Bioactive substance contents and therapeutic potential for skin inflammation of an herbal gel containing Derris reticulata and Glycyrrhiza glabra

Abstract

Context

Derris reticulata Craib. and Glycyrrhiza glabra L., of the Fabaceae, have been used as active components in Thai herbal formulas for the treatment of fever and skin diseases.

Objective

To evaluate the physicochemical and pharmacological properties of the developed herbal gel formulation containing the combined extract from D. reticulata stem wood and G. glabra root (RGF).

Materials and methods

The potential of the herbal gel formulation containing RGF (8% w/w) as the active ingredient was studied by evaluating the anti-inflammatory, antioxidant, and anti-Staphylococcus aureus activities using quantitative reverse transcription-polymerase chain reaction assay, spectrophotometric method, and broth microdilution technique, respectively. The reference standards for the biological testing included Nω-nitro-L-arginine (L-NA), ascorbic acid, catechin, and penicillin G. The stability study of the RGF herbal gel was performed by a heating-cooling test (at 45 °C for 24 h and at 4 °C for 24 h/1 cycle; for 6 cycles), and the bioactive marker compounds in the herbal gel were investigated by the HPLC technique.

Results

RGF showed promising pharmacological effects, particularly on its anti-inflammatory property (IC₅₀ 73.86 µg/mL), compared to L-NA (IC₅₀ 47.10 µg/mL). The RGF-containing gel demonstrated anti-inflammatory (IC₅₀ 3.59 mg/mL) and free radical scavenging effects (IC₅₀ 0.05–4.39 mg/mL), whereas it had no anti-S. aureus activity (MIC > 10 mg/mL). The active ingredient in the developed herbal gel significantly inhibited lipopolysaccharide-induced nitric oxide production by downregulating iNOS mRNA levels. The contents of the bioactive markers in the RGF gel (lupinifolin and glabridin) did not change significantly after stability testing.

Discussion and conclusions

The RGF-containing gel has potential to be further developed as an herbal product for the treatment of skin inflammation.

Introduction

The use of herbal medicines continues to expand rapidly across the world, with not less than 80% of people worldwide relying on these products to prevent and treat a variety of health- and life-threatening diseases (Ali et al. 2021; Department of International Trade Promotion [DITP] 2021). In traditional Thai medicine, Derris reticulata Craib. and Glycyrrhiza glabra L., two medicinal plants with the same local name ‘Cha-Em’” from the family Fabaceae, have been documented for their combined utilization as an active component in antipyretic formulations and for the treatment of skin diseases, sore throats, and mouth ulcers (Department of Curriculum and Instruction Development [CID] 1999). Modern pharmacological investigations have demonstrated that both medicinal species exhibit a variety of promising biological properties (Vongnam et al. 2013; Yang et al. 2017; Issarachot et al. 2021). G. glabra root extracts and compounds, especially flavonoids such as chalcones, isoflavans, and isoflavones, were found to have potent anti-inflammatory activity via a key mechanism involving the suppression of numerous inflammatory mediators (Simmler et al. 2013; Yang et al. 2017). In addition, it was reported that D. reticulata extract and its major active compounds belonging to prenylated flavanones displayed therapeutic potential for inflammatory-related disorders in several preclinical studies (Ganapaty et al. 2006; Vongnam et al. 2013; Issarachot et al. 2021). In this regard, the inhibition of iNOS gene expression, COX-2 enzyme, and numerous pro-inflammatory cytokines has been described earlier for its anti-inflammatory mechanisms (Vongnam et al. 2013). In traditional Thai medicine, the formulating drug principle is generally based on combinations of herbal remedies for synergy or minimizing the toxicity of a herbal preparation (Medicinal Registration Division [MRD] 1998). To our knowledge, there is no previous report on the development of an herbal gel formulation that comprises the combined extract of D. reticulata and G. glabra (RGF) as an active ingredient for treating inflammatory skin diseases, in particular contact dermatitis.

Contact dermatitis is an inflammatory eczematous skin disease related to a response of the immune system to contact irritants or contact allergens (Novak-Bilić et al. 2018). Atopic dermatitis, on the other hand, is a multifactorial, complex chronic inflammatory skin disease that causes skin barrier dysfunction with different aetiologies and prognoses (Weidinger et al. 2018). Staphylococcus aureus is a facultative pathogen found on the skin and can promote skin inflammation in patients with atopic dermatitis (Bitschar et al. 2020). Individuals with a history of atopic dermatitis are more susceptible to contact dermatitis (Litchman et al. 2023). Some drugs applied externally during the treatment of skin inflammation, such as corticosteroids, immunomodulators, and antimicrobials, are still limited, however, because of their unpleasant topical side effects (Abraham and Roga 2014). Herbal products derived from medicinal plants that possess anti-inflammatory actions can be beneficial as an alternative treatment for inflammatory skin conditions, especially contact dermatitis and atopic dermatitis (Ghasemian et al. 2016; Goh et al. 2022). There have been various pharmacological studies on plant-based herbal remedies that support their therapeutic potential in clinical applications for inflammatory skin diseases (Dawid-Pać 2013; Wu et al. 2021). Preclinical pharmacological evaluations have been recommended among factors affecting the integration of herbal medicine into modern medical practices. Herbal medicines being considered for integrative therapy must first be tested for safety and efficacy in preclinical models (Ekor 2014).

