Plant material
Neem leaves and Rosemary herbs were obtained from the Medicinal, Aromatic, and Poisonous Plants Experimental Station -Faculty of Pharmacy/Cairo University-October 2022). A flowering plant of both samples was identified and approved by Mrs. Therese Labib; Botanical Specialist and consultant at Orman and Qubba Botanical Gardens/Giza/Egypt. Voucher samples were submitted to the Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Giza, Egypt (voucher specimen RO-10-2021) for rosemary herb, and (voucher specimen AI-10-2021) for neem leaves.
Chemicals
Rutin hydrate HPLC powder grade, rosmarinic acid, Folin-Ciocalteu reagent, 2,2-Diphenyl-1-picrylhydrazyl (DPPH), 2,2′ Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid; ABTS) in the crystallized diammonium salt form, 2,2′-Azobis (2-amidinopropane) dihydrochloride (AAPH), 6-hydroxy-2,5,7,8-tertamethylchroman-2-carboxylic acid (Trolox), fluorescein disodium salt, sodium, and TPTZ were purchased from (Sigma-Aldrich, St. Louis, MO, USA). Gallic acid and aluminum chloride were obtained from (Misr Company for Pharmaceutical Industry, Mataria, Cairo, Egypt). HPLC grade solvents, including methanol (MeOH) and acetonitrile, (Sigma-Aldrich, Steinheim, Germany) were employed, orthophosphoric acid (SD Fine Chem Ltd., Mumbai, India).
Preparation of ethanolic extracts
Two kg of each plant was dried in the shade and ground to yield (1kg) powder. Each powder underwent separate extractions using repeated cold maceration in 90% ethanol for a 7-days duration until exhaustion was reached. The extracts were then filtered, combined, and evaporated to dryness via rotary evaporation under reduced pressure, maintaining a temperature below 50 °C. The final residue was about 61g (6.1% W/W) and 52.1g (5.2% W/W) from Rosemary and Neem ethanolic extract, respectively. Aliquots were used for phytochemical and biological studies.
Evaluation of total phenolic and flavonoid contents
The total phenolic content of the extracts was quantified using a rapid microtiter plate Folin-Ciocalteu assay, as described by Attard et al.41. Additionally, the aluminum chloride assay was employed to screen the flavonoid content42. Gallic acid and rutin served as the standard references for phenolic acid and flavonoid, respectively. Briefly, sample preparation involved a 4 mg/mL solution in a 9:1 (v/v) methanol/water mixture. Calibration curves were established for gallic acid (25–1000 µg/mL) and rutin (5–160 µg/mL) through serial dilutions of their respective standards. Each curve was derived from the average of six replicates, achieving high correlation coefficients (R2) of 0.9994 for gallic acid and 0.9991 for rutin. (See Supplementary Fig. S1 online). Total phenolic content analysis involved the oxidation of samples with the Folin-Ciocalteu reagent at room temperature in sodium carbonate solution for 90 min. For flavonoid content, samples were reacted with aluminum chloride solution at room temperature. The resulting-colored products were measured using a microplate reader (FluoStar® Omega, BMG Lab tech, Germany) at 630 nm for gallic acid and 420 nm for rutin. Absorbance values were compared to standard curves generated using known concentrations of gallic acid and rutin. Results are expressed as µg gallic acid equivalents (GAE) per mg of dried sample for total phenolics and µg rutin equivalents per mg of dried sample for total flavonoid concentration. All data represent the mean ± SD of three independent replications.
UPLC standardization
Stock preparation
Rosemary and neem ethanolic extract (10 mg) was accurately weighed and dissolved in methanol in 10 volumetric flasks to obtain a stock solution of 1 mg/mL and the same was done for the rutin and rosmarinic acid standards, yet 1 mL from the stock solution for each extract was transferred into 100 mL flask obtaining 100 µg/mL working stock solution. All samples were filtered through a 0.22 µm syringe filter and 10 µl was introduced to the column through autosampler (WPS-3000TRS, Thermo scientific USA).
