Effect of Streblus Asper Root Extract on Anti-cancer Activities of Osteosarcoma Cells

1.2.1 General objective

To study the effectiveness of Streblus asper root extract on chemopreventive activities of osteosarcoma cells.

1.2.2 Specific objective

1. To determine the inhibitory concentration doses (IC₅₀) which inhibit 50% cell proliferation from Streblus asper root extract.
2. To identify the anti-proliferative effects from Streblus asper root extract on osteosarcoma cells.
3. To identify of the effectiveness of Streblus asper root extract on apoptosis of osteosarcoma cells.

Research operational definition

Natural plant - Streblus asper Lour

Cancer cell - Osteosarcoma cells

Research hypothesis

Streblus asper root extract have chemopreventive activities on osteosarcoma cells

1.3 Literature review

Streblus asper is well known for its medicinal usage. It is used traditionally in leprosy, piles, diarrhea, dysentery, elephantiasis (Kirtikar & Basu, 1993) and cancer (Bhakuni et al., 1969). It finds place in the Ayurvedic Pharmacopoeia of India and has also been described in some monographs (Gupta et al., 2005). Streblus asper is used in Ayurveda and its use in the Indian traditional folk medicine is also well documented. For instance, the root is used as an application to unhealthy ulcers and sinuses and as antidote to snake bite, in epilepsy and obesity. While the stem bark is given in fever, dysentery and diarrhea, stomach ache and urinary complaints, useful in piles, edema and wounds, decoction effective against lymphadema, chylurea and other effects of filariasis. Apart from that, the ethanolic extract of the root bark of Streblus asper found to indicate interesting activity on blood pressure which it induces a positive ionoptropic effect in 10^-5 dilution and a systolic response in 10^-4 dilution. Streblus asper also has been reported to possess anticancer activity. KB (cell line derived from a human carcinoma of the nasopharynx) cytotoxicity was found to be concentrated sequentially in the methanol and dichloromethane extracts of S. asper stem bark. Two cytotoxic cardiac glycosides, strebloside andmansonin, were isolated which displayed significant activity in KB cell culture system with ED₅₀ values of 0.035 and 0.042 µg ml^-1, respectively. Then, the volatile oil from fresh leaves of Streblus asper showed significant anticancer activity (ED₅₀ -1) from cytotoxicity primary screening tests with P388 (mouse lymphocytic leukemia) cells but no significant antioxidant activity (IC₅₀ values -1) in a DPPH radical scavenging assay. As the usage and chemopreventive of Streblus asper is known, it is now to know it potentiality on osteosarcoma cells.

1.4 Methodology

1.4.1 Study design: In vitro experimental study

1.4.2 Experiment details

Objective 1: To determine the inhibitory concentration doses (IC₅₀) which inhibit 50% cell proliferation from Streblus asper root extract.

Inhibitory Concentration (IC₅₀) of the cells will be conducted in 6 well plates. Total number of cells will be seeded on day 