This study explores the scientific proof for using a mixture of extracts from D. reticulata and G. glabra as the active ingredient in a topical herbal gel to alleviate the symptoms of inflammatory skin conditions, in particular contact dermatitis. The developed herbal gel formulation was tested for anti-inflammatory, antioxidant, and anti-S. aureus activities using LPS-induced NO production in RAW264.7 macrophage model, spectrophotometric method, and broth microdilution assay, respectively. A real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to gain a deep understanding of the anti-inflammatory potential of the active ingredient RGF in the herbal gel. In addition, a heating-cooling test was performed to assess the thermal stability of the gel, and HPLC was conducted to analyze the bioactive marker compounds contained in the developed topical herbal formulation.

Materials and methods

Drugs and chemicals

The following equipment was utilized in this study: HPLC (Agilent 1100, Agilent Technologies, USA); a microplate reader (Spectrostar Nano, BMG Labtech, USA); real-time qRT-PCR (Stratagene Mx3005P, Agilent Technologies, Germany); UV-vis spectrophotometer (UV-1700 PharmaSpec, Shimadzu, Japan); Stability chamber (KBF 115, Binder, USA); pH meter (Lab845, Xylem Analytics, Germany), Viscometer (Dv2t, Brookfield, USA). The standard substances, such as glabridin (purity ≥ 98%), gallic acid, catechin, Nω-nitro-L-arginine (L-NA), and indomethacin (purity > 98.5%) were purchased from Sigma-Aldrich (USA). Chemicals and reagents for biological activity tests including complete Dulbecco’s modified Eagle’s medium (cDMEM), lipopolysaccharide (LPS), phosphate buffer saline, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), naphthylethylene diamine dihydrochloride (NED), tryptic soy agar (TSA), and Meuller-Hinton broth (MHB) were from Sigma-Aldrich (USA), while Griess’s reagent and sodium nitroprusside were from Merck (Germany) and Himedia (India), respectively. Foetal bovine serum (FBS) and antibiotic-antimycotic were bought from Gibco (USA). Poloxamer 407, disodium EDTA, and phenoxyethanol which are used for herbal gel preparation were from commercial sources in Thailand. Lupinifolin (purity > 95%) was obtained from previous work (Issarachot et al. 2021).

Plant material and preparation of the active ingredient in herbal gel formulation

The wood of D. reticulata and the root of G. glabra were obtained from a Thai herbal pharmacy in Hat Yai District, Songkhla Province, in October 2020. Botanical identification was carried out preliminary by a licenced traditional Thai pharmacist and further confirmed by Associate Prof. Dr. Oratai Neamsuvan, an ethnobotany specialized lecturer at Prince of Songkla University, Thailand. The authentic samples of each plant material (WS-S0963 for D. reticulata and WS-S1063 for G. glabra) have been deposited in the herbarium of the Traditional Thai Medicine Faculty, Prince of Songkla University. Each dried herb (D. reticulata wood and G. glabra root) was coarsely pulverized, extracted by maceration with 95% ethanol (dried plant material:solvent, 1:10; 3 times at room temperature), concentrated in a rotary evaporator at 45 °C, and evaporated to dry on a water bath. The crude ethanol extract of each plant was re-extracted using solvent partitioning method to obtain its bioactive-rich fraction (hexane fraction for D. reticulata and dichloromethane fraction for G. glabra). The active ingredient, RGF, in the herbal gel was prepared by combining the bioactive-rich fractions from D. reticulata and G. glabra in a 1:1 ratio, and it was kept at 4 °C for further examination.

Preparation and physicochemical characterization of herbal gel formulation

The topical herbal formulation was prepared as a hydrogel, which consisted of a mixture of RGF (8% w/w) and gel base. The gel base was made using the following components: poloxamer 407 (gelling agent), propylene glycol (dispersing agent), ethanol 70% (solvent), lime water (liquid vehicle), and phenoxyethanol (preservative). Briefly, poloxamer was dispersed in a cold-water phase including distilled water, lime water, and phenoxyethanol, stirring until homogeneous. Then the solution of RGF in 70% ethanol and propylene glycol was thoroughly incorporated into the poloxamer suspension and stirred again until homogeneous. The formulation was then stored at 4 °C to achieve a clear formulation. Standard gel was prepared in the same manner as herbal gel, except that indomethacin (1% w/w) was added instead of RGF. The physicochemical characteristics to be evaluated for the prepared herbal gel included organoleptic properties (colour, odour, texture), pH, viscosity, and skin sensation (spread, stickiness, moisture, or dryness) after applying the gel to the arm area. The herbal gel was maintained at room temperature in the opaque container until further evaluation for its pharmacological activities and stability.

Quantitative analysis of active substances in herbal gel

Determination of the total phenolic content

The total phenolic content in the developed herbal gel was determined spectrophotometrically using Folin-Ciocateu method as described previously by Hsieh et al. (2008), with slight modifications. Briefly, the gel was prepared and diluted with DMSO to obtain final concentrations of 25–5000 µg/mL, and then 30 μL of the test solution was left to react with 150 μL of tenfold diluted Folin-Ciocalteu reagent in the dark for 5 min. Afterward, 7.5% sodium carbonate (120 μL) was added to this mixture, which was further incubated in the dark for another 30 min. A microplate reader was used to measure absorbance at 750 nm. Gallic acid was used as the reference standard for a calibration curve. The result is presented as mg gallic acid equivalent per mg of herbal gel (mg GAE/mg RGF gel).

Determination of the total flavonoid content

The total flavonoid content in the developed herbal gel was evaluated using aluminium chloride spectrophotometric method as described previously by Zhishen et al. (1999), with slight modifications. Briefly, various concentrations of the gel were prepared in DMSO (final conc. 25–5000 µg/mL, and a 20 µL aliquot of test solution was mixed with 6 μL of 5% sodium nitrite, and after 5 min, 6 μL of 10% aluminium chloride was added. After incubation for 6 min, 40 μL of 4% sodium hydroxide was added to this mixture. The absorbance was measured at 510 nm in a microplate reader. Catechin was used as the reference standard for a calibration curve. The result is presented as mg catechin equivalent per gram of herbal gel (mg CAE/g RGF gel).

