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INTRODUCTION
Skin is the largest organ of our body which serves as a protective barrier against environmental hazards and various pathogens. It also saves us from oxidative stressor and toxic substances. The barrier function of the skin is essential for maintaining homeostasis and protecting internal organs from microbial invasion and mechanical injury (Jiao et al., 2024). But in various autoimmune and chronic inflammatory skin disorders such as Psoriasis, this barrier becomes weakened and it results in transepidermal water loss, immune imbalance, and persistent inflammation (Dong et al., 2024). In this disease, the patient not only suffers from the trauma but also it rises a financial burden globally. The recent investigation indicates that the number of individuals living with psoriasis has nearly doubled over the past three decades. A study conducted by (Li et al., 2025) reported number of individuals living with psoriasis reached approximately 43 million cases worldwide in 2021, with more than 5 million new diagnosed that year. This increase is reflected in growing disabilities and comorbidities such as psoriasis arthritis (Damiani et al., 2021). The Global economic costs for curing and managing the disease were estimated at nearly USD 149 billion in 2021(Wei et al., 2025). Asia accounts for more than half of global incident and prevalent cases and a substantial proportion of psoriasis-related DALYs, highlighting the need for research focused on Asian populations where disease burden continues to rise (Xiong et al., 2025). As Pakistan lies within Asia, where the Southern coastal region, covering the coastal belt of Sindh and Balochistan. This represents a distinct phytogeographical zone characterized by arid to semi-arid climate, saline soils, high temperatures, and low rainfall (Haider et al., 2025). Plants growing under these region frequently accumulate higher levels of secondary metabolites, including flavonoids, terpenoids, and phenolic acids, as compensatory biochemical responses (Zahedi et al., 2021). According to Ethnobotanical surveys, the coastal region reports several local plants that are being used for various skin diseases as traditional or complementary medicine. This reflects both the cultural reliance on natural remedies as well as phytochemical richness of the regional flora (Zainab et al., 2025). Despite this potential, rigorous studies on the chemical composition and dermatological applications of coastal medicinal plants remain limited. We selected Tagetes erecta flower for our research due to its local availability, traditional usage, environmental conditions and rich phytoconstituents that support to reduce inflammation and oxidative stress.
There are various medicinal plants such as Aloe barbadensis (Jales et al., 2022), Mahonia aquifolium (Anheyer et al., 2025), Salvia officinalis (Hamzic et al., 2022), Glycyrrhiza glabra (Khorshidian et al., 2024), Centella asiatica (Lin et al., 2023) that have been explored for the treatment and management of psoriasis. Many herbs have been reported for their anti-inflammatory and anti-proliferative potential in psoriasis-related models but clinical findings remain uneven and still limited (Ojha et al., 2024). According to previous studies it has been noted that that only a few extracts, including Mahonia aquifolium, Indigo naturalis, and Aloe vera, which have progressed to evaluation in randomized clinical trials but there are limited evidence for routine clinical use and formulation challenges (Anheyer et al., 2025). Despite encouraging preclinical results, translation into meaningful clinical benefit has been slow, underscoring the ongoing need for well-designed studies on promising plant-derived agents that can target the key pathological pathways of psoriasis.
Tagetes erecta L. (T. erecta), a member of the Asteraceae family, is native to Mexico and Central America but is now cultivated worldwide. Among the 56 species of the Tagetes genus, T. erecta has gained prominence because of its diverse applications in the food industry as a natural colorant and condiment. It is also used for medicinal ornamental purpose (Estrada et al., 2025; Bohatu et al., 2024). Various phytoconstituents including flavonoids, terpenoids, phenols, tannins, alkaloids, quinones, coumarins, and carbohydrates have been reported as antibacterial, antifungal, anti-inflammatory, insecticidal, and antioxidant properties (Tomar et al., 2025; Javed et al., 2025).
Traditionally in many Asian countries, the flowers of T. erecta have been used to treat wounds, menstrual irregularities, and various skin conditions (Chaudhari et al., 2024). The highest carotenoid such as lutein reported in managing visual impairments, age-related macular degeneration, and cancer prevention (Estrada et al., 2025). The highest phenolic content emphasize its strong free radical scavenging activity (Rivas-García et al., 2023) that exhibit the suppression of LPS-induced inflammatory responses through regulation of the TLR4/MD2 signaling pathway (Sanjaya et al., 2024). This potential makes it a strong candidate for Psoriasis management.
According to (Vaz et al., 2024), the flowers of T. erecta extracts shows regulation of cytokine release, migration of neutrophil, and inflammatory markers in in vivo studies. However, no prior study has investigated on T. erecta formulation effect on psoriasis such as keratinocyte hyper proliferation.
Psoriasis is driven by persistent autoimmune activation, characterized by cytokines such as TNF-α, IL-17, and IL-23, along with downstream signaling molecules including NF-κB and STAT3, which together stimulate keratinocyte proliferation and recruit inflammatory cells (Chhabra et al., 2022). Flavonoids such as Quercetin and kaemferol in T. erecta possess inhibitory effects on pro-inflammatory mediators, it can interfere with inhibition of NF-κB and TLR4-associated pathways that mitigate the oxidative stress and inflammatory signaling in psoriatic tissue (El-Gazzar et al., 2024).