Instrument
The analyses of rosemary herb and neem leaf extract were performed on Thermo Fisher UPLC Ultimate 3000–Complete UPLC (USA), equipped with a quaternary pump, degasser G1322A, and coupled with UV Detector (PDA -3000 RS, USA). Separation was achieved using a Hypersil Gold column (250 × 4.6 mm i.d., particle size, 5µ) at 25 °C. Agilent ChemStation software was used for data acquisition and processing.
Establishment of a standard calibration curve
Each standard compound (rutin and rosmarinic acid) was dissolved in methanol and used to establish calibration curves at concentration range (1–500 µg/mL). UV chromatographic profiles were analyzed, showing a rutin peak at 17 min (270 nm) while a rosmarinic acid peak was observed at 25.31 min (320 nm).
Method of the analysis
A mobile phase consisting of 0.1% (v/v) orthophosphoric acid in water as a solution (A) and Acetonitrile as a solution (B) was used at a flow rate of 1 ml/min for a 10 μL injection volume. Gradient elution ran for 55 min as follows: 0–3 min (15% B), 3–40 min (35% B), 40–50 min (15% B), followed by (100% B) for 5 min. The mobile phase in gradient flow was optimum to get uniform peak resolution of plant extract. The same gradient condition was used to determine rutin and rosmarinic acid calibration standards. Chromatographic profiles were UV detected at wavelength 270 nm for rutin in neem ethanolic extract and 320 nm for rosmarinic acid in rosemary ethanolic extract, each sample was injected in triplicates. The identification of rutin and rosamarinic acid peaks in the ethanolic extract of neem and rosemary was assessed depending on their retention time compared to that obtained for their corresponding standard as well as their UV spectra. Quantification of rutin and rosmarinic acid in the ethanolic extract of neem and rosemary ethanolic extract was done by comparing the peak areas of the samples with the calibration curves of corresponding standard solutions.
Anti-microbial activity
Microorganisms
The fungal species utilised in this investigation included Trichophyton rubrum (RCMB 025002) and Malassezia furfur (RCMB 003), whereas the bacterial species encompassed Staphylococcus epidermidis RCMB 009. The microbes were acquired from the Regional Centre for Mycology and Biotechnology, Al-Azhar University (RCMB).
Agar well diffusion method
Susceptibility testing followed NCCL recommendations (National Committee Standards 1993). Using the agar well diffusion method, the antimicrobial activity of neem leaves, rosemary herb, and rosemary-neem extract combinations (RN-E 2:1) and (RN-E 1:1) was determined.
Trichophyton rubrum and Malassezia furfur cultures were spread over malt agar plates, while Staphylococcus epidermidis culture was spread over Mueller–Hinton agar plates. After spreading the culture, wells of 6mm diameter were cut in the agar surface with a sterile cork borer, and 100 µl of the tested extracts dissolved in dimethyl sulfoxide (DMSO) was added to these wells in a concentration of 50 mg/mL. ketoconazole (100 µg/ml) in DMSO was used as Positive control for fungi while, gentamycin (4 µg/ml) in DMSO for bacteria. DMSO alone was tested in wells. The plates were incubated for 48 h at 28 °C for fungi and 24 h at 37 °C for bacteria. To determine the antimicrobial activity, the diameter of the zone of inhibition was measured in mm.
Minimum inhibitory concentration assay (MIC)
The MIC of individual/combined rosemary-neem extracts that gave the highest zone of inhibition was evaluated by the broth microdilution method following CLSI recommendations (CLSI, 2008). Serial dilution of extract dissolved in DMSO was prepared in a range from (25–0.125 mg/mL). Following incubation (48 h at 28 °C for fungi and 24 h at 37 °C for bacteria), the minimum inhibitory concentration (MIC) of the extract was determined. The MIC was established as the lowest extract concentration exhibiting no visible microbial growth in the wells43.
