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BMC Complementary and Alternative Medicine

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Cell cycle arrest and apoptosis induction by methanolic leaves extracts of four Annonaceae plants

  • Kitti Pumiputavon1,
  • Tanawat Chaowasku1,
  • Chalermpong Saenjum2,
  • Maslin Osathanunkul1,
  • Boonsong Wungsintaweekul3,
  • Kriangkrai Chawansuntati4,
  • Jiraprapa Wipasa4Email author and
  • Pathrapol Lithanatudom1Email author
Contributed equally
BMC Complementary and Alternative MedicineBMC series – open, inclusive and trusted201717:294

https://doi.org/10.1186/s12906-017-1811-3

Received: 23 March 2017

Accepted: 26 May 2017

Published: 5 June 2017

Abstract

Background

Uvaria longipes (Craib) L.L.Zhou, Y.C.F.Su & R.M.K.Saunders, Artabotrys burmanicus A.DC, Marsypopetalum modestum (Pierre) B.Xue & R.M.K.Saunders and Dasymaschalon sp. have been used for traditional medicine to treat cancer-like symptoms in some ethnic groups of Thailand and Laos.

Methods

We evaluated the anti-cancer activity of these Annonaceae plants against several human cancer cell lines. The apoptosis induction was detected by Annexin/propidium iodide (PI) staining. Phytochemical screening was tested by standard protocols and bioactive compounds were determined by HPLC.

Results

The crude extracts from leaves of U. longipes, Dasymaschalon sp., A. burmanicus, and M. modestum showed particular effects that were found to vary depending on the cancer cell line, suggesting that the effect was in a cell-type specific manner. Interestingly, the induction of apoptotic cell death was prominent by the leaves-derived crude extract of M. modestum. This crude was, therefore, subjected to cell cycle analysis by PI staining. Results showed that this crude extract arrested cell cycle and increased the percentage of cells in the SubG1 phase in some cancer cell lines. The phytochemical screening tests indicated that all crude extracts contained tannins and flavonoids. HPLC of flavonoids using standards identified rutin as an active compound in U. longipes and Dasymaschalon sp., whereas quercetin was found in U. longipes and M. modestum.

Conclusions

These crude extracts provide a new source for rutin and quercetin, which might be capable of inducing cancer cell apoptotic death in a cell-type specific manner. This suggests, by analyzing the major bioactive compounds, the potential use of these crudes for chemotherapy in the future.

Keywords

AnnonaceaeAnti-cancer activityCell cycle arrestApoptosisQuercetinRutin

Background

Cancer refers to a group of cells with growth disorder arising from aberrant cell division. The resulting phenotypes involve uncontrollable cell growth with the possibility of the disorder spreading throughout the body [1]. To date, more than 100 types of cancer have been reported [2]. A number of studies have shown that cancer is one of the primary causes of death worldwide, second only to cardiovascular disorders [3]. Furthermore, morbidity and mortality associated with cancer are expected to increase annually, thus becoming a serious global public health problem.

Accumulating evidence suggests that development of cancer is mostly triggered by external factors or environmental factors (90%). These include smoking (30%), diet and obesity (35%), infection (20%), radiation (10%), and stress and environmental pollution (5%), while the genetic factor contributes only 10% to the cause of cancer [1]. Definitive cancer treatment is crucial and urgently required for future management of the disease. Traditional remedial methods of cancer, comprising surgery, chemotherapy, and radiotherapy, although proven to be effective, have several major drawbacks such as detrimental side effects, cancer resistance, and relapses [4]. Hence, acquisition of novel alternative cancer treatment methods is necessary.

Induction of apoptosis, a programmed cell death mechanism for wiping out unwanted cells in tissue, is one of the effective strategies to kill cancer cells [5]. This procedure is mainly associated with morphological change, heterochromatin condensation, cell shrinkage and budding, loss of organelles in the cytoplasm, and formation of apoptotic bodies [6]. Apoptosis can be induced through extrinsic and/or intrinsic pathways. The extrinsic pathway is initiated by the interaction of death receptors and specific signaling molecules, while the intrinsic pathway is primarily stimulated by cellular sensing of extracellular and/or intracellular stresses, both of which require appropriate stimuli to trigger [7]. Therefore, continual pursuit of natural compounds having the ability to trigger apoptosis pathways in cancer cells is currently gathering much interest and becoming more attractive in the field of oncology [8].

The Annonaceae is a tropical plant family of trees, shrubs, and lianas. There are 109 validly described and recognized genera and approximately 2440 species in this family [9]. Interestingly, numerous bioactive compounds have been isolated from the Annonaceae [10]. Alkaloids are among the most important natural compounds of the Annonaceae family. A previous study has reported the isolation of isoquinoline alkaloids from this family [11]. Additionally, terpenoids [12] and acetogenins [13] can also be isolated from some species of the Annonaceae family. Certain types of alkaloids, such as jerantinine B [14], liriodenine [15], and vinoreline [16], exhibit the ability to induce apoptosis and block the cell cycle in the G1 phase. Moreover, rutin and squamocin B were reported as bioactive flavonoids in Annona squamosal [17]. There are plenty of species from the Annonaceae waiting to be studied with the potential to uncover novel anti-cancer bioactivity compounds.

In this study, we used three groups of cancer cell lines representing the three major cancer types observed with significantly high incidence worldwide, including human cervical carcinoma, human hepatocellular carcinoma, and human hematopoietic cell lines, as in vitro experimental models to evaluate the anti-cancer activity of four genera (four species) of the Annonaceae, viz. Uvaria longipes (Craib) L.L.Zhou, Y.C.F.Su & R.M.K.Saunders, Dasymaschalon sp., Artabotrys burmanicus A.DC., and Marsypopetalum modestum (Pierre) B.Xue & R.M.K.Saunders. These species were chosen because there has been no previous study on anti-cancer activity, but certain species in the genus Uvaria L. [18, 19], Dasymaschalon Dalla Torre & Harms [20, 21], and Artabotrys R.Br. [22] had been shown to exhibit considerable anti-cancer activities. High performance liquid chromatography (HPLC) was also performed to identify bioactive components in all studied crude extracts.

