0PHCOG RES
ORIGINAL ARTICLE
Antiproliferative and phytochemical analyses of leaf extracts of ten Apocynaceae species
Siu Kuin Wong, Yau Yan Lim, Noor Rain Abdullah1, Fariza Juliana Nordin1
School of Science, Monash University Sunway Campus, 46150 Petaling Jaya, Selangor, 1Herbal Medicine Research Centre, Institute for Medical Research, 50588 Kuala Lumpur, Malaysia
Submitted: 29-10-2010 Revised: 21-12-2010 Published: 08-06-2011
ABSTRACT
Background: The anticancer properties of Apocynaceae species are well known in barks and roots but less so in leaves. Materials and Methods: In this study, leaf extracts of 10 Apocynaceae species were assessed for antiproliferative (APF) activities using the sulforhodamine B assay. Their extracts were also analyzed for total alkaloid content (TAC), total phenolic content (TPC), and radical scavenging activity (RSA) using the Dragendorff precipitation, Folin-Ciocalteu, and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays, respectively. Results: Leaf extracts of Alstonia angustiloba, Calotropis gigantea, Catharanthus roseus, Nerium oleander, Plumeria obtusa, and Vallaris glabra displayed positive APF activities. Extracts of Allamanda cathartica, Cerbera odollam, Dyera costulata, and Kopsia fruticosa did not show any APF activity. Dichloromethane (DCM) extract of C. gigantea, and DCM and DCM:MeOH extracts of V. glabra showed strong APF activities against all six human cancer cell lines. Against breast cancer cells of MCF-7 and MDA-MB-231, DCM extracts of C. gigantea and N. oleander were stronger than or comparable to standard drugs of xanthorrhizol, curcumin, and tamoxifen. All four extracts of N. oleander were effective against MCF-7 cells. Extracts of Kopsia fruticosa had the highest TAC while those of Dyera costulata had the highest TPC and RSA. Extracts of C. gigantea and V. glabra inhibited the growth of all six cancer cell lines while all extracts of N. oleander were effective against MCF-7 cells. Conclusion: Extracts of C. gigantea, V. glabra, and N. oleander therefore showed great promise as potential candidates for anticancer drugs. The wide-spectrum APF activities of these three species are reported for the first time and their bioactive compounds warrant further investigation.
Key words: Antiproliferative, Apocynaceae, radical scavenging, total alkaloid content, total phenolic content
Access this article online
Website:
www.phcogres.com DOI:
10.4103/0974-8490.81957 Quick Response Code:
INTRODUCTION
The family Apocynaceae consists of about 250 genera and 2000 species of tropical trees, shrubs, and vines.[1] With the inclusion of species of Asclepiadaceae, the family has now been enlarged from two to five subfamilies.[2] Characteristic features of the family are that almost all species produce milky sap; leaves are simple, opposite, or whorled; flowers are large, colorful, and slightly fragrant with five contorted lobes; and fruits are in pairs.[1,3]
In traditional medicine, Apocynaceae species are used to treat gastrointestinal ailments, fever, malaria, pain, and
Address for correspondence:
Ms. Siu Kuin Wong, School of Science, Monash University Sunway Campus, 46150 Petaling Jaya, Selangor, Malaysia. E-mail: nywsk@yahoo.com
diabetes.[1] Of the 10 species studied, leaves of Allamanda cathartica are used as a purgative or emetic in Southeast Asia.[4] Leaves are also used as an antidote, and for relieving coughs and headaches. Stems, leaves, and latex of Alstonia angustiloba are used for gynecological problems and skin sores in Indonesia.[5] Leaves are externally applied to treat headache in Malaysia.[6] Roots and leaves of Calotropis gigantea are used to treat skin and liver diseases, leprosy, dysentery, worms, ulcers, tumours, and earache.[7] Its latex has wound-healing properties.[8] A decoction of all parts of Catharanthus roseus is used to treat malaria, diarrhea, diabetes, cancer, and skin diseases.[9] The species is also well known as an oral hypoglycemic agent. Extracts prepared from leaves have been used as an antiseptic agent for healing wounds and as a mouthwash to treat toothache. In Southeast Asia, leaves of Cerbera odollam are used in aromatic bath by women after childbirth.[10] Leaves, bark and latex are emetic and purgative, and seeds are toxic and
strongly purgative. Leaves and bark of Dyera costulata have been used for treating fever, inflammation, and pain.[11] Kopsia fruticosa has cholinergic effects and is used to treat sore and syphilis.[12] Nerium oleander is highly poisonous with no reported benefits in traditional medicine. In Asia, a decoction of leaves of Plumeria obtusa is used for treating wounds and skin diseases.[13] Its latex and bark are known to have purgative and diuretic properties. VaUaris glabra is well known in Thailand because the scent of its flowers is similar to that of pandan leaves and aromatic rice.[14] Its use in traditional medicine is not known.