Determination of lupinifolin and glabridin content

The amount of lupinifolin and glabridin in the developed herbal gel was analyzed by the HPLC-VWD method modified from previous work (Joycharat et al. 2016). A standard stock solution (100 mg/L) was prepared and diluted with methanol to obtain five concentrations ranging from 1 to 50 mg/L. The test solution was prepared by dissolving 200 mg of the formulation in 1 mL of methanol and vigorously shaking. HPLC conditions used in the present work are as follows: stationary phase, Hypersil ODS (4.0 × 250 mm, 5 µm); mobile phase, isocratic system (methanol and 15% acetic acid in DI water, 80:20 v/v); flow rate, 1.0 mL/min; detector, VWD; wavelength, 281 nm (0-6 min) and 254 nm (6.1-20 min). A calibration curve was plotted for the value of the peak area versus that of the corresponding concentration of standard solution. The validation of the analytical method was evaluated by linear regression analysis. To eliminate matrix effects from a measurement, the standard addition HPLC calibration technique was also included herein for quantification analysis of glabridin in the developed herbal gel.

Cell culture

The murine macrophage cell lines (RAW 264.7, ATCC No. TIB-71) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultured in complete Dulbecco’s modified Eagle’s medium (cDMEM) containing DMEM high glucose medium, L-glutamine, sodium pyruvate, 10% fetal bovine serum (FBS), and 1% antibiotic-antimycotic (100 units/mL of penicillin, 100 μg/mL of streptomycin, and 250 ng/mL of amphotericin B) at 37 °C in a 5% CO₂ incubator.

Cytotoxicity assay

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to obtain noncytotoxic concentrations of the test samples (RGF, herbal gel, and gel base) before performing the nitrite assay, as previously described (Sae-Wong et al. 2011). Briefly, the murine macrophage RAW 264.7 cell lines were seeded into a 96-well plate at a density of 1 × 10⁵ and incubated at 37 °C for 24 h. Following incubation, the adherent cells were treated with different concentrations of the test samples (6.25–200 µg/mL for RGF and 25–10,000 µg/mL for RGF gel) and incubated at 37 °C for 24 h with 5% CO₂. The culture media containing the samples were removed and MTT was added to a final concentration of 0.5 mg/mL. After 4 h of incubation, the MTT solution was removed and DMSO was added to dissolve the formazan crystals. The optical density (OD) values were read at 595 nm using a microplate reader. The percentage of cell viability was calculated according to the equation: [OD value of test sample/OD value of control well] × 100.

Assay for anti-inflammatory activity

The anti-inflammatory effects of the test samples (RGF, herbal gel, and gel base) on the inhibition of LPS-induced NO production by RAW264.7 cells were carried out using nitrite assay described previously (Sae-Wong et al. 2011). In brief, the suspension of the RAW264.7 cells was seeded into a 96-well microplate at 5 × 10⁵ cells/100 µL/well in complete medium, and plates were incubated at 37 °C for 24 h in a CO₂ incubator. Then, the cells were treated with various noncytotoxic concentrations of the test samples (25–200 µg/mL for RGF and 25–10,000 µg/mL for RGF gel) and stimulated with 1 µg/mL of LPS for 24 h. The nitrite formation in cell culture supernatant was measured with Griess reaction. In this case, Griess’s reagent was mixed with the culture supernatant at a 1:1 ratio and the plate was incubated at room temperature for 10 min. The absorbance was measured at 570 nm using a microplate reader. The amount of nitric oxide in the supernatant was calculated from the linear regression equation of nitrite standard curve. The percentage of NO inhibition was calculated using the following formula:

$$\mathit{\text{Inhibition}}\hspace{0.17em}\left(\text{\%}\right)\text{\hspace{0.17em}=\hspace{0.17em}}{\text{[(NO}}_{\text{LPS}}-{\text{NO}}_{\text{sample}}{\text{)/(NO}}_{\text{LPS}}-{\text{NO}}_{\text{control}}\text{)]}\times 100$$

where NO_LPS, NO_sample, and NO_control are the NO production levels (µM) of LPS-stimulated group (LPS+, Sample−), treatment group (LPS+, Sample+), and untreated group (LPS−, Sample−), respectively. Indomethacin gel and L-NA were used as reference standards. The IC₅₀ values were determined by non-linear regression analysis in Prism software.

Assay for inhibition of iNOS mRNA expression

The mechanism of the anti-inflammatory effect of RGF was investigated by a real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay. RAW264.7 macrophages were seeded into a 24-well plate at a density of 2 × 10⁵ cells/well and incubated at 37 °C for 24 h. The following day, cells were stimulated with LPS (1 µg/mL) and treated with different concentrations of RGF (25–200 µg/mL). Total mRNA from the treated cells was extracted using TRIZOL reagent (Invitrogen, USA) according to the manufacturer’s directions. The cell culture was homogenized with TRIZOL before being mixed with chloroform. The aqueous containing RNA was precipitated with isopropanol and the concentration of the purified RNA was determined by measuring absorbance at 260 nm. To create single-stranded complementary DNA (cDNA), reverse transcriptase (Thermo Scientific, USA) was used as recommended by the manufacturer. The first strand of cDNA was generated from 1 µg of total RNA and real-time PCR analysis was conducted using qPCRBIO SyGreen Blue Mix (PCR Biosystems, USA) with a real-time PCR instrument. Primer sequences were utilized for amplification of specific genes, as described previously (Jakkawanpitak et al. 2021). Sequences of the forward (F) and reverse (R) primers are as follows: iNOS, 5′-TTGGAGCGAGTTGTGGATTGTC-3′′ (F) and 5′-GCAGCCTCTTGTCTTTGACCCAG-3′′ (R); and Actb 5′CATTGCTGACAGGATGCAGAAGG-3′′ (F) and 5′-TGCTGGAAGGTGGACAGTGAGG-3′′ (R). The quantitative PCR assays were run at 95 °C for 2 min by 1 cycle, followed by 40 cycles of 5 s at 95 °C and 20 s at 60 °C. Data analysis was performed using the 2^−ΔΔC_t method, with β-actin as the internal control.

