of twelve bacopa saponins in Bacopa monnieri extracts and formulations by high-performance liquid chromatography. Chemical Pharmacy Bullitin. 2016; 54(1): 907–911.

3. Singh N, Kumar S, Singh P, Raj HG, Piper longum Linn. extract inhibits TNF-alpha-induced expression of cell adhesion molecules by inhibiting NF-kappaB activation and microsomal lipid peroxidation. Phytomedicine. 2018; 15(2): 284–291.

4. Rastogi S, Pal R, Kulshreshtha DK, Bacoside a triterpenoid saponin from Bacopa monnieri. Phytochemistry. 1994; 36(1): 133–137.

5. Chonpathompikunlert P, Wattanathorn J, Muchimapura S. Piperine, the main alkaloid of Thai black pepper, protects against neurodegeneration and cognitive impairment in animal model of cognitive deficit like condition of Alzheimer's disease. Food Chem Toxicol. 2020; 48(2): 798–802.

6. Rajmane, Akash D, Komal P. A review on hplc method development and validation as per ICH Guidelines 2023; 13(2): 143-151.

7. Daharwal SJ, Suman S. HPTLC Method Development and Validation for Quantification of Quercetin in Thuja occidentalis Mother tincture. Research Journal of. Pharmacy and Techlogy. 2019; 12(9): 4102-4106.

8. Giang DT, Hoang VD. Comparative study of RP-HPLC and UV spectrophotometric techniques for the simultaneous determination of amoxicillin and cloxacillin in capsules. Journal of Young Pharmacist. 2020; 2: 190-195.

9. Thiruvengada RV. Mohamed ST, Ramkanth S. A simple RP-HPLC method for quantitation of itopride HCl in tablet dosage form. Journal of Young Pharmacist. 2021; 2: 410–413.

10. Nagalekshmi R., Menon A., Chandrasekharan D.K., Nair C.K. Hepatoprotective activity of Andrographis paniculata and Swertia chirayita. Food Chem Toxicol. 2011; 49: 3367–3373.

11. Saran S. Menon SK, Shailajan S. Validated RP-HPLC method to estimate eugenol from commercial formulations like Caturjata Churna, Lavangadi Vati, Jatiphaladi Churna, Sitopaladi Churna and clove oil. Journal Pharmacy and Research. 2013; 6: 53-60.

12. Gubbannavar JS, Chandola H, Harisha CR, Kalyani R, Shukla VJ. Analytical profile of Brahmi Ghrita: a polyherbal Ayurvedic formulation. Ayush. 2022; 33: 289–293.

13. Satyanarayana L, Naidu SV, Narasimha Rao M, Ayyanna C, Alok Kumar. The Estimation of Raltigravir in Tablet dosage form by RP-HPLC. Asian Journal of Pharmaceutical Analysis 2011; 1(3): 56-58.

14. Meghal Patel. Development and Validation of Simultaneous Estimation of Two Catecholamines in Combine Dosage Form by HPTLC Method. Asian Journal of Pharmaceutical Analysis. 2014; 4(2): 57-77.

15. Pooja J, Parimal K. The Wonder of Herbs to Treat Alopecia. Asian Journal of Pharmacy and Technology. 2022; 12(2): 151-158.

16. Kshirsagar VB, Deokate UA, Bharkad VB, Khadabadi SS. HPTLC Method Development and Validation for the simultaneous Estimation of Diosgenin and Levodopa in marketed formulation. Asian Journal of Research in Chemistry. 2008; 1(1): 36-39.

17. Nitin Kumar, Satyendra Singh, Manvi, Rajiv Gupta. Hair Growth Activity of Trichosanthes dioica R. Leaves. Research Journal of Pharmacognosy and Phytochemistry. 2011; 3(1): 30-33.

18. Agrawal MR, Aher AN, Pal sc. Isolation, Characterization of Phytoconstituents from petroleum ether extract of Momordica balsamina fruits and its quantitative analysis by HPTLC. Research Journal of Pharmacognosy and Phytochemistry. 2020; 12(3): 156-161.

19. Mankar SD, Bhosale MS, Mohini, Pankaj S. A Review on Murraya koenigii: for Hair Growth Promoter. Research Journal of Pharmacognosy and Phytochemistry. 2021; 13(1): 39-43.

20. Dolih Gozali, Resmi Mustarichie. Hair Tonic Formulation of Anti-alopecia of Angiopteris evecta Extract. Research Journal of Pharmacognosy and Phytochemistry. 2019; 12(3): 1079-1085.

21. Nafisah A, Tendri A, Elita Puspita S, Nur Fitriana Muhammad Ali, Suryani. Hair Growth-Promoting Activity of Hair Tonic containing Delipidated Ethanol Extract of Capsicum frutescens L. Leaves on Male Rabbit (Oryctolagus cuniculus). Research Journal of Pharmacy and Technology. 2023; 16(7): 3305-3310.

Received on 01.12.2023 Revised on 22.04.2024

Accepted on 30.07.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):5851-5854.

DOI: 10.52711/0974-360X.2024.00888