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Cytotoxic activity screening of some indigenous Thai plants

Article  in  Fitoterapia · August 2008

DOI: 10.1016/j.fitote.2008.06.007 · Source: PubMed

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 Fitoterapia 79 (2008) 598 – 601
www.elsevier.com/locate/fitote

Short report
Cytotoxic activity screening of some indigenous Thai plants
P. Prayong a , S. Barusrux b , N. Weerapreeyakul c,⁎
a
Graduate School and Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand
b
Centre for Research and Development of Medical Diagnostic Laboratories (CMDL), Faculty of Associated Medical Sciences,
Khon Kaen University, Khon Kaen 40002, Thailand
c
Center for Research and Development of Herbal Health Products (CRD-HHP), Faculty of Pharmaceutical Sciences,
Khon Kaen University, Khon Kaen 40002, Thailand
Received 3 September 2007; accepted 30 June 2008
Available online 11 July 2008

Abstract

The 50% ethanolic extracts from 14 plant species used in Thai traditional folklore were screened for cytotoxic activity against a
malignant human hepatoma (HepG2) cell line and a normal African green monkey kidney (Vero) cell line. The extracts of Polyalthia
evecta and Erythroxylum cuneatum showed potent anticancer activity in the HepG2 cell line with IC50 of 70 ± 3 µg/ml and 64 ± 4 µg/ml,
respectively. P. evecta demonstrated more selectivity to the HepG2 than the Vero cell (selectivity index N 14.3) indicating its potential for
biopharmaceutical use.
© 2008 Elsevier B.V. All rights reserved.

Keywords: Cytotoxic screening; HepG2; Vero; Thai plants

1. Plants

Fourteen plants, listed in Table 1 were collected during 2002 to 2003 from Khon Kaen Thailand, where is a
sanctuary area for Plant Genetics Conservation Project under the Royal Initiation of her Royal Highness Princess Maha
Chakri Sirindhorn. Voucher specimens were deposited in the Center for Research and Development of Herbal Health
Products (CRD-HHP), Khon Kaen University, Thailand.

2. Uses in traditional medicine

See Table 1.

3. Previously isolated class of constituents

See Table 1.

⁎ Corresponding author. Tel.: +66 43 202 378; fax: +66 43 202 379.
E-mail address: natthida@kku.ac.th (N. Weerapreeyakul).

0367-326X/$ - see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2008.06.007
 P. Prayong et al. / Fitoterapia 79 (2008) 598–601 599

4. Tested material

50% Ethanol extracts (yields in Table 2). Stock samples of each extract were dissolved in dimethyl sulfoxide at a
concentration of 100 mg/ml.

5. Cytotoxic activity

The stock samples were diluted with Dulbecco's Modified Eagle's Medium (DMEM) to desired concentrations
ranging from 62.5 to 1000 µg/ml. The final concentration of DMSO in each sample did not exceed 1%v/v. The
cytotoxic activity of the extracts were tested in malignant human hepatoma (HepG2) cell line and normal African green
monkey kidney (Vero) cell line by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
method [26] with minor modification. Melphalan was used as a standard anticancer drug for a comparison.
Briefly, the cell were seeded in 96 well-plates (100 µl/well at a density of 3 × 105 cells/ml) and treated with various
concentrations of the sample solutions for 24 h. At 20 h, cells were washed with sterile phosphate buffer (PBS) 3 times and the
supernatant was discarded. MTT solution (20 µl of 5 mg/ml) was added to each well and incubated at 37 °C for another 4 h.