Assay for antioxidant activity

DPPH assay

The DPPH assay was carried out according to a previously published protocol with some modifications (Brand-Williams et al. 1995). Briefly, the test samples (RGF, herbal gel, and gel base) were prepared in 70% ethanol, and a 20 µL aliquot of each sample (final conc. 25–6250 µg/mL) was mixed with 180 µL of DPPH-methanol solution (0.031 g/L). The resulting mixture was left at 25 °C for 30 min, and absorbance was measured in a spectrophotometer at 517 nm in a microplate reader. Ascorbic acid was used as the reference standard. The results are performed as the IC₅₀ values.

FRAP assay

The FRAP assay was carried out according to a previously described methodology by Butsat and Siriamornpun (2010), with some modifications. Briefly, a 100 µL aliquot of the test samples (RGF, 62.5 µg/mL; herbal gel and gel base, each 62.5 mg/mL in DMSO) was separately mixed with 900 µL of the fresh FRAP solution in test tubes and the resulting solution was incubated for 30 min. The absorbance was measured at 596 nm in a UV-vis spectrophotometer. Ferrous sulphate (FeSO₄) was used as the standard for a calibration curve. The results are expressed as FRAP values (mM Fe (II)/mg).

NO radical scavenging assay

The NO radical scavenging activities of the test samples (RGF, herbal gel, and gel base) were assessed using the modified assay mentioned previously (Sun et al. 2014). Briefly, the test samples were prepared in 70% ethanol, an 80 μL aliquot of serial 2-fold diluted test samples (final conc. 3.90–500 µg/mL) was separately allowed to react with 40 μL of sodium nitroprusside solution under dark condition for 150 min. An 80 μL aliquot of Griess’s reagent was added into the reaction mixture, which was further incubated for 20 min. The absorbance was recorded at 546 nm in a microplate reader. Indomethacin gel and catechin were used as reference standards. The IC₅₀ values were analyzed with Prism software by using linear regression method and dose-response-inhibition model.

Anti-Staphylococcus aureus assay

Staphylococcus aureus ATCC 29213 was obtained from the Division of Biological Science, Faculty of Science, Thailand. The bacterial strain was stored in tryptic soy broth (TSB) (Difco, Bordeaux, France) with 20% glycerol at −80 °C until use. The strain was cultured on TSA and incubated at 37 °C for 24 h. Minimum inhibitory concentration (MIC) values of the test samples (RGF, herbal gel, and gel base) were determined using a broth microdilution method according to CLSI procedures (Clinical and Laboratory Standards Institute [CLSI] 2020). Briefly, log phase culture of S. aureus was adjusted to obtain approximately 1 × 10⁶ CFU/mL. Two-fold-serial dilutions of the sample concentrations were carried out in a 96-well microtiter plate, resulting in a final concentration between 0.25 and 64 µg/mL. Penicillin G was included as a positive control. DMSO was used as a negative control, and its final concentration was ≤ 1% in all assays. The lowest concentration of the test samples required to completely inhibit bacterial cell growth after incubation at 35–37 °C for 24 h was recorded as the MIC value.

Stability test

The developed herbal gel formulation was subjected to an accelerated stability test in the stability chamber using a heating-cooling cycle (at 45 °C for 24 h and at 4 °C for 24 h/1 cycle) for 6 cycles (Thanasakdecha and Tewtrakul 2021). The physicochemical characteristics of the formulation, including appearance, odour, pH value, viscosity, phase separation, and number of bioactive constituents, were examined both before and after accelerated stability testing.

Statistical analysis

All experiments were performed in triplicate. The results of active substance content and ferric-reducing antioxidant power (FRAP value) were expressed as the means ± SD. IC₅₀ was determined by non-linear regression analysis in Prism software, and its value was shown as the mean and 95% confidence interval (95% CI) [min to max value]. Statistical analysis was performed using GraphPad Prism version 8.0 (GraphPad Software Inc.). Student’s t-test and one-way analysis of variance (ANOVA) were used to evaluate the significant differences (p < 0.05) between the means.

Results

Physicochemical properties of herbal gel formulation containing RGF

The RGF herbal gel was prepared successfully using 20% poloxamer 407 as the gelling agent. Phenoxyethanol (0.64%) and lime water (q.s. to 100%) were used as a preservative and liquid vehicle in the RGF gel, respectively. Our preliminary results (Sangkaew 2021) of the developed gel preparations containing 1-10% RGF revealed that the formulation with 8% RGF dissolved in a 12% ethanol solution (70% v/v) and 18% propylene glycol was the most effective in preserving the physicochemical properties at day 0 (Figure 1A). Consequently, the topical preparation with RGF (8% w/w) is the most suitable candidate for the formulation of herbal gel proposed in this study. The physicochemical properties of the formulation containing 8% RGF are shown in Table 1. Upon addition of RGF at 8% w/w, the homogeneous transparency of the gel base (Figure 1B) changed to a yellowish brown. The gel had a light herbaceous scent, a smooth texture, and no phase separation. Considering skin sensation, it was revealed that the herbal gel, after drying, produced a film that spread well on the skin and was highly moisturizing. The gel had a pH of 6.62 ± 0.10, which was within the usual range for skin pH, and a viscosity of 38,387 ± 2100 cP.

