SupplementaryFile

Medicinal Chemistry Research

Bioassay directed isolation of a novel anti-inflammatory cerebroside from the leaves of Aerva sanguinolenta

Anurup Mandala, DurbadalOjhab, Asif Laleea, Sudipta Kaitya, Mousumi Dasa, Debprasad Chattopadhyayb* , Amalesh Samantaa*

aDivision of Microbiology, Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, India

bICMR virus unit, ID & BG Hospital, Beliaghata, Kolkata 700010, India

*Corresponding authors:

Amalesh Samanta Debprasad Chattopadhyay

Division of Microbiology, ICMR virus unit,

Department of Pharmaceutical Technology, ID & BG Hospital, Beliaghata,

Jadavpur University, Kolkata 700032, India Kolkata 700010, India

E-mail: E-mail:

Ph: +91-9432315461 Ph: +91-9433331293

Preliminary evaluation of extracts for anti-inflammatory activity

Preliminary screening of the anti-inflammatory activity of ASP, ASC and ASE was done in acetic acid induced protein exudation model. The study was carried out following the method of Koo et al. (2006) with minor modifications.Eleven groups of Swiss albino mice were taken (n = 6). The control group was treated with the vehicle (0.3% sodium carboxy methyl cellulose in water), test groups with extracts (ASP, ASC, ASE) (100, 200 and 400 mg/kg b.w. each) and the positive control group with indomethacin (10 mg/kg b.w.), orally. Dilute acetic acid (0.7% solution in normal saline) was administered (0.1ml/10g b.w) to each animal (i.p.) one hour after the administration of the test drug or vehicle. Three hours later, the animals were sacrificed by cervical dislocation. Physiological saline (3 ml) was injected into the peritoneal cavity and the washing solutions were collected. The protein content in the peritoneal fluid was determined (Nag Chaudhuriet al. 2005).

Intraperitoneal administration of acetic acid produces an increase in capillary permeability, resulting in increased cellular infiltration and protein exudation leading to severe inflammatory conditions. Petroleum ether extract (ASP) (400 mg/kg b.w.) as also both chloroform (ASC) and ethanol (ASE) extracts (200 and 400 mg/kg b.w.) produced (Table S1)significant inhibition of peritoneal protein exudation in acetic acid-induced inflammation in mice. However indomethacin caused relatively higher degree of reduction.

As ASE was the most effective extract (53.78% inhibition of peritoneal protein exudation at 400 mg/k.gb.w.), so, further study was conducted with ethanol extract only.

Table S1 Effect of different extracts on exudative inflammation.

Treatment / Dose (mg/kgb.w.) / protein (mg/ml) / Inhibition (%)
Control (vehicle) / - / 14.775±0.121 / -
ASP / 100 / 14.460±0.087 / 2.13
200 / 14.400±0.185 / 2.54
400 / 10.280±0.122** / 30.42
ASC / 100 / 14.169±0.183 / 4.1
200 / 10.94±0.123** / 25.95
400 / 8.040±0.115** / 45.58
ASE / 100 / 14.11±0.102 / 4.5
200 / 9.999±0.121** / 32.32
400 / 6.829±0.146*** / 53.78
Indomethacin / 10 / 4.755±0.136*** / 67.82

Values are expressed as mean ± S.E.M. (n=6); **P < 0.01, ***P < 0.001versus control (one way ANOVA followed by Dunnett‘t’ test)

References

Koo HJ, Lim KH, Jung HJ, Park EH (2006) Anti-inflammatory evaluation of gardenia extract, geniposide and genipin. J Ethnopharmacol 103: 496-500

Nag Chaudhuri AK, Karmakar S, Roy D, Pal S, Pal M,Sen T (2005) Anti-inflammatory activity of Indian black tea (Sikkim variety). PharmacolRes 51: 169-175

Fig. S1a Mass spectrum of ASE-1

Fig. S1b IRspectrum of ASE-1

Fig. S1c 1H NMR spectrum (0.7-2.6 ppm) of ASE-1

Fig.S1d 1H NMR spectrum (3.8-5.6 ppm) of ASE-1

Fig.S1e 1H NMR spectrum (6.1-8.8 ppm) of ASE-1

Fig. S1f 1H NMR spectrum (full region) of ASE-1

Fig. S1g 13C NMR spectrum (14-36 ppm) of ASE-1

Fig. S1h 13C NMR spectrum (50-85 ppm) of ASE-1

Fig. S1i 13C NMR spectrum (105-180 ppm) of ASE-1

Fig. S1j 13C NMR spectrum (full) of ASE-1

Fig. S1k DEPT 90 NMR spectrum of ASE-1

Fig. S1l DEPT 135 NMRspectrum (14-36 ppm) of ASE-1

Fig. S1m DEPT 135 NMRspectrum (full) of ASE-1

Fig. S2 Effect of ASE on carrageenan induced rat paw oedema. Values are expressed as mean ± S.E.M. (n= 6); *P < 0.01 versus control (one way ANOVA followed by Dunnett ‘t’ test)

Fig. S3 Effect of ASE on dextran induced rat paw oedema. Values are expressed as mean ± S.E.M. (n= 6); *P < 0.01 versus control (one way ANOVA followed by Dunnett ‘t’ test)

Fig. S4 Effect of ASE on histamine induced rat paw oedema. Values are expressed as mean ± S.E.M. (n= 6); *P < 0.01 versus control (one way ANOVA followed by Dunnett ‘t’ test)