Methods

Chemicals

RPMI (Roswell Park Memorial Institute) medium, FBS (fetal bovine serum), Penicillin–Streptomycin, L-Glutamine, Fungizone, and 0.25% Trypsin-EDTA were purchased from Gibco-BRL, USA. Annexin V-FITC (fluorescein isothiocyanate) was purchased from ImmunoTools GmbH, Germany. HEPES was purchased from Merck Millipore, Germany. Sodium chloride, sodium bicarbonate, and calcium chloride were purchased from RCI LABSCAN, Thailand. Dimethyl sulfoxide (DMSO), Triton X-100, and propidium iodide (PI) were purchased from Sigma-Aldrich, USA. Ribonuclease A (RNase A) was purchased from Worthington Biological Corporation, USA. All solvents and chemicals used were either HPLC grade or analytical grade and were purchased commercially from Sigma Chemical Co. (St. Louis, MO), Fluka Chemical Co. (Switzerland), and Merck (Darmstadt, Germany).

Cell lines and culture

The human cancer cell lines used in this study consisted of human cervical carcinoma (HeLa, SiHa, and CaSki) (a kind gift from Assoc. Prof. Tipaya Ekalaksananan, Khon Kaen University, Thailand), human hepatocellular carcinoma (HepG2 and Hep3B) (a kind gift from Prof. Duncan R. Smith, Mahidol University, Thailand), and human myeloid leukemia (K562, U937, and RAJI) (a kind gift from Prof. Sumalee Tungpradabkul, Mahidol University, Thailand). All the cell lines were maintained in the RPMI medium containing 10 mM of HEPES, 1 mM of sodium bicarbonate, 10% fetal bovine serum (FBS), penicillin (100 IU/ml), and streptomycin (100 μg/ml) (RPMI complete media) at 37 °C in a humidified 5% CO2 atmosphere.

Plant materials

Uvaria longipes (collection no.: Chaowasku 132), Dasymaschalon sp. (collection no.: Chaowasku 120), and Marsypopetalum modestum (collection no.: Chaowasku 164) were collected from private residences at coordinates 13.790384, 100.372378; and Artabotrys burmanicus (collection no.: Chaowasku 163) was collected from a private garden at coordinates 13.919300, 99.952555. All the voucher specimens were deposited in the Chiang Mai University Biology (CMUB) herbarium. It should be noted that the Dasymaschalon sp. used in our study is roughly identified as Dasymaschalon lomentaceum and is currently authenticated for its potentially new species as evidenced by both morphological and molecular data (manuscript in preparation). The collection, preparation and identification of all plant specimens used in this study was performed by Dr. Tanawat Chaowasku.

Preparation of crude extracts

Leaves of these plants were washed, air dried at 25-30 °C and crushed to powder. Dried and powdered leaves of U. longipes, Dasymaschalon sp., A. burmanicus and M. modestum were incubated in methanol at room temperature for 24 h. Then, the solution was collected and evaporated under vacuum in a rotary evaporator. All the methanolic extracts were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 100 mg/ml. Finally, these extracts were serially diluted into 1000 μg/ml, 500 μg/ml, 250 μg/ml, and 125 μg/ml in RPMI complete media.

Annexin V staining assay

Human cancer cell lines (HeLa, SiHa, CaSki, HepG2, Hep3B, K562, U937, and RAJI) at 1 × 105 cells/ml were cultured in 24-well plates in the presence of various concentrations of methanolic extracts (1000 μg/ml, 500 μg/ml, 250 μg/ml, and 125 μg/ml) for 24 h. The quantification of the apoptotic cells was measured by Annexin V-FITC (fluorescein isothiocyanate)/PI (propidium iodide) co-staining assay. Briefly, at the end of the 24 h incubation, the cells were harvested and centrifuged at 1800 rpm for 8 min. The pellet was resuspended in 50 μl binding buffer containing 0.5 μl Annexin V-FITC and then incubated at 4 °C for 30 min in the dark. PI (50 μg/ml) in 200 μl binding buffer was added to each of the tubes and incubated for 5 min. Finally, the cells were analyzed by flow cytometry (CyAn ADP Analyzer, Beckman Coulter, USA).

Cell cycle analysis

Human cancer cell lines at 1 × 106 cells were cultured in 6-well plates in the presence of leaves of M. modestum methanolic extracts (1000 μg/ml, 500 μg/ml, 250 μg/ml, and 125 μg/ml) for 24 h. After treatment, the cells were washed and centrifuged at 1800 rpm for 8 min. The cells were resuspended and fixed with 70% ethanol at 4 °C for 2 h. After fixing, the cells were washed with PBS and centrifuged at 1800 rpm for 8 min. The pellet was broken up by vortexing and then resuspended in 250 μl PBS containing PI (20 μg/ml), RNase A (20 μg/ml), and Triton X-100 (0.1%), and incubated for further 30 min in the dark. Finally, the cells were analyzed by flow cytometry (BD FACSCalibur, BD Biosciences, USA).

Phytochemical screening tests

The four crude leaf extracts were tested for constituents such as alkaloids, sterols, cardiac glycosides, anthaquinone glycosides, saponins, flavonoids, and tannins by using standard methods as described previously [2325]. The qualitative results are expressed as (−) for the absence and (+) for the presence of constituents.