Apocynaceae species have been reported to possess anticancer properties. They include those of A//amanda,[15] A/ston/a,[16'17] Calotropis,[1820] Catharanthus,[21] Cerbera,[22'23] Nerium,[24,25] P/umeria,[26] and Tabernaemontana.[27]
Prompted by the anticancer properties found in many species of Apocynaceae, leaf extracts of 10 species were assessed for antiproliferative activity against six human cancer cell lines. Their extracts were also analyzed for total alkaloid content, total phenolic content, and radical scavenging activity.
MATERIALS AND METHODS
Plant materials
The 10 Apocynaceae species studied were A. cathartica, A/stonia angusti/oba, Ca/otropis gigantea, Catharanthus roseus, Cerbera odo//am, Dyera costu/ata, Kopsia fruticosa, Nerium oleander,, P/umeria obtusa, and Va/larisg/abra [Figure 1]. Their common or vernacular names and brief descriptions are given in [Table 1]. Leaves of the species were collected from Sunway, Puchong, or Kepong, all in the state of Selangor, Malaysia. Identification of species was based on
documented descriptions and illustrations.[1,3] The voucher specimens of these species were deposited in the herbarium of Monash University Sunway Campus.
Extraction of leaves
For crude extraction, fresh leaves of each species (40 g) were cut into small pieces and freeze-dried overnight. Dried samples were blended and extracted with 250 ml of methanol (MeOH) three times for 1 h each time. Samples were filtered and the solvent was removed using a rotary evaporator (Eyela). The dried crude extracts were stored at —20oC for further analysis. For sequential extraction, fresh leaves of each species (40 g) were freeze-dried, ground, and extracted successively with hexane, dichloromethane (DCM), DCM:MeOH (1:1), and MeOH (HmbG Chemicals). For each solvent, the suspension of ground leaves in 250—300 ml of solvent was shaken for 1 h on the orbital shaker. After filtering, the samples were extracted two more times for each solvent. Solvents were removed with a rotary evaporator to obtain the dried extracts, which were stored at —20oC for further analysis.
Antiproliferative activity
Antiproliferative (APF) activity of extracts (25 ¡¡g/ml) was initially screened for growth inhibitory activity against three human cancer cell lines (MCF-7, MDA-MB-231, and HeLa) using the sulforhodamine B (SRB) assay.[28-30] Growth inhibitory activity with less than 50% cell growth was considered positive while that with more than 50% cell growth was considered negative. Extracts with positive growth inhibition were further tested against six human cancer lines (MCF-7, MDA-MB-231, HeLa, HT-29, SKOV-3, and HepG2) using six different extract concentrations. Human cancer cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The
Figure 1: The ten Apocynaceae species studied Pharmacognosy Research | April 2011 | Vol 3 | Issue 2
Table 1: Common or vernacular names and brief description of Apocynaceae species studied
Species (common/vernacular Brief description name)
Allamanda cathartica L. (Common allamanda) Alstonia angustiloba Miq. (Pulai) Calotropis gigantea (L.) Aiton (Giant milkweed) Catharanthus roseus (L.) G. Don (Madagascar periwinkle) Cerbera odollam Gaertner (Pong-pong)
Dyera costulata Hook (Jelutong)
Kopsia fruticosa (Ker.) A.
DC. (Pink kopsia)
Nerium oleander L. (Oleander)
Plumería obtusa L. (Frangipanni) Vallaris glabra Kuntze (Kesidang)
A shrub with trumpet-shaped yellow flowers similar in size as leaves which are in whorls
A medium-sized tree with leaves in whorls and having fine secondary veins A shrub with pale green leaves and white or lilac flowers with a crown rising from the center
A common ornamental shrub with oval to oblong leaves and white, pink or purple flowers with a dark colored center
A tree bearing white flowers in clusters and rounded fruits that are green when young and red when mature
A tall timber tree with straight columnar bole, leaves in whorls and latex which was an important source of chewing gum
A shrub with large glossy leaves and clusters of light pink flowers resembling those of Ixora
An ornamental shrub with thick narrow leaves in pairs or whorls and bearing clusters of pink, red, or purple flowers
A tree producing dark green, glossy and oval leaves and white fragrant flowers with a yellow center A woody climber producing clusters of white flowers with a scent characteristic of pandan leaves or fragrant rice
cells were seeded 24 h prior to treatment in 96-well plates at densities of 10,000—20,000 cells/well. Each cell line was designated one plate. Initial cell population of each cell lines prior to addition of extracts was determined by fixing with trichloroacetic acid (TCA) (Sigma). Extracts were dissolved in dimethyl sulfoxide (DMSO) (Sigma) and serially diluted from 8—25 fg/ml. Control cultures were treated with the same volume of DMSO. The concentration of DMSO was kept within 1% to avoid any interference with cell viability. After the addition of extracts, the plates were incubated for 48 h. After incubation, the cells were fixed with 50 fl of cold 50% TCA and incubated for 1 h at 4°C. The plates were then washed with tap water and air dried. Cells were stained with 100 fl of 0.4% SRB solution (Sigma) diluted with 1% acetic acid followed by incubation for 10 min at room temperature. Unbound dye was removed by washing with 1% acetic acid. Bound stain was then solubilized with 200 fl of 10 mM trizma base (Sigma). Absorbance of each well at 505 nm was obtained using a microplate reader. Dose—response curves were constructed to obtain GI50 or growth inhibition of cell lines by 50%. Activity is considered to be effective when GI50 value <20 ^g/ml.[31]