Given the absence of previous research examining T. erecta within psoriasis-relevant biological systems there is need for novel plant therapies with validated mechanisms. This study aims to formulate and evaluate a T. erecta flower extract-based topical cream for the management of psoriasis.
Methods for standardization and quality parameter determination
Collection of flowers
The flowers of Tagetes erecta L. were collected from the botanical garden of Ziauddin University located at the southern coastal region of Pakistan. The plant was identified and authenticated by Dr. Muhammad Mohtasheem Ul Hasan. A voucher specimen (No. TEF-11-24) was deposited at the Department of Botany, University of Karachi. The flowers selected for the investigation were thoroughly washed under running tap water, followed by rinsing with distilled water. They were then allowed to air dry for a period at room temperature before being shade dried for approximately 3-4 weeks. The dried plant material was ground via an electric grinder (Anex AG-6042).
Physicochemical evaluation
Determination of extractive value
The dried powder of T. erecta (500 g) was measured and macerated with analytical grade ethanol at a sample/ solvent ratio of 1:20 (w/v) for seven days. The flask was shaken at regular interval during maceration. The macerate was then filter by muslin cloth and then filter by means of Whatman No. 41 filter paper with a pore size of 20 to 25 µm. The solvent was removed from filtrate by means of rotary evaporator (IKA RV05) and the extractive value was determined. The semi solid extract was stored in an air tight container at 4ºC (Mohammedi et al., 2019).
Determination of loss on drying
The degree of loss during drying was determined by heating the sample to a temperature below its melting point, which included water and volatile substances such as alcohol. 2 g of powdered drug was weighed and heated for 3 hr at 105ºC. From the difference in weight, the percentage loss of drying with respect to the air-dried substance was calculated via the following formula (Sagar et al., 2022).
Total ash
2 g of powder drug was taken from preweighed crucibles and placed in a muffle furnace (SX-2.5-10) for 5 hr at 600ºC until all the carbon from the powder drug was completely removed. The crucible and the amount of ash were weighed. The procedure was repeated for three samples to determine the mean reading (Mandal et al., 2017).
pH
For the determination of pH, an aqueous solution of the powder drug was prepared by weighing 1 g of the powder drug with 10 mL of water (10% w/v), filtering the solution and determining the pH via a digital pH meter (Mushtaq et al., 2014).
Phytochemical investigation
Qualitative screening was performed for the identification of various primary and secondary metabolites, such as carbohydrates, fat/oil, alkaloids, flavonoids, phytosterols, terpenoids, glycosides, tannins and polyphenols, following standard methods.
Test for alkaloids
Hager test
When alkaloid react with Hager reagent it forms yellow or yellowish orange ppt. For the identification of alkaloid 1-2 mL of the extract was taken in a test tube, and few mL of Hager’s reagent was added, which yielded yellow precipitate indicating the presence of alkaloids (Maheshwaran et al., 2024).
Test for flavonoids
Shinoda’s Test
When magnesium reacts with flavonoids and concentrated hydrochloric acid it undergoes a reduction reaction that converts the flavonoids into the aglycone forms resulting in the formation of colored complex. For the identification of flavonoid, a small piece of magnesium ribbon is added to the sample in the presence of concentrated HCl, resulting in appearance of red color (Maheshwaran et al., 2024).
Test for Phenols and Tannin
Ferric chloride test
The phenolic OH group react when react with FeCl3, a colored complex is formed between the ferric ions and OH- group. For the flavonoid test, 1-2 mL of extract was added to a few millilitres of FeCl3, resulting in the formation of green precipitate, which indicates the presence of flavonoids (Maheshwaran et al., 2024).
Test for Cardiac Glycoside
Keller-Kilani test
In this test, the Ferric Ions (Fe⁺³) in the reagent react with the deoxy sugars (cardiac glycosides) in the plant extract to form colored complex, while glacial acetic acid stabilizes the glycoside. The addition of sulfuric acid produces distinct color changes at the interface in form of ring.
T. erecta flower extract (2 mL) was added to 1 mL of glacial acetic acid, followed by 1-2 drops of iron chloride solution and 2 mL of concentrated sulfuric acid. A brown ring was appeared at interphase indicated the presence of cardiac glycosides (Maheshwaran et al., 2024).
Test for Phytosterols
Hesse response
Add 5 mL of extract to 2 mL of chloroform and 1 mL of sulphuric acid. The red color was appeared in the chloroform layer which indicates the presence of phytosterols (Shaikh and Patil, 2020).
Test for Quinone
Conc. Acid test
In this test, the acid undergoes deprotonation when react with the quinones, resulting in a color change. 1-2 mL of extract was added to 1 mL of concentrated HCl. A green-brown color appeared, indicating the presence of Quinone (Maheshwaran et al., 2024).
Test for Carbohydrates
Molish test
In this test when concentrated acid react with the carbohydrates present in plant, it undergoes a dehydration reaction followed by a condensation reaction with phenol molecules, resulting in the formation of violet colored ring. 2 mL of plant extract was added to 1 mL of Molish reagent, followed by few drops of conc. H2SO4 at the inner surface of the test tube. A violet ring formed at the interface, indicating the presence of carbohydrates (Maheshwaran et al., 2024).
Test for Terpenoids
A few millilitres of extract was first dissolved in 2 mL of CCl4 and allowed to evaporate. To this mixture, 2 mL of concentrated H2SO4 was added, and the mixture was heated for approximately 2 min. A grayish color indicates the presence of terpenoids (Shaikh and Patil, 2020).