Determination of rutin and quercetin by reversed-phase HPLC

The content of quercetin and rutin in the four crude extracts was determined by using a reversed-phase HPLC (RP-HPLC) system (Shimadzu Corporation, Japan) including LC-10AV VP pumps and SPD-10AV VP with UV detector. The column for the separation was 250 × 4.6 mm in diameter (SymmetryShield® RP18 C18; Water Co., Ltd.). The mobile phases used for the determination of quercetin and rutin consisted of 5 mM KHPO4:acetonitrile:methanol in the ratio of 49:40:11% v/v and de-ionized H2O:Methanol:Triethylamine in the ratio of 60:40:0.1, respectively. The flow rate and the detection wavelengths for quercetin and rutin were 0.7 and 0.5 mL/min, and 350 and 256 nm, respectively. All the crude extracts were subjected to the RP-HPLC system in parallel with known concentrations of quercetin dihydrate and rutin (GmBH, Germany). The concentrations of quercetin and rutin were calculated from the peak area using the calibration curves. The assays were performed in triplicates.

Statistical analysis

Statistical analysis was performed using statistical analysis program (SPSS, 16.0, International Business Machines, USA). Comparisons between groups (controls and treatments) were performed by one-way ANOVA with Tukey’s HSD post hoc test. Statistical significance was accepted at P value lower than 0.05.

Results

Apoptosis assessment

To investigate the anti-cancer activity of the crude extracts on eight human cancer cell lines, we employed Annexin V and PI co-staining to examine possible induction of cell death (necrosis and/or apoptosis). The percentages of cells analyzed by flow cytometry following Annexin V and PI staining could be classified into four categories. The populations of cells residing in the Annexin V+/PI- and the Annexin V+/PI+ quadrants were determined as early and late apoptotic cells, respectively. The Annexin V−/PI - and the Annexin V−/PI+ quadrants were determined as living cells and necrotic cells, respectively. Both the untreated control (UT-C) and the DMSO-treated cells (DMSO-C) showed the percentages of the apoptotic cells as ranging from 3% to 12% after 24 h of incubation (Table 1). All treatments of crude extracts (leaves from Uvaria longipes: LUL, Dasymaschalon sp.: LD, Artabotrys burmanicus: LAB, and Marsypopetalum modestum: LMM) showed varying degrees of percentages of apoptotic cells (the percentages of the early apoptotic cells and the late apoptotic cells combined) depending on the type of crude extract. Consistently, changes in cell morphology were observed under an inverted microscope. This was corroborated by the decreases in the intensity of the forward light scatter when analyzed by flow cytometry (data not shown). Interestingly, the apoptotic effects from individual crude extracts exhibited activity in a cell-type specific manner. For example, the percentages of apoptotic HepG2 cells treated with LUL were significantly higher than those of SiHa cells treated with the same extract (Table 1). A dose-dependent manner was also observed in some crude extracts in particular cell types, for example, RAJI cells treated with LAB and LMM (Table 1 and Fig. 1). In our study, the most prominent apoptotic effect on cancer cell lines was observed in LMM and LAB crude extracts (Table 1), particularly against the RAJI cell line (Fig. 1). The half maximal inhibitory concentrations (IC50) of all crude extracts on cell death are shown in Table 2.
Table 1

The average (±SD) percentages of apoptotic cells identified from the dot plots of Annexin V vs PI with statistical analysis

 

Concentrations of crude extracts (μg/ml)

Controls

1000

500

250

125

UT-Ca

DMSO-Cb

HeLa

LULc

50.9 ± 4.8**

45.3 ± 1.7**

37.3 ± 3.7**

16.2 ± 6.8**

3.1 ± 1.5

3.2 ± 1.3

LDd

22.8 ± 1.6**

22.9 ± 1.7**

22.1 ± 0.0**

17.9 ± 0.5**

  

LABe

13.4 ± 2.1**

6.0 ± 1.0**

5.9 ± 2.7*

6.6 ± 1.9**

  

LMMf

70.2 ± 5.0**

33.7 ± 6.9**

2.8 ± 0.4

2.1 ± 0.1

  

SiHa

LULc

17.0 ± 1.3**

30.0 ± 2.0**

18.8 ± 1.3**

7.9 ± 0.0*

5.2 ± 1.7

8.6 ± 1.4

LDd

93.1 ± 1.3**

77.1 ± 2.1**

19.2 ± 0.1**

3.2 ± 0.4**

  

LABe

87.9 ± 4.8**

84.5 ± 3.7**

37.2 ± 6.2**

4.5 ± 0.4**

  

LMMf

96.6 ± 0.1**

15.3 ± 1.5**

3.5 ± 1.1**

3.9 ± 0.2**

  

CaSki

LULc

37.5 ± 1.5**

16.1 ± 3.7**

20.6 ± 0.4**

6.0 ± 0.8

6.0 ± 0.2

6.4 ± 1.4

LDd

88.2 ± 1.8**

77.9 ± 2.2**

51.9 ± 4.4**

7.6 ± 0.0

  

LABe

89.5 ± 4.6**

51.9 ± 3.1**

38.0 ± 1.0**

3.9 ± 0.6**

  

LMMf

76.1 ± 2.6**

34.4 ± 0.8**

5.6 ± 0.4

7.0 ± 0.9

  

HepG2

LULc

87.1 ± 4.7**

65.2 ± 1.1**

36.7 ± 2.2**

38.9 ± 2.4**

11.1 ± 6.6

12.4 ± 6.6

LDd

84.6 ± 3.8**

75.1 ± 1.9**

45.0 ± 0.3**

42.8 ± 3.8**

  

LABe

80.1 ± 2.4**

70.5 ± 4.6**

76.7 ± 0.4**

16.4 ± 0.4

  

LMMf

93.7 ± 2.1**

56.5 ± 4.0**

46.1 ± 7.3**

40.2 ± 2.6**

  

Hep3B

LULc

51.1 ± 0.1**

27.3 ± 2.0**

12.0 ± 0.9**

10.5 ± 1.0**

8.1 ± 0.6

6.9 ± 0.9

LDd

43.1 ± 2.5**

13.3 ± 7.9*

7.5 ± 2.6

5.9 ± 0.3

  