Analysis of TAC, TPC, and RSA
Total alkaloid content (TAC) of extracts was determined using the Dragendorff precipitation assay.[32] For each species, extracts (15 mg) were dissolved in 1 ml of distilled water that was acidified to pH 2.0-2.5 with 0.01 M HCl. Analysis was conducted in triplicate. Alkaloids were then precipitated with 0.4 ml of Dragendorff reagent. Washed with 0.5 ml of distilled water to remove traces of the reagent, the precipitate was later treated with 0.4 ml of 1% sodium sulfide, resulting in a brownish-black precipitate. Precipitates formed at each stage were recovered by centrifugation at 14,000 rpm for 1 min. The resulting
precipitate was dissolved in 0.2 ml of concentrated nitric acid and diluted to 1 ml with distilled water. Addition of 2.5 ml of 3% thiourea to 0.5 ml aliquots of this solution resulted in a yellow-colored complex. Absorbance was measured at 435 nm and TAC was expressed as boldine equivalent in milligram per gram of extract. The calibration equation for boldine (Sigma) was y = 1.068.x (R2 = 0.9959) wherey is absorbance and x is mg/ml of boldine. Dragendorff reagent was prepared by dissolving 0.8 g of bismuth nitrate (Sigma) in 40 ml of distilled water and 10 ml of glacial acetic acid. The resulting solution was mixed with 20 ml of 40% potassium iodide.
Total phenolic content (TPC) of extracts was determined using the Folin—Ciocalteu (FC) assay.[33] Extracts (300 fil in triplicate) were introduced into test tubes followed by 1.5 ml of FC reagent (Fluka) at 10 times dilution and 1.2 ml of sodium carbonate (Fluka) at 7.5% w/v. The tubes were allowed to stand for 30 min in the dark before absorbance was measured at 765 nm. TPC was expressed as gallic acid (GA) equivalent in milligram per gram of extract. The calibration equation for GA (Fluka) was y = 0.0111x - 0.0148 (R2 = 0.9998) where j is absorbance and x is mg/ml of GA.
Radical scavenging activity (RSA) of extracts was determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay.[33] Different dilutions of extracts (1 ml in triplicate) were added to 2 ml of DPPH (Sigma). The concentration of DPPH used was 5.9 mg in 100 ml of methanol. Absorbance was measured at 517 nm after 30 min. RSA was calculated as IC50, the concentration of extract to scavenge 50% of the DPPH radical. RSA was then expressed as ascorbic acid equivalent antioxidant capacity (AEAC) using the equation of AEAC (mg ascorbic
ic50 of
acld/g of extract) - IC50(ascorbate)/IC50(sample) X 105
ascorbic acid used for calculation of AEAC was 0.00387 mg/ml.
RESULTS AND DISCUSSION
Initial screening of leaf extracts of 10 Apocynaceae species against three human cancer cell lines (MCF-7, MDA-MB-231, and HeLa) showed that extracts of A. angusti/oba, C. gigantea, C. roseus, N. o/eander, P. obtusa, and V. g/abra displayed positive growth inhibitory activity, that is, inhibition with <50% cell growth [Table 2]. DCM extract of C. gigantea, and DCM and DCM:MeOH extracts of N. o/eander and V. g/abra inhibited all three cancer cell lines. All four extracts of N. o/eander were effective against MCF-7 cells. Only DCM:MeOH extract of C. roseus was active against MCF-7 and HeLa cells. Hexane extract of N. o/eander inhibited MCF-7 cells while hexane extract of P. obtusa inhibited MCF-7 and HeLa cells. MeOH extracts of N. o/eander and V. g/abra inhibited MCF-7 cells. In general, DCM and DCM:MeOH extracts of these species were the most effective. All extracts of A. cathartica, C. odo//am, D. costu/ata, and K. fruticosa did not show any APF activity.
Contrary to findings of this study, cytotoxic activities have been reported in species of Cerbera, A//amanda, and Kopsia. Methanol extract of leaves of C. odo//am strongly inhibited MCF-7 and T47D cells.[23] Cardenolides from seeds of C. odo/lam had cytotoxic activity against KB, BC, and NCI-H187 cells.[34] Potent cytotoxic activity was reported in ethanol extracts of fruits and leaves of Cerbera
Table 2: Leaf extracts of Apocynaceae species with positive growth inhibitory activity against three human cancer cell lines
Species Leaf extract3 Growth inhibitory activity1
MCF-72 MDA-MB-2312 HeLa2
Alstonia DCM - + +
angustiloba
Calotropis DCM + + +
gigantea DCM:MeOH + - +
Catharanthus DCM:MeOH + - +
roseus
Nerium HEX + - -
oleander DCM + + +
DCM:MeOH + + +
MeOH + - -
Plumeria HEX + - +
obtusa DCM - - +
Vallaris DCM + + +
glabra DCM:MeOH + + +
MeOH + - -
1Growth inhibitory activity with <50% cell growth is considered positive (+) while that with >50% cell growth is considered negative (-); 2MCF-7 and MDA-MB-231 are human breast cancer cells, and HeLa are human cervical cancer cells; 3HEX = Hexane, DCM = Dichloromethane, and MeOH = Methanol.