Table 1
Thai plants investigated for cytotoxic activity
No Species (Voucher number)
1 Ellipeiopsis cherrevensis
(Pierre ex Finet and Gapnep.)
R.E.Fr. (CRD-HHP-018L)
2 Polyalthia debilis
(Pierre) Finet and Gapnep.
(CRD-HHP-010L)
3 Polyalthia evecta
(Pierre) Finet and Gapnep.
(CRD-HHP-011L)
4 Aganosma marginata (Roxb.)
G. Don (CRD-HHP-017)
5 Cassytha filiformis L.
(CRD-HHP-025)
6 Parinari anamense Hance
(CRD-HHP-015)
7 Garcinia cowa Roxb. ex DC.
(CRD-HHP-007)
8 Cratoxylum formosum
(Jack) Dyer. (CRD-HHP-006)
9 Erythroxylum cuneatum
(Miq.) Kurz (CRD-HHP-012)
10 Aporosa villosa (Wall.ex Lindl.)
Baill (CRD-HHP-016)
11 Flacourtia indica
(Burm.f.) Merr. (CRD-HHP-020)
12 Rhodamnia dumetorum
(DC.) Merr. and L.M. Perry
(CRD-HHP-024)
13 Dioecrescis erythroclada (Kurz)
Tirveng. (CRD-HHP-022)
14 Harrisonia perforata
(Blanco) Merr. (CRD-HHP-023)
⁎ Information was asked from local person.
Families Known chemical constituents Thai traditional uses
Annonaceae Ferrudiol, zeylenol, ellipeiopsol A, B, C [1], ellipeiopsol D, Blood and kidney tonic⁎
2V,4V-dihydroxy-3V-(2-hydroxybenzyl)-6V-methoxychalcone,
flavonoids, tiliroside and kaempferol 3-O-rutinoside and an
oxoaporphine alkaloid [2]
Annonaceae Clerodan diterpenes, triterpenes, benzopyrans, Abdominal pain treatment [3]
polyacetylenes, azaanthracenes, aporphines, bisaporphines,
indolosesquiterpenes, seco-benzyltetrahydroisoquinolines,
oxoprotoberberines.and bidebilines A-D [3].
Annonaceae Evectic acid [4,5]. Galactagogue [4]
Apocynaceae Not found. Laxative and anti-pyretic [24a]
Cassythaceae Neolitsine, dicentrine, cassythine and Anti-pyretic, diuretic,
actinodaphnine [6–8]. antihypertension and anti-
inflammation [25a]
Rosaceae Not found Antipruritic and antiashmatic
[25b]
Guttiferae Xanthones, flavonoids, benzophenones, Anti-pyretic and antimicrobial
lactones, and phenolic acids [9–12]. agent [11]
Guttiferae Chlorogenic acid,xanthones and Anti-diarrhea, internal bleeding
triterpenoids [13–15]. and food poisoning [14].
Erythroxylaceae (+)-Catechin, quercetin 3-O-alpha-L-rhamnoside, Not available.
apocynol B, (6S,9R)-roseoside, vomifoliol 9-O-alpha-L-
arabinofuranosyl (1-->6)-beta-D-glucopyranoside,
inamoside, and citroside A [16]
Euphorbiaceae Not found. Anti-pyretic [25c]
Flacourtiaceae Daucosterol, (-)- flacourtin, ramontoside Anti-itching, antihelmintic agent,
and β-sitosterol [17,18]. anti-diarrhea and dysentery [23]
Myrtaceae Not found. Anti-pyretic [25d]
Rubiaceae Not found Laxative and anti-pyretic [24b]
Simarubaceae Haperforine, perforamone [19–21]. Anti-pyretic, anti-diarrhea and
dysentery [22]
600 P. Prayong et al. / Fitoterapia 79 (2008) 598–601