LABe

54.2 ± 2.3**

57.6 ± 3.6**

43.0 ± 1.3**

36.8 ± 1.3**

  

LMMf

45.6 ± 6.9**

14.0 ± 2.2**

7.0 ± 8.6

9.1 ± 8.4

  

K562

LULc

44.2 ± 0.7**

8.2 ± 0.0**

6.7 ± 1.1

6.8 ± 0.9

5.6 ± 1.3

5.0 ± 0.1

LDd

25.9 ± 4.2**

7.4 ± 0.7**

7.5 ± 2.1

5.6 ± 0.1

  

LABe

77.1 ± 6.6**

37.3 ± 3.2**

22.9 ± 1.7**

7.3 ± 1.6

  

LMMf

79.6 ± 1.3**

13.5 ± 1.3**

5.7 ± 0.6

5.5 ± 0.7

  

U937

LULc

94.2 ± 0.9**

10.1 ± 0.0**

6.0 ± 1.7

6.2 ± 0.0

7.4 ± 1.4

5.7 ± 0.7

LDd

88.8 ± 0.3**

8.8 ± 0.4*

8.0 ± 0.3

7.4 ± 1.2

  

LABe

29.1 ± 4.1**

8.6 ± 4.2*

5.4 ± 0.9

6.2 ± 0.2

  

LMMf

84.6 ± 3.0**

54.9 ± 1.2**

5.3 ± 4.9

5.0 ± 3.7

  

RAJI

LULc

71.8 ± 3.4**

32.6 ± 1.6**

29.9 ± 2.6**

23.8 ± 0.5**

5.6 ± 2.2

7.0 ± 3.1

LDd

81.0 ± 2.9**

26.9 ± 0.3**

21.1 ± 0.2**

19.4 ± 2.2**

  

LABe

98.6 ± 0.1**

93.3 ± 0.6**

41.1 ± 0.4**

24.5 ± 6.1**

  

LMMf

98.5 ± 0.3**

88.5 ± 3.0**

33.7 ± 1.0**

25.3 ± 2.4**

  

Symbols * and ** represent statistically significant differences less than 0.05 and 0.01, respectively, when compared to DMSO-treated control

aUntreated control

bDMSO-treated control

cCrude extract from leaf of U. longipes

dCrude extract from leaf of Dasymaschalon sp

eCrude extract from leaf of A. burmanicus

fCrude extract from leaf of M. modestum

Fig. 1

Effect of crude extracts from the leaves of U. longipes a, Dasymaschalon sp. b, A. burmanicus c, and M. modestum d on the induction of apoptosis in the sample RAJI cell. RAJI cells were cultured in the presence of various concentrations of the crude extracts for 24 h. The cells were then stained with Annexin V and PI, and analyzed by flow cytometry

Table 2

IC50 values of leaf crude extracts against cancer cell lines

 

IC50 value (μg/ml)

HeLa

SiHa

CaSki

HepG2

Hep3B

K562

U937

RAJI

LULa

661.16

2985.13

1219.53

386.32

394.94

1262.15

620.56

621.21

LDb

1370.02

590.28

465.71

329.88

1125.20

2364.85

1720.44

645.34

LABc

796.46

370.50

298.29

79.15

16.45

419.86

441.49

329.83

LMMd

445.91

619.31

669.85

358.68

871.80

726.92

716.84

362.51

aCrude extract from leaf of U. longipes.

bCrude extract from leaf of Dasymaschalon sp

cCrude extract from leaf of A. burmanicus.

dCrude extract from leaf of M. modestum.

Table 1. Cancer cell lines were incubated for 24 h in the presence of 125–1000 μg/ml of crude extracts (leaf of U. longipes: LUL, leaf of Dasymaschalon sp.: LD, leaf of A. burmanicus: LAB, and leaf of M. modestum: LMM). Untreated cells (UT-C) and DMSO-treated cells (DMSO-C) were used as the controls. Statistical comparisons were analyzed by the one-way ANOVA with Tukey’s HSD post hoc test.

Table 2. IC50 values of leaf crude extract against cancer cell lines was calculated from all types of cell death (Annexin V+/PI-, Annexin V−/PI+ and the Annexin V+/PI+ quadrants).

Effect of LMM on cell cycle analysis

To test whether the apoptotic cell death was due to the inhibitory effect of the crude extracts on the cell cycle process, cell cycle analysis was then carried out. Although both LMM and LAB showed maximum potent effect on most of the cancer cell lines, LMM was chosen for the cell cycle analysis since there have been no reports on its anti-cancer activity, whereas the crude extract from A. siamensis (closely related to A. burmanicus) was reported having inhibitory effect on three other cell lines [26]. Furthermore, A. burmanicus is a rare species in the genus Artabotrys and resources for raw material were limited, making it difficult to study for therapeutic uses in the long run. Cancer cell lines were treated with various concentrations of LMM for 24 h, stained with propidium iodide, and subsequently analyzed by flow cytometry. All the cancer cell lines except for SiHa and Hep3B showed an increase in the DNA fragmentation in a dose-dependent manner as reflected by the increase in the percentages of cells in the sub G1 phase compared to the untreated or the DMSO-treated cells (Table 3 and Fig. 2).
Table 3

The average (±SD) percentages of cells in the sub-G1 phase obtained from the histogram of PI staining with statistical analysis

 

Concentration of crude extracts (μg/ml)