manghas.[35] Cardenolides from roots of C. manghas also showed APF activity.[22] Ethanol root extracts of A. schottii and A. b/anchetti displayed stronger cytotoxicity against K-562 cells than leaf and stem extracts.[15] Valparicine from the stem bark of Kopsia arborea showed cytotoxic effects against KB and Jurkat cells[36] while kopsimaline from leaves and stem bark of Kopsia singapurensis was found to inhibit KB cells.[37]
Extracts of the six species were further tested against six human cancer cell lines (MCF-7, MDA-MB-231, HeLa, HT-29, SKOV-3, and HepG2). Results showed that DCM extract of A. angusti/oba inhibited only MDA-MB-231, HeLa, and SKOV-3 cells with GI50 values of 20, 20, and 16 ¡g/ml, respectively [Table 3]. DCM extract of C. gigantea strongly inhibited all cancer cell lines with GI50 values ranging from 1.3—3.3 ¡¡g/ml. Against MCF-7 and MDA-MB-231 cells, GI50 of DCM extract of C. gigantea (1.9 and 1.3 ¡g/ml) was much stronger than that of xanthorrhizol (11 and 8.7 ¡¡g/ml), curcumin (4.1 and 8.7 ¡¡g/ml), and tamoxifen (8.3 and 4.6 ¡¡g/ml), respectively.[30] DCM:MeOH extract of C. roseus strongly inhibited MCF-7 and HeLa cells with GI50 of 3.5 and 4.7 ¡¡g/ml, respectively. All four extracts of N. o/eander were effective against MCF-7 cells with GI50 ranging from 3.7—12 ¡¡g/ml. DCM and DCM:MeOH extracts inhibited all cell lines except HepG2 cells. Hexane extract of P. obtusa was effective against MCF-7 and HeLa cells, while its DCM extract was effective against HeLa cells. DCM and DCM:MeOH extracts of Vg/abra inhibited all cell lines with GI50 values ranging from 7.5—12 ¡¡g/ml and 5.8—13 ¡g/ml, respectively. In addition, MeOH extract of V. g/abra also inhibited the growth of MCF-7 and HepG2 cells. Against MCF-7 cells, GI50 of DCM and DCM:MeOH extracts of V glabra (7.7 and 7.0 ¡g/ml) was stronger than xanthorrhizol (11 ¡¡g/ml) and comparable to tamoxifen (8.3 ¡¡g/ml), respectively.[30]
To the best of our knowledge, this study represents the first report of APF activities from leaf extracts of A. angusti/oba, P. obtusa, and V. g/abra. Earlier studies have reported cytotoxic activity from the root bark of A/stonia macrophy//a16] and from the stem bark of A/stonia scho/aris.[17] Iridoids isolated from the bark of P/umeria rubra were cytotoxic.[26] A recent study reported potent cell growth inhibition of cardenolide glycosides isolated from VaUaris so/anacea'P8] The finding of strong APF activities from DCM and DCM:MeOH extracts of C. gigantea from this study is supported by earlier reports that leaf and root extracts of C. gigantea had strong inhibitory activity against cancer cells.[18-20] Isolated from leaves of N. o/eander, pentacyclic triterpenoids were cytotoxic to KB cells.[24] Against HL60 and K562 cells, the stem extract of N. o/eander displayed stronger cytotoxic activity than leaf and root extracts.[39]
Table 3: Antiproliferative activity of leaf extracts of Apocynaceae species with positive growth inhibitory activity against six human cancer cell lines
Species Leaf extract3 Growth inhibition (GI50)1
MCF-72 MDA-MB-2312 HeLa2 SKOV-32 HT-292 HepG22
Alstonia angustiloba DCM - 20 ± 1.7 20 ± 1.1 16 ± 1.4 - -
Calotropis gigantea DCM 1.9 ± 0.2 1.3 ± 0.3 2.5 ± 0.5 2.5 ± 0.2 3.3 ± 0.2 1.8 ± 1.7
DCM:MeOH 13 ± 0.3 - 15 ± 1.0 20 ± 2.3 24 ± 0.7 16 ± 3.5
Catharanthus roseus DCM:MeOH 3.5 ± 0.1 - 4.7 ± 0.6 - - -
Nerium oleander HEX 11 ± 0.9 - - 12 ± 2.8 - -
DCM 3.7 ± 0.1 5.2 ± 0.7 5.1 ± 0.7 4.5 ± 1.0 6.1 ± 0.9 -
DCM:MeOH 4.3 ± 0.2 18 ± 2.3 6.8 ± 0.9 7.6 ± 2.1 9.2 ± 2.2 -
MeOH 12 ± 1.1 - - - - -
Plumeria obtusa HEX 5.7 ± 0.8 - 10 ± 1.4 - - -
DCM - - 19 ± 4.0 - - -
Vallaris glabra DCM 7.7 ± 1.3 12 ± 2.0 9.8 ± 1.5 7.5 ± 4.5 9.3 ± 2.0 7.6 ± 0.2
DCM:MeOH 7.0 ± 2.5 13 ± 6.3 8.5 ± 2.9 7.7 ± 2.4 12 ± 1.2 5.8 ± 1.2
