In Vitro Anticancer Studies on Petroleum Ether Fraction of Methanolic Extract of Leucas Aspera

Medicinal plants have proved to be an important natural source of anticancer therapy for many years due to their low side effects, low cost, and ease of availability ^[1-4]. Phytochemicals play an important role as cancer chemotherapeutic drugs. There were numbers of studies dealing with cytotoxicity screening of plant extracts ^[5-7]. The whole plant of Leucas aspera is traditionally important because it has many therapeutic values. Its leaves, flowers, stem and root of has the pharmacological activities ^[8-10]. In vitro cell viability and cytotoxicity assays with cultured cells are widely used for cytotoxicity tests of chemicals and for drug screening. HeLa cells, the human cervical cancer cells were the first type of human cancer cell to be cultured continuously for experiments. A great number of in vitro and in vivo methods have been developed to measure the efficiency of natural anticancer compounds either as pure compounds or as plant extracts. This study validates the potential of phytochemical research in cancer therapy and experimental evidence confirming the anticancer potential of this specific fraction and also validation of traditional medicine uses of Leucas aspera. By using bioassays, fractions of active compounds can be tested in a specific cell line, to evaluate whether or not they possess a cytotoxic effect. Our LC-MS study using petroleum ether fraction identified phytochemicals with anticancer property especially ?-sitosterol. Hence HeLa cells were selected to evaluate the anticancer property of MELA and its active fraction. In the present study in vitro cytotoxicity and anti-cancer effect of MELA and its active fraction (petroleum ether) against the HeLa cell line was evaluated by MTT assay method. The mode of HeLa cell death was investigated using active fraction of MELA by morphological analysis (fluorescent microscopy). LDH release assay was conducted to study whether the extract is causing lysis. To evaluate whether the extract from the plant L.aspera induce apoptosis in the HeLa cells, morphological analysis by microscopic examination of acridine orange/ethidium bromide - stained target cells was performed. Changes in the cell cycle of HeLa cells treated with the fraction was analyzed by Flow Cytometer.

Objective of the present work was to study the antioxidant activity of the plant extract, to investigate the anticancer potential of the petroleum ether fraction of methanolic extract of Leucas aspera, to identify the bioactive compounds responsible for the observed anticancer activity using LC/MS analysis, to validate the traditional medicinal uses of Leucas aspera and explore its potential as a source of novel anticancer therapeutics.

MATERIALS AND METHODS

Chemicals

Fetal bovine serum (FBS), acridine orange (AO), ethidium bromide (EB) were purchased from sigma chemical Co. St. Louis, MO, USA. EDTA, Dimethyl sulfoxide was obtained from High media, India. All other chemicals used were also of high purity grade.

Cell Culture

HeLa cells were purchased from NCCS Pune was maintained in Dulbecco’s modified Eagles media (Hi media) supplemented with 10?S and grown to confluence at 37°C in 5% CO₂ in a humidified atmosphere in a CO₂ incubator (Eppendorf, Germany). The cells were trypsinized (500µl of 0.025% Trypsin in PBS/0.5mM EDTA solution (Hi media) for 2 minutes and transferred to T flasks.

Preparation of plant extract

The whole plant of L. aspera was collected from Kottayam, Kerala and authenticated. A voucher specimen (SBSBRL04) was maintained in the Institute. A 100gm each of dried powder of the whole plant of L. aspera was subjected to successive Soxhlet extraction using a series of solvents of increasing polarity starting from petroleum ether, chloroform and methanol respectively for 72 hrs. The extracts were filtered using Whatman filter paper (No.1), while hot and concentrated in a rotary evaporator.

Preparation of Leucas aspera methanolic extract and its sub fraction

A 100g of dried powder of whole plant of Leucas aspera was Soxhlet extracted with           500 ml of methanol for 24hr. The solvent was concentrated in a rotary evaporator. The percentage yield was approximately 4.6% (w/v). The methanolic extract thus obtained was subjected to activity guided fractionation, where the plant extracts were sequentially fractionated and each fraction tested for activity. The active fraction was taken for LC-MS analysis.

In vitro antioxidant studies

The plant extracts were subjected to free radical scavenging activity such as DPPH assay ^[11]. The plant extracts were subjected to quantification of phytoconstituents such as total phenolic ^[12] and flavonoid contents ^[13].