Controls

1000

500

250

125

UT-Ca

DMSO-Cb

HeLa

11.2 ± 1.4**

4.0 ± 0.0

3.9 ± 0.3

4.8 ± 0.1

4.0 ± 1.2

6.2 ± 2.7

SiHa

3.5 ± 0.5**

2.4 ± 0.1**

1.0 ± 0.0

1.0 ± 0.3

0.6 ± 0.0

0.7 ± 0.0

CaSki

46.7 ± 0.5**

11.3 ± 1.2**

10.0 ± 1.8**

8.5 ± 1.2

5.6 ± 1.3

6.6 ± 0.5

HepG2

17.2 ± 1.0**

12.6 ± 1.7**

6.9 ± 1.6**

7.1 ± 1.9**

6.2 ± 1.6

2.8 ± 0.7

Hep3B

7.9 ± 1.6**

6.5 ± 1.1*

5.3 ± 0.0*

6.5 ± 0.4*

4.2 ± 4.2

5.4 ± 0.8

K562

46.7 ± 0.5**

11.3 ± 1.2**

10.0 ± 1.8**

8.5 ± 1.2

5.6 ± 1.3

6.6 ± 0.5

U937

55.5 ± 2.4**

10.5 ± 0.5**

5.4 ± 0.1**

3.3 ± 0.1**

1.4 ± 0.0

1.4 ± 0.0

RAJI

43.9 ± 1.6**

41.4 ± 1.3**

15.0 ± 0.3**

8.0 ± 0.2**

4.4 ± 0.0

4.1 ± 0.3

Symbols * and ** represent statistically significant differences less than 0.05 and 0.01, respectively, when compared to DMSO-treated control

aUntreated control

bDMSO-treated control

Fig. 2

Cell cycle analysis of RAJI cells treated with 125–1000 μg/ml of crude extract from the leaves of M. modestum (LMM). Untreated (UT-C) cells and DMSO were used as the controls. a shows the gating strategy and b histograms show the patterns of cells stained with PI

Table 3. Cancer cell lines were cultured in the presence of 125–1000 μg/ml of crude extract from the leaves of M. modestum for 24 h, stained with PI, and then analyzed by flow cytometry. Statistical comparisons were analyzed by the one-way ANOVA with Tukey’s HSD post hoc test.

Phytochemical screening

All four leave-derived crude extracts were tested for phytochemicals. The results showed the presence of tannins and flavonoids in all the crude extracts (Table 4). Alkaloids were found in U. longipes and A. burmanicus, and saponins were found in U. longipes and Dasymaschalon sp. The phytochemicals in the Annonaceae family observed in our study was consistent with that in a previous report [27].
Table 4

Phytochemical analysis of Crude Methanolic Extract

Biochemicals

U. longipes

Dasymaschalon sp.

A. burmanicus

M. modestum

Alkaloids

+

+

Sterols

Tannins

+

+

+

+

Saponins

+

+

Anthraquinone glycosides

Cardiac glycosides

Flavonoids

+

+

+

+

Coumarins

Note: (−): Absence; (+): Presence

High-performance liquid chromatography (HPLC)

Quercetin and rutin have been reported for their numerous biological activities, such as anti-inflammatory, free radical scavenging, immunomodulatory, and cancer chemoprevention [28, 29]. Interestingly, quercetin and rutin have been reported as present in a number of species of the Annonaceae family [30]. Therefore, all the methanolic leaves extracts were analyzed for quercetin and rutin by reversed-phase HPLC. The HPLC chromatogram demonstrated that the leaf methanolic extracts of U. longipes (Fig. 3b) and Dasymaschalon sp. (Fig. 3c) contain rutin amounting to 3.29 ± 0.22 mg/g extract and 2.05 ± 0.10 mg/g extract, respectively. While, the leaf methanolic extract of U. longipes and M. modestum contained quercetin amounting to 1.52 ± 0.13 mg/g extract and 0.43 ± 0.11 mg/g extract, respectively (Fig. 4b and c).
Fig. 3

HPLC chromatogram of rutin a, crude extracts of Uvaria longipes b and Dasymaschalon sp. (c). Arrows show the peaks corresponding to the standard rutin

Fig. 4

HPLC chromatogram of quercetin a, crude extracts of Uvaria longipes b and Marsypopetalum modestum c. Arrows show the peaks corresponding to the standard quercetin

Discussion

Medicinal plants are plants that can synthesize certain chemical compounds and produce biological activities that protect the plants from insects, fungi, and other herbivores. For a long time in history, human beings have been utilizing medicinal plants for curing a number of diseases [31]. Although revolutions in the modern drug industry have given rise to a trend of replacing herbal remedies with modern drugs, studies of natural compounds from herbal plants and their activities are still of interest as far as discovering novel drugs is concerned. According to a WHO report (2002), 60% of medicinal drugs are isolated from natural sources, including anti-cancer drugs [32].

In this study, we evaluated the anti-cancer activity of leaf methanolic extracts of U. longipes, Dasymaschalon sp., A. burmanicus, and M. modestum. These plants belong to the Annonaceae family, a family of particular interest for its secondary metabolites [33]. All selected species have never been reported for any therapeutic application in cancer treatment. Interestingly, M. modestum, locally known as the Lao traditional herb, has been shown recently to possess anti-tuberculosis activity in vitro [34]. We evaluated the anti-cancer activity of these four species in three groups of cancer cell lines, including human cervical carcinoma (HeLa, SiHa, and CaSki), human hepatocellular carcinoma (HepG2 and Hep3B), and human myeloid leukemia (K562, U937, and RAJI). The results showed that the leaf methanolic extracts of U. longipes, Dasymaschalon sp., A. burmanicus, and M. modestum induced apoptotic cell death in dose-dependent and cell-type dependent manner. Moreover, the leaf methanolic extract of M. modestum chosen for cell cycle analysis induced accumulation of cells in the subG1 phase, reflecting apoptotic cell death population. This effect was also dose-dependent and cell-type dependent.