Total phenolic content
The total phenolic content was assessed via a modified version of the method described by (Rizvi et al., 2023). 1 mL sample extract (0.10 g/2.5 mL) was combined with 2.5 mL of 10% Folin reagent and incubated for 5 min. Next, 2.5 mL of 7.5% Na2CO3 was added with continuous shaking. The solution was kept in the dark for 2 hr, and the absorbance was measured at 765 nm via a V-730 spectrophotometer. Gallic acid (10-100 µg/mL) was used as a standard, and the results are expressed as Gallic Acid Equivalent (GAE) per 100 g of dry extract.
Total flavonoid content
The total flavonoid content was assessed via the method outlined by Farooqi et al., (2024) with some modifications (Farooqi et al., 2024). 1 mL of sample extract (0.10 g/2.5 mL) was combined with 0.5 mL of 10% Aluminum Chloride (AlCl3), 0.5 mL of 1 M potassium acetate (C2H3O2K), and 4.15 mL of distilled water, and the absorbance was measured at 415 nm via a UV spectrophotometer, with methanol and distilled water serving as blanks. Rutin (10-100 µg/mL) was utilized as the standard.
Determination of antioxidant activity
DPPH radical scavenger for the extract
The antioxidant activities of the TE extract were assessed by measuring the DPPH radical scavenging activity, following the methods of Meda et al., and Liu et al., (2007), with slight modifications. In this method, 0.5 mL of flower extract was mixed with 3.5 mL of DPPH solution prepared in methanol. The concentration of the sample extracts ranged from 60 to 100 mg/mL. Each sample was transferred to a separate test tube, wrapped in aluminum foil to prevent light exposure, and placed in the dark for 30 min. The absorbance of each sample was measured at 517 nm in triplicate via UV-visible spectrophotometer. Methanol was used as the blank, and the control consisted of DPPH solution and methanol without extract. A calibration curve was constructed using ascorbic acid solutions ranging from 1 mg/mL to 10 mg/mL. The antioxidant activity of the extracts was calculated in terms of Ascorbic Acid Equivalents (AAE), expressed as g/100 g of dry extract. The free radical scavenging activity of each extract was expressed as the percentage inhibition and IC50 values (Siddiqa et al., 2025).
where Tc is the absorbance of the control sample and Ts is the absorbance of the sample.
Formulation of anti-psoriatic cream
The flowers of T. erecta were selected for the formulation of a cream intended for the treatment of psoriasis. The cream was formulated via the Oil-in-Water (O/W) emulsion method and consisted of two phases: an oil phase and an aqueous phase. The water-soluble ingredients, including glycerol, propylene glycol, triethanolamine, and methyl paraben, were added to one beaker. Similarly, the oil-phase ingredients stearic acid, beeswax, cetyl alcohol, and the preservative propyl paraben were placed in separate beakers. Both phases were heated in a water bath at 72±2ºC. The aqueous phase was then gradually added to the oil phase with continuous stirring until a smooth emulsion formed (Dhamale et al., 2023).
Experimental design and optimization of anti-Psoriatic cream
A 3² factorial design was employed to investigate the combined effect of Tagetes erecta extract (Factor A) and stearic acid (Factor B) on the physicochemical properties of a topical cream intended for psoriasis treatment. A total of 4 experimental formulations were prepared. The formulations were coded F1 to F4 accordingly. Each factor was studied at three levels-low, medium, and high coded as −1, 0, and +1, respectively. Finally, TE was incorporated into the cream base (Okafo et al., 2023).
Characterization of the formulated cream
Organoleptic properties
The organoleptic test of a cream preparation was performed visually, including color, texture, odor, appearance and consistency.
Stability of a formulation
The stability of each formulation was evaluated by placing the formulation at different storage conditions ranging from 25ºC to 40ºC. The parameters used were pH, homogeneity, viscosity and spreadability at different time intervals (Singh et al., 2024).
pH of the cream
To determine the pH of the cream, 1 g of the formulation was dispersed in 10 mL of distilled water, and the pH was measured via a calibrated digital pH meter (Sirsat et al., 2022).
Homogeneity
The prepared cream formulation was evaluated for its visual appearance because of its homogeneity and because its texture was compressed between fingers (Ijaz et al., 2022).
Viscosity
The viscosity of the creams was evaluated with a Rotary viscometer (NDJ-8S) at a temperature of 25ºC±2. Spindle 3 was inserted into the creams and rotated at 30 rpm. The displayed values were measured (Okafo et al., 2023).
Spreadability
The method was used with slight modifications. A quantity of 0.1 g of cream was placed between two glass slides. A weight of 100 g was placed on top of the upper plate for 5 min. Spreadibility was assessed by measuring the diameter of the cream in both lengthwise and widthwise directions using a digital caliper, and the average of these measurements was taken for each reading (SE et al., 2022).
HPLC quantification of querecitin in anti-psoriatic cream
The quantification of quercetin in the formulated cream of Tagetes erecta was performed via a validated High-Performance Liquid Chromatography (HPLC) method adapted from the standard method of Deshmukh et al., 2024. HPLC analysis was carried out via a Shimadzu SPD-20A Prominence UV/vis detector with an LC-20 AT pump, a manual injector with a loop volume of 20 µL (Rheodyne), and a Welchorm C18 column (4.6 × 250 mm, 5 µm). GC-10 software (Shimadzu Japan) was used to acquire and process the data. The mobile phase consisted of HPLC-grade acetonitrile and 0.1% (v/v) orthophosphoric acid in deionized water at a 1:1 ratio and was delivered at a flow rate of 0.8 mL/min, and the column was maintained at room temperature (25ºC). The detection wavelength was set to 370 nm.
