Preliminary evaluation for cancer chemopreventive and cytotoxic potential of naturally growing ethnobotanically selected plants of Pakistan

Figures & data

Table 1.  Summary of the ethanobotanical uses and literature review of the selected plants.

S. no	Plant name	Local name	Family	Plant part used	Purpose of use	Major phytochemicals	References
1.	E. wallichii	Dodal	Euphorbiacea	Roots	Skin diseases such as frunkles, cutaneous anthrax, exanthema and edema	Terpenoids	Wang et al., 2004; Pan et al., 2006a, 2006b; Ali et al., 2008, 2009; Haq et al., 2012
2.	B. ciliate	Zakham e Hayat	Saxifragaceae	Rhizome	Dissolution of kidney and bladder stones, treatment of piles, leucorrhea, pulmonary infections, heart diseases, hemorrhoids, diabetes, stomach disorders, antiviral, antibacterial, anti-tussive, anti-inflammatory, antitussive, anti-HIV, neuroprotective, Anti-neoplastic	Berginin, β-sitosterol-d-glucoside, Pashano lactone, α-afzelechin, methyl gallate, catechin, leucocyanidin and gallic acid	Asolkar et al., 1992; Kapur, 1993; Singh et al., 2007; Rajkumar et al., 2011
3.	A. oblongifolium	Kaeen	Aceraceae	Aerial parts	Cytotoxic, antitumor, phytotoxic	-	Inayatullah et al., 2007
4.	A. thomsonii		Asteraceae	Aerial parts	Anti-cough, expectorant, anti-tumor, antibacterial, diuretic, antiulcer, anti-viral, anti-diarrheal	Phenolic esters, triterpenes, simple phenol, indoles, flavonoids, quinic acid derivatives, phenyl propanoids, phenyl propanoid glycosides	Shirota et al., 1997; Meneghetti, 1997; Wang and Yu, 1998; Lin et al., 2007; Bibi et al., 2011
5.	O. dillenii	Anaar phali	Cactaceace	Fruit	Gastric ulcer, cholesterol lowering, diabetes, analgesic, anti-inflammatory, anti-spermatogenic	Betalain, betacyanins, betaxanthins, ascorbic acid, betanin, isobetanin, kaempherol 3-O-arrabinoside, isorhamnetin 3-O-glucosidesteroids, isorhamnitin 3-O-rutinoside	Loro et al., 1999; Gupta et al., 2002; Ahmed et al., 2005, Jiang et al 2006; Chang et al., 2008
6.	W. coagulans	Panir	Solanaceae	Berries, arial parts, roots	Antimicrobial, anthelmentic, anti-tumor, anti-inflammatory, hepatoprotective, diuretic, sedative, anti-asthmatic, blood purifier, wound healer, antirheumatic, antidyspepsia, antidropsy, antiulcer, antidiabetic, cardiovascular treatment, toothache, opthalmia, immunomodulation	Withanolides, estrases, alkaloids, lignans, essential oil, fatty oil	Rehman et al., 2003; Chattopadhyay et al., 2007; Hemalatha et al., 2008; Huang et al., 2009; Maurya, 2010

Table 2.  Detail of sample preparation scheme of selected plants for biological evaluation.