Our results imply that these crude extracts might have some active compounds that function against human cancer cell lines. In this regard, all crude extracts were subsequently screened for certain chemical components such as alkaloids, flavonoids, etc. by phytochemical screening. Results from phytochemical screening showed that tannins and flavonoids were present in all the crude extracts. The leaf methanolic extracts of U. longipes and Dasymaschalon sp. contained saponins, whereas the leaf methanolic extracts from U. longipes and A. burmanicus contained alkaloids. All phytochemicals observed in our study have been previously reported about their anti-cancer activity in several studies. Flavonoids have the ability to induce apoptosis, block the cell cycle [35] by demolishing the structure of the spindle fiber [36], and inhibit angiogenesis [37]. Saponins are natural glycosides which have been previously proposed as anti-inflammatory, vaso-protective, hypocholesterolemic, antifungal, antiparasitic, and anti-cancer agents [38]. Alkaloids which can be isolated from plant sources [39] also have cytotoxicity and anti-cancer activity. For example, berberine can inhibit the proliferation of cancer cell lines by interfering with cell proliferation [40] and inducing apoptotic cell death [41]. Evodiamine or quinolone alkaloid showed anti-cancer activities by inducing cell cycle blocking in the K562 erythroleukemic cell line [42], causing DNA damage in MCF-7 breast cancer cells [43], inducing apoptosis in U937 human leukemic cells [44], interfering with angiogenesis [45], and interfering with cell metastasis in Lewis lung carcinoma (LLC) and B16-F10 melanoma [46].

When all the crude extracts were analyzed by HPLC, we found that rutin was an active compound in U. longipes and Dasymaschalon sp., and quercetin was an active compound in U. longipes and M. Modestum. These active compounds have been reported for numerous biological activities, such as anti-inflammatory, free radical scavenging, immunomodulatory, and cancer chemotherapy [28, 29]. Quercetin can promote the pro-apoptotic gene (Bax), enhance the anti-apoptotic gene (Bcl-2 and ERK) in leukemia cells, and activate caspase in osteosarcoma and oral cavity cancer cells [47, 48]. Rutin has been reported as having the ability to induce apoptosis in murine leukemia WEHI-3 cells in vitro and human leukemia HL-60 cells in vivo (murine xenograft model) [49]. To our knowledge, this is the first report showing rutin in leaf-derived crude extracts of U. longipes and Dasymaschalon sp., and quercetin in U. longipes and M. modestum. This finding suggests new sources for chemical compounds capable of inducing apoptotic cell death in vitro. However, these results are from preliminary screening of crude extracts; toxicity tests against normal cells, such as human mononuclear cells, hepatocytes etc., and an in-depth phytochemical analysis are necessary to guarantee the suitability of these extracts in therapeutic application against cancer.

Conclusions

The crude extracts from leaves of U. longipes, Dasymaschalon sp., A. burmanicus, and M. modestum showed particular effects that were found to vary depending on the cancer cell lines examined. The leaves-derived crude extract of M. Modestum increased the percentage of the SubG1 phase in some cancer cell lines. Moreover, crude extracts from leaves of U. longipes, Dasymaschalon sp. and M. modestum provide a new source for rutin and quercetin, which might be capable of inducing cancer cell apoptotic death in a cell-type specific manner.

Declarations

Acknowledgements

The authors express our gratitude to Kritsadee Rattanathammethee, and Narumon Techawong for their excellent technical assistance, and to Hi-Tech Outsourcing Services for editing the manuscript.

Funding

This study was supported by Chiang Mai University Research Group Grant (JW), The Research University Program of Chiang Mai University under Thailand’s Office of Higher Education Commission (JW) and the Office of the Higher Education Commission/Thailand Research Fund (TRF) MRG5680080 (PL). The funding sources had no role in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

Availability of data and materials

All data generated or analysed during this study are included in this published article.

Authors’ contributions

JW and PL were responsible for conception, experimental designs, performed the experiments, data analysis and manuscript preparation. KP performed the experiments, analyzed the primary data and drafted the manuscript. TC and BW collected plant materials, prepared crude extracts and authenticated plant samples. CS performed phytochemical analysis. KC and MO performed the experiments and data analysis. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable.

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Authors’ Affiliations

(1)
Department of Biology, Faculty of Science, Chiang Mai University
(2)
Department of Pharmaceutical Science, Faculty of Pharmacy, Chiang Mai University
(3)
School of Pharmacy, Walailak University
(4)
Research Institute for Health Sciences, Chiang Mai University