MeOH 16 ± 2.1 - - - - 19 ± 0.9
*GI50 ([g/ml) is growth inhibition of cancer cell lines by 50% and inhibition is not effective (-) if values >20 [g/ml; 2MCF-7 and MDA-MB-231, HeLa and SKOV-3, HT-29, and HepG2 are human breast, cervical, colon, and liver cancer cells, respectively; 3HEX = Hexane, DCM = Dichloromethane, and MeOH = Methanol
It is interesting to note that out of four leaf extracts of C. roseus tested against six cell lines, only DCM:MeOH extract inhibited MCF-7 and HeLa cells. The species is well known for its indole alkaloids notably vinblastine and vincristine, which are used to treat Hodgkin's disease and acute leukemia in children, respectively.[1] It can be inferred that the APF activities of C. roseus may be cell line specific unlike those of C. gigantea, N. oleander, and V. glabra which are wide spectrum, inhibiting most or all cell lines tested.
Of the 10 species analyzed, MeOH crude and DCM extracts of K. fruticosa had the highest TAC (100 and 129 mg BE/g of extract), respectively [Table 4]. Other species with moderately high TAC were D. costulata, C. roseus, and A.. angustiloba with MeOH crude and DCM:MeOH extracts having values ranging from 23—58 and 58—68 mg BE/g of extract, respectively. Based on TAC, the species can be ranked as high (K. fruticosa), moderate (D. costulata,
C. roseus, and A. angustiloba), and low (C. odollam, C.gigantea, V.glabra, P. obtusa, A. cathartica, and N. oleander). Extracts of
D. costulata had the highest TPC and strongest RSA. MeOH crude, DCM:MeOH, and MeOH extracts of D. costulata yielded TPC values of 319, 354, and 279 mg GAE/g of extract, and RSA values of 377, 349, and 278 mg AA/g of extract, respectively. Compared to D. costulata, extracts of other species can be categorized as moderate to low.
The high TAC of leaf extracts of K. fruticosa may be attributed to the presence of alkaloids identified as fruticosamine, fruticosine, and kopsine.[40] The presence of flavonols identified as 3'7-dimethoxyquercetin and quercetin-3-0-a-L-rhamnopyranoside[11] may contribute to the high TPC and RSA of leaf extracts of D. costulata.
Overall, there is a strong correlation between TPC and RSA of extracts (R2 = 0.992) but not with TAC (R2 =
0.135). The strong correlation between TPC and RSA of extracts affirms that Apocynaceae species with higher concentration of phenolic compounds in the leaves also have stronger radical scavenging capacity. This would mean that the phenolic compounds are the main contributors to the antioxidant potential of leaves. Similar findings have been reported in medicinal plants and herbs,[41-43] plants of industrial interest,[44] wild edible fruits,[45] and mushrooms. [46] The correlation of results of phytochemical analysis with APF activities remains unclear. Extracts of C. gigantea, N. oleander, and V. glabra which showed strong APF activities had low TAC, and moderate to low TPC and RSA.
CONCLUSION
Out of 10 species of Apocynaceae, leaf extracts of A. angustiloba, C. gigantea, C. roseus, N. oleander, P. obtusa, and V. glabra displayed positive APF activities. DCM extract of C. gigantea, and DCM and DCM:MeOH extracts of V. glabra inhibited the growth of all six human cancer cell lines. Against MCF-7 and MDA-MB-231 breast cancer cells, DCM extracts of C. gigantea and N. oleander were stronger than or comparable to standard drugs of xanthorrhizol, curcumin, and tamoxifen. All four extracts of N. oleander were effective against MCF-7 cells. With wide-spectrum APF activities, leaves of these three species are therefore promising candidates as alternative resources for anticancer drugs. Their wide-spectrum APF activities are reported for the first time and this warrants further investigation into their bioactive compounds.
