Proximate Analysis Antiproliferative And Apoptotic Effects Biology Essay

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Introduction

Natural products have played an important role throughout the world in treating and preventing human diseases. Natural product medicines have come from various natural materials including terrestrial plants, terrestrial microorganisms, marine organisms, and terrestrial vertebrates and invertebrates[1].

Scrutiny of different medical indications using various sources of compounds has demonstrated that natural products and related drugs are used to treat 87% of all categorized human diseases, including as antibacterial, anticancer, anticoagulant, antiparasitic, and immunosuppressant agents, among others[2]. Terrestrial plants, especially those with ethnopharmacological uses, have been the primary sources of natural product medicines for drug discovery. Recent analysis by Fabricant and Farnsworth (2001) showed that the uses of 80% of 122 plant-derived drugs were related to their original ethnopharmacological purposes[3].

Current drug discovery from terrestrial plants have mainly relied on bioactivity-guided isolation methods, which, for example, have led to discoveries of the important anticancer agents, paclitaxel from Taxus brevifolia and camptothecin from Camptotheca acuminata [4].

Cancer is a broad group of various diseases, all involving unregulated cell growth (a shift in the control mechanisms that govern cell proliferation and differentiation)[5]. They retain the ability to undergo repeated cycles of proliferation as well as to migrate to more distant parts of the body through the lymphatic system or blood stream and colonize various organs in the process called metastasis [6]. Cancer related morbidity and mortality have been on the increase in the developed and developing world over the past few decades. In 2008 approximately 12.7 million cancers were diagnosed (excluding non-melanoma skin cancers and other non-invasive cancers) and 7.6 million people died of cancer worldwide [7]. Cancers as a group account for approximately 13% of all deaths each year with the most common being: lung cancer (1.4 million deaths), stomach cancer (740,000 deaths), liver cancer (700,000 deaths), colorectal cancer (610,000 deaths), and breast cancer (460,000 deaths) [8]. This makes invasive cancer the leading cause of death in the developed world and the second leading cause of death in the developing world[7]. The incidence, geographic distribution and behavior of specific types of cancer are related to multiple factors, including sex, age, race, genetic predisposition and exposure to environmental carcinogens[6].

The most significant risk factor for developing cancer is old age. Some of the association between aging and cancer is attributed to immunosenescence errors accumulated in DNA over a lifetime, and age-related changes in the endocrine system[9].

Lately, numerous research groups are actively involved in the search for new antitumor agents. Although thousands of plant extracts have already been submitted to various screening methods, only a very few species have produced valuable drugs for the chemotherapy of cancer. Notable examples are Catharanthus roseus (Apocynaceae) alkaloids, Podophyllum peltatum (Berberidaceae) podophyllotoxin derivatives and, more recently, Taxus brevifolia (Taxaceae) diterpenes [10].

Jatropha multifida otherwise known as coral bush is a fast growing evergreen shrub or small tree with belonging to the euphorbiaceae family[11-12]. The roots, stems, leaves, seeds and oil of the plant have been widely used in Africa folk medicine for the treatment of various diseases. In Nigeria, leaf juice extract is used for treatment of oral candidiasis[13]. Leaves and leaf sap are used as purgative, the leaves and fruits are boiled, used internally or externally in a bath for fever[14]. Poultice of root bark and roots are used as wound dressing, the roots are taken internally, for worms and the latex are used for wounds and skin infections[15]. Previous scientific based investigations have revealed the anti-bacterial activity of extracts of the roots of J. multifida against Bacillus subtilis and Staphylococcus aureus[16]. The haemostatic potential of the sap have also been demonstrated[17].

Labaditin, a cyclic decapeptide and biobollein, a cyclic nonapeptide were isolated from the latex of J. multifida on the basis of immunomodulatory activity-guided purification and both peptides selectively inhibited the classical pathway of human complement activation[18]. Also, Multifidone isolated from the stems was measured on four different cancerous cell lines[19].

Experimental

Plant collection and processing

Fresh roots of Jatropha multifida were collected from Uzebba, Owan West local government area of Edo State in April, 2012. The plant was identified and authenticated in the Forest Research Institute of Nigeria, Ibadan where a herbarium specimen was prepared and a voucher specimen number FHI109573 was deposited. The roots were washed off earthy materials, air dried and reduced to fine powder using a mechanical grinder.

Proximate analysis

The following quantitative parameters were carried out using standard methods[20-21].

