Evaluation of Cuscuta reflexa seed essential oil on TPA-induced inflammation in mice

2.1 Chemicals and reagents

Acetone, dimethyl sulfoxide (DMSO), Griess reagent kit, malondialdehyde, thiobarbituric acid (TBA), trichloroacetic acid (TCA), 12-O-tetradecanoylphorbol-13-acetate (TPA), and indomethacin were purchased from Sigma-Aldrich (USA), phosphate buffer saline tablet (PBS) was taken from Himedia, Mumbai and from Thermo Scientific (USA) TNF-α, IL-6 and IL-1β biochemical kit. Every material used in present work was of analytical grade.

2.2 Plant material

The seed of C. reflexa were purchased in August 2018 from the local market in Lucknow, India and identified by the Scientist, Botany and Pharmacognosy, Department, CSIR-CIMAP, Lucknow. Voucher no. 774 has been deposited with Institute’s herbarium.

2.3 Isolation of essential oil

The essential oil of crushed seed of C. reflexa (500 g) was obtained by hydro-distillation in a Clevenger-type apparatus. The yield of the essential oil was 0.05% (w/w) on a dry weight basis. The oil was dried over anhydrous sodium sulfate and stored in a sealed glass vial at low temperature until further analysis.

2.4 Gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analysis

GC analysis of the samples was done with a Agilent gas chromatograph (7890 B), equipped with a HP-5 fused silica capillary column (5% phenyl)-polymethyl siloxane stationary phase; 30 m × 0.32 mm internal diameter; film thickness 0.25 µm) and a flame ionization detector (FID). Hydrogen was the carrier gas at 1.7 mL min⁻¹. Oven temperature was programmed from 60 °C to 240 °C with increase of 3 °C min⁻¹ and final hold time of 2 min. Injector and detector temperatures were set at 280 °C and 290 °C, respectively. The samples (0.01 µl, neat for essential oil and 0.1 µl, diluted in hexane for absolute) were injected in the spit mode (1:100).

Gas chromatography-Mass spectroscopy of C. reflexa essential oil was performed on a Clarus 680 GC interfaced with a Clarus SQ 8C Quadrupole mass spectrometer of Perkin Elmer fitted with Elite-5 MS fused-silica capillary column (30 m × 0.25 mm i.d., film thickness 0.25 µm). Temperature of oven was planned from 60 °C to 240 °C, at 3 °C min^-1to 270 °C at 5 °C min⁻¹; Temperature of injector was 250 °C; transfer line and source temperatures were 220 °C; 0.03 µl neat of injection size; 1:50 split ratio; Helium as a carrier gas at 1.0 mL min⁻¹;70 eV ionization energy and 40–450 amu mass range scan.

The constituents of the essential oil were identified on the basis of retention index, determined using a homologous series of n-alkanes (C₇-C₃₀ hydrocarbons, Supelco Bellefonte, PA USA), To compare data of sample and literature data from MS Library search (NIST and WILEY). The relative quantity of individual chemical compounds was determined by optimizing the peak area of GC (FID response) without employing correction factor (Adams, 2007).

2.5 Experimental animals

Present study was conducted on female Swiss albino mice (18–22 g). PSIT Kanpur has Animal House Facility to bred and maintain. The animals were fed with standard rodent pellet diet and water was given to the animals. Permission and approval for animal studies was obtained from CPCSEA (Reg. No. 1273/PO/RE/S/09/CPCSEA), Government of India through the Institutional Animal Ethics Committee.

2.6 Cell viability assay

Cell viability was determined by using MTT assay. RAW 264.7 were washed by buffer (PBS), decant the solution and seeded the cell in DMEM in 96-well microplates for 24 hrs. at 8X 10⁴ densities per cell. Test samples of C. reflexa essential oil (CREO) 1,2 and5% were dissolved in DMSO. After incubation of 48 h 10 µl of MTT mg/mL in a PBS buffer were added again plates were incubated at 37 ͦC for 4 h., removed the medium and dissolved formazan crystals in 100 mL DMSO. ELISA plate reader from BioRad was used to determine the optical density at 540 nm. Cell viability was calculated by following equation from six replicate well. $$\%\text{Viability}=\left(\text{OD treated cells}/\text{OD control cells}\right)\times 100$$

2.7 In-vivo study

Swiss albino female mice (7–8 week; 25 ± 2 g) were acclimatized in controlled environmental conditions (55 ± 10% RH and 12 h day/ night cycle), 7 days before commencement of experiment. During experiment mice were divided into six group (n = 6). Right ear of the mouse was used for testing. Sample was applied topically followed by TPA after 30 min (Yadav et al., 2009). Experiment were performed under the guidelines of Institutional Animal Ethics Committee approved by CPCSEA, Government of India.