Sample code	Extract/fraction name	Plant name	Plant part	Preparation method	Solvent/mobile phase
1	EWR CME	E. wallichii	Root	Maceration	Methanol
2	EWR NHF	E. wallichii	Root	Sol-Sol Ext	n-Hexane
3	EWR NBF	E. wallichii	Root	Sol-Sol Ext	n-Butanol
4	EWR CHF	E. wallichii	Root	Sol-Sol Ext	Chloroform
5	EWR EAF	E. wallichii	Root	Sol-Sol Ext	Ethyl acetate
6	EWR AQF	E. wallichii	Root	Sol-Sol Ext	Water
7	EWA CME	E. wallichii	Aerial	Maceration	Methanol
8	EWA FCC-1	E. wallichii	Aerial	FCC	DCM
9	EWA FCC-2	E. wallichii	Aerial	FCC	DCM
10	EWA FCC-3	E. wallichii	Aerial	FCC	DCM
11	EWA FCC-4	E. wallichii	Aerial	FCC	DCM
12	EWA FCC-5	E. wallichii	Aerial	FCC	Ethyl acetate
13	EWA FCC-6	E. wallichii	Aerial	FCC	Ethyl acetate
14	EWA FCC-7	E. wallichii	Aerial	FCC	Ethyl acetate
15	EWA FCC-8	E. wallichii	Aerial	FCC	Methanol
16	EWA FCC-9	E. wallichii	Aerial	FCC	Methanol
17	EWA FCC-10	E. wallichii	Aerial	FCC	Methanol:Water::1:1
18	EWA FCC-11	E. wallichii	Aerial	FCC	Water
19	BCR CME	B. ciliata	Rhizome	Maceration	Methanol
20	BCR NBF	B. ciliata	Rhizome	Sol-Sol Ext	n-Butanol
21	BCR EAF	B. ciliata	Rhizome	Sol-Sol Ext	Ethyl acetate
22	BCR AQFsp	B. ciliata	Rhizome	Sol-Sol Ext	Water
23	BCR AQFppt	B. ciliata	Rhizome	Sol-Sol Ext	Water
24	AOA CME	A. oblongifolium	Aerial	Maceration	Methanol
25	AOA DCM	A. oblongifolium	Aerial	SP Ext	DCM
26	AOA MeOH	A. oblongifolium	Aerial	SP Ext	Methanol
27	AOA FCC-1	A. oblongifolium	Aerial	FCC	Methanol:DCM::1:1
28	AOA FCC-2	A. oblongifolium	Aerial	FCC	Methanol:DCM::4:1
29	AOA FCC-3	A. oblongifolium	Aerial	FCC	Methanol:DCM::4:1
30	AOA FCC-4	A. oblongifolium	Aerial	FCC	Methanol:DCM::4:1
31	AOA FCC-5	A. oblongifolium	Aerial	FCC	Methanol
32	AOA FCC-6	A. oblongifolium	Aerial	FCC	Methanol
33	AOA FCC-7	A. oblongifolium	Aerial	FCC	Methanol:water::4:1
34	ATA CME	A. thomsonii	Aerial	Maceration	Methanol
35	ATA NHF	A. thomsonii	Aerial	Sol-Sol Ext	n-Hexane
36	ATA AQF	A. thomsonii	Aerial	Sol-Sol Ext	Water
37	OD CME	O. dillenii	Fruit	Maceration	Methanol
38	OD FCC-1	O. dillenii	Fruit	FCC	Ethyacetate: Methanol::1:1
39	OD FCC-2	O. dillenii	Fruit	FCC	Ethyacetate: Methanol::1:1
40	OD FCC-3	O. dillenii	Fruit	FCC	Ethyacetate: Methanol::1:1
41	OD FCC-4	O. dillenii	Fruit	FCC	Methanol
42	OD FCC-5	O. dillenii	Fruit	FCC	Methanol
43	OD FCC-6	O. dillenii	Fruit	FCC	Methanol:Water::1:1
44	OD FCC-7	O. dillenii	Fruit	FCC	Methanol:Water::1:1
45	OD FCC-8	O. dillenii	Fruit	FCC	Water
46	OD FCC-9	O. dillenii	Fruit	FCC	Water
47	WCA CME	W. coagulans	Aerial	Maceration	Methanol
48	WCF CME	W. coagulans	Fruit	Maceration	Methanol
49	WCA&F CME	W. coagulans	Aerial with fruit	Maceration	Methanol
50	WCA&F FCC-1	W. coagulans	Aerial with fruit	FCC	Ethyl acetate
51	WCA&F FCC-2	W. coagulans	Aerial with fruit	FCC	Ethyl acetate
52	WCA&F FCC-3	W. coagulans	Aerial with fruit	FCC	Ethyl acetate
53	WCA&F FCC-4	W. coagulans	Aerial with fruit	FCC	Ethyl acetate: Methanol::1:1
54	WCA&F FCC-5	W. coagulans	Aerial with fruit	FCC	Ethyl acetate: Methanol::1:1
55	WCA&F FCC-6	W. coagulans	Aerial with fruit	FCC	Methanol
56	WCA&F FCC-7	W. coagulans	Aerial with fruit	FCC	Methanol
57	WCA&F FCC-8	W. coagulans	Aerial with fruit	FCC	Methanol: Water::1:1
58	WCA&F FCC-9	W. coagulans	Aerial with fruit	FCC	Methanol: Water::1:1
59	WCA&F FCC-10	W. coagulans	Aerial with fruit	FCC	Water

DCM, dichloromethane; FCC, flash column chromatography; Sol-Sol Ext, solvent-solvent extraction; SP Ext, solid phase extraction.