Moisture content/Water loss on drying

The powdered root bark (2 g) was weighed into a clean, dry crucible of known weight. The crucible with its content was oven dried at 105oC until a constant weight was reached. The average percentage weight loss (moisture content) with reference to the air dried powdered sample was determined for six replicates.

Total ash

The powdered root bark (2 g) was weighed into a crucible. The crucible with its content was heated in the furnace at 600oC for 6 hours. After the temperature was allowed to drop, the crucible was removed, cooled in a dessicator and reweighed. The percentage ash was calculated for six replicates.

Acid insoluble ash

The ash from the experiment above was transferred into a beaker containing 25 mL of dilute HCl. The content of the beaker was boiled for 5 minutes, filtered through an ashless filter paper . The filter paper with the residue was completely ashed. The weight of the residue was determined and the percentage acid insoluble ash calculated based on the initial weight of the air dried powdered drug. The percentage acid insoluble ash was calculated for six replicates.

Water insoluble ash

The ash was transferred into a beakers containing 25 mL of distilled water. The content of the beaker was boiled for 5 minutes and filtered through an ashless filter paper. The filter paper with the residue was completely ashed. The weight of the residue was determined and the percentage acid insoluble ash calculated based on the initial weight of the air dried powdered drug. The percentage acid insoluble ash was calculated for six replicates.

Alcohol Soluble Extractive Value

The powdered root bark (5 g) was weighed into a 250 mL stoppered conical flask and was macerated with 100 mL of 98% ethanol for 24 hours, shaking frequently for the first 6 hours and then allowed to stand for 18 hours. The extract was filtered by suction filtration using a Buckner funnel. 20 mL of the filtrate was taken into a clean, dried and weighed crucible. The filtrate was evaporated to dryness. The residue was dried to constant weight and the final weight noted. The alcohol extractive was then calculated with reference to the initial weight of the powdered drug and expressed as percentage.

Water Soluble Extractive Value

The above experiment was repeated using chloroform:water 1:400.

Extraction of Crude Powdered Sample

The powdered plant material (760 g) was extracted with 5 L of methanol by maceration at room temperature for 7 days. The extract was concentrated to dryness using a rotary evaporator at reduced pressure (-80 Kpa). The extract was stored in an air-tight container and kept in the refrigerator at 4oC until further experiment.

Anticancer Screening

Cell culture

The oestrogen-sensitive human breast adenocarcinoma cell line (MCF-7) were cultured in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and 1% gentamycin, regulated at 37oC, in a 5% CO2 atmosphere. Confluent Cells were treated with 0.05% trypsin and 0.02% EDTA. The medium was replaced every two days.

Treatment with plant extract

Cells (0.5 x 106) were seeded in regular culture medium for one day. Thereafter the cells were washed with phosphate buffered saline (PBS) and adapted to phenol-red-free Dulbecco's modified Eagle's medium with 10% charcoal stripped fetal bovine serum for 48 hours to avoid unspecific stimulation of endogenous hormones in the assay medium. Treatment with J. multifida extract (final concentrations 10 µg/mL and 50 µg/mL) were carried out for 48 hours in assay medium. The vehicle dimethylsulfoxide (0.1%) was used in the same manner as control.

Flow Cytometric Measurement of Cell Proliferation

The extent of cell cycle progression and apoptosis in the cells were estimated by flow cytometric analysis after propidium iodide staining of cells according to standard method[22].

After plant extract treatment, cells were trypsinized with 0.05% trypsin, 0.02% EDTA for 10 minutes. Cell suspension was transferred to FACS tubes and fixed in 70% ethanol for 12 hours at -20oC. After washing with PBS, cells were incubated with RNase (1 mg/mL) at 37oC for 30 minutes. Finally, cells were re-suspended in propidium iodide (50 mg/mL) for at least 3 hours at 8oC protected from light until flow cytometric analysis.

Flow cytometric measurement was performed on flow cytometer equipped with an argon-ion laser of wavelength 488 nm. For data acquisition, the software CellQuest Pro 4.0.1 (BD Biosciences, USA) was used. A minimum of 15,000 ungated events was recorded. Double and clumps were excluded by gating on the DNA pulse width versus pulse area displays. For the analysis of cell proliferation, the cell cycle phases G0/G1, S and G2/M were calculated in percentages using ModFIT LT 3.0 for Power Mac G4 (BD Biosciences, USA). For statistical analysis, the S-phase and G2/M-phase cells were defined as proliferative cells.

Statistical Analysis

Every experiment was done in triplicate and data sets were expressed as means ± stardard deviations (SD). Statistical significance was determined by unpaired t-test (***P < 0.001, **P < 0.01, *P < 0.1).

