INTRODUCTION:

Hair is considered to be a signature of aesthetic appeal in almost every society. There has caused in the expanding of cosmetic products for hair and scalp maintenance. Shampoos are applied for the purposes of personal hygiene and cleansing, which are eliminate the accumulation of dirt, sebum, fragments, and various environmental contaminants within the hair.

A wide array of shampoo products is found in the marketplace, and these products are categorized based on cosmetic purposes and functions. Various types of shampoos are displayed on the shelves including daily shampoos, conditioning shampoos, anti-dandruff shampoos, anti-hair fall shampoos, baby shampoos, and dry shampoos. However, the majority of commercial are contained synthetic ingredients. Health risks and adverse effects associated with synthetic chemical exposure are concerned, such as skin irritation, scalp flaking, hair follicle corrosion, allergic reactions, hair dryness, dandruff problems, and other related risks. Hence, natural organic ingredients contained products are increasing the demand from consumers. Furthermore, hair shampoos derived from natural renewable sources are more environmentally sustainable, easily accessible, and possess bioactivities, such as antioxidants, anti-microbials, anti-fungal, anti-inflammation, and other medicinal effectiveness ^1-9.

Hair disorders can exert influence especially on socio-psychological well-being of an individual. Alopecia is one of most hair losses caused by several factors, including genetic, social, metal, and hormonal factors. Androgenetic alopecia (AGA) and alopecia areata (AA) are the most prevalent forms of hair loss, which are roughly 50% of both genders, while AA is observed in approximately 2% of the entire population. Whereas, this hair loss may potentially occur up to 70% of in males during their lifespan ^10,
11. AGA is correlated with increased 5-α-reductase activity, elevated levels of 5α-dihydrotestosterone (DHT), and disrupted signalling of transforming growth factor-β (TGF-β). It has been suggested that the purpose of preventing or treating AGA is inhibited 5-α-reductase activity. phenolics are anticipated to assume significant functions in the management alopecia ^{12, 13, 14}. Saponins are natural surfactant for hair cleansing by amphiphilic property. There are consisting of hydrophilic heads and hydrophobic tails, which are function as non-ionic surfactants by reducing the surface tension of water and interfacial tensions; and solubilize the oils and dirt from hair strands and scalp ^{15, 16}. The maintenance of healthy hair is referred to promotion of hair. The hair follicles can be strengthened from the antioxidant properties of bioactive constituents contained in natural herbs such as, tannins, phenolic compounds, and flavonoids. Additionally, tannins are natural astringents, which can be eliminating excessive sebum from the scalp, fortifying hair roots, and prevention of hair loss ^17-20. Hair colour is determined by the extent and arrangement of melanin pigments in the outer hair layer. The process of hair colour fading is due to exposure to UVA, UVB, and visible light differs depending on the specific wavelength range. UVB and UVA radiation have an adverse impact on the proteins in the hair, whereas visible light contributes to the breakdown of melanin granules. Natural polyphenols, such as tannins, exhibit significant antioxidant properties and have the potential to mitigate the fading of hair colour ²¹.

The utilization of Suaeda maritima root is acknowledged in Thai traditional medicine as treatment of some symptoms and diseases i.e., hair loss, abscesses, allergy, and dermatitis. In addition, S. maritima root has locally developed to the herbal shampoo, which is believe that this root can be prevent hair loss and maintain hair colour. Nevertheless, there is a lack of scientific finding that has elucidating its possible applications in the realms of hair treatment. Our objectives of this study were to screen the active components contained in root extract; to assess its in vitro biological properties for prevention of hair loss and hair care, including the 5α-reductase inhibitory activity, antioxidants, anti-inflammation and anti-fungal properties, which were regarded to its application in the field of haircare products.