Table 3.  Bioassay results.

Sample code^a	NFκB^b		Aromatase^c		Nitrite^b		QR1^d		RXRE^e	Cytotoxicity^f
										MCF-7		Lu		MDA-MB-231
	% inhibition	IC50 (µg/mL)	% inhibition	IC50 (µg/mL)	% inhibition	IC50 (µg/mL)	IR	CD (µg/mL)	FI	% survival	IC50 (µg/mL)	% survival	IC50 (µg/mL)	% survival	IC50 (µg/mL)
1	0.0		7.1		19.1		1.0		0.9	46.1		60.1		57.6
2	0.0		20.2		71.8	4.8 ± 0.5	1.3		1.4	47.8		102.1		70.5
3	0.0		2.7		22.5		1.2		1.0	47.1		48.2		54.9
4	0.0		0.0		13.6		1.2		0.8	46.2		64.6		63.6
5	0.0		20.1		28.4		0.8		1.4	48.0		49.9		71.1
6	0.0		2.8		0.0		1.0		0.6	47.0		65.0		94.2
7	0.0		16.0		61.8		1.4		1.0	45.6		51.7		77.5
8	0.0		13.5		0.0		1.4		1.1	73.2		101.9		94.7
9	0.0		51.7		86.0	15.6 ± 4.4	1.7	18.1 ± 1.8	0.7	27.4	7.5 ± 0.2	61.7		3.4	2.5 ± 0.3
10	0.0		73.4	17.7 ± 2.2	95.6	11.5 ± 1.0	0.1	20.6 ± 2.9	0.8	7.7	2.9 ± 0.7	11.2	5.1 ± 1.0	5.4	3.4 ± 1.2
11	0.0		52.7		94.7	9.9 ± 1.2	1.1		0.8	19.2	6.3 ± 1.1	29.9	7.1 ± 2.2	42.8
12	0.0		36.1		83.2	9.2 ± 1.3	1.1		1.1	34.6		57.9		46.6
13	0.0		25.9		37.7		0.8		1.5	53.2		52.0		68.5
14	0.0		26.4		23.8		0.9		1.3	54.5		61.2		77.8
15	22.4		9.0		1.1		1.0		1.3	73.9		81.8		95.1
16	0.0		5.9		0.0		0.9		0.7	47.1		56.6		101.9
17	2.2		4.4		0.0		1.1		0.6	100.0		81.3		101.7
18	6.9		3.2		0.0		0.9		0.8	79.1		87.0		101.8
19	0.0		25.7		0.0		1.3		1.0	71.7		61.1		97.5
20	0.0		28.5		0.0		1.1		0.7	66.4		59.5		83.5
21	0.0		26.6		16.8		0.9		0.6	104.9		51.0		69.9
22	0.0		7.5		0.0		1.0		0.7	105.8		88.2		95.9
23	0.2		11.5		0.0		0.9		0.9	109.3		83.6		103.1
24	20.9		23.4		48.6		1.5		1.0	74.7		83.8		73.5
25	35.9		50.8		99.2	9.8 ± 0.7	0.1	9.6 ± 1.4	1.0	93.9		77.8		60.7
26	26.7		27.1		40.2		1.1		0.9	74.8		82.1		71.4
27	0.0		32.9		41.1		1.3		0.6	82.0		58.2		85.0
28	14.5		22.3		0.0		1.2		0.7	60.9		95.0		78.3
29	9.8		18.7		0.0		1.3		0.9	56.6		92.5		85.3
30	0.0		11.5		0.0		0.9		1.0	71.6		89.3		92.8
31	15.9		7.2		0.0		0.9		1.6	113.6		101.1		102.5
32	16.1		6.3		0.0		1.0		1.2	108.9		105.3		106.5
33	0.6		7.1		0.0		1.0		1.0	115.0		108.4		88.8
34	15.6		20.8		25.3		1.2		1.2	90.5		105.2		100.8
35	0.0		56.6		98.0	12.5 ± 0.7	0.4	10.8 ± 2.1	0.8	46.5		75.6		84.9
36	2.8		16.7		0.0		1.1		0.9	94.9		99.1		105.8
37	7.5		12.5		0.0		1.1		1.1	119.0		107.6		106.0
38	18.2		11.9		0.0		1.1		1.0	103.4		100.7		104.7
39	0.0		10.2		0.0		1.0		0.6	91.2		105.9		111.1
40	0.0		8.6		0.0		1.1		0.8	81.1		105.8		104.5
41	0.0		4.4		0.0		1.0		1.3	115.9		98.0		100.2
42	0.8		7.3		0.0		0.9		0.8	112.4		110.5		104.3
43	6.9		10.5		0.0		1.0		0.8	114.1		114.2		106.5
44	3.0		0.0		0.0		1.1		0.6	126.8		116.1		112.1
45	8.1		0.0		0.0		1.2		0.9	123.6		109.4		113.9
46	9.5		0.0		0.0		1.2		0.7	118.6		109.6		109.3
47	53.4		31.2		100.1	5.0 ± 0.5	2.7	2.1 ± 0.7	0.9	44.7		68.4		29.0	7.8 ± 1.1
48	49.1		19.0		61.9		2.4	19.9 ± 1.6	1.0	42.1		75.9		37.8
49	52.2		20.5		89.3	9.0 ± 1.0	3.4	7.8 ± 0.9	1.0	35.4		78.9		33.3
50	57.9		100.0	17.0 ± 1.8	100.0	3.1 ± 0.4	0.0	2.2 ± 0.4	0.8	29.2	3.4 ± 0.3	58.6		19.3	5.4 ± 0.4
51	95.5	2.6 ± 1.2	43.5		99.0	1.3 ± 0.2	0.0	1.0 ± 0.2	1.4	9.3	1.2 ± 0.5	18.5	3.1 ± 1.2	5.4	4.2 ± 0.5
52	91.5	4.3 ± 0.8	30.3		98.6	3.0 ± 0.1	0.3	1.1 ± 0.5	1.6	18.2	2.2 ± 0.6	54.6		18.7	5.1 ± 0.6
53	60.3		8.2		79.7	11.7 ± 2.4	3.2	6.0 ± 1.3	0.9	37.2		75.8		28.5	5.9 ± 1.0
54	52.8		10.2		55.2		2.7	7.2 ± 0.8	0.6	43.1		80.8		37.5
55	54.1		10.3		60.6		2.3	6.1 ± 0.5	0.8	45.2		75.6		43.7
56	58.6		11.8		61.5		2.7	7.3 ± 1.1	1.0	30.6		61.0		38.3
57	22.1		13.1		15.1		1.2		1.3	76.0		97.9		61.5
58	27.7		11.3		8.1		1.2		1.0	89.8		93.2		79.2
59	7.7		11.0		0.0		1.2		1.0	59.8		104.8		97.6

