SUPPLEMENTARY MATERIAL Chemical profiling and anti-psoriatic activity of methanolic extract of Andrographis nallamalayana J.L.Ellis

Andrographis nallamalayana is being widely used as tribal medicine in the treatment of leucoderma and mouth ulcers. Chemical profiling of methanolic extract of the whole plant (PE), using GC-MS and LCMS, revealed the presence of compounds viz., α-tocopherol, β-sitosterol, tetradecanoic acid, monostearin, flavones/flavanones and their glycosides, chromones etc. Topical application of imiquimod on the dorsal portion of male BALB/C mice resulted in the development of psoriatic symptoms (erythema, scaling, thickening and folding) with a mean disease activity index (DAI) of >7.0. Topical treatment with 100 μL PE (~6.4%/12.8%) formulations, for 12-days, resulted in the alleviation of disease symptoms. Compared to water-based formulations, emu oil-based formulation, PE400EO was found more effective in reducing the mean DAI (>84%), keratinocyte count (>65%) (p < 0.01) and Interleukin-22 (~70%) (p < 0.05). We report, for the first time, anti-psoriatic activity of A. nallamalayana having great potential in developing a potent phytomedicine against psoriasis.


Preparations of plant extract (PE)
Plants of A. nallamalayana were collected from Nallamala hills of Kurnool district in the month of April 2013 after obtaining the permission from the Department of Forestry, Government of Andhra Pradesh.
Identification of the species was done based on the taxonomic literature and authentication given by Deccan Circle, Botanical Survey of India and deposited the specimen of A. nallamalayana (Voucher no. CNS01AN2013). Leaves, stems and roots of the plant were shade dried and cut into small pieces. The dried material was ground into fine powder. Extraction was carried out at 65 o C using soxhlet apparatus (Oyedeji et al. 2012;Vijayalakshmi et al. 2013) by taking 10 g of the fine powder in 300 mL of methanol.
The extract was filtered using whatman filter paper and vacuum dried to remove methanol.

GC-MS analysis
The plant extract was subjected to derivatization prior to GC-MS analysis (Mamza et al. 2012;Muthulakshmi et al. 2012;Eddy et al. 2011). The sample was silylated using N,Obis(trimethylsilyl)trifluoroacetamide (BSTFA). About 2 mg of dried methanolic extract of A.
nallamalayana was taken in 1 mL of methanol and vortex mixed for 5 min. The mixture was centrifuged for 5 min at 10,000 rpm to remove the particles. The supernatant was transferred to a fresh vial and evaporated to dryness using scanvac (Denmark). The residue was dissolved in 200 L of acetonitrile (ACN) and 200 µL of BSTFA was added. The vial was sealed with parafilm and kept at 60 o C for one hour in water bath. The reaction mixture was concentrated to 100 L by nitrogen purging. The same procedure was also applied for generating solvent blank with pure methanol. Samples were sealed with parafilm and stored at -20 o C until subjected to GC-MS analysis.
The GC-EIMS analyses were performed using an Agilent 6890 gas chromatograph (Agilent Technologies, Palo Alto, CA) equipped with a 5973N mass selective detector and a HP-5MS capillary column of length 30 m, 250 μm i.d. and 0.25 μm of film thickness. One L of the sample (containing 20 µg PE) was injected into the GC-MS under split injection mode with 20:1 split ratio. The column oven was programmed initially from 50 o C with 2 min holdup time to the final temperature of 280 o C with 10 o C/min ramp and the final temperature holdup time was 5 min with a total runtime of 30 min. Helium was used as the carrier gas in constant flow mode at a flow rate of 1.0 mL/min. The inlet and GC/MS interface temperatures were kept at 250 o C and 280 o C, respectively. EI source and quadrupole analyzer were kept at 230 o C and 150 o C, respectively. MS scanning was kept in full scan mode from m/z 29 to 600.

LC-MS Analysis
The sample was subjected for direct ESI-HRMS analyses under positive and negative ion modes. (Song et al. 2013; Barrios et al. 2010) The number of signals and intensities were found to be better in negative ion mode. Hence, the sample was subjected for LC-MS analysis under negative ion mode. Prior to the sample run, a solvent blank run was performed with the methanol/water (65:35, v/v) to know the peaks related to system background. The LC-PDA-MS analysis of methanolic extract of A. nallamalayana was performed on Exactive Orbitrap mass spectrometer (Thermo Scientific, Waltham, MA, USA) in negative ion mode connected to Thermo Accela 600 ultra high performance liquid chromatography (UHPLC) with quaternary gradient pump. Accela autosampler (Thermo Scientific, USA) was used to inject the samples.
After separation, samples were allowed to enter Photo Diode Array detector (Accela PDA, 80 Hz) operated at the range of ultraviolet-visible wavelength (200 nm to 600 nm) using deuterium and tungsten lamps. Two component mobile phase consisting of 0.02% formic acid in methanol (A) and 0.02% formic acid in 5 mM ammonium formate buffer (B), with gradient mode of elution was employed for the separation with a flow rate of 0.6 mL/min. The column temperature was set at 40 o C and 5 µL of sample (containing 100 µg PE) was injected and subjected to 30 min run. MS source was equipped with heated electrospray ionization probe and the typical operational conditions include, 300 o C probe temperature, capillary temperature 280 o C, capillary voltages of 85 V, spray voltage of 4.5 kV, tube lens voltage of 175 V. Nitrogen was used as both auxiliary and sheath gas at a flow rate of 45 and 15 mL/min, respectively.
Mass range of 100 to 1000 m/z with a resolution of 40,000 of full width at half maximum was used.