Sample preparation for quantification
For sample preparation, 1 g of cream was mixed with 10 mL of HPLC-grade methanol and subjected to sonication (Elmasonic E-60 H) for 30 min to ensure complete solubilization of the bioactive compound. The sample was then centrifuged at 4,500 rpm for 10 min (DUAB DM 0412S). The resulting supernatant was carefully separated and filtered through a 0.45 µm syringe filter. The filtered extract was transferred into HPLC vials for analysis. Identification of the bioactive compound was achieved by comparing the Retention Time (RT) and UV absorption spectra of the sample peak with those of the standard quercetin (Deshmukh and Patil, 2024).
Limit of Detection (LOD) and Limit of Quantification (LOQ)
The Detection Limit and Quantification Limit was determined using the standard deviation of the regression line (σ) from the calibration curve and the slope, based on ICH Q2 (R1) guidelines (Guideline, 2022).
In vitro anti-inflammatory test
Membrane stabilization assay
The anti-inflammatory effect of the extract on membrane stabilization (human red blood corpuscles) was evaluated following the methods of Sakkaa et al., (2022) (Sakkaa et al., 2022) with slight modifications. Diclofenac sodium was used as a standard drug, and the anti-inflammatory activity was expressed as the percentage of human red blood cell lysis. As the membrane of red blood cells resembles the lysosomal membrane, its stabilization by the extract suggests the stabilization of the lysosomal membrane as well. The absorbance was measured with a spectrophotometer in the 560 nm range to estimate the RBC content in the test sample suspension. Blood was collected in anticoagulant test tubes (EDTA tubes) from healthy volunteer (the author) who had not used any anti-inflammatory drugs for two weeks under the ethical approval no 2023-07/SK/FoP. The blood sample was centrifuged (centrifuge DUAB DM 0412S) for 10 min at 3000 rpm, and the plasma was separated and skimped to obtain packed red blood cells. The RBC mixture was then washed 3 times with normal saline solution (0.9% w/v) by removing the supernatant via centrifugation at 4500 rpm for 5 min. A 10% (v/v) suspension of the RBC pack was prepared by adding 1 mL of washed packed RBCs to 9 mL of Phosphate Buffered Saline (PBS).
Acid Hemolysis-Induced RBC Membrane Stabilization Assay
Extracts at various concentrations (200 μg/mL, 400 μg/mL, 600 μg/mL, 800 μg/mL and 1000 μg/mL) were mixed with 5 mL of PBS (pH 7.4) in centrifuge tubes. A total of 0.5 mL of the stock RBC suspension (10% v/v) was mixed with 2 mL of the hyposaline solution (0.36% NaCl, 4.5 pH), 1.0 mL of PBS (0.15 M) and 1 mL of test sample. The control sample was mixed by adding hyposaline solution (pH 5.0) and an RBC suspension without extract. All the samples were incubated for 30 min at 37ºC. The whole mixture was centrifuged at 4500 rpm for 5 min, and the absorbance of the supernatant liquid was measured at 560 nm with a UV spectrophotometer. Different concentrations of diclofenic sodium were used as standard drugs following the same procedure (Sakkaa et al., 2022). The hemolysis inhibition rate (%) was calculated via the following equation:
ROS Generation Inhibition Assay (Egg Albumin-H₂O₂ Model)
Reduction of Reactive Oxygen Species (ROS) by a test sample indicates antioxidant and anti-inflammatory activities, which suggests the potential anti-psoriatic effect of the sample. The anti-inflammatory potential of Tagetes erecta (TE) extract was evaluated using an in vitro egg albumin-Hydrogen Peroxide (H₂O₂) model, which mimics oxidative protein damage (antioxidant activity). The assay was adapted from (Osman et al., 2016) with some minor modification, as described in recent studies (Madhuranga and Samarakoon, 2023). Hydrogen Peroxide (H₂O₂) was used as an oxidizing agent to generate ROS and induce protein denaturation (Hambly and Gross, 2009). We prepared 0.5% (w/v) H₂O₂ stock solution by diluting 0.5 mL of H₂O₂ in 29.5 mL phosphate buffer. TE extract was prepared at varying concentrations of 100, 200, 400, and 800 µg/mL. For the assay, 0.2 mL of fresh egg albumin was mixed with 2.8 mL phosphate buffer in each test tube. H₂O₂ (0.5 mL, 0.5%) was added to all tubes except the normal control, and test samples (1.5 mL) were added according to the experimental group. Each test tube contains final volume of 5 mL of the reaction mixture. Ascorbic acid served as a positive standard, while albumin along with H₂O₂ served as the negative control. Tubes were gently mixed by inversion and incubated at 37°C for 20 min. After cooling to room temperature, absorbance was measured at 660 nm using a UV-vis spectrophotometer, with buffer used as the blank.
The percentage inhibition of ROS-induced protein denaturation was calculated as:
Where, TE1= Control sample; TE2 = Test sample.