Initial test concentration in each of the assays was 20 µg/mL. All data shown above are the mean of duplicate/triplicate tests and have standard error in an acceptable range.

All IC₅₀s are in µg/mL.

CD, concentration (µg/mL) at induction ratio two; FI, fold increase; IC₅₀, concentration at 50% inhibition; IR, induction ratio; NT, Not tested.

^aSample number: Taxonomic identification and method of preparation are given in Table 2.

^bCytotoxicity was also run in parallel to avoid false positives, and none of the samples were cytotoxic at the IC₅₀. The samples which showed ≥70% inhibition at 20 µg/mL were considered active and were tested at lower concentrations to find the IC₅₀.

^cThe samples which showed ≥70% inhibition at 20 µg/mL were considered active and were tested at lower concentrations to find the IC₅₀.

^dCytotoxicity was also run in parallel to avoid false negatives, and no sample was cytotoxic at the CD except 9 and 10. The samples which showed IR ≥2 or were cytotoxic at 20 µg/mL were tested at lower concentrations to find the CD.

^eThe samples which show ≥4 fold increase were considered active. 9-cis-retinoic acid was used as positive control and it had a 13.1-fold increase.

^fThe samples which show ≤30% survival at 20 µg/mL were considered active and were tested at lower concentrations to find the IC₅₀.

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