ACKNOWLEDGMENTS
The authors are thankful to Monash University Sunway Campus and Institute of Medical Research for funding and supporting
Table 4: Total alkaloid content, total phenolic content, and radical scavenging activity of leaf extracts of Apocynaceae species
Species MeOH crude extract1 Sequential extract1
HEX DCM DCM:MeOH MeOH
TAC (BE mg/g extract)2 Kopsia fruticosa Dyera costulata Catharanthus roseus Alstonia angustiloba Cerbera odollam Calotropis gigantea Vallaris glabra Plumeria obtusa Allamanda cathartica Nerium oleander TPC (GAE mg/g extract)2 Dyera costulata Vallaris glabra Plumeria obtusa Kopsia fruticosa Alstonia angustiloba Nerium oleander Catharanthus roseus Allamanda cathartica Cerbera odollam Calotropis gigantea RSA (AA mg/g extract)2 Dyera costulata Vallaris glabra Plumeria obtusa Kopsia fruticosa Nerium oleander Alstonia angustiloba Catharanthus roseus Allamanda cathartica Cerbera odollam Calotropis gigantea
100 ± 4.2 58 ± 2.5 37 ± 2.8
23 ± 0.3 2.8 ± 0.9 2.7 ± 0.3 2.7 ± 0.4
2.3 ± 0.3 2.0 ± 0.1
1.4 ± 0.7
319 ± 5.5 99 ± 3.8 85 ± 0.9
83 ± 1.1 68 ± 2.0 56 ± 3.8 53 ± 2.5 37 ± 0.8 30 ± 4.1 28 ± 0.8
377 ± 25
84 ± 0.5 66 ± 3.5 63 ± 3.2 42 ± 1.3 29 ± 0.9
24 ± 0.8 17 ± 1.1 15 ± 1.0 7.6 ± 0.4
63 ± 1.1 129 ± 4.0 99 ± 2.5 46 ± 1.6
2.4 ± 0.6 11 ± 1.8 68 ± 2.7 41 ± 2.4
18 ± 0.9 9.1 ± 0.8 76 ± 5.5 35 ± 3.3
13 ± 2.3 27 ± 3.1 58 ± 1.2 27 ± 1.0
3.6 ± 0.5 3.0 ± 0.5 5.0 ± 1.5 3.2 ± 0.4
3.7 ± 1.0 8.6 ± 1.2 9.6 ± 1.9 9.2 ± 2.7
4.4 ± 0.8 8.9 ± 0.6 9.2 ± 0.2 8.7 ± 2.2
2.3 ± 0.4 2.5 ± 0.1 4.3 ± 1.3 5.8 ± 1.7
4.4 ± 1.3 8.7 ± 0.9 12 ± 1.0 8.7 ± 2.1
1.6 ± 0.0 2.8 ± 0.8 3.9 ± 0.7 8.3 ± 0.5
21 ± 0.2 23 ± 0.1 354 ± 6.0 279 ± 3.0
15 ± 0.9 24 ± 0.5 134 ± 1.0 164 ± 13
21 ± 1.7 52 ± 1.3 104 ± 4.0 134 ± 3.0
39 ± 0.5 20 ± 0.5 129 ± 1.0 84 ± 1.0
17 ± 0.6 24 ± 0.6 96 ± 1.1 94 ± 1.1
18 ± 0.3 23 ± 0.1 57 ± 0.3 29 ± 0.3
24 ± 0.5 46 ± 1.4 75 ± 0.7 61 ± 1.1
11 ± 0.4 27 ± 0.2 39 ± 0.9 42 ± 0.5
19 ± 0.7 26 ± 0.5 30 ± 0.6 40 ± 0.9
14 ± 0.6 44 ± 1.7 42 ± 0.8 33 ± 0.5
15 ± 0.3 9.3 ± 0.5 349 ± 21 278 ± 5.4
6.0 ± 0.3 8.4 ± 0.4 77 ± 2.2 119 ± 6.8
4.9 ± 0.2 10 ± 0.9 58 ± 0.1 115 ± 5.3
12 ± 1.4 7.5 ± 0.3 70 ± 2.0 48 ± 1.5
6.4 ± 0.1 8.5 ± 0.6 48 ± 1.2 33 ± 0.2
10 ± 0.2 5.7 ± 0.7 50 ± 1.2 46 ± 2.2
13 ± 0.5 21 ± 0.5 33 ± 1.1 31 ± 0.3
11 ± 0.1 7.9 ± 0.8 18 ± 1.4 24 ± 2.0
16 ± 0.4 5.5 ± 0.1 14 ± 1.3 25 ± 0.7
5.6 ± 0.3 6.1 ± 0.4 8.0 ± 0.6 14 ± 1.3
1HEX = Hexane, DCM = Dichloromethane, and MeOH = Methanol; 2BE = Boldine equivalent, GAE = Gallic acid equivalent, AA = Ascorbic acid, TAC = Total alkaloid content, TPC = Total phenolic content, and RSA = Radical scavenging activity
the project. The assistance of Dr HT Chan (Former Division
Director, Forest Research Institute Malaysia) in identifying and
locating the plant species is gratefully acknowledged.
REFERENCES
1. Wiart C. Medicinal Plants of Asia and the Pacific. Boca Raton: CRC Press/Taylor and Francis; 2006.
2. Endress ME, Bruyns PV. A revised classification of the Apocynaceae. Bot Rev 2000;66:1-56.
3. Ng FS. Tropical Horticulture and Gardening. Kuala Lumpur, Malaysia: Clearwater Publications; 2006.
4. Rahayu SS. Allamanda cathartica L. In: Valkenburg JL, Bunyapraphatsara N, editors. Plant Resources of South-East Asia No. 12(2): Medicinal and poisonous plants 2. Leiden, Netherlands: Backhuys Publisher; 2001. p. 51.