2.8 Acute dermal irritation test

Determination of acute dermal irritation study was performed on the swiss albino mice. 1-inch square area was shaved on (n = 6). 5% sample was applied on the shaved skin while acetone as the vehicle. Assessment of dermal vulnerability, itchiness, redness, skin irritation and skin reactions were observed at 0, 4, 24, 48, 72 h. Skin oedema and erythema were recorded on the basis of arbitrary grading scale of 1–5. For ear erythema the score was taken as 1erythema formation, 2 very slight erythema (barely perceptible), 3 average erythema, 4 well-developed erythema, 5serious erythema (meat redness). Similarly, weight of both the ears (left & right) were taken and calculate the difference. On last day 6 hr after treatment the animals were sacrificed by cervical dislocation method and ears of each mice were collected. With the help of 8 mm biopsy punch ears were excised, weighed and calculate the difference. Right ear part of every ear was retaining in PBS buffer (PH 7.4) till further experiment. For ear oedema,1 no oedema, 2 very slight oedema, 3 well-defined oedema, 4 moderate oedema, 5severe oedema (beef raise than 1 mm). Similarly, primary irritation score was assessed for all animals of the study by cumulative irritation index (Aroonrerk and Kamkaen, 2009).

2.9 TPA induced skin inflammation in mice

Present study was done as described in Kumar et al., 2018. TPA (disease control) was applied to both the ears (inner and outer side) as 2.5/ear solubilize in 20 µl acetone. Test sample of C. reflexa essential oil (CREO) along with positive control was applied on the ear after 30 min of TPA application. All the test samples and positive control sample should be properly dissolved in acetone. In vehicle control group only, acetone was applied whereas in toxin group TPA was introduced only and maximum inflammation was observed.

2.10 Tissue weight measurement

Thickness of ear was measured by digital micrometer before and after induction of inflammation. After sacrificing the mice 6.5 mm diameter disc was used to remove ear by help of metal punch and weighed ear. Tissue (ear) homogenates (10% PBS) were prepared collect the supernatant and proceed for further investigations for the effect of CREO for pro-inflammatory cytokines.

2.11 Evaluation of pro-inflammatory cytokines

Previously collected mice ear was homogenized by using tissue homogenizer (Pro 200; Pro Scientific, USA) with 10% PBS and placed in cold ice and centrifuged for 15 min at 4 °C and 10,000 rpm. Supernatant fluid was collected for further experiments.

Estimation of cytokines TNF- α, IL-6 and IL-1β were done as prescribed in the ELISA kits (Thermo Scientific, USA) (Deepika et al., 2014).

2.12 Measurement of malondialdehyde (MDA) and nitric oxide (NO) production

1. Lipid peroxidation assay: 200 μl of w/v was mixed with 200 μl of ear supernatant followed by mixing. Prepared solution was centrifuged for 5 min at 5000 rpm 200 μl aliquot solution was taken and 0.67% TBA inequal amount was added. Boil the solution on water bath for 10 min. Absorbance was measured at 600 and 535 nm including blank with TCA and TBA

2. 75 μl of Griess reagent taken with equal amount of ear supernatant in a micro titer plate and mix well followed by incubation for 10 min at 37 °C. At 540 nm absorbance was taken and nitrite concentration was determined by standard curve plotted by sodium nitrite serial dilution method

2.13 Histopathology

Ear of animals was kept in 10% formalin solution. Each sample was treating with xylene and then fixed with paraffin. Cut into section of 5 µm and stained with hematoxylin-eosin and examined the image under light microscope at magnification of 10X and 40X for finding the inflammatory cellular response.

2.14 Statistical analysis

Experimental data was recorded as mean ± standard error. One-way analysis of variance (ANOVA) was used to summarize the results. GraphPad PRISM® version 5.01(GraphPad Software, Inc., USA). p < 0.05 was considered indicative of significance.

