Antiproliferative Effects of Paronychia argentea Lam. and Tamarix aphylla (L.)

H.Karst. Grown in Jordan and Evaluation of Their Volatile Oils Composition

Noor Taisir M. Alhourani

MSc. in pharmaceutical sciences

The University of Jordan - School of Pharmacy

Introduction. Methods. Results. Conclusions. Future recommendations.

Introduction

Medicinal plants and drug discovery

Ancient era

• Plant-derived remedies utilized in folk medicine have directed the research for the isolation of several bioactive compounds.

Nowadays

• Many of secondary metabolites are isolated, structurally identified and their bioactivities are investigated.

Medicinal plants in Jordan

Of the widely distributed plant families in Jordan are:

Tamaricaceae

• Represented by four genera and 78 species.

Caryophyllacaea

• Represented by 81 genera and 2,625 species.

Tamarix aphylla (L.)

H.Karst

Paronychia argentea

Lam.

Tamarix aphylla (L.) H.Karst. (Tamaricaceae)

Known as “tamarisk” in English and “األثيل” in Arabic.

It is an evergreen tree with tiny, triangular and scale-like leaves .

Used traditionally for eczema, wounds and liver conditions.

Paronychia argentea Lam. (Caryophyllaceae)

Known as “Whitlow Wort” in English and in Jordan as “ الحمامه رجل ” in Arabic.

It is an herbaceous hairy plant with branching stems.

Used traditionally for diabetes, urinary tract infections and kidney stones.

Medicinal plants and cancer

Cancer represents the second leading cause of death in Jordan after cardiovascular diseases.

To date, numerous commercially available anticancer agents are from plant origin; such as etoposide, paclitaxel, and the vinca alkaloids.

Currently, screening plants for anticancer activity is of a critical importance in research for introducing new chemical agents with cytotoxic activities.

Aims and objectives

To provide qualitative and quantitative analysis of volatile oils composition obtained from flowering tops of Paronychia argentea and aerial parts of Tamarix aphylla using GC and GC-MS techniques.

To assess the anti-proliferative activity of the ethanol and water extracts of both plants using Methylthiazol Tetrazolium (MTT) assay against selected cancer cell lines;

oBreast adenocarcinoma (MCF-7),

oPancreatic carcinoma (Panc-1),

oColon adenocarcinoma (Caco-2),

o and selective cytotoxicity on normal human fibroblasts.

Methods

Phytochemical analysis

Plant selection

and preparation

Extraction EO

Identification

Plant material selection

In-vitro cytotoxicity assay (MTT)

Kovat’s retention index (RI)

Gas chromatography-mass spectroscopy (GC-MS)

Ethanol extract (EE)

Aqueous extract (AE)

Essential oils (EO) extraction

Grinding

Drying

Extraction

Electron Impact MS (EI-MS)

Essential oil Hydrodistillation Clevenger-type apparatus

10 mg of each crude extract was dissolved in suitable solvent then the required concentrations prepared .

AE and EE crude plant extracts

Essential oil Identification GC-MS system

Retention Index(RI) vs Mass Spectral(MS) Search

Retention -Kovàts-Index (RI):

Kovàts retention index (RI) has been considered as an intelligent contribution to the chromatographic science.

This index, in Temp-Programmed GC-Regimes is defined as:

RIx = 100n + 100 [(Tx – Tn) / (Tn+1 – Tn)]

= 1420

The combined use of both the mass spectral- and RI-library searching has been elaborated to be as a powerful tool for structural confirmation of the

separated components

RI

Compound

1410

-Gurjunene

1420

-Caryophyllene

1440

Aromadendrene

1480

Germacrene D

50 100 150 200 250

m/z

50

100

Re

lative

Ab

un

da

nce

Beta-caryophyllene

91.0

105.1133.1

161.179.0

189.177.0204.1

The Mass Spectrum (MS):

unknown

The Unknown is -Caryophyllene

EI-MS

• Identified essential oil components were analyzed quantitatively by calculating relative peak area of

each oil principle in the total ion current

chromatogram, assuming a unity response by all

components.

In-vitro cytotoxicity

Cells were cultured in suitable media.

Seeded in 96-well plates overnight.

Treated with increasing concentrations of AE or EE (0.1- 800 µg/mL) or reference drugs (0.1-200 µg/mL).

Incubated at 37 ˚C in a 5% CO2 with 95% humidity for 72 h.

Measurement of cells viability by MTT assay.