Results

The value (%) of the proximate parameters of the powdered roots of J. multifida is given in table 1.

Table 1: Percentage proximate parameters of the root bark of J. multifida

Parameter

Mean value ± SD (%)

Moisture content

9.48 ± 1.28

Total ash

10.65 ± 0.0331

Acid insoluble ash

5.9833 ± 0.0310

Water insoluble ash

8.083 ± 0.0204

Alcohol extractive index

0.64 ± 0.0239

Water extractive index

1.404 ± 0.0238

At a dose of 10 µg/mL, the crude methanol extract of J. multifida did not inhibit the proliferation of MCF-7 cells but rather increased the proliferative phase (G2 + S phase) by 160.10 ± 1.29 % compared with DMSO control (100 %). However, the extract inhibited the proliferation of human breast cancer cell line (MCF-7) at a dose of 50 µg/mL. When compared with the control (DMSO), the proliferative phase (G2 + S phase) of extract treated MCF-7 cells was significantly reduced (figure 1).

Figure 1: Percentage proliferative cells (G2 + S phase) after treatment with 10 µg/mL and 50 µ/mL J. multifida root extract.

The percentage apoptosis (cell death) after 48 hour exposure to plant extract (50 µg/mL) was found to be 211 ± 3.86% compared to DMSO (100 %). This effect was significant with P-value < 0.001(figure 2).

Figure 2: Percentage apoptotic cells after treatment with 50 µ/mL J. multifida root extract.

Discussion

The present study evaluated the antiproliferative and apoptotic activities of the crude methanol extract of Jatropha multifida on human breast cancer cell line (MCF-7) via flow cytometry. MCF-7 cells are useful for in vitro breast cancer studies because the cell line has retained several ideal characteristics particular to the mammary epithelium. These include; (i) the ability for MCF-7 cells to process estrogen in the form of estradiol, via estrogen receptors in the cell cytoplasm. This makes the MCF-7 cell line an estrogen receptor (ER) positive control cell line. (ii) the MCF-7 cells are also an excellent in vitro model for studying the mechanisms of chemo-resistance because of its susceptibility to apoptosis and (iii) its ability to go through DNA fragmentation[23].

Cell cycle analysis via flow cytometry distinguishes between different cell cycle phases and detects apoptotic DNA fragmentation simultaneously so that the proliferative (S + G2/M) as well as the apoptotic (degraded DNA) effects of the extracts can be measured[24].

In this study, proximate analysis was carried out for the purpose of authentication of the crude powdered plant material. The moisture content shows the susceptibility of crude drug samples to microbial attack especially fungi, and also to degradation due to hydrolysis of the crude powdered sample. The maximum permissible range for a crude drug is between 6 - 8% [19]. The total ash is a measure of the non-volatile inorganic constituents remaining after ashing. The acid insoluble ash is the residue obtained when total ash is boiled with 1N hydrochloric acid, it is a measure of the sandy matter in crude samples.

The study revealed that there was a significant decrease in percentage proliferation (P < 0.001) of MCF-7 cells treated with J. multifida root extract (50 µg/mL) when compared to the control (DMSO).

The extent of apoptosis for the MCF-7 cells treated with 50 µg/mL extract was significant (P < 0.001) when compared with DMSO treated cells.

The concentrations of the extract used were 10 µg/mL and 50 µg/mL. However, 50µg/ml was found to be the effective concentration for both antiproliferative and apoptotic effects of the methanol root extract of J. multifida.

Conclusions

The roots of J. multifida have both antiproliferative and apoptotic effects at 50 µg/mL concentration against human breast cancer cell line (MCF-7). Therefore, the roots of J. multifida could potentially become a natural source of anticancer chemotherapeutic agent particularly against breast cancer. Ongoing work is investigating the fractions for antiproliferative and apoptotic activities with a view to isolating the bioactive compound(s).

Acknowledgements

The authors would like to acknowledge the Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Benin, Nigeria and the Department of Cell Biology, University Medical Centre, Rostock, Germany for the facility. The STEP-B grant of the Federal Ministry of Education, Nigeria is greatly appreciated.

Conflict of Interest

No conflict of interest associated with this work.

Contribution of Authors

We declare that this work was done by the authors named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by the authors.

Abiodun Falodun: Conceived and designed the study, coordinated all laboratory activities.

Nadja E. Lutz: Anticancer screening of the extracts.

Osayemwenre Erharuyi: Preparation of extract and data analysis.

Sylvester Ukor: Sample collection and proximate analysis.

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