Bioassay guided active fractionation of MELA

If an extract shows promising biological activity in the bioassays, the extract was subjected to activity guided fractionation, where the plant extracts were sequentially fractionated and each fraction tested for activity. The extract needs to be separated into its active constituents and each component tested for bioactivity, in order to isolate the agents responsible for the bioactivity. MELA was subjected to further sub fractionation using various solvents such as chloroform, petroleum ether, ethyl acetate and methanol. The different extracts obtained were tested for its DPPH radical scavenging activity. IC- 50 value of different extracts was also determined.

Liquid Chromatography-Mass Spectrophotometry (LC-MS) analysis

The phytochemical profiling of methanolic extract of Leucas aspera (MELA) and its active fraction were carried out using LC-MS 2010A instrument (Schimadzu, Kyoto, Japan). 10µl of the filtered sample was injected to the manual injector using a micro syringe (1-20µl, Schimadzu). The mobile phase used was acetonitrile: 0.1% OPA in methanol (70:30) in an isocratic mode. The column and pump used were reverse phase C-18 (25cm X 2.5mm) (Phenomenex) and SPD 10 AVP-RD respectively. The separated components were then ionized using electro spray ionization method (ESI). The flow rate was maintained to 1.6ml/min with a temperature of 25^oC and spectral data were collected at 315nm. Mass analysis was performed in the range 50-800m/z, under both positive and negative ion mode. The class VP integration was used for the analysis. The constituents of the extract were identified by referring the LC-MS library, Metwin 2010 (Version 2.1).

Microculture Tetrazolium Testing (MTT Assay)

Cell viability was determined by use of the micro culture tetrazolium technique (MTT) ^[14]. Percentage inhibition was calculated using the formula,

Percentage growth inhibition =

(Mean abs of the control cells)- (Mean abs of treated cells) X 100

Mean absorbance of the control cells

Determination of apoptosis by acridine orange (AO) and ethidium bromide (EB) double staining

DNA-binding dyes AO and EB (Sigma, USA) were used for the morphological detection of apoptotic and necrotic cells ^[15]. The cells were divided into four categories as follows: living cells (normal green nucleus), early apoptotic (bright green nucleus with condensed or fragmented chromatin), late apoptotic (orange-stained nuclei with chromatin condensation or fragmentation) and necrotic cells (uniformly orange-stained cell nuclei).

Lactate dehydrogenase leakage

Lactate dehydrogenase is used as a quantitative marker enzyme for the intact cell, its activity providing information on cellular glycolytic capacity. Measurement of LDH release (leakage) is an important and frequently applied test for severe irreversible cell damage. LDH leakage assay was performed with cell free supernatant collected from tissue culture plates ^[16].

Analysis of DNA content and cell cycle distribution by Flow cytometry

Monitoring a cell’s ability to proliferate is critical for assessing a cell’s health during toxicity studies. The most accurate method of doing this is by directly measuring DNA synthesis ^[17].

Statistical analysis

Results were expressed as mean ± S.D and all statistical comparisons were made by means of one way ANOVA test followed by Tukey’s post hoc analysis and p values less than or equal to 0.05 were considered significant

RESULTS AND DISCUSSION

Preliminary phytochemical screening revealed the presence of tannins, proteins, steroids, glycosides, carbohydrates, saponins, terpenoids, flavonoids and alkaloids in different extracts of L.aspera ^[18].

DPPH radical scavenging activity of Leucas aspera

The petroleum ether, chloroform and methanol extracts of L.aspera exhibited a significant increase in DPPH activity especially at a concentration of 500µg/ml. A concentration dependent assay was carried out with these extracts and the results are shown in Figure 1. Among five different concentrations used in the study (5 to 500µg/ml), methanol extract showed maximum scavenging activity with IC- 50 value of 180.46µg/ml.

Determination of total phenolics and flavonoid content of MELA

MELA showed a high phenolics and flavonoid content. The results are given in Table 1.

Bioassay guided active fractionation of MELA

Bioassay guided active fractionation of MELA analysis showed (Figure.2) that the concentration was expressed with respect to the dried weight of the respective extracts. The DPPH activity was followed in all cases. IC₅₀ was calculated for each extract with respect to the absorption given by the UV-spectrophotometer. Petroleum ether fraction of MELA was the active fraction with IC₅₀ of 25mg/ml (Figure.2).