Animal housing
Five weeks-old BALB/C male mice were procured from Sainath agencies. All animal procedures have been approved by the Animal Ethical Committee (No: 033/C/07/CPCSCA) in accordance with animal experimentation and care guidelines provided by IAEC/CPCSEA, India. The animals were acclimatized to laboratory conditions prior to initiation of experiment. They were given feed in the form of pellets and water ad libitum. Forty two mice were randomized according to their body weight and divided into 7 groups (G1-G7), comprising of 6 mice each with a mean body weight variation not exceeding + 20 % between the groups. Hair on the dorsum portion (nearly 2×2 cm 2 ) of each mouse was removed by using depilatory cream from Reckitt Benckiser Company, U.S.A.

Preparation of test drug formulations
Stock solutions of various test drug formulations taking 200 mg PE or 400 mg PE/kg body weight of the animal were prepared freshly (Jiao et al. 2013). Considering the average body weight of each mouse as 32 g, formulations of PE200/PE200EO (determining the dose 200 mg/kg body weight) were constituted by dissolving 38.4 mg of dried methanolic extract in 180 µL of DMSO and made up the volume to 600 µL by adding 420 µL of water/emu oil. Daily each animal was administered with 100 µL of formulation containing 6.4 mg of test drug. Similarly PE400/PE400EO formulations (determining the dose 400 mg/kg body weight) were prepared by doubling the content of plant extract and 100 µL of formulation consisting of 12.8 mg of PE was administered daily to each animal.

Induction of psoriasis
Group1 (G1) served as control and topically administered with 50 mg of vaseline on the shaved dorsal surface of the skin of each mouse once daily for 11 days. Psoriasis was induced in the groups 2-7 (G 2-G7) by topical application of 50 mg of IMQ cream (Glenmark pharmaceuticals) once daily for 11 days.
Mice were evaluated daily, 4 hours prior-and post-application of IMQ. To score the severity of inflammation of the dorsum, clinical Psoriasis Area and Severity Index (PASI) scoring system was used (Sun et al. 2013). Signs of psoriasis, erythema, scaling, thickening and folding of the skin were scored independently on a scale from 0 to 3 (0-without any disease symptoms, 1-mild, 2-Severe and 3-Very severe symptoms of disease). Disease Activity index (DAI) was calculated using cumulative scores of four disease parameters (Erythema + Scaling + Thickening + Folding) on a scale 0 to 12 and served as a measure of severity of the induced-disease condition.

Treatment of psoriasis
All the animals were re-randomized based on mean DAI and subjected to treatment daily with the topical application of test drug formulations for 12 days. Application of IMQ (50 mg) was continued throughout the treatment phase, to all the mice from G2-G7 groups, to ensure that the recovery, if any, in the mice is due to the test drug formulations. In addition to IMQ, animals of groups 2, 3, 4, 5 and 6 were administered with 100 μL of PE200, PE400, PE200EO, PE400EO and commercial drug BMT, respectively. Group 7 without any treatment served as untreated disease-induced mice. Mice from control group (G1), without disease, were administered with vaseline (50 mg) and 100 μL 30 % DMSO.
Body weight, food and water intake of each animal were recorded daily. Erythema, thickness, scaling and folding was scored daily 4 hours pre-and post-application of the test drug formulation and IMQ.
Reduction in the signs of psoriasis was measured by PASI scoring, histopathological observations of skin sections and quantification of plasma Interleukin-22 (IL-22).

Collection of samples
At the end of experiment, blood samples were collected by retro orbital into the vials containing EDTA.
Plasma was separated from blood by centrifugation and was stored at -70 ºC. Skin tissues of all the treated mice were excised, washed in Phosphate Buffered Saline (PBS) (9.64 mM phosphate buffer + 0.8 % NaCl pH: 7.4) and fixed in 10 % neutral buffered formalin.

Histological studies
After one week of fixation, wax embedded blocks of excised skin tissue was prepared in routine process.
The sections (3 μ) were made using microtome (Leica RM 2245) on to glass slides. After complete removal of wax, the sections were subjected to Hemotoxylin and Eosin (H&E) staining (Riedel et al. 2015) and observed under microscope. Number of keratinocytes was counted under 40X magnification of H&E stained skin sections of normal, disease-induced and disease-induced mice treated with test drug formulations.

Enzyme linked immunosorbant assay (ELISA)
Plasma samples were used for the quantification of IL-22 in all the groups by ELISA using the kit ELISA MAX Deluxe Sets (Cat. No. 436304) from Biolegend, Inc., U.S.A and performed the assay according to the manufacturers protocol using recombinant IL-22 of mouse as standard. The experiment was performed in duplicates and the means and standard deviations were calculated.

Statistical analysis
To evaluate recovery of mice from induced psoriasis, statistical significance was calculated for DAI, keratinocyte proliferation and IL-22 levels between the disease-induced group vs respective treatments.
Data are presented as mean ± standard error mean (SEM). Statistical significance of data was performed by unpaired-t test using graphpad software (www.graphpad.com) (Priyanka et al. 2012).