Figure 1.

The developed gel formulation at day 0; A: RGF (8%w/w) gel and B: Gel base.

Table 1.

Physicochemical characterization of developed gel formulation.

Parameters	RGF (8% w/w) gel
	Day 0	After accelerated testing
Color	Yellowish brown	Yellowish brown
Odor	Light herbaceous aroma	Herbaceous aroma
pH	6.62 ± 0.10	6.13 ± 0.18
Viscosity (cP)	38,387 ± 2191	56,079 ± 10,223
Texture	Homogeneous, translucent	Homogeneous, translucent
Skin feels	Film formed after drying and highly moisturizing	Film formed after drying and highly moisturizing

The pH and viscosity had no significant difference between before and after the heating-cooling test, p > 0.05 (mean ± S.D., n = 3).

Active substances of herbal gel formulation containing RGF

The quantification of total phenolic and total flavonoid contents in the RGF herbal gel is shown in Table 2. The gel presented a concentration of total phenolic compounds of 0.07 ± 0.00 mg GAE/mg RGF gel measured using the Folin-Ciocalteu method. Total flavonoid content was evaluated at 3.06 ± 0.10 mg CAE/g RGF gel based on the aluminium chloride colorimetric assay. The active substances belonging to flavonoids present in RGF gel were further studied using HPLC (Figure 2). By comparing the peak areas of lupinifolin and glabridin in the RGF gel chromatogram to those in the calibration curves of their respective authentic standards, the amounts of lupinifolin and glabridin were determined to be 0.2160 ± 0.0011 and 0.0910 ± 0.0011 mg/g RGF gel, respectively (Table 2).

Table 2.

The content of active substances in RGF (8% w/w) gel formulation.

RGF gel/ext.	Active substances
	TPC (mg GAE/mg)	TFC (mg CAE/g)	Glabridin (mg/g)	Lupinifolin (mg/g)
Gel (Day 0)	0.07 ± 0.00	3.06 ± 0.10	0.0910 ± 0.0011	0.2160 ± 0.0011
Gel (Acc. cond.)	0.02 ± 0.00	0.95 ± 0.03	0.0941 ± 0.0011	0.2315 ± 0.0011
RGF	791.25 ± 5.51	76.52 ± 4.83	14.53 ± 0.18	48.66 ± 0.08

All the active substance contents had no significant difference between before and after the heating-cooling test, p > 0.05 (mean ± S.D., n = 3). Acc. cond.: accelerated condition. Ext.: extract.

Figure 2.

HPLC chromatogram of the combined standards glabridin and lupinifolin. Detection: VWD (wavelength; 281 nm at 0–6 min, 254 nm at 6.1–20 min). Mobile phase: Isocratic system (methanol:15% acetic acid in DI water, 80:20 v/v).

Anti-inflammatory activity of herbal gel formulation containing RGF

The RGF (8%) herbal gel was assessed for its ability to inhibit the cellular NO production in LPS-activated macrophage model. As shown in Table 3 (Figure 3), we found that the NO release in LPS-treated RAW264.7 cells was moderately suppressed by the formulated herbal gel with IC₅₀ value of 3589 µg/mL, whereas that of the gel base was 5322 µg/mL. The RGF gel and its gel base did not exhibit any significant toxicity against macrophage cells at the highest tested concentration of 10,000 µg/mL.

Table 3.

Pharmacological activities of RGF (8% w/w) gel formulation.

Gel/ext./std.	Anti-inflammation		Antioxidant		Anti-S aureus
	IC50 (µg/mL), 95% CI	NO IC50 (µg/mL), 95% CI	DPPH IC50 (µg/mL), 95% CI	FRAP (mM Fe 2+/ mg)	MIC (mg/mL)
RGF gel (Day 0)	3589* [2887, 4445]	59.47 [26.89, 93.77]	4385 [3757, 5227]	0.43 ± 0.00	> 10
RGF gel (Acc. cond.)	4730* [3967, 5663]	35.17 [15.21, 55.24]	5199 [4886, 5549]	0.38 ± 0.02	ND
Gel base (Day 0)	5322 [4306, 6734]	66.51 [36.77, 125.60]	NA	NA	> 10
Gel base (Acc. cond.)	6061 [4934, 7772]	194.70# [99.06, 289.10]	NA	NA	ND
Std. gel	8696 [8311, 9128]	35.23 [21.21, 42.55]	ND	ND	ND
RGF	73.86 [65.84, 82.84]	7.26 [4.44, 10.56]	51.89 [46.94, 58.23]	3.64 ± 0.11	0.128
L-NA	47.10 [43.78, 50.80]	ND	ND	ND	ND
AA	ND	ND	1.53 [1.02, 3.38]	ND	ND
CA	ND	2.64 [2.34, 2.99]	ND	ND	ND
Pen G	ND	ND	ND	ND	0.001

The IC₅₀ values in this table show the mean and 95% confidence interval (95% CI) [min to max value] of a triplicate determination. FRAP values are mean ± SD, n = 3. *Significant difference from the value of gel base at day 0 (p < 0.05). #Significant difference from the value at day 0 (p < 0.05). Acc. cond.: accelerated condition. ND: not determined. NA: not active at the tested concentration. L-NA: Nω-nitro-L-arginine. AA: ascorbic acid. CA: catechin. Pen G: penicillin G.