3.1 GC and GCMS analysis

A total of 29 chemical constituents were identified in the seed essential oil by retention index and mass spectral data (Fig. 1). The concentrations of the volatile components are presented in Table 1, according to their elution order on an HP-5 fused silica capillary column. Components of the seed essential oil were Hexahydro farnesyl acetone (14.2%), β-Bisabolene (13.7%), and Carotol (9.8%), cis-Chrysanthenyl acetate (4.7%), Caryophyllene oxide (4.9%), α-Asarone (3.0%), trans-α-Bergamotene (3.2%), (E)-caryophyllene (3.1%).

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Fig. 1

Gas chromatographic profile (TIC) of C. reflexa essential oil as a) Cis-chrysanththenol acetate, b) trans-sabinyl acetate, c) carvacrol, d) (E)-β-Damascenone, e) (E)-caryophyllene, f) α-trans-bergamotene, g) (E)-β-farnesene, h) β-Bisabolene, i) spathnlenol, j) caryophyllene oxide, k) carotol, l) Daucol, m) α-asarone, n) Hexahydrofarnesyl acetone, o) (E)-phytol.

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3.2 Cytotoxicity assessment

The cell viability of RAW 264.7 macrophages was evaluated. Fig. 2 shows that CREO at concentrations of 1%, 2% and 5% did not affect cell viability. There is no significant change in live cell population when compared with normal control.

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Fig. 2

Cell viability of CREO using MTT assay in RAW 264.7 Data are mean ± SEM; n = 3 normal versus test.

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3.3 In-vivo anti-inflammatory

3.3.2

3.3.2 TPA induced skin inflammation

Initially no erythema on the ear of animals was observed. After 4, 24, 48, and 72 hrs of treatment significant increase in auricle blood flow was recorded as compared to the vehicle control as 0.33 and 0.5 respectively. Ear thickness was recorded before and after application of TPA at the duration of 4 and 24 h with acetone. The vehicle and positive control group did not showed edema when compared to toxin group. Ear weight 6.5 mm discs biopsy punch are used to calculate the weight of each ear and found that there is tremendous increased in weight in TPA induced group while reduction in positive control or also showed dose dependent decrease in the ear weight (Fig. 3).

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Fig. 3

Effect of CREO on TPA-induced ear inflammation. Data are mean ± SEM; n = 5^. (a) Ear redness, (b) Ear weight, ANOVA; Tukey test p < 0.05.

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3.3.4

3.3.4 Measurement of IL-1β, IL-6, TNF-α level in serum

After the completion of experiment the serum was collected and measure IL-6, IL-1β, and TNF-α in mice serum by ELISA kits according to the instructions (Fig. 4). This experiment TPA induced group clearly showed increased in cytokines level whereas the concentration of 5% showed significantly reduced the in production of IL-6, TNF-α and IL1-β and these results were compared to TPA (negative) control and positive control groups (Table 3).

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Fig. 4

Effect of CREO on inflammatory mediators in the ears tissue isolated from TPA induced inflammation. Data are mean ± SEM; n = 5 ^#Normal versus disease control, *Disease control versus treated, ANOVA; Tukey test p < 0.05.

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Table 3 Effect of CREO on pro-inflammatory cytokines.

Parameter	Normal control	Disease control	Indomethacin	1% CREO	2% CREO	5% CREO
TNF- α	43.64 ± 5	113.34 ± 3	51.28 ± 1	86.18 ± 2	74.7 ± 3*	60.2 ± 2**
IL-1 β	27.65 ± 2	150.76 ± 6	51.27 ± 2	136.81 ± 5	106.24 ± 3	78.43 ± 1**
IL-6	175.37 ± 14	1466.08 ± 147	195.40 ± 8	1156.18 ± 27	671.18 ± 27*	443.07 ± 13**

Data are mean ± SEM; N = 5.

^#Normal versus toxin, * Toxin versus treatment (ANOVA; Tukey test), p < 0.05.

3.4 Histopathology

In present study the vehicle used during the experiment showed no effect in the cutaneous morphology of the ear of mice, while the TPA control group showed severe increase in the diameter of ear. Leucocyte infiltration and ear edema are observed vigorously but after the treatment with 5% CREO there is significantly reduce in the elevated interleukins Fig. 5.

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Fig. 5

Histopathological profile of ear tissues under microscopy (a) Normal control, (b) Disease control, (c) Indomethacin, (d) 1%CREO, (e) 2%CREO (f) 5%CREO.

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