RESULTS
Physicochemical evaluation
The extractive values, effects of loss on the drying and pH values of the flower extracts are shown in Table 1.
| Sl. No, | Parameters Value (w/w) | Mean±SD |
|---|---|---|
| 1 | Extractive value | 47.66±0.57 |
| 2 | Loss on drying | 0.133±0.03 |
| 3 | pH of 1% aqueous solution | 4.61±0.04 |
| 4 | pH of 10% aqueous solution | 4.23±0.03 |
Phytochemical investigation of different solvent extracts
The ethanolic extract of Tagetes erecta showed the strong presence of the various phytoconstituents including flavonoid, phenolic compounds and tannins, phytosterol, Quoinone and Terpenoid, while showed a weak presence of alkaloid. It also demonstrates a significant presence of carbohydrate as shown in Table 2.
| Sl. No. | Phytoconstituents | Result |
|---|---|---|
| 1 | Alkaloid | |
| 2 | Hager test | + |
| 3 | flavonoids | |
| 4 | Shinoda’s Test | +++ |
| 5 | Phenols and Tannin | |
| 6 | Ferric chloride test | +++ |
| 7 | Cardiac Glycoside | |
| 8 | Keller-Kilani test | +++ |
| 9 | Phytosterol | |
| 10 | Hesse response | +++ |
| 11 | Quinone | |
| 12 | Conc. Acid test | +++ |
| 13 | Test for Carbohydrates | |
| 14 | Molish test | +++ |
| 15 | Test for Terpenoids | +++ |
Total phenolic content and Total flavonoid content
The data presented in the table indicate that the Total Phenolic Content (TPC) of the extract was 0.389 mg GAE/g, indicating a moderate presence of phenolic compounds, which are known for their antioxidant properties (Figure 1). These findings may contribute to the therapeutic potential of the extract. The TFC value was significantly greater, at 17.80 mg RE/g, suggesting that the extract is rich in flavonoids (Figure 2). Flavonoids such as quercetin and rutin derivatives are key bioactive compounds with potent antioxidant, photoprotective, and skin-protecting effects see Table 3.
| Assay | Sample | Absorbance | Dilution factor | C (mg/mL) | V (mL) | M (mg) | Result (mg/g) |
|---|---|---|---|---|---|---|---|
| TPC | TE | 0.62 | 2 | 0.778 | 10 | 40 | 0.389 GAE/g |
| TFC | TE | 0.769 | 2 | 35.601 | 10 | 40 | 17.80 RE/g |
Formulation of anti-psoriatic cream (Tagetes erecta)
The flower extract at different concentrations are used in the formulations in the range of 1-5% as the active ingredient. The quantity of all the ingredients are mentioned in Table 4. The concentrations of T. erecta extract and stearic acid were optimized based on a factorial design.
| Ingredients | Quantity (%) | Function |
|---|---|---|
| Phase A (oil phase) | ||
| T. erecta extract | 1-5% | Active ingredient; provides therapeutic and antioxidant effects for psoriasis treatment. |
| Beeswax | 1.5% | Emollient and contributes to cream thickness and stability. |
| Stearic Acid | 2-6% | Emulsifying agent and thickener. |
| Cetyl Alcohol | 5% | Emollient and improves texture and smoothness. |
| Propyl Paraben | 0.15% | Lipophilic preservative. |
| Phase B (Aqueous phase) | ||
| Glycerol | 5% | Humectant and moisturizer. |
| Propylene Glycol | 5% | Humectant. |
| Methyl Paraben | 0.25% | Hydrophilic preservative. |
| Triethanolamine (TEA) | 1% | Emulsifying and pH-adjusting agent. |
| Distilled Water | q.s | Vehicle and dispersion medium. |
Factorial design matrix
A total of four cream formulations (F1 to F4) were developed via a factorial design approach, where the concentrations of TE and stearic acid were systematically varied, while the rest of the formulation components were held constant shown in Table 5. The goal was to evaluate the effects of these two variables on the physicochemical properties, viscosity, and spreadability of the herbal cream
| Ingredient | F1 | F2 | F3 | F4 |
|---|---|---|---|---|
| Extract | 1 | 3 | 5 | 5 |
| Stearic acid | 2 | 4 | 6 | 2 |
| Bees wax | 1.5 | 1.5 | 1.5 | 1.5 |
| Cetyl alcohol | 2.5 | 2.5 | 2.5 | 2.5 |
| Triethanolamine | 1 | 1 | 1 | 1 |
| M.paraben | 0.15 | 0.15 | 0.15 | 0.15 |
| p. paraben | 0.25 | 0.25 | 0.25 | 0.25 |
| Glycerol | 5 | 5 | 5 | 5 |
| Propylene glycol | 5 | 5 | 5 | 5 |
| Water | q.s | q.s | q.s | q.s |
Organoleptic and physicochemical properties of the anti-psoriatic cream
Organoleptic characteristics provide crucial preliminary insights into the aesthetic and sensory qualities of topical formulations, which influence patient acceptability and compliance. The evaluated parameters included color, texture, odor, appearance, and consistency, as summarized in the table below. All formulations presented shades of yellow consistent with the natural pigmentation of T. erecta extracts, indicating stable incorporation of plant constituents. F2 appeared slightly brighter, possibly due to differences in the extracts. F1 to F3 presented a uniform texture, suggesting effective emulsification and blending of the cream base with the active ingredients. However, F4 exhibited a nonuniform texture, indicating inadequate homogenization during the formulation process. All formulations had a characteristic floral aroma, likely attributable to the volatile aromatic compounds inherent in T. erecta. All formulations were described as nondripping, which is optimal for topical creams. This indicates a suitable viscosity that ensures ease of application. In terms of thermal stability, all formulations remained stable at 25ºC, indicating good shelf stability under accelerated conditions. The pH values ranged from 6.29 to 6.79, all within the acceptable range for skin application, ensuring compatibility with the natural pH of the skin. Viscosity measurements at 30 rpm revealed that F4 had the highest viscosity and poor spreadibility followed by all remaining formulations, F3 is best overall it shows optimal balance of extract, stearic acid which make the formulation stable, uniform and spreadable (Table 6).