Assessment of antioxidant activity
DPPH free radical scavenging assay
The DPPH free radical scavenging assay was conducted on the two individual extracts and RN-E (2:1) combined extract according to the procedure previously stated by Chen et al.44. Briefly, the individual extracts and (RN-E 2:1) extract were prepared to obtain a final concentration of 0.05 mg/mL in methanol, a standard stock solution of Trolox at a concentration of (100 µM) in methanol was serially diluted to six concentrations; (5–40 µM). Freshly prepared DPPH reagent 100 µL (0.1% in methanol) was added to 100 µL of each sample in a 96-well plate (n = 6), and the reaction was incubated at room temp for 30 min in the dark. Following incubation, the reduction in DPPH radical activity was quantified by measuring the decrease in absorbance at 540 nm using a microplate reader45. The results were expressed as micromolar (μM) Trolox equivalents per milligram (mg) of the sample, calculated using the linear regression equation derived from the calibration curve (see Supplementary Fig. S2).
Oxygen radical absorbance capacity (ORAC) assay
The oxygen radical absorbance capacity (ORAC) assay, adapted with slight modifications from the method described by Liang et al.46, was employed to assess the antioxidant activity of the samples. Briefly, the assay was conducted in black 96-well plates (Corning Scientific, Corning, NY). Each well contained 10 µL of prepared samples at a concentration of 200 µg/mL in methanol, or 10 µL of Trolox standard solution (final concentration 2 mM, with a range of 50–800 µM). Additionally, 30 µL of fluorescein (final concentration 100 nM) was added to each well. The plates were then incubated at 37 °C for 10 min. Fluorescence measurements were performed for background assessment using three cycles (90 s each) at an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Subsequently, 70 µL of freshly prepared 2,2′-Azobis(2-amidinopropane) dihydrochloride (AAPH) solution (240 mM) was promptly added to each well. Fluorescence measurement was then continued for 60 min at the same excitation and emission wavelengths, employing 40 cycles (90 s each).
ORAC values, expressed as µM Trolox equivalents (TE) per milligram (mg) of the sample, were calculated using the linear regression equation derived from the calibration curve (See Supplementary Fig. S3 online), depicting the linear dose-inhibition curve of Trolox). Data are presented as mean ± SD, based on triplicate measurements.
Determination of ferric reducing antioxidant power (FRAP) assay
This method leverages the ability of the samples, at low pH, to reduce ferric (Fe3+) ions to ferrous (Fe2+) ions. In the presence of TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine) (Sigma Aldrich, USA), the formed Fe3+-TPTZ complex is reduced to the Fe2+-TPTZ complex, generating an intense blue color with a maximum absorbance at 593 nm. The assay is adapted from the method of Benzie and Strain47, with slight modifications for microplate compatibility. Briefly, samples were prepared at 25 mg/mL in methanol, while the standard Trolox solution (2 mM in methanol) was serially diluted within the range of 1500–50 µM. Each well of a 96-well plate was filled with 190 µL of freshly prepared TPTZ reagent (prepared by mixing 300 mM acetate buffer (pH 3.6), 10 mM TPTZ in 40 mM HCl, and 20 mM FeCl3 in a 10:1:1 v/v/v ratio) followed by 10 µL of the sample. Triplicates were prepared and measured for each sample. The reaction mixture was incubated in the dark for 30 min at room temperature. After incubation, the absorbance of the resulting blue color was measured at 593 nm using a microplate reader. The ferric-reducing ability of the samples was expressed as µM Trolox equivalents (TE) per mg of sample, calculated using the linear regression equation derived from the calibration curve (see Supplementary Fig. S4 online). Data are presented as mean ± SD.