MTT assay

Results

In-vitro cytotoxic activity of P. argentea EE, T. aphylla AE and EE, cisplatin and doxorubicin tested against

Caco-2 colorectal cancer cell line

In-vitro cytotoxic activity of P. argentea EE and T. aphylla EE, cisplatin and doxorubicin tested against

Panc-1 pancreatic cancer cell line

In-vitro cytotoxic activity of T. aphylla AE and EE, cisplatin and doxorubicin tested against

MCF-7 breast cancer cell line.

In-vitro cytotoxic activity of P. argentea AE and EE, T. aphylla AE and EE, cisplatin and doxorubicin tested

against normal Fibroblast cells.

According to The American National Cancer Institute (NCI) guidelines, it sets the limit of activity for crude extracts at 50% inhibition (IC50) of proliferation to be < 30 µg/mL after the exposure time of 72 h (Kuete, et al., 2013).

Kuete, V. Fankam, A.G. Wiench B. and Efferth, T. (2013), Cytotoxicity and modes of action of

the methanol extracts of six cameroonian medicinal plants against multidrug-resistant tumor

cells. Evidence Based Complementary and Alternative Medicine, 2013: ID 285903.

Cytotoxic potential

Cytotoxic potential

Cytotoxicity IC50 values of P. argentea and T. aphylla extracts, cisplatin and doxorubicin tested in a panel of cancer cell lines

Treatment Cytotoxicity (IC50 value: mean ± SD; μg/mL)

MCF-7 Caco-2 Panc-1

Periodontal Fibroblasts

Doxorubicin 0.01±0.001 0.10 ± 0.01 0.06 ± 0.01 0.14 ± 0.02

Cisplatin 1.11 ± 0.15 5.97 ± 0.57

P. argentea AE Non-toxic Non-toxic Non-toxic 427.38 ± 0.38

P. argentea EE Non-toxic 134.70±6.27 160.97± 10.76

T. aphylla AE 479.76±54.99 Non-toxic

T. aphylla EE 130.55±12.25 26.65±3.09 154.90 ± 3.29

2.17 ± 0.10

1.17 ± 0.13

79.99 ± 4.90

9.08 ± 0.29

68.91 ± 7.52

88.74 ± 2.44

9% 11%

10%

51%

19%

Hydrocarbons MT

Oxygenated MT

Hydrocarbons ST

Oxygenated ST

Non-aromaticcompounds

MT: Monoterpenes ST: Sesquiterpenes

Phytochemical analysis of P. argentea EO

GC-MS chromatogram of P. argentea EO

O-cymene 7.53%

6,10,14-trimethyl-2-pentadecanone 16.14%

Isolongifolanol 5.19%

Isobicyclo-germacrenal

4.33%

9-epi-e-caryophyllene

4.89%

2% 4% 2%

27%

53%

2% 10%

Hydrocarbons MT

Oxygenated MT

Hydrocarbons ST

Oxygenated ST

Non-aromaticcompoundsNon-terpenoid aromaticcompoundsTraces compounds

Phytochemical analysis of T. aphylla EO

MT: Monoterpenes ST: Sesquiterpenes

GC-MS chromatogram of T. aphylla EO

6,10,14-trimethyl-2-pentadecanone 32.39%

β-ionone 13.74%

Dodecanoic acid 6%

Conclusions

No antiproliferative potential against Caco-2 or Panc-1 cancer cells were detected at concentrations less than 30 μg/mL.

Exceptionally T. aphylla AE and EE showed potent cytotoxic effects against MCF-7 cells, with IC50 values (2.17±0.10 and 26.65±3.09; μg/mL) respectively.

T. aphylla AE demonstrated a comparable cytotoxic activity to cisplatin’s (IC50 value of 1.17±0.13 μg/mL), with selectively less cytotoxic effects against normal fibroblast.

In-vitro cytotoxicity

Phytochemical analysis

Essential oils obtained by hydro-distillation from flowering tops of Paronychia argentea Lam. was found to be rich in sesquiterpenes (60.96%), while aerial parts of Tamarix aphylla (L.) H.Karst. was found to be rich in non-aromatic hydrocarbons (53.06%).

6,10,14-trimethyl-2-pentadecanone, a non-aromatic ketone, was found to be the predominant principle in both oils.

Future recommendations

Study of ontogentic (time of collection), inter-organ, geographical, and environmental effects on composition of EO hydro-distilled from either plant species is of particular future interest.

The chemical composition of T. aphylla different extracts that is responsible for the activity has to be analyzed. Also, the action mechanism by which this inhibition takes place has to be revealed.

Co-incubation of promising T. aphylla extracts with conventional drugs used for breast cancer may be screened for overcoming chemoresistance and subsequent chemosensitisation of tumor cells to chemotherapeutic agents.