Figure 3.

Anti-inflammatory effect of RGF gel, gel base, standard indomethacin gel, and standard L-NA on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in murine macrophage RAW264.7 cells: A herbal gel at day 0, B herbal gel after accelerated testing, C gel base at day 0, D gel base after accelerated testing, E std. indomethacin gel, and F std. L-NA. IC₅₀ was determined by non-linear regression analysis in Prism software.

To have a deep understanding on the anti-inflammatory potential of RGF, the active ingredient in the herbal gel, its capacity to modulate the LPS-triggered transcription of iNOS gene was analyzed by qRT-PCR. The RAW 264.7 cells were incubated with different concentrations (25–200 μg/mL) of RGF for 24 h, and cell viability was evaluated. MTT assay showed RGF alone did not decrease cell viability (Figure 4). It was found that 1 μg/mL LPS-treated macrophage cells resulted in overexpression of iNOS. As shown in Figure 5, treatment of the LPS-stimulated macrophages with RGF at all doses revealed significant downregulation of iNOS mRNA level (***p < 0.001 and ^****p < 0.0001) compared to LPS-stimulated control cells. Downregulation activity of RGF on iNOS mRNA level was concentration-dependent (14.10% by 25 µg/mL, 31.55% by 50 µg/mL, 54.82% by 100 µg/mL, and 83.02% by 200 µg/mL). β-actin served as the internal control. The inhibitory effect of RGF on LPS-induced production of NO can be attributed to the decreased expression of iNOS at the transcriptional level.

Figure 4.

Inhibition of nitric oxide production of different concentrations of the extract (6.25-200 µg/mL) on RAW 264.7 cells stimulated by lipopolysaccharides (LPS). The viability was investigated using MTT assay (A). The nitric oxide levels in the cell culture supernatant were measured by nitrite assay (B). Values are representative of three independent experiments performed in triplicate and are expressed as mean ± SD (error bars). **** significant difference between treatment and control group (^****p < 0.0001).

Figure 5.

Inhibitory effects of the extract on LPS-stimulated iNOS expression in RAW 264.7 cells. The expression levels of the gene are shown after normalization with the housekeeping actin gene β-actin. Values are representative of three independent experiments performed in triplicate and are expressed as mean ± SD (error bars). ***, **** significant difference between treatment and control group (p < 0.001 and 0.0001, respectively).

Antioxidant activity of herbal gel formulation containing RGF

The radical-scavenging capacities of RGF (8%) herbal gel were investigated using DPPH and NO assays. The RGF gel showed DPPH radical scavenging activity with an IC₅₀ of 4385 μg/mL, whereas the gel base did not exhibit % inhibition at the highest concentration tested of 6250 µg/mL. For NO radical scavenging activity, the IC₅₀ of the formulated gel was at 59.47 µg/mL, whereas that of the gel base was at 66.51 µg/mL. Ascorbic acid and catechin were used as positive standards (Table 3). The RGF herbal gel was also investigated for reducing power using FRAP assay. The result demonstrated that the herbal gel possessed antioxidant property with a FRAP value of 0.43 mM Fe (II)/g, whereas the gel base lacked activity at the maximum tested concentration of 6250 µg/mL (Table 3).

Antibacterial activity of herbal gel formulation containing RGF

The antibacterial activities of the RGF (8%) gel and the gel base against S. aureus are presented in Table 3. Both the herbal gel and its gel base were inactive at the highest tested concentration of 10 mg/mL, while the active ingredient (RGF) in the formulated gel had MIC of 0.128 mg/mL. The pathogenic S. aureus ATCC 29213 strain was sensitive to penicillin G, served herein as the positive control, with MIC of 1 µg/mL.

Stability of herbal gel formulation containing RGF

Physical characteristics of the RGF gel after accelerated stability testing

Observing changes in the gel after undergoing a heating-cooling test compared with day 0 revealed that the colour (Figure 6) and odour remained unchanged, the texture was of similar consistency, and the pH became slightly more acidic (Table 1). When applied to the skin, the RGF gel provided good spreading characteristic and a well-moisturized feeling.

Figure 6.

RGF-containing herbal gel; A: at day 0 and B: after the heating-cooling test.

The content of lupinifolin and glabridin in RGF gel after accelerated stability testing

The amount of lupinifolin and glabridin in RGF (8%) gel after the heating-cooling test as determined by HPLC method is detailed in Table 2. The amount of glabridin per 1 g of the prepared gel (day 0) and after accelerated stability testing was 0.0910 ± 0.0011 and 0.0941 ± 0.0011 mg/g RGF gel, respectively, while those of lupinifolin were 0.2160 ± 0.0011 and 0.2315 ± 0.0011 mg/g RGF gel, respectively (Table 2). The total phenolic and total flavonoid contents of the RGF herbal gel showed a non-statistically significant decrease after a heating-cooling test (Table 2).

Anti-inflammatory and antioxidant activities of RGF gel after accelerated stability testing

The results of the biological activity stability test of the RGF (8%) gel after the heating-cooling test revealed that its antioxidant activity decreased as measured by the DPPH and FRAP methods, while it was found to be better than on day 0 in the NO scavenging test (Table 3). However, the RGF herbal gel developed herein after the heating-cooling test showed no statistically significant differences in all antioxidant assays compared to that on day 0 (Table 3). Additionally, our study has shown that the anti-inflammatory effect of the formulated herbal gel was not significantly lower than that on day 0 (Table 3).