5. Mulyoutami E, Rismawan R, Joshi L. Local knowledge and management of simpukng (forest gardens) among the Dayak people in East Kalimantan, Indonesia. For Ecol Manage 2009;257:2054-61.
6. Lin KW. Ethnobotanical study of medicinal plants used by the Jah Hut people in Malaysia. Indian J Med Sci 2005;59:156-61.
7. Rajakaruna N, Harris CS, Towers GH. Antimicrobial activity of
plants collected from serpentine outcrops in Sri Lanka. Pharm Biol 2002;40:235-44.
8. Nalwaya N, Pokharna G, Deb L, Jain NK. Wound healing activity of latex of Calotropis gigantea. Int J Pharm Pharm Sci 2009;1:176-81.
9. Sutarno H, Rudjiman SU. Catharanthus roseus (L.) G. Don. In: de Padua LS, Bunyapraphatsara N, Lemmens RH, editors. Plant Resources of South-East Asia No. 12(1): Medicinal and poisonous plants 1. Leiden, Netherlands: Backhuys Publisher; 1999. p. 185-90.
10. Khanh TC. Cerbera odollam Gaertner. In: Valkenburg JL, Bunyapraphatsara N, editors. Plant Resources of South-East Asia No. 12(2): Medicinal and poisonous plants 2. Leiden, Netherlands: Backhuys Publisher; 2001. p. 154-5.
11. Subhadhirasakul S, Jankeaw B, Malinee A. Chemical constituents and antioxidative activity of the extract from Dyera costulata leaves. Songkla J Sci Technol 2003;25:35-7.
12. Johnson T. CRC Ethnobotany Desk Reference. CRC Press LLC; 1999.
13. Burkill IH. A Dictionary of the Economic Products of the Malay Peninsula. Vol 2. (I-Z). London: Crown Agents for the Colonies; 1935.
14. Wongpornchai S, Sriseadka T, Choonvisase S. Identification and quantitation of the rice aroma compound, 2-acetyl-1-
pyrroline, in bread flowers (Vallaris glabra Ktze). J Agric Food Chem 2003;51:457-62.
15. Schmidt DF, Yunes RA, Schaab EH, Malheiros A, Filho VC, Franchi GC Jr, et al. Evaluation of the anti-proliferative effect of extracts of Allamanda blanchetti and A. schottii on the growth of leukemic and endothelial cells. J Pharm Pharm Sci 2006;9:200-8.
16. Keawpradub N, Eno-Amooquaye E, Burke PJ, Houghton PJ. Cytotoxic activity of indole alkaloids from Alstonia macrophylla. Planta Med 1999;65:311-5.
17. Jagetia GC, Baliga MS. Evaluation of anticancer activity of the alkaloid fraction of Alstonia scholaris (Sapthaparna) in vitro and in vivo. Phytother Res 2006;20:103-9.
18. Lhinhatrakool T, Sutthivaiyakit S. 19-Nor- and 18,20-epoxy-cardenolides from the leaves of Calotropis gigantea. J Nat Prod 2006;69:1249-51.
19. Wang ZH, Wang MY, Mei WL, Han Z, Dai HF. A new cytotoxic pregnanone from Calotropis gigantea. Molecules 2008;13:3033-9.
20. Seeka C, Sutthivaiyakit S. Cytotoxic cardenolides from the leaves of Calotropis gigantea. Chem Pharm Bull 2010;58:725-8.
21. Siddiqui MJ, Ismail, Z, Aisha AF, Abdul Majid AM. Cytotoxic activity of Catharanthus roseus (Apocynaceae) crude extracts and pure compounds against human colorectal carcinoma cell line. Int J Pharmacol 2010;6:43-7.
22. Chang LC, Gills JJ, Bhat KP, Luyengi L, Farnsworth NR, Pezzuto JM, et al. Activity-guided isolation of constituents of Cerbera manghas with anti-proliferative and anti-estrogenic activities. Bioorg Med Chem Lett 2000;10:2431-4.
23. Nurhanan MY, Asiah O, Mohd Ilham MA, Siti Syarifah MM, Norhayati I, Lili Sahira H. Anti-proliferative activities of 32 Malaysian plant species in breast cancer cell lines. J Trop For Sci 2008;20:77-81.
24. Siddiqui BS, Begum S, Siddiqui S, Lichter W. Two cytotoxic pentacyclic triterpenoids from Nerium oleander. Phytochemistry 1995;39:171-4.
25. Pathak S, Multani AS, Narayan S, Kumar V, Newman RA. Anvirzel™1, an extract of Nerium oleander, induces cell death in human but not murine cancer cells. Anticancer Drugs 2000;11:455-63.
26. Kardono LS, Tsauri S, Padmawinata K, Pezzuto JM, Kinghorn AD. Cytotoxic constituents of the bark of Plumeria rubra collected in Indonesia. J Nat Prod 1990;53:1447-55.