Free radical scavenging activity (ABTS) assay
The assay was carried out adopting the method of Arnao et al.48, with minor modifications. Briefly, an ABTS stock solution was prepared by dissolving ABTS (Sigma Aldrich, USA) in DW and adjusting the volume to 25 mL. Subsequently, 1 mL of this solution was mixed with 17 µL of 140 mM potassium persulfate and incubated in the dark for 24 h. The reaction mixture was then diluted with methanol to a final volume of 50 mL to obtain the working ABTS solution. In a 96-well plate, 190 µL of the freshly prepared ABTS solution was mixed with 10 µL of the samples (prepared at 0.5 mg/mL in ethanol) or Trolox standard solutions (2 mM, diluted in methanol to a range of 600–50 µM) (n = 6) (see Supplementary Fig. S5 online). The reaction mixture was incubated in the dark for 30 min at room temperature. After incubation, the decrease in absorbance of the ABTS radical cation was measured at 734 nm using a microplate reader.
The percentage inhibition of ABTS radical cation by each extract was calculated using the following equation and expressed as mean ± SD:
$$ \% {\text{ Inhibition}}\,\left( {I\% } \right)\, = \,(\left( {{\text{Average}}\,{\text{absorbance}}\,{\text{of}}\,{\text{blank}}\, – \,Average\,absorbance\,of\,average\,absorbance\,of\,bank \, } \right) \times 100 $$
Anti-inflammatory in vitro activity in Murine macrophage cell line RAW 264.7 cells activated with lipopolysaccharide
Cell culture and media
Murine macrophage RAW264.7 cells (ATCC®) were cultured in a humidified incubator containing 5% CO2. The culture medium consisted of complete DMEM (Corning, USA) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin sulfate, and 2 mM L-glutamine. For subculture and treatments, cells were detached from the flasks using sterile cell scrapers.
Inhibition of LPS-induced NO release
RAW 264.7 cell stock (0.5 × 106 cells/mL) was inoculated into 96-well microwell plates and incubated overnight. On the next day, cells received either DMSO, 0.1% v/v considered as the negative control group (LPS−), lipopolysaccharide (Sigma-Aldrich, from Escherichia coli serotype O111: B4) at a concentration of (100 ng/mL) assigned as inflammation group (LPS +), LPS in the presence of the combined extract (RN-E 2:1) at screening concentrations of (10 and 100 µg/mL) dissolved in DMSO and diluted into culture media (final concentration of DMSO = 0.1%, by volume) that was considered as sample group, or indomethacin (IM, 0.25 mM) for anti-inflammatory positive control group. Using the Griess reaction49 test for the determination of nitric oxide levels in quadrants of culture supernatants after 24 h of exposure in all groups (n = 3).
NO assay
Briefly, equal volumes (100 µL) of culture supernatants and Griess reagent, prepared as described previously49 containing (1% sulfanilamide, 0.1% naphthyl ethylene diamine dihydrochloride, and 2.5% phosphoric acid), were mixed and incubated at room temperature for 10 min. The resulting colored diazonium salt was measured for absorbance at 540 nm using a microplate reader (Tecan Sunrise™, Austria). Nitric oxide (NO) inhibition percentage (%) of the tested extract (RN-E 2:1) was calculated relative to the LPS-stimulated control group ((LPS +)) and normalized to viable cell number, as determined by the Alamar Blue™ reduction assay50.
Rosemary-Neem combined extract concentration-dependent iNOS
RAW 264.7 cells stock (0.75 × 106 cells/mL) were seeded into 6-well plates and incubated overnight. Following the protocol described above, triplicate wells in the sample groups received increasing concentrations (1.25, 2.5, 5, and 10 µg/mL) of the dissolved sample (RN-E 2:1) in DMSO, diluted into culture medium containing LPS (final DMSO concentration 0.1% v/v). After 24 h of incubation, the NO content in all wells was determined using the Griess assay, as previously described49 The concentration inhibiting 50% of LPS-induced NO release (IC50) was statistically calculated using GraphPad Prism software, employing a non-linear regression model curve fit.