Discussion

The medicinal plants used in traditional medicine are reasonable alternatives for discovering the active pharmacological agents and implementing them into herbal products for the treatment of various diseases. The use of medicinal plants has made a significant contribution to primary health care for people worldwide (World Health Organization [WHO] 2010). One of the dosage forms of herbal remedies is an extract derived from one or more medicinal plants, which can be easy to prepare and hence useful in primary health care (Bakasatae et al. 2018). Scientific evidence proving the therapeutic properties of the medicinal plants used traditionally, as well as herbal products composed of active ingredients derived from herbal plants, is valuable for promoting sustainable and healthy communities. It has been documented that anti-inflammatory, antioxidant, and antibacterial properties play significant roles in the pharmacological effects of D. reticulata and G. glabra extracts (Vongnam et al. 2013; Yang et al. 2017; Issarachot et al. 2021). However, there is no report on preclinical studies of the topical gel containing the combined extract of these medicinal plants for its efficacy in treating inflammatory skin conditions, particularly contact dermatitis. Hence, the topical herbal gel (RGF 8% w/w) was herein developed using the combined extract from the stem wood of D. reticulata and the root of G. glabra and evaluated for its anti-inflammatory, antioxidant, and antibacterial properties. Our findings demonstrated that RGF, the active ingredient in RGF gel, possessed marked anti-inflammatory, antioxidant, and antibacterial effects (Table 3). The quantitative analysis of RGF by HPLC revealed that it contained considerable amount of lupinifolin and glabridin, as well as a high level of total phenolic and total flavonoid contents. Phenolics and flavonoids were previously reported as the key pharmacological agents in both plants. Glabridin is the most abundant isoflavane in the roots of G. glabra and it has the potential to serve as a bioactive marker for G. glabra (Simmler et al. 2013). Regarding lupinifolin, previous research indicated that it could be used as a bioactive marker for D. reticulata (Issarachot et al. 2021). Both glabridin (Singh et al. 2015; Yang et al. 2017) and lupinifolin (Sianglum et al. 2019; Issarachot et al. 2021) are known to have anti-inflammatory, antioxidant, and antimicrobial activities.

It has been well documented that the induction of iNOS expression is regulated by transcription factors such as nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), and Janus kinase/signal transducers and activators of transcription (JAK/STAT) (George et al. 2021). The observed decrease in LPS-stimulated NO production caused by RGF is due to its capacity to limit iNOS gene transcription, hypothetically by interfering with the above-mentioned inflammatory signalling pathways. Studies have shown that D. reticulata stem wood and G. glabra root possess several flavonoids, including glabridin and genistein, possibly responsible for the anti-inflammatory effect (Yang et al. 2017; Issarachot et al. 2021). Glabridin significantly decreased STAT3 mRNA expression in mouse skin under imiquimod induction (Li et al. 2018). Genistein remarkably inhibited LPS-induced activation of MAPK and NF-κB in BV2 microglial cells (Du et al. 2018). Additionally, a prenylated flavanone derivative that has a closely related structure to lupinifolin has been documented to suppress pro-inflammatory mediators through the NF-κB signalling pathway in LPS-stimulated macrophages (Han et al. 2021). All these studies confirmed the potential of RGF for further study on its development into an anti-inflammatory herbal product.

Poloxamer-based gels represent a convenient choice in pharmaceutical technology, and they are considered non-toxic and non-irritant (Russo and Villa 2019; An et al. 2021). Poloxamer 407 was used as the gelling agent to formulate RGF herbal gel in this study. The bioactivity study of the 8% RGF gel at day 0 showed that the gel containing the active substances lupinifolin and glabridin, 0.2160 and 0.0910 mg/g, respectively, had a strong NO scavenging property while exhibiting marked antioxidant activity with DPPH and FRAP methods as well as anti-inflammatory activity in LPS-induced NO production in the RAW264.7 macrophage cell model. The potential pharmacological benefits related to the anti-inflammatory actions of the herbal gels were well documented in previous studies (Kola-Mustapha et al. 2020; Thanasakdecha and Tewtrakul 2021; Bakasatae et al. 2022; Perera et al. 2023). This is the first study to evaluate in vitro models the anti-inflammatory, antioxidant, and anti-S aureus effects of a poloxamer-based gel containing the combined extract of D. reticulata and G. glabra as the active ingredient. However, the herbal gel developed herein exhibited no antibacterial activity against S. aureus when tested at the highest concentration of 10 mg/mL. According to a previous study, the concentration variation of poloxamer in the herbal gel might affect the antibacterial activity of the developed formulation (An et al. 2021). It is also important to note that there are other pathogenic skin microbes associated with promoting dermatitis, including those belonging to fungi and viruses (Findley and Grice 2014; Ong and Leung 2016). Additional investigations into the potential of RGF gel against these pathogenic microorganisms may be of interest to clarify whether the herbal gel developed herein is beneficial for non-bacterial infections. In addition, since flavonoids are the major active substance group and possess the pharmacological effects of RGF, the isolation and characterization of a flavonoid-rich extract from RGF for further evaluation on different testing models for distinct skin conditions would be interesting for the future development of a high-quality anti-inflammatory herbal gel, especially for the treatment of contact dermatitis.

Stability evidence plays an important role in supporting the shelf life proposed for finished herbal products, whereas chemical markers are valuable in defining the recommended shelf life and monitoring product quality and stability over time (González-González et al. 2022). Herbal products can degrade due to a variety of factors, such as physical, chemical, and environmental, which can result in toxic substances and the loss of therapeutic efficacy. The stability study of the RGF herbal gel was evaluated by a heating-cooling test. The physical stability test of the gel showed no changes in colour or odour, while the pH value remained within the optimal pH range of the skin. In comparison to the gel viscosity on day 0, the gel viscosity increased by more than 10% after the heating-cooling test. However, the gel texture remained homogeneous, did not precipitate, and retained its potential to spread well according to skin sensation following application to the arm area. Changes in the chemical composition of the gel after a thermal cycling stability test may affect the interactions between poloxamer polymer chains leading to increase the viscosity (Fakhari et al. 2017). A previous study revealed that drug penetration was largely unaffected by hydrogel viscosity, while moderately enhanced viscosity is advisable for hydrogels to allow for convenient application (Binder et al. 2019). This is in accordance with the result of the statistical analysis of both the pH and viscosity values of the gel in the present work, showing no significant difference between before and after stability testing (p > 0.05). The biological activity stability of the gel indicated that its antioxidant activity decreased in the DPPH and FRAP tests but increased in the NO scavenging assay after the heating-cooling test. In this case, the NO scavenging activity of the RGF gel (IC₅₀ 35.17 µg/mL) after stability testing was comparable to that of the standard indomethacin gel (IC₅₀ 35.23 µg/mL). Changes in functional groups in the chemical structures of antioxidant compounds in RGF may account for the different antioxidant effects among the methods used after the herbal gel was tested in an accelerated condition (Siddeeg et al. 2021). In the present study, the quantity of total phenolic and total flavonoid contents in the RGF gel was found to be non-statistically lower after stability testing. Additionally, HPLC analysis showed that the amount of lupinifolin and glabridin, the bioactive marker compounds in the RGF herbal gel, did not change significantly after stability testing (p > 0.05). It has been demonstrated that there are various other components belonging to phenolics and flavonoids in D. reticulata or G. glabra that can contribute to their therapeutic potential for skin inflammation. In this regard, previous studies showed that genistein, among the major flavonoids from Derris species, possessed strong anti-inflammatory activity through inhibition of various mediators in inflammatory signalling pathways such as iNOS, prostaglandins, proinflammatory cytokines, and reactive oxygen species (Siddeeg et al. 2021; Goh et al. 2022). In G. glabra, besides glabridin, other isoflavonoids such as licoricidin, licorisoflavan A, isoangustone A, and dehydroglyasperin C and D were also primarily responsible for the anti-inflammatory activity via a variety of mechanisms, particularly the downregulation of mediators such as TNF-α, MMPs, and prostaglandin E2 (Simmler et al. 2013; Yang et al. 2017). The potential efficacy on anti-inflammatory and antioxidant activities of the herbal gel were diminished after undergoing accelerated conditions in this study, possibly due to changes in the contents of other active compounds in the polyphenol group, which might be synergistic to lupinifolin and glabridin or additive to their effects. However, it is worth noting that the pharmacological properties of the RGF gel in all assays performed herein did not weaken significantly after the heating-cooling test compared to those on day 0. Our findings provided preliminary stability data for RGF gel using accelerated stability testing in the heating-cooling cycle model described previously (Thanasakdecha and Tewtrakul 2021). This testing protocol gives early stability data of a herbal product, allowing for the comparison and evaluation of different formulations. It also in line with general principles for stability testing of herbal medicinal products and traditional herbal medicinal products. Following the ICH guidelines for accelerated stability studies (40 ± 2 °C/75% RH ± 5% RH as general cases), it would take at least 6 months of experimental laboratory work to obtain preliminary stability results, whereas a thermal cycling stability model (at 45 °C for 24 h and at 4 °C for 24 h/1 cycle; for 6 cycles) carried out herein can shorten the time of stability testing and also be helpful to facilitate decision-making during the preclinical phase development of herbal products (Thanasakdecha and Tewtrakul 2021; González-González et al. 2022). Additional testing performed using real-time stability testing conditions may be beneficial to confirm the long-term stability of the developed herbal gel in this study (González-González et al. 2022).

Conclusions

This study concluded that the anti-inflammatory and antioxidant efficacy of the herbal gel developed herein was attributed to the potent pharmacological potential of its active ingredient, RGF, giving support to traditional claims regarding the therapeutic effects of D. reticulata and G. glabra. The active compounds belonging to the polyphenol group, including lupinifolin and glabridin, are characterized as the bioactive markers of the poloxamer-based gel containing RGF (8% w/w). The potency of this herbal gel as a promising topical anti-inflammatory product could be partly mediated by the modulatoreffect of RGF on iNOS mRNA expression. The physicochemical and pharmacological properties of the RGF-containing herbal gel reported herein are novel results. Our findings suggest that the RGF gel has potential for development as an herbal medicinal product, especially for reducing the symptoms of dermatitis. Additional research is required to confirm its safety in preclinical skin irritation models.

Author’s contributions

The authors confirm their contribution to the paper as follows: conceptualization: W. Sangkaew, W. Sianglum, S. Wunnoo, S.P. Voravuthikunchai, N. Joycharat; data curation: W. Sangkaew, N. Joycharat; formal analysis, investigation, and methodology: W. Sangkaew, W. Sianglum, S. Wunnoo, N. Joycharat; writing – original draft: N. Joycharat. The project administrator, N. Joycharat, received funding for this study. All authors reviewed the results and approved the final version of the manuscript.

Funding Statement

This work was financially supported by National Science Research and Innovation Fund (NSRF) and Prince of Songkla University (Ref. No. TTM6701279S).

Disclosure statement

No potential conflict of interest was reported by the author(s).