27. Lee CC, Houghton P. Cytotoxicity of plants from Malaysia and Thailand used traditionally to treat cancer. J Ethnopharmacol 2005;100:237-43.
28. Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, et al. New colorimetric cytotoxicity assay for anticancer drug screening. J Natl Cancer Inst 1990;82:1107-12.
29. Vichai V, Kirtikara K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat Protoc 2006;1:1112-6.
30. Cheah YH, Nordin FJ, Tee TT, Azimahtol HL, Abdullah NR, Ibrahim Z. Antiproliferative property and apoptotic effect of xanthorrhizol on MDA-231 breast cancer cells. Anticancer Res 2008;28:3677-90.
31. Gaidhani SN, Lavekar GS, Juvekar AS, Sen S, Singh A, Kumari S. In-vitro anticancer activity of standard extracts used in Ayurveda. Phcog Mag 2009;5:425-9.
32. Ribeiro B, Lopes R, Andrade PB, Seabra RM, Goncalves RF, Baptista P, et al. Comparative study of phytochemicals and
antioxidant potential of wild edible mushroom caps and stipes. Food Chem 2008;110:47-56.
33. Wong SK, Lim YY, Chan EW. Antioxidant properties of Hibiscus: Species variation, altitudinal change, coastal influence and floral colour change. J Trop For Sci 2009;21:307-15.
34. Laphookhieo S, Cheenpracha S, Karalai C, Chantrapromma S, Rat-a-pa Y, Ponglimanont C, et al. Cytotoxic cardenolide glycoside from the seeds of Cerbera odollam. Phytochemistry 2004;65:507-10.
35. Ali AM, Mackeen MM, Ei-Sharkawy SH, Hamid JA, Ismail NH, Ahmad FB, et al. Antiviral and cytotoxic activities of some plants used in Malaysian indigenous medicine. Pertanika J Trop Agric Sci 1996;19:129-36.
36. Lim KH, Hiraku O, Komiyama K, Koyano T, Hayashi M, Kam TS. Biologically active indole alkaloids from Kopsia arborea. J Nat Prod 2007;70:1302-7.
37. Subramaniam G, Hiraku O, Hayashi M, Koyano T, Komiyama K, Kam TS. Biologically active aspidofractinine alkaloids from Kopsia singapurensis. J Nat Prod 2008;71:53-7.
38. Ahmed F, Sadhu SK, Ohtsuki T, Khatun A, Ishibashi M. Glycosides from Vallaris solanacea with TRAIL resistance overcoming activity. Heterocycles 2010;80:477-88.
39. Turan N, Akgün-Dar K, Kuruca SE, Kilipaslan-Ayna T, Seyhan VG, Atasever B, et al. Cytotoxic effects of leaf, stem and root extracts of Nerium oleander on leukemia cell lines and role of the p-glycoprotein in this effect. J Exp Ther Oncol 2006;6:31-8.
40. Kam TS, Lim KH. Alkaloids of Kopsia. In: Cordell GA, editor. The Alkaloids: Chemistry and Biology. Amsterdam, Netherlands: Elsevier Inc; 2008. p. 1-111.
41. Akinmoladun AC, Obuotor EM, Farombi EO. Evaluation of antioxidant and free radical scavenging capacities of some Nigerian indigenous medicinal plants. J Med Food 2010;13:444-51.
42. Guo DJ, Cheng HL, Chan SW, Yu PH. Antioxidative activities and the total phenolic contents of tonic Chinese medicinal herbs. Inflammopharmacology 2008;16:201-7.
43. Raj JX, Bajpjpai PK, Kumar PG, Murugan PM, Kumar J, Chaurasia OP, et al. Determination of total phenols, free radical scavenging and antibacterial activities of Mentha longifolia Linn. Hudson from the Cold Desert, Ladakh, India. Phcog J 2010;2:470-5.
44. Dudonne S, Vitrac X, Coutiere P, Woillez M, Merillon JM. Comparative study of antioxidant properties and total phenolic content of 30 plant extracts of industrial interest using DPPH, ABTS, FRAP, SOD, and ORAC assays. J Agric Food Chem 2009;57:1768-74.
45. Lamien-Meda A, Lamien CE, Compaore MM, Meda RN, Kiendrebeogo M, Zeba B, et al. Polyphenol content and antioxidant activity of fourteen wild edible fruits from Burkina Faso. Molecules 2008;13:581-94.
46. Cheung LM, Cheung PC, Ooi VE. Antioxidant activity and total phenolics of edible mushroom extracts. Food Chem 2003;8: 249-55.
Cite this article as: Wong SK, Lim YY, Abdullah NR, Nordin FJ. Antiproliferative and phytochemical analyses of leaf extracts of ten Apocynaceae species. Phcog Res 2011;3:100-6.
Source of Support: Monash University Sunway Campus and Institute of Medical Research, Conflict of Interest: None declared.
Copyright of Pharmacognosy Research is the property of Medknow Publications & Media Pvt. Ltd. and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.