       
           
       
Figure. 2: Bioassay guided active fractionation of MELA

The mass spectrum of the active fraction by LC- MS was given in Figure. 3.  The constituent of petroleum ether fraction of MELA identified using LC-MS analysis that possess potent anticancer property was ?-sitosterol (Molecular mass 414.70). ?-sitosterol reported to possess antiproliferative and apoptotic potential in several cancer models ^[19,20]. Anticancer efficacy of MELA may be due to this identified compound. The list of pharmacologically active phytochemicals in the active fraction was given in Table 2.

       
           
       
Figure.3 : A) Mass spectrum of positive ionization B) Mass spectrum of negative ionization

The isolated fractions were further tested for their cytotoxic and apoptotic studies. The cytotoxic activity of L.aspera (100-1000µg/ml) against HeLa cells was assessed in vitro by MTT proliferation assay. 0ne hundred to 1000µg/ml of L.aspera significantly reduced the viability of HeLa cells. As shown in Figure.4 percentage of   viable cells remained more than 57% even when cells were treated with 100µl of L.aspera for 24 hr. But when the doses were increased, the percentage of viable cells was decreased and finally at a dose of 1000µl of L.aspera, only 26 ?lls were viable in the active fraction. These results indicate that L.aspera showed significant potentiality against the viability and proliferation of cervical carcinoma cell (HeLa cell) line. The above result affirms that cytotoxicity of L.aspera substantially  increased with increase in concentration. Morphological analysis was given in Figure.5.

       
           
       
Figure. 4: Graphical representation of MELA concentration of petroleum ether fraction against % viability

       
           
       
Figure.5 : Microscopic images of HeLa cells after 24 hr incubation with  petroleum ether fraction of MELA

Lactate dehydrogenase leakage assay

Lactate dehydrogenase leakage assay of active fraction of MELA was carried out and the results were provided in Figure.6. The release of lactate was monitored after 48 hrs of treatment in this study. Lactate release was monitored to measure the glycolysis rate and by-product formation from cell growth. Increased LDH leakage confirms   increased membrane damage which is directly proportional to cytotoxicity.

       
           
       
Figure.6 : Graphical representation of LDH leakage of HeLa cells treated with petroleum ether fraction of MELA

Determination of apoptosis by acridine orange (AO) and ethidium bromide (EB) double staining

To evaluate whether the extract from the plant L.aspera induce apoptosis in the HeLa cells, morphological analysis by microscopic examination of acridine orange/ethidium bromide stained target cells was performed. The morphological characteristic of apoptotic cell death, such as cell shrinkage, condensation and even fragmentation of nucleus, as well as the presence of orange - red stained cells at late stages of apoptosis or secondary necrosis (It was observed in extract treated HeLa cells) and apoptotic bodies. Microscopic examination revealed that the extract induced apoptosis in target HeLa cells after 24 hr treatment (Figure 7). The plant extract cause apoptosis

at the concentration of 1000µg/ml. These analysis confirmed that the cytotoxicity of the extract was based on their prominent pro-apoptotic effects.

Examination of changes in the cell cycle phase distribution of Hela cells treated with extract for 24 hours was done to elucidate the mechanisms of the observed cytotoxic action (Figure 8 and Figure 9). Flow cytometric analysis of HeLa cells treated with methanolic extracts showed significant inhibition of cells at Go/G₁ phase. Therefore the G₁ phase arrest was one of the possible mechanisms of anti-proliferative activities of the extracts.  There was 45% increase in cells arrested at G_O/G₁ phase when compared with untreated control whereas the S phase and M Phase cells decreased proportionally. These results indicated that MELA active fraction blocks the cell cycle at the G₁ phase transition and causes HeLa cells to remain at the G₁ phase.

Figure.8: Changes in cell cycle phase distribution of HeLa cells treated with  control after 24 hr

Figure.9: Changes in cell cycle phase distribution of HeLa cells treated with petroleum ether fraction of MELA after 24 hr

In the present study apoptotic activity exhibited by petroleum ether fraction of MELA might be attributed to the presence of identified class of phytochemicals such as ?-sitosterol, chrysoeriol, dotriacontanol, leucasperone A and leucasperone B by LC-MS. ?-sitosterol reported to possess antiproliferative and apoptotic potential in several cancer models ^[20].