References

  1. Anand P, Kunnumakara AB, Sundaram C, Harikumar KB, Tharakan ST, Lai OS, et al. Cancer is a preventable disease that requires major lifestyle changes. Pharm res-Dord. 2008;25(9):2097–116.View ArticleGoogle Scholar
  2. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100(1):57–70.View ArticlePubMedGoogle Scholar
  3. Yusuf S, Wood D, Ralston J, Reddy KS. The world heart Federation's vision for worldwide cardiovascular disease prevention. Lancet. 2015;386(9991):399–402.View ArticlePubMedGoogle Scholar
  4. Gottesman MM, Fojo T, Bates SE. Multidrug resistance in cancer: role of ATP-dependent transporters. Nat rev Cancer. 2002;2(1):48–58.View ArticlePubMedGoogle Scholar
  5. Wong RS. Apoptosis in cancer: from pathogenesis to treatment. J Exp Clin Cancer res. 2011;30:87.View ArticlePubMedPubMed CentralGoogle Scholar
  6. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 1972;26(4):239–57.View ArticlePubMedPubMed CentralGoogle Scholar
  7. Kroemer G, Galluzzi L, Vandenabeele P, Abrams J, Alnemri ES, Baehrecke EH, et al. Classification of cell death: recommendations of the nomenclature committee on cell death 2009. Cell Death Differ. 2009;16(1):3–11.View ArticlePubMedGoogle Scholar
  8. Fulda S. Modulation of apoptosis by natural products for cancer therapy. Planta med. 2010;76(11):1075–9.View ArticlePubMedGoogle Scholar
  9. Couvreur TLP, Pirie MD, Chatrou LW, Saunders RMK, Su YCF, Richardson JE, et al. Early evolutionary history of the flowering plant family Annonaceae: steady diversification and boreotropical geodispersal. J Biogeogr. 2011;38(4):664–80.View ArticleGoogle Scholar
  10. Aminimoghadamfarouj N, Nematollahi A, Wiart C. Annonaceae: bio-resource for tomorrow's drug discovery. J Asian Nat Prod res. 2011;13(5):465–76.View ArticlePubMedGoogle Scholar
  11. Rabelo SV, Costa EV, Barison A, Dutra LM, Nunes XP, Tomaz JC, et al. Alkaloids isolated from the leaves of atemoya (Annona cherimola x Annona squamosa). Rev Bras Farmacogn. 2015;25(4):419–21.View ArticleGoogle Scholar
  12. Moreira IC, Lago JHG, Roque NF. Alkaloid, flavonoids and terpenoids from leaves and fruits of Xylopia Emarginata (Annonaceae). Biochem Syst Ecol. 2003;31(5):535–7.View ArticleGoogle Scholar
  13. Bermejo A, Figadere B, Zafra-Polo MC, Barrachina I, Estornell E, Cortes D. Acetogenins from Annonaceae: recent progress in isolation, synthesis and mechanisms of action (vol 22, pg 269, 2005). Nat Prod rep. 2005;22(3):426–6.Google Scholar
  14. Qazzaz ME, Raja VJ, Lim KH, Kam TS, Lee JB, Gershkovich P, et al. In vitro anticancer properties and biological evaluation of novel natural alkaloid jerantinine B. Cancer Lett. 2016;370(2):185–97.View ArticlePubMedGoogle Scholar
  15. Nordin N, Majid NA, Hashim NM, Rahman MA, Hassan Z, Ali HM. Liriodenine, an aporphine alkaloid from Enicosanthellum pulchrum, inhibits proliferation of human ovarian cancer cells through induction of apoptosis via the mitochondrial signaling pathway and blocking cell cycle progression. Drug des Devel Ther. 2015;9:1437–48.PubMedPubMed CentralGoogle Scholar
  16. Rabbani-Chadegani A, Paydar P, Amirshenava M, Aramvash A. An in vitro study on the effect of vinca alkaloid, vinorelbine, on chromatin histone, HMGB proteins and induction of apoptosis in mice non-adherent bone marrow cells. Drug Chem Toxicol. 2015;38(2):220–6.View ArticlePubMedGoogle Scholar
  17. Himesh S, Sarvesh S, Sharan PS. Preliminary phytochemical screening and HPLC analysis of flavonoid from methanolic extract of leaves of Annona squamosal. International Research Journal of Pharmacy. 2011;2(5):242–6.Google Scholar
  18. Nguyen TH, Ho VD, Do TT, Bui HT, Phan VK, Sak K, et al. A new lignan glycoside from the aerial parts and cytotoxic investigation of Uvaria rufa. Nat Prod res. 2015;29(3):247–52.View ArticlePubMedGoogle Scholar
  19. Macabeo AP, Martinez FP, Kurtan T, Toth L, Mandi A, Schmidt S, et al. Tetrahydroxanthene-1,3(2H)-dione derivatives from Uvaria valderramensis. J Nat Prod. 2014;77(12):2711–5.View ArticlePubMedGoogle Scholar
  20. Hongthong S, Kuhakarn C, Reutrakul V, Jariyawat S, Piyachaturawat P, Nuntasaen N, et al. Cytotoxic alkaloids from leaves and twigs of Dasymaschalon sootepense. Nat Prod Commun. 2014;9(7):929–32.PubMedGoogle Scholar
  21. Jaidee A, Promchai T, Trisuwan K, Laphookhieo S, Rattanajak R, Kamchonwongpaisan S, et al. Cytotoxic and antimalarial alkaloids from the twigs of Dasymaschalon obtusipetalum. Nat Prod Commun. 2015;10(7):1175–7.PubMedGoogle Scholar
  22. Murphy BT, Cao S, Brodie PJ, Miller JS, Ratovoson F, Birkinshaw C, et al. Antiproliferative compounds of Artabotrys Madagascariensis from the Madagascar rainforest. Nat Prod res. 2008;22(13):1169–75.View ArticlePubMedPubMed CentralGoogle Scholar
  23. Kumar RS, Venkateshwar C, Samuel G, Rao SG. Phytochemical screening of some compounds from plant leaf extracts of Holoptelea integrifolia (planch.) and Celestrus emarginata (Grah.) used by Gondu tribes at Adilabad District, Andhrapradesh, India. International Journal of Engineering Science Invention. 2013;2(8):34–8.Google Scholar
  24. Hossain MA, KA AL-R, ZH AL-M, Weli AM, Al-Riyami Q: Study of total phenol, flavonoids contents and phytochemical screening of various leaves crude extracts of locally grown Thymus vulgaris. Asian Pac J Trop Biomed 2013, 3(9):705–710.Google Scholar