| Organoleptic analysis | F1 | F2 | F3 | F4 |
|---|---|---|---|---|
| Color | Light golden yellow | Slighter brighter yellow | Golden yellow | Golden yellow |
| Texture | Uniform | Uniform | Uniform | Non uniform |
| Odor | Characterstic floral aroma | Characterstic floral aroma | Characterstic floral aroma | Characterstic floral aroma |
| Appearance | Glossy | Glossy | Glossy | Glossy |
| Consistency | Non dripping | Non dripping | Non dripping | Non dripping |
| Thermal Stability at room temperature | Stable | Stable | Stable | Stable |
| pH | 6.35±0.01 | 6.76±0.05 | 6.29±0.04 | 6.79±0.1 |
| Viscosity (30 rpm) | 1937.78±563.37 | 994.41±207.94 | 1108.62±166.17 | 2179.14±57.04 |
| Homogeneity | Uniform | Uniform | Uniform | Non uniform |
| Spreadibility | 2.56±0.10 | 2.5±0.08 | 2.26±0.14 | 2.25±0.05 |
DPPH activity
The antioxidant potential of both the TE extract and the anti-psoriatic cream formulation was evaluated via the DPPH assay, and their IC50 values and Ascorbic Acid Equivalent (AAE) capacities were determined (Figure 3). The TE extract exhibited an IC50 of 11.88 mg/mL, with a corresponding AAE of 46.3 mM/100 g, indicating that the extract retained a significant level of free radical scavenging potential, comparable to that of standard ascorbic acid. In contrast, the anti-psoriatic cream formulation had an IC50 value of 30.83 mg/mL, and the calculated AAE for the cream was 17.83 mM/100 g, indicating a lower antioxidant capacity than that of the raw extract (Table 7).
| Extract | Conc (mg/mL) | Percent inhibition % | IC50 (mg/mL) | AAE (mM/100 g) |
|---|---|---|---|---|
| TE | 8 | 31.5 | 11.88 | 46.3 |
| 10 | 42 | |||
| 12 | 50.5 | |||
| 14 | 52 | |||
| 16 | 54.5 | |||
| 18 | 53.25 | |||
| Anti-psoriasis cream F3 | 20 | 43.06 | 30.83 | 17.83 |
| 40 | 55.88 | |||
| 60 | 61.17 | |||
| 80 | 65.76 | |||
| 100 | 77.39 |
HPLC method for Quercetin determination in T. erecta anti-psoriatic cream
The method development for Quercetin as marker was based on HPLC. To get the better separation and resolved peak, different types of columns and mobile phase were employed. The column was selected on the basis of good resolutions of peaks. Absorbance was selected using UV spectra of standard and extract samples overlaid. The best response was found at 370 nm.
The aim of this study is to establish an accurate, precise and reproducible method with sensitivity and cost effectiveness to quantify Quercetin in Tagetes erecta cream.
Method Validation
The developed method was validated following ICH guidelines.
Linearity
A standard calibration curve for quercetin was established using five concentrations ranging from 200 µg/mL to 1000 µg/mL. The relationship between the concentration and peak area was linear, following the equation:
With a correlation coefficient of R2=0.9901, indicating excellent linearity (Figure 4).
Precision
In this study the intraday variations were evaluated at different concentration. The % RSD values for the standard concentrations ranged between 0.17% and 1.32%, demonstrating the acceptable repeatability of the method (Table 8). The percentage recovery of quercetin ranged between 97.74% and 101.63%, which falls within the acceptable range (98-102%) as per ICH Q2 (R1) guidelines, confirming the accuracy of the developed method.
| Quercetin conc. (µg/mL) | Mean (AUC) | %RSD | % Recovery |
|---|---|---|---|
| 200 | 118,949 | 0.17 | 100.35 |
| 400 | 136047 | 0.49 | 101.01 |
| 600 | 149854 | 0.56 | 99.34 |
| 800 | 163236 | 1.32 | 97.74 |
| 1000 | 186145 | 0.48 | 101.63 |
Accuracy
The accuracy of the developed HPLC method was determined through the analysis of quercetin at three concentration levels corresponding to 100±20% of the target concentration (200 µg/mL). The percentage recovery was calculated by comparing the measured concentration with the nominal concentration, while the precision was expressed as % RSD.