MATERIALS AND METHODS:

Plant collection, preparation and extraction:

S. maritima plants (5 kg) were collected from Samut Songkhram Campus, Suan Sunandha Rajabhat University, Muang district, Samut Songkhram, Thailand (100.0370572°E, 13.4200603°N). Botanical identity of the plant was confirmed from botanists who affiliated in the Provincial Agricultural Office in Samut Songkhram, Thailand. The root was cut, cleaned, shade-dried, and grinded in fine powder form. Dried S. maritima root powder (500 g) was macerated in 95% ethanol (450 ml) at 25 °C for 72 h (×3). The ethanol extract was concentrated with a rotary vacuum evaporator, water bath warming and air-dried. The yield of extraction was estimated as following: yield of extraction (%) = (weight of extract/ weight of plant material) × 100

Phytochemical quantification:

Total phenolic content

The ethanol extract from S. maritima root (SMRE) was quantitated for total phenolic content (TPC) by colorimetric assay. SMRE (20 mg) was dissolved in ethanol (RCI Labscan, Thailand) and concentration was 0.2 mg/ml. 1.0 ml of extract or standard solution was mixed with 0.3 ml of saturated sodium bicarbonate (RCI Labscan, Thailand), and 0.1 ml of Folin-Ciocalteu reagent (Loba, Chemie, India), respectively. Volume of mixture was adjusted by distilled water (4.6 ml) and incubated at room temperature in the dark for 1 h. Absorbance of TPC was determined by using a UV-visible spectrophotometer at 765 nm. Gallic acid (Sigma-Aldrich, USA) was diluted within dimethylsulfoxide, DMSO (RCI Labscan, Thailand) for standard curve plotting (0.00012, 0.00024, 0.00049, 0.00098, 0.00195 and 0.00391 mg/ml). TPC was informed as mg of gallic acid equivalent (GAE) per gram ²².

Total flavonoid content:

SMRE was quantitated for total flavonoid content (TFC) by colorimetric assay. 50 ml of SMRE (2.0 mg/ml) in ethanol or standard solution was deposited in a 96-well microplate and each well was contained aluminum chloride (Loba Chemie, India), ethanol, and sodium acetate (10: 96: 10). The mixture was incubated at room temperature in the dark for 40 min and absorbance of TFC was measured at 415 nm. Quercetin (HWI Analytik GmbH, Germany) was applied to calibration curve and TFC was informed as mg of quercetin equivalent (QE) per gram ²³.

Total tannin content:

SMRE was determined total tannin content (TTC) by using a spectrophotometric-based method (lmax = 760 nm). This analytical approach was involved the use of Folin-Denis reagent (Sigma Alrich, USA) in accordance with the established by the Association of Official Analytical Chemists, AOAC (2005) 952.03. A standard curve was performed by utilizing of known concentration of tannic acid (Fluka, Switzerland) and TTC was informed as mg of Tannic acid equivalent (TE) per gram ²⁴.

Total saponin content:

The determination of the total saponin content (TSC) in SMRE was carried out using the vanillin colorimetric method, and the colour of reaction mixture was developed by the Lieberman-Burchard reagent. The coloured absorbance was assessed by using of spectrophotometer at 528 nm. A range of saponin (Sigma-Aldrich, USA) concentrations were applied to calibration curve and TSC was reported as mg of saponin equivalent (SE) per gram ²⁵.

Antioxidant assays:

The SMRE was diluted using absolute ethanol, which was adjusted to 0.001, 0.01, 0.1, 1.0, and 10 mg/ml. A range of antioxidant assays, such as the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and nitric oxide (NO) radical scavenging assays, and the ferrous iron-ferrozine complex method and ferric iron-thiocyanate complex method, were included in this study. The methods were monitored the reduction of DPPH radical, the reduction of NO radical from Griess reagent (Sigma-Aldrich, USA), the ability of metal chelation, and inhibition of lipid peroxidation, respectively. Micro-titer plate reader (BIO-RAD, USA) was employed in order to monitor the absorbance of their color of mixture at maximum wavelength (lmax). Antioxidant activity from each assay was reported a 50% inhibitory concentration (IC₅₀) of SMRE, which was calculated from triple measurements. Positive control of DPPH and NO radical scavenging assays was vitamin C, ascorbic acid (Sigma-Aldrich, USA). While, positive control of ferric iron-thiocyanate complex method or inhibition of lipid peroxidation was vitamin E, a-tocopherol (Sigma-Aldrich, Germany). The metal chelating agent, ethylenediaminetetraacetic acid, EDTA (Sigma-Aldrich, USA) was positive control for ferrous iron-ferrozine complex method ^{26, 27, 28}.