  25. Bhandary SK, Kumar S, Bhat VS, Sharmila KP, Bekal MP. Preliminary phytochemical screening of various extract of Punica granatum peel, whole fruit and seeds. Nitte University Journal of Health Science. 2012;2(4):2249–7110.Google Scholar
  26. Tanechpongtamb W, Khawsak P. Cytotoxicity of crude extracts from Allamanda cathartica, Guaiacum officinale, and Artabotrys Siamensis to cancer cells. Journal of Medicine and Health Sciences. 2005;12(2):59–69.Google Scholar
  27. Florence AR, Joselin J, Shynin Brintha TS, Sukumaran S, Jeeva S. Preliminary phytochemical studies of selected members of the family Annonaceae for bioactive compounds. Biosci Discov. 2013;5(1):85–96.Google Scholar
  28. Jimenez-Aliaga K, Bermejo-Bescos P, Benedi J, Martin-Aragon S. Quercetin and rutin exhibit antiamyloidogenic and fibril-disaggregating effects in vitro and potent antioxidant activity in APPswe cells. Life Sci. 2011;89(25–26):939–45.View ArticlePubMedGoogle Scholar
  29. Azevedo MI, Pereira AF, Nogueira RB, Rolim FE, Brito GA, Wong DV, et al. The antioxidant effects of the flavonoids rutin and quercetin inhibit oxaliplatin-induced chronic painful peripheral neuropathy. Mol Pain. 2013;9:53.View ArticlePubMedPubMed CentralGoogle Scholar
  30. Santos DY, Salatino ML. Foliar flavonoids of Annonaceae from Brazil: taxonomic significance. Phytochemistry. 2000;55(6):567–73.View ArticlePubMedGoogle Scholar
  31. Cragg GM, Newman DJ. Plants as a source of anti-cancer agents. J Ethnopharmacol. 2005;100(1–2):72–9.View ArticlePubMedGoogle Scholar
  32. Wang CZ, Calway T, Yuan CS. Herbal medicines as adjuvants for cancer therapeutics. Am J Chinese med. 2012;40(4):657–69.View ArticleGoogle Scholar
  33. Castillo LE, Jiménez JJ, Delgado MA. Secondary Metabolites of The Annonaceae, Solanaceae and Meliaceae Families Used asS Biological Control of Insects. Tropical and Subtropical Agroecosystems. 2010;12(1):445–62.Google Scholar
  34. Elkington BG, Sydara K, Newsome A, Hwang CH, Lankin DC, Simmler C, et al. New finding of an anti-TB compound in the genus Marsypopetalum (Annonaceae) from a traditional herbal remedy of Laos. J Ethnopharmacol. 2014;151(2):903–11.View ArticlePubMedGoogle Scholar
  35. Kuntz S, Wenzel U, Daniel H. Comparative analysis of the effects of flavonoids on proliferation, cytotoxicity, and apoptosis in human colon cancer cell lines. Eur J Nutr. 1999;38(3):133–42.View ArticlePubMedGoogle Scholar
  36. Beutler JA, Hamel E, Vlietinck AJ, Haemers A, Rajan P, Roitman JN, et al. Structure-activity requirements for flavone cytotoxicity and binding to tubulin. J med Chem. 1998;41(13):2333–8.View ArticlePubMedGoogle Scholar
  37. Mojzis J, Varinska L, Mojzisova G, Kostova I, Mirossay L. Antiangiogenic effects of flavonoids and chalcones. Pharmacol res. 2008;57(4):259–65.View ArticlePubMedGoogle Scholar
  38. Podolak I, Galanty A, Sobolewska D. Saponins as cytotoxic agents: a review. Phytochem rev. 2010;9(3):425–74.View ArticlePubMedPubMed CentralGoogle Scholar
  39. Lu JJ, Bao JL, Chen XP. Huang M. Alkaloids Isolated from Natural Herbs as the Anticancer Agents. Evid-Based Compl Alt: Wang YT; 2012.Google Scholar
  40. Sun Y, Xun K, Wang Y, Chen X. A systematic review of the anticancer properties of berberine, a natural product from Chinese herbs. Anti-Cancer Drugs. 2009;20(9):757–69.View ArticlePubMedGoogle Scholar
  41. Eom KS, Kim HJ, So HS, Park R, Kim TY. Berberine-induced apoptosis in human Glioblastoma T98G cells is mediated by endoplasmic reticulum stress accompanying reactive oxygen species and mitochondrial dysfunction. Biol Pharm Bull. 2010;33(10):1644–9.View ArticlePubMedGoogle Scholar
  42. Sun C, Zhang G, Luan S, Luan C, Shao H, Dong F, et al. Evodiamine inhibits the proliferation of leukemia cell line K562 by regulating peroxisome proliferators-activated receptor gamma (PPARgamma) pathway. J Recept Signal Transduct res. 2016;36(4):422–8.View ArticlePubMedGoogle Scholar
  43. Chan ALF, Chang WS, Chen LM, Lee CM, Chen CE, Lin CM, et al. Evodiamine stabilizes topoisomerase I-DNA cleavable complex to inhibit topoisomerase I activity. Molecules. 2009;14(4):1342–52.View ArticlePubMedGoogle Scholar
  44. Lee TJ, Kim EJ, Kim S, Jung EM, Park JW, Jeong SH, et al. Caspase-dependent and caspase-independent apoptosis induced by evodiamine in human leukemic U937 cells. Mol Cancer Ther. 2006;5(9):2398–407.View ArticlePubMedGoogle Scholar
  45. Shyu KG, Lin SK, Lee CC, Chen E, Lin LC, Wang BW, et al. Evodiamine inhibits in vitro angiogenesis: implication for antitumorgenicity. Life Sci. 2006;78(19):2234–43.View ArticlePubMedGoogle Scholar
  46. Ogasawara M, Matsunaga T, Takahashi S, Saiki I, Suzuki H. Anti-invasive and metastatic activities of evodiamine. Biol Pharm Bull. 2002;25(11):1491–3.View ArticlePubMedGoogle Scholar
  47. Dajas F. Life or death: neuroprotective and anticancer effects of quercetin. J Ethnopharmacol. 2012;143(2):383–96.View ArticlePubMedGoogle Scholar
  48. Duraj J, Zazrivcova K, Bodo J, Sulikova M, Sedlak J. Flavonoid quercetin, but not apigenin or luteolin, induced apoptosis in human myeloid leukemia cells and their resistant variants. Neoplasma. 2005;52(4):273–9.PubMedGoogle Scholar
  49. Lin JP, Yang JS, Lin JJ, Lai KC, Lu HF, Ma CY, et al. Rutin inhibits human leukemia tumor growth in a murine xenograft model in vivo. Environ Toxicol. 2012;27(8):480–4.View ArticlePubMedGoogle Scholar

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© The Author(s). 2017

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