The obtained results, as mentioned in Table 9, reveal that the %RSD values ranged from 0.18-0.25%, which indicate excellent repeatability, and the %Recovery values were between 99.58% and 100.60%, confirming the accuracy and reliability of the developed analytical method for the quantification of quercetin in the cream formulation.
| Concentration (%) | Mean conc. (µg/mL) | % RSD | % Recovery |
|---|---|---|---|
| 80 | 160 | 0.25 | 99.75 |
| 100 | 200 | 0.18 | 100.55 |
| 120 | 240 | 0.22 | 99.67 |
Quercetin content in the F1 and F3 formulations
The F1 and F3 T. erecta cream formulations were analyzed under identical HPLC conditions. The F1 cream yielded a mean AUC of 161576.7with % RSD of 0.98%, and the F3 cream showed a mean AUC of 234717.3 with % RSD of 0.55%. On the basis of the calibration equation represented in Figure 5, the quercetin content was calculated to be 0.732 mg/g in the F1 cream and 1.638 mg/g in the F3 cream (Table 10). Both formulations were above the LOQ and therefore, considered reliably quantifiable represented in Table 11.
| Cream formulation | AUC | %RSD | Quercetin Content (µg/g) | Quercetin Content (mg/g) |
|---|---|---|---|---|
| F1 | 161576.7 | 0.98 | 732.822 | 0.732 |
| F3 | 234717.3 | 0.55 | 1638.131 | 1.638 |
| SD | LOD (µg) | LOQ (µg) | |
|---|---|---|---|
| Calculated from Residual standard deviation (from regression) | 2949.724 | 120.486 | 365.108 |
Acid Hemolysis-Induced RBC Membrane Stabilization Assay
Comparative analysis of the standard and treated samples at various concentrations (200-1000 µg/mL) revealed a concentration-dependent trend toward inhibition. Compared with the standard group, the 1000 µg/mL and 800 µg/mL groups presented highly significant reductions in absorbance values, with % inhibition values of 74.78% (p<0.0001) and 70.0% (p=0.0000223), respectively. A statistically significant inhibition of 12.05% was also observed at 600 µg/mL (p=0.00303). In contrast, at 400 µg/mL and 200 µg/mL, no significant difference was noted between the standard and treated samples (p>0.05), and no inhibition was recorded. These results are summarized in Table 12 and Figure 6.
| Concentration (µg) | Standard Mean ± SD | Treated Mean Mean ± SD | % inhibition |
|---|---|---|---|
| 1000 | 0.2023 | 0.0510* | 74.78 |
| 800 | 0.2228 | 0.0671* | 70 |
| 600 | 0.2307 | 0.2029 | 12.05 |
| 400 | 0.2317 | 0.2311 | 0.25 |
| 200 | 0.2352 | 0.2471 | - |
ROS Generation Inhibition Assay (Egg Albumin-H₂O₂ Model)
The ROS generation inhibition assay demonstrated that Tagetes erecta extract exhibits a concentration-dependent inhibition of H₂O₂-induced albumin denaturation. At 800 µg/mL, TE extract achieved 81.445% inhibition, indicating significant antioxidant and anti-inflammatory potential, approaching the activity of the standard antioxidant (ascorbic acid) mention in Table 13. These findings suggest dose-dependent increase in % inhibition for both the Standard and Extract. Two-way ANOVA revealed that concentration had a highly significant effect (p<0.01), while treatment type also differed significantly (p<0.05). IC₅₀ analysis further confirmed the superior potency of the Standard (IC₅₀=391.38 µg/mL) relative to the Extract (IC₅₀=561.20 µg/mL) as shown in Figure 7.
| Conc (µg/mL) | Absorbance of Extract (mean ±SD) | Absorbance of standard (mean ±SD) | % inhibition of Extract | IC50 Standard | % inhibition of Standard | IC50 Extract |
|---|---|---|---|---|---|---|
| 100 | 0.175 ±0.05 | 1.330±0.04 | 25.354 | 391.38 | 42.855 | 561.2 |
| 200 | 0.150±0.06 | 1.111±0.20 | 36.119 | 52.257 | ||
| 400 | 0.110±0.00 | 0.843±0.47 | 53.258 | 63.754 | ||
| 800 | 0.150±0.05 | 0.391±0.06 | 81.445 | 83.163 |
DISCUSSION
Tagetes erecta flowers are medicinal herbs that have therapeutic effects on a number of diseases because of their high flavonoid and phenolic contents (Burlec et al., 2021). The solvent Ethanol, extract polar compounds such as flavonoids, phenolics, tannins and glycosides (Ng et al., 2020, Bartnik and Facey, 2024). This explains why the antioxidant and antimicrobial potential of medicinal plants varies according to the extraction solvent used (Agidew, 2022). The total phenolic content measured for TE was 0.389 mg GAE/g, suggesting the traditional use of T. erecta in the treatment of various skin diseases, such as wound healing (Vaz et al., 2024), allergies and eczema, as phenolic compounds are reported to be involved in fibroblast-mediated responses, collagen production, and tissue regeneration, help in wound healing and inhibit scar formation (Fernandes et al., 2023). These phenolic compounds protect against UV-induced skin damage, reduce signs of aging, and reduce the risk of skin carcinogenesis (Baloghová et al., 2023, Carrara et al., 2021). Notably, the Total Flavonoid Content (TFC) in the extract was significantly greater, i.e., 17.80 mg RE/g, which represents a substantial presence of flavonoid compounds, predominantly quercetin derivatives (Kusumiyati K, 2025), which are known for their broad-spectrum pharmacological activities. A high total flavonoid content in plant extracts is closely associated with amended management of skin disorders and psoriasis, mainly via antioxidant, anti-inflammatory, and immunotherapeutic actions (Rivera-Yanez CR, 2021). Flavonoids reduce the activation of the proinflammatory cytokines IL-6, IL-17A, and TNF-α and downregulate signaling pathways that help reduce the symptoms of psoriatic lesions (Baloghová et al., 2023). In a previous study, the extract of Scrophularia desrti 16.85 mg QE was shown to help reduce inflammatory mediator levels