Anti-inflammatory assays:

Albumin degradation test

The SRME and diclofenac diethyl ammonium (Sigma-Aldrich, Germany) were solubilized in 10% (v/v) DMSO and in distilled water, respectively. Sample solution was centrifuged at 150 rpm and its supernatant was serial diluted at 0.625, 1.25, 2.5, 5.0 and 10.0 mg/ml. The range of sample or control was performed with 0.2 % albumin solution at 70 ±2 °C for 5 min. Anti-inflammatory activity of SRME or diclofenac diethyl ammonium was monitored measured the reduction of albumin absorbance at 278 nm by UV-visible spectrophotometer. Result was represented as IC₅₀ of SMRE compared with diclofenac diethyl ammonium as positive control ²⁹.

Reduction of NO production from inflammatory cells:

Macrophage cells are important inflammatory cells and play a role of inflammation via the mediators and cytokines production including NO. In this study, anti-inflammatory activity of SRME was monitored the reduction of NO releasing from activated macrophages in present of the extract. Mouse macrophage cell (RAW264.7) was cultured in Dulbecco ¢s modified Eagle ¢s medium, DMEM (Invitrogen, USA), containing foetal bovine serum, FBS (10%) and penicillin/streptomycin (1%) at appropriated condition, which was deposited to a 24-well plate and cell volume was adjusted to 1 × 10⁵ cells with 500 μl of medium/well. The cell suspension was incubated with SRME or control for 1 h and activated with lipopolysaccharide, LPS (Sigma, USA) for 24 h. The supernatant from treated cell suspension was transferred to a 96-well plate and Griess reagent (Sigma-Aldrich, USA) was added. The reduction of NO production was performed by colorimetric method at 540 nm. Result was represented as IC₅₀ of SMRE compared with triamcinolone acetonide as positive control ³⁰.

Anti-fungal assay:

Anti-fungal activity of SMRE (0.05, 0.5 and 5 mg) was evaluated by Kirby-Bauer method ³¹. Common pathogenic fungi, Candida albicans and Malassezia furfur were obtained from Thai Institute of Scientific and Technological Research (TISTR), Pathum Thani, Thailand, and maintained in potato dextrose agar, PDA (HiMedia Laboratories, India). A 6 mm filter paper disc (Macherey-Nagel, Germany), petri dishes (Union Science, Thailand), a laminar flow biohazard class II (Renovation Technology, Thailand), and the incubator (SLIO-600ND, EYELA, Japan) were included in this study. The result was measured the diameter of the inhibition zone (mm) surrounding sample disc compared with control, and calculated as mean±SD from triplicate measurements. A 0.025 mg fluconazole and 0.2 mg ketoconazole dices (Oxoid, UK) were the positive controls for C. albicans and M. furfur, respectively. 95% ethanol contained disc (7 mg) was negative control.