iNOS by western blot analysis
Overnight culture of RAW264.7 (6-well plates, initially seeded as 1.5 × 106 cells/well) was treated with (RN-E 2:1) combined extract (5 and 25 µg/mL), or indomethacin (IM positive control, 250 μM). After 24 h of incubation, Raw 264.7 cells were washed using cold PBS (phosphate buffered saline) and scraped from each well in cold lysis buffer (RIPA buffer containing protease inhibitor cocktail, 100 µl/well). Cell lysates were incubated on ice for 15 min with an occasional vortex every 5 min. Cell lysates were centrifuged at 10,000×g for 10 min (4 °C). Total protein was determined in the supernatants using a BCA kit (Thermofisher Scientific, USA). Samples (20 µg total proteins) were resolved using polyacrylamide gel electrophoresis on a 10% acrylamide/bis-acrylamide gel using the OmiPAGE TETRAD system (Cleaver Scientific, UK) at 150 V for 1.5 h. A nitrocellulose membrane was electro-transferred with resolved proteins at 100 V for 70 min. Membranes were blocked using skim milk (5% w/v in TS buffer saline, Tween 20, TBST) for 1 h at room temperature. Membranes were probed overnight (4 °C) with a rabbit iNOS antibody (Biomatik, Canada) and β-actin (Thermofisher Scientific, USA) under gentle shaking. Probed membranes were washed with TBST for 3 × 5 min under shaking. Membranes were then incubated for 1 h at room temperature with matched horseradish peroxidase-conjugated secondary antibodies under gentle shaking. Using an enzyme chemiluminescence reagent (Clarity, Bio-Rad, USA), protein bands were observed following a TBST washing period of 3 × 5 min.
Preparation of the pharmaceutical formulae
Materials
Carbopol 934, methylparaben, and propylparaben of analytical grade and sodium hydroxide pellets were purchased from Adwic Co. (Cairo, Egypt). Propylene glycol, Jasmine oil, and absolute ethanol were purchased from Agitech Co. (Cairo, Egypt).
Formulation of herbal gel, and leave-in hair tonic
Two types of formulae were made using plant extract (RN-E 2:1) as shown in (Table 2). The herbal gel was formulated by adding the extract to propylene glycol till a completely homogenous mixture was obtained. On a magnetic stirrer, 1% of carbopol 934 was sprinkled on the surface of the previous mixture and left till swelling of the polymer. The obtained gel was then neutralized using a few drops of triethanolamine till the desired consistency was achieved.
Table 2 Plant extract formulation composition (gel and hair tonic).
As for the leave-in hair tonic, RN-E was dissolved in jasmine oil (Table 2). The solution was then mixed with propylene glycol. After that, it was added portion-wise to distilled water and stirred on a magnetic stirrer to obtain a homogenous one-phase leave-in hair tonic. Finally, a few mL of absolute ethyl alcohol was added to the hair tonic to enhance the homogeneity of the tonic.
Evaluation of herbal gel, and leave-in hair tonic
To evaluate the formulated herbal gel and leave-in hair tonic, several quality tests were conducted including organoleptic properties, viscosity, and pH determination. Furthermore, Ex-vivo skin deposition testing for hair tonic and herbal gel was done. While the spreadability testing was done for herbal gel formula only.
The organoleptic examination
Both plant extract formulations were subjected to an organoleptic evaluation, which included assessing their color, odor, and homogeneity through visual observation.
PH measurement
The pH of the RN-E (2:1) herbal gel and leave-in hair tonic were determined using a pH meter (Jenway, 3510 pH meter) at room temperature.
Viscosity determination
The viscosity of the formulated gel and leave-in hair tonic was determined using Brookfield Viscometer (Ametek, DV-1 viscometer) at room temperature.
Spreadability evaluation of herbal gel
The spreadability of the formulated gel was determined by spreading 0.5 gm of the gel on a circle having a 2 cm diameter predetermined on a glass plate and another glass plate was placed on top. 50 g weight was applied on the upper glass plate and allowed to rest for 5 min and then the spreadability of the gel was determined by measuring the new diameter of the circle.