and enhance skin repair (Patidar et al., 2024). Phytotherapeutics high in flavonoids are promising for both prophylaxis and the management of such dermatological disorders. The antioxidant activity of TE and the formulated anti-psoriatic cream was further proven through a DPPH free radical scavenging assay. TE presented a relatively low IC₅₀ value of 11.88 mg/mL, accompanied by an Ascorbic Acid Equivalent (AAE) concentration of 46.3 mM/100 g, revealing efficient suppression of oxidative stress. However, the anti-psoriatic cream formulation demonstrated a higher IC₅₀ of 30.83 mg/mL and an AAE value of 17.83 mM/100 g. These results support the antioxidant-rich nature of T. erecta, with the extract offering stronger free radical scavenging activity than the final formulation does (Okafor et al., 2023), although both have substantial antioxidant potential that is helpful for managing psoriasis (Zhu, 2024). A factorial design-based approach was adopted to optimize the anti-psoriatic cream formulation by varying the concentrations of the TE extract and stearic acid in the four formulations (F1-F4). The assessment of physicochemical parameters revealed significant differences in the ratios of these components. All formulations demonstrated appropriate organoleptic properties, including a uniform and stable color with the natural pigmentation of T. erecta, homogenous texture (except F4), floral essence, and glossy appearance. These parameters are critical for patient adherence to and acceptance of topical dermatological preparations (Imaz et al., 2020). Viscosity measurements indicated formulation-dependent variation, with F4 exhibiting the highest viscosity (40.5%), likely due to the stearic acid content and potential interaction with the extract concentration. In contrast, F3 exhibited relatively lower viscosity and better handling properties, possibly making it a more favorable formulation from a consumer perspective (Imaz et al., 2020; Shaikh et al., 2020). The pH values of all formulations are within the range of the skin’s natural range (6.36-6.9), which falls within the dermatologically acceptable range for topical products and the matrix (Burlec et al., 2021). For the analysis of the delivery and stability of the principle active constituents in post formulation, the quercetin content was quantified in selected formulated creams (F1 and F3) via a validated HPLC method (U, 2024; S, 2024). The calibration curve displayed excellent linearity (R²=0.9949). Quercetin levels were found to be 0.746 mg/g and 1.625 mg/g in F1 and F3, respectively, with both values exceeding the method’s LOQ (489.41 µg), confirming reliable quantification. The higher concentration in F3 is consistent with its increased extract percentage, verifying that quercetin is retained effectively in the final formulation (Chaudhari et al., 2024). These results highlight that formulation parameters directly influence the physicochemical characteristics and phytoconstituent retention of the final product. Compared with all formulations, F3 demonstrated an optimal balance of quercetin content, viscosity, texture, and pH, suggesting its superiority for further pharmacological validation and clinical consideration (Lin et al., 2023; Anheyer et al., 2025). The anti-inflammatory potential of the Tagetes erecta-based formulation was further evaluated via an acid-induced hemolysis assay, which serves as a model for evaluating the membrane-stabilizing properties of therapeutic agents (Ibrahim et al., 2023). The extract clearly inhibited red blood cell destruction in a concentration-dependent manner, with significant therapeutic effects at relatively high concentrations. At 1000 µg/mL and 800 µg/mL, the treated samples presented highly significant reductions in hemoglobin release, with % inhibition values of 74.78% and 70.0% (p<0.0001), respectively. These findings revealed strong membrane-stabilizing and anti-inflammatory properties, potentially linked to the presence of flavonoids and phenolic compounds such as quercetin, which are known to inhibit lysosomal enzyme release and prevent red blood cell lysis under acidic conditions (Ajazuddin, 2024). These results support the role of the extract in membrane stabilization and justify its inclusion in natural topical anti-psoriatic formulations.
In in vitro study Egg Albumin-H₂O₂ Model, Hydrogen peroxide was used to generate oxidative stress, as H₂O₂ is an established oxidant for inducing protein oxidation and structural modification. Previous studies have shown that millimolar concentrations of H₂O₂ (e.g., ~75-100 mM) cause significant protein oxidation in model proteins. This supporting the use of a 0.5% H₂O₂ working solution in our assay to induce albumin denaturation (Davies, 2016).
CONCLUSION
The results of the present study indicate that Tagetes erecta is a rich source of valuable phytochemicals, namely, flavonoids and phenolics that have antioxidant and anti-inflammatory effects. The extract demonstrated significant DPPH scavenging activity and membrane stabilization activity and thus warrants application in the treatment of oxidative stress and inflammation-associated cutaneous disorders. Experiments with formulations confirmed that the concentration of the extract influences the texture, pH, and quercetin recovery of the final formulation. HPLC analyses further ensured the precise quantification of the quercetin content in the finished formulation. These findings support the use of T. erecta as a promising candidate herbal topical cream formulation for the management of psoriasis.