Assessment of 5-α-reductase inhibitory activity:

SMRE dissolved in 10% DMSO was added to Roswell Park Memorial Institute (RPMI) Medium contained fetal bovine serum, FBS (10%) and penicillin/streptomycin (1%) and sterile by 0.2 mm membrane filtration. SMRE in culture medium was prepared for serial ten-fold dilution (0.1-1,000 mg/ml). Positive control was dutasteride (Sigma, USA) and prepared similar to SMRE. Prostate cancer cell DU-145 (ATCC HTB-81) was suspended for 2.0-3.0 × 10⁴ cells/ml and incubated with each concentration of sample or control within 48 h. The cell viability was performed by sulforhodamine B (Biobasic, USA) staining for adjustment of the appropriate range of SRME or dutasteride (control) concentration. In this study, the nontoxic concentrations were maintained up to 80% of stained cell (or viable cell), which were included in study of 5α-reductase inhibitory activity ³². This enzymatic inhibitory activity test was conducted as cell viability; therefore, the range of concentration of test or control was narrower and lack of staining process. Each treated prostate cancer cell DU-145 was harvested and RNA was extracted. RT-PCR was conducted and the DNA amplified product was measured by the Qubit fluorometer. The 5α-reductase DNA of SMRE-, dutasteride- treated and untreated cell line were separated by agarose-gel electrophoresis, and visualized by ethidium bromide staining. The pictures of 5α-reductase DNA band (700 bp) were demonstrated by Gel Documentation System (BIO-RAD laboratories, UK). The inhibition of 5α-reductase activity (%) of SRME or dutasteride was calculated from band intensity of 5α-reductase DNA compared with untreated cell line ³³.

RESULT:

SMRE was darkish brown resin-like characteristic and odorless, and the yield of extraction was 9.87%. The TPC, TFC, TTC and TSC contained in SRME were 13.26±0.21 mg GAE/g, 67.41±0.27 mg QE/g, 92.34±2.56 mg TE/g and 364.67±2.97 mg TS/g, respectively. SMRE was exhibited DPPH radical scavenging (IC₅₀ = 0.29±0.02 mg/ml) and inhibition of lipid peroxidation (IC₅₀ = 0.08±0.03 mg/ml), whereas it was poorly NO radical scavenger and metal chelator (Table 1). SMRE was strongly inhibited the albumin degradation (IC₅₀ = 0.81±0.03 mg/ml) and the NO releasing from LPS-induced macrophage (at 0.1 mg/ml, 20.5±2.8%), which was preferable anti-inflammation when compared with diclofenac and triamcinolone acetonide (Table 1 and 2). However, SMRE was lack of anti-fungal activity against C. albicans and M. furfur when used of Kirby-Bauer method (Table 3). Dutasteride (³ 10 mg/ml) was more toxic against the prostate cancer cell DU-145 rather than SMRE (>100 mg/ml). At toxic level, treated cells were degraded and clumped (Figure 1). Hence, maximum concentration of non-toxic levels of SMRE (100 mg/ml) and dutasteride (10 mg/ml) were assessed on inhibitory activity of 5α-reductase. The 5α-reductase inhibition was represented the reduction of DNA intensity from treated cell compared with controls (Figure 2). SMRE (100 mg/ml) and dutasteride (10 mg/ml) were inhibited 19.71±0.48 and 51.91±3.35% of DNA intensity of 5α-reductase, respectively.

Figure 1. The cytotoxicity of SMRE and dutasteride against prostate cancer cell DU-145 was represented the degraded cells and cell clumping at 1,000 and 10 to 1,000 mg/ml, respectively (dashed red line)

Figure 2. The band intensity of 5α-reductase DNA (700 bp) from SMRE treated-, dutasteride (DU) treated-, and untreated- (C) prostate cancer cell DU-145. Anti-5α-reductase activity was the reduction of band intensity from treated cells when compared with untreated cell.

DISCUSSION:

Recently, Manojkumar et al. (2024) had extracted whole plant of S. maritima by Soxhlet extraction and its methanol extract is highest yielded (7.3%). Saponin is the most of active compound contained in hexane, acetone and methanol extract ³⁴. In our study, we were concerned on chemical toxicity, and used ethanol as less toxic solvent. In addition, we were selected only S. maritima root and extracted by simple maceration as traditional preparation. The result was found that SMRE (ethanol extract) was gained higher yield (9.87%) than methanol. In the root part, higher polarity of ethanol can extract more phytochemicals content, which was affected by solvent efficiency and temperature. Saponins were 67% of all active component contained in SMRE. Thus, our finding was comparable to previous study that solvent for S. maritima should more polarity and saponin is also the major active components ³⁴. Therefore, in this study we were extracted only root part, because we were simulated traditional decoction and proved its utilization by scientific explaining. SMRE was possessed significant DPPH radical scavenging activity and inhibition of lipid peroxidation comparable to vitamin C and vitamin E with lower IC₅₀ values. Antioxidant activity of SMRE was corresponded to previous studies that study on whole plant extract, which is scavenge DPPH and ABTS radicals, and inhibited iron oxidation ^34-37. Therefore, SMRE was unable to trap NO radical and metal. SMRE was exhibited anti-inflammatory activity in both of in vitro assays. There was strongly inhibited albumin degradation and NO releasing of LPS-induced macrophage in preferable level when correlated with diclofenac and triamcinolone acetonide, respectively. There had sparsely reported anti-inflammatory activity of S. maritima. Recently, Um et al. (2023) had reported that xinghamide A, the nonapeptide is active metabolite from Streptomyces xinghaiensis habited in S. maritima. This compound is exhibited NO secretion and cyclooxygenase-2, COX-2 expression from LPS-stimulated RAW264.7 cells, and it is similar to our finding in this study ³⁸. SMRE was lack of anti-fungal activity against C. albicans and M. furfur. Previous studies had reported that S. maritima extracts from leaf and stem had anti-microbial activity against only pathogenic bacteria ^{35, 36}. Thus, the uses of part of plant, solvent and extraction method and type of pathogens for anti-microbial test were considered. As the results, SMRE was lesser exhibited 5α-reductase inhibitory activity than dutasteride. Therefore, SMRE was safer than dutasteride due to less toxicity.

We were proved the uses of S. maritima root on hair care by scientific findings. SMRE was prepared similar to traditional utilization, determined major active constituents, and evaluated their biological activities. SMRE was contained saponins, tannins, flavonoids and phenolics, respectively. Hence, saponins are natural surfactants, which was mild on hair and scalp, less likely to cause irritation and dryness, and more suitable for sensitive skin ^{15, 16, 39}. The natural surfactants are also maintaining the moisture balance of the hair, while they can effectively hair cleansing. In addition, this capably cleansing is acted by displacing the dirt, oil and impurities, therefore some of them may less to produce foam ^39-41. Tannins, phenolics, and flavonoids are possessed antioxidant activity and they can strengthen the hair follicles. The antioxidant capacity of phenolic compounds is positively associated with the quantity of its phenolic hydroxyl groups, which are reduce the free radicals by acted as electron donor. There can scavenged radicals and chelated transition metals that ceased the progression of oxidative damage ⁴². Tannins are also exhibited astringent property on removing of excessive sebum on the scalp, which is lead to the strengthen of hair roots and prevention of hair falls ^{18-20, 42}. In our study, tannins were second most of bioactive compounds contained in SRME following with flavonoids and phenolics, respectively. There was implied that antioxidant and anti-inflammation of SMRE were possessed from these active constituents. Seborrheic dermatitis is an inflammatory dermatological condition in sebum-rich areas, and dandruff is categorized as its mild form affected in the scalp. The pathogenesis of the disease is not fully clarified; therefore, it has been linked to an inflammatory reaction triggered by yeasts. Malassezia spp. are largely yeast on the scalp, which are producing lipases, catalysed triglycerides to free fatty acid, and cause an inflammatory response and epidermal hyperproliferation ^{43, 44}. In addition, Tinea capitis, folliculitis, seborrheic dermatitis, and pediculosis capitis are four prevalent dermatological conditions affected to the scalp. Since, SMRE was lack of anti-fungal activity, which was unable to inhibited C. albicans and M. furfur. Therefore, it can inhibit the inflammation on scalp area, reducing the excessive fatty acid/sebum, and indirectly preventing development of dandruff and relieve the symptoms of scalp from other conditions.

