Chemical composition and cytotoxic properties of Clinacanthus nutans root extracts

Figures & data

Figure 1. The anti-proliferative effect of C. nutans root extracts. Treatments were performed on (a) MCF-7, (b) HeLa and (c) 3T3 cells for three days. Data represent three independent experiments performed in triplicate. Asterisks denote differences with statistical significances compared to untreated cells (*, ** and *** represent p < 0.05, p < 0.005 and p < 0.0005 respectively). p-values were obtained from a two-tailed t-test.

Figure 2. Morphological changes caused by C. nutans extracts on MCF-7. Cells were treated with (a) ethyl acetate and (b) methanol root extracts of C. nutans at their respective IC₅₀ value for up to three days. Cells were stained with methylene blue and observed under microscope with 40x magnification. Arrows indicate some cells with morphological changes when compared with untreated cells.

Figure 3. Apoptotic effects of C. nutans root extracts on MCF-7 cells. Cells were treated with C. nutans root extracts and camptothecin for three days at the indicated concentrations. Data represent three independent experiments performed in triplicate. Asterisks denote differences with statistical significances compared to untreated cells (*, ** and *** represent p < 0.05, p < 0.005 and p < 0.0005 respectively). p-values were obtained from a two-tailed t-test.

Figure 4. The effect of C. nutans root extracts on the expression of ACTB (a), BCL2 (b) and BAX (c) in MCF-7 breast cancer cell line by RT-PCR. Lane 1: 100 bp DNA marker, lane 2: negative control, lane 3: without treatment, lane 4: methanol root extract and lane 5: acetyl acetate root extract.

Figure 5. GC-MS chromatogram of ethyl acetate root extract of C. nutans.

Figure 6. GC-MS chromatogram of methanol root extract of C. nutans.

Table 1. Cytotoxic compounds identified in the ethyl acetate root extract of C. nutans.

No.	Retention time (min)	Bioactive compound	Nature of compound	Peak area (%)	Molecular formula	Molecular weight	Reported cytotoxic effect on cancer cells
1	3.675	Oleic acid	Fatty acid	0.22	C18H34O2	282.46	Nelson 2005; Menendez et al. 2006
2	20.374	Squalene	Triterpenoid	0.34	C30H50	410.71	Nakagawa et al.1985; Rao et al. 1998; De Los Reyes et al. 2015
3	24.956	Vitamin E	Fat soluble vitamin	0.34	C29H50O2	430.70	Ramdas et al. 2011
4	26.191	Campesterol	Phytosterols	0.37	C28H48O	400.68	Awad et al. 2003
5	26.658	Stigmasterol	Phytosterols	1.50	C29H48O	412.69	Ghosh et al. 2011; Lee et al. 2014
6	27.532	gamma-Sitosterol and beta-Sitosterol	Phytosterols	1.15	C29H50O	414.70	Awad et al. 2003; Chai et al. 2008; Sundarraj et al. 2012
7	28.441	Lup-20(29)-en-3-one	Triterpenoid	1.69	C30H48O	424.70	Hata et al. 2002, 2008
8	28.500	Lup-20(29)-en-3-one	Triterpenoid	1.10	C30H48O	424.70	Hata et al. 2002, 2008
9	28.806	beta-amyrin	Triterpenoid	1.15	C30H50O	426.71	Lee et al. 2014
10	28.972	Lupeol	Triterpenoid	26.40	C30H50O	426.71	Saleem 2009; Saleem et al. 2009; Pitchai et al. 2014; Lee et al. 2014
11	29.065	Lupeol	Triterpenoid	19.50	C30H50O	426.71	Saleem 2009; Saleem et al. 2009; Pitchai et al. 2014; Lee et al. 2014
12	29.182	Lupeol	Triterpenoid	33.15	C30H50O	426.71	Saleem et al. 2009; Pitchai et al. 2014; Lee et al. 2014
13	30.621	Lup-20(29)-en-3-ol, acetate	Triterpenoid	1.50	C32H52O2	468.75	Lee et al. 2014
14	36.199	Betulin	Triterpenoid	0.96	C30H50O2	442.73	Kommera et al. 2011

Table 2. Cytotoxic compounds identified in the methanol root extract of C. nutans.

No.	Retention time (min)	Bioactive compound	Nature of compound	Peak area (%)	Molecular formula	Molecular weight	Reported cytotoxic effect on cancer cells
1	24.898	Vitamin E	Fat soluble vitamin	0.39	C29H50O2	430.70	Ramdas et al. 2011
2	26.139	Campesterol	Phytosterols	0.39	C28H48O	400.68	Awad et al. 2003
3	26.600	Stigmasterol	Phytosterols	1.33	C29H48O	412.69	Ghosh et al. 2011; Lee et al. 2014
4	27.445	gamma-Sitosterol and beta-Sitosterol	Phytosterols	1.01	C29H50O	414.70	Awad et al. 2003; Chai et al. 2008; Sundarraj et al. 2012
5	27.987	beta-amyrin	Triterpenoid	0.82	C30H50O	426.7174	Lee et al. 2014
6	28.943	Lupeol	Triterpenoid	94.21	C30H50O	426.71	Saleem 2009; Saleem et al. 2009; Pitchai et al. 2014; Lee et al. 2014
7	36.065	Betulin	Triterpenoid	1.38	C30H50O2	442.73	Kommera et al. 2011

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