Ex-vivo skin deposition testing of herbal gel and leave-in hair tonic
Animals
Female Swiss albino mice (20–25 g)/Wistar rats weighing (150–200 g) were obtained from the animal house (Faculty of Pharmacy/The British University in Egypt). Mice were housed in a temperature-controlled environment (25 °C) with a light–dark cycle of 12 h every day and were allowed free access to standard laboratory food (standard diet and tap water ad libitum). This study is approved by the ethics committees of the Committee of Animal Care and Use of NRC. All procedures and experiments were performed according to the approved protocol, and animals were treated in accordance with ARRIVE guidelines (https://arriveguidelines.org). The British University in Egypt’s Faculty of Pharmacy and Ethical Committee accepted the experiment under reference number EX-2207.
Removal of skin
Two mice were used for each formula, the skin was carefully removed after euthanizing the female mice (humane killing) using a CO2 chamber (See Supplementary Fig. S6 online). The skin was removed from both dorsal and ventral sides, and subcutaneous tissues and adhering fats were removed by rubbing with a cotton swab. Before beginning the experiment, the excised full-thickness skin samples were equilibrated by soaking them in phosphate buffer saline (PBS) solution with a pH of 7.4 at 4–8 °C for approximately one hour. Each skin was kept separately in the refrigerator for further ex-vivo skin deposition study. The dead bodies were discarded by incineration.
Ex-vivo skin deposition test
Ex-vivo skin testing was carried out using static vertical Franz diffusion cells with a diffusion area of 1.77 cm2. The receptor compartment of the Franz cells was filled with pH 7.4 phosphate buffer and maintained at 37.5 °C water bath. The dorsal skin of mice was used as the membrane. The skin was placed with the stratum corneum facing the donor compartment. After equilibration for 30 min. A 0.5 g of gel or 500 uL of hair tonic containing the same concentration of plant extract was applied to the stratum corneum and the Franz cells were left for 24 h on a magnetic stirrer at 100 rpm. At the end of deposition testing, the dorsal skin of the mice was removed from the Franz cells and gently wiped to remove formulation residue. The skin was then stripped 20 times using an adhesive tape to remove the stratum corneum layer. Afterward, the epidermis and dermis were separated using a pair of forceps, and each layer was cut into small pieces. Each skin layer was then soaked using 20 mL of methanol and the collected samples were subjected to analysis using validated UPLC-PDA (as previously mentioned), subsequently, the samples were filtrated through 0.22 µm filter membrane to determine the drug concentration hence deposition in each layer. The experiment was conducted in triplicate and the results were calculated as mean ± SD.
Statistical analysis
GraphPad Prism software (GraphPad Software Tools, Inc., La Jolla, CA, USA) was used in statistical analyses. Results were compared using Two-way ANOVA followed by Sidak’s post hoc test with a statistically significant difference at p < 0.05.
Hair growth activity test on rats
Twenty rats were randomly divided into four treatment groups, each group consisting of 5 rats. The first group (Gp-1) was left untreated (control group), The second group (Gp-2) was treated with (RN-E 2:1) hair gel 1%, the third group (Gp-3) was treated with (RN-E 2:1) leave-in hair tonic 1%, and the fourth group (Gp-4) (positive control group), that applied minoxidil spray 2% as a hair tonic. Following hair removal by shaving 5 × 5 cm2 of the dorsal skin of the rats, the residual hairs were removed using depilatory cream. In each testing area, a square measuring 2 × 2 cm2 was outlined. Then, the rats were left for 24 h. After that, samples were applied topically to the test area once daily for 30 consecutive days at a volume of 2 mL (see Supplementary Fig. S8 online). The rats’ hair was evaluated by measuring the average hair’s length using a caliper and inverted microscope (in μm) to measure the thickness.