AGA is a persistent condition of hair loss, which is distinguished by the miniaturization of hair follicles and inflammation of the hair follicle surrounding. DHT is an active metabolite of testosterone and steroid 5α-reductases is the key enzyme for AGA progression ^{10, 11}. In addition, NO is mediated radical inflammation of the hair follicle surrounding, which is responded by DHT level ⁴⁵. The use of anti-androgens (finasteride, dutasteride, and spironolactone), corticosteroids, and tretinoin (anti-seborrheic) had relieve AGA progression, while the side effects had concerned ⁴⁶. Quercetin has been documented to exhibit anti-inflammatory properties and anti-androgenic effects by inhibition of steroid 5α-reductases and suppression of androgen receptors. Quercetin-enriched extracts from Ginkgo biloba, Camellia sinensis, and Cuscuta reflexa have demonstrated the promoting of hair growth through inhibit steroid 5α-reductases ^{47, 48}. Moreover, procyanidin and flavonoids in present of TGF-β-triggered cell apoptosis may inhibit 5α-reductase, involve the antioxidant pathway, and enhance the anti-apoptotic factors like Bcl-xL ⁴⁹. In our study, SMRE was contained preferable amount of antioxidant, possessed anti-inflammation especially inhibition of NO releasing from induced-macrophage, and inhibited 5α-reductase with less toxicity. Thus, we were summarized that SMRE can use to modulate AGA progression by hair follicle protection and stimulation of hair growth.

CONCLUSION:

We were concluded that S. maritima root is a notable hair care herb according by Thai traditional medicine. This S. maritima root was traditionally prepared as SMRE, which was contained its active constituents including saponins, tannins, flavonoids and phenolics. SMRE was exhibited antioxidant property by scavenging DPPH radical and inhibiting lipid peroxidation; anti-inflammation by inhibiting albumin degradation and NO releasing from LPS-induced macrophage; and anti-hair loss by inhibiting 5a-reductase with less toxicity. Therefore, SMRE was lack of anti-fungal activity against C. albicans and M. furfur. The use of S. maritima root as ingredient of hair cleansing and hair care products was considered by high content of saponins, antioxidant content and activity, and hair growth promotion.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

We express our sincere gratitude to Suan Sunandha Rajabhat University in Bangkok, Thailand for their generous research funding and technical assistance. Our appreciation also extends to the team at the College of Allied Health Sciences, Samut Songkhram Campus, Suan Sunandha Rajabhat University, provided invaluable support in the identification of herbal specimens and the collection of application data in Thai Traditional Medicine.

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Received on 10.04.2024 Revised on 14.08.2024

Accepted on 24.10.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):6008-6014.

DOI: 10.52711/0974-360X.2024.00912

Sample / Assay	DPPH	NO	ILP	MC	IAD
SMRE	0.29±0.02	ND	0.08±0.03	>1,000	0.81±0.03
Vitamin C	0.01±0.00	0.11±0.00	-	-	-
Vitamin E	-	-	0.01±0.00	-	-
EDTA	-	-	-	0.01±0.00	-
Diclofenac	-	-	-	-	0.55±0.03

Sample (mg/ml)	Inhibition of NO release (%)
Sample (mg/ml)	0.0001	0.001	0.01	0.1
SMRE	ND	17.6±2.8	19.7±2.3	20.5±2.8
Triamcinolone acetonide	27.8±4.9	34.3±3.3	31.6±4.9	30.0±3.3

Pathogenic yeast	Extract (mg)	Diameter of inhibition zone (mm)
Pathogenic yeast	Extract (mg)	0.05	0.05	5.0
*C. albicans*	SMRE	NA	ND	ND
	Fluconazole (0.025 mg)	20.63±0.37
	Ethanol (7 mg)	ND
*M. furfur*	SMRE	ND	ND	ND
	Ketoconazole (0.2 mg)	28.46±0.59
	Ethanol (7 mg)	ND