Table 2
Cytotoxic activity result in malignant human hepatoma (HepG2) and normal African green monkey kidney (Vero) cell of 14 plants ethanol extracts
No. Sample Part used Yield (%) IC50 (µg/ml) SI c
HepG2 Vero
0 Melphalan a 12.4 ± 3 57 ± 10 4.6
1 Ellipeiopsis cherrevensis Leaves 10.2 104 ± 18 86 ± 11 0.8
2 Polyalthia debilis Leaves 8.4 304 ± 43 Inactive b N3.3
3 Polyalthia evecta Leaves 10.4 70 ± 3 Inactive b N14.3
4 Aganosma marginata Vine 13.0 866 ± 125 Inactive b N1.2
5 Cassytha filiformis Vine 3.7 143 ± 5 Inactive b N7.0
6 Parinari anamense Branches 1.8 546 ± 65 431±22 0.8
7 Garcinia cowa Leaves 15.8 430 ± 34 Inactive b N2.3
8 Cratoxylum formosum Leaves 24.8 108 ± 19 859 ± 100 8.0
9 Erythroxylum cuneatum Leaves 7.7 64 ± 4 366 ± 9 5.8
10 Aporosa villosa Branches 14.6 Inactive b Inactive b 1.0
11 Flacourtia indica Branches 4.8 Inactive 658 ± 21 b0.7
12 Rhodamnia dumetorum Branches and roots 5.3 386 ± 66 186 ± 26 0.5
13 Dioecrescis erythroclada Branches 4.0 Inactive b Inactive b 1.0
14 Harrisonia perforata Branches 3.8 Inactive b 276 ± 31 b0.3
Data were expressed as the means of three determinations.
a
Standard anticancer drug.
b
IC50 N 1000 µg/ml is considered to be inactive.
c
SI referred to Selectivity Index, when SI value N 3 indicates high selectivity.

Then the medium was aspirated. In each well, the formed formazan crystals were dissolved with 200 µl of DMSO. An
absorbance of formazan was detected by a dual wavelength UV spectrometer at 570 nm with 650 nm reference wavelength.
The percentage of cytotoxicity compared to the untreated cells was determined with the equation given below. The plot of %
cytotoxicity versus sample concentration was used to calculate the concentration which exhibited 50% cytotoxicity (IC50).
ðAbsorbance of cell without treatment  Absorbance of cell with treatmentÞ  100
k Cytotoxicity ¼ :
Absorbance of cell without treatment
The selectivity index (SI) was also calculated from the IC50 ratio in Vero cell versus HepG2 cells [27]. SI value indicates
selectivity of the sample to the cell lines tested. Any sample which has SI value higher than 3 will be considered to have high
selectivity.

6. Results and conclusions

Cytotoxicity results are summarized in Table 2. In consideration of the cytotoxicity and selectivity result, the samples
could be classified into 5 categories. Firstly, potentially cytotoxic (IC50 in HepG2 b 100 µg/ml) and high selectivity (SI ≥ 3)
are melphalan, P. evecta and E. cuneatum. Secondly, moderate cytotoxic (100 µg/ml b IC50 in HepG2 b 1000 µg/ml) and
high selectivity (SI ≥ 3) are P. debilis, C. filiformis and G. cowa. Thirdly, moderate cytotoxic (100 µg/ml b IC50 in
HepG2 ≤ 1000 µg/ml) but less selectivity (SI b 3) are E. cherrevensis, A. marginata, P. anamense, G. cowa and
R. dumetorum. Fourthly, toxic to only Vero cells are F. indica and H. perforata. Finally, non toxic in both cell lines
(IC50 N 1000 µg/ml) are A. villosa, and D. erythroclada. P. evecta and E. cuneatum possess high cytotoxicity with the
IC50 b 100 µg/ml in the HepG2 cell line, at 70 ± 3 and 64 ± 4 µg/ml, respectively. The crude samples in the first two
categories could be taken for further bioassay guided experiment. Interestingly, P. evecta possessed the highest selectivity
(SI N 14.3) to the HepG2 cell line indicating its ultimate potential for biopharmaceutical use among the sample tested.

Acknowledgements

Pokpong Prayong is grateful for the Graduate School, Khon Kaen University for the financial support (Grant
No. 50111113). The authors thank to the Plant Genetics Conservation Project under the Royal Initiation of her Royal Highness
Princess Maha Chakri Sirindhorn for permission of conducting the research and to Dr. Chanvit Leelayuwat, Centre for
Research and Development of Medical Diagnostic Laboratories (CMDL), Khon Kaen University for providing the cell lines.
 P. Prayong et al. / Fitoterapia 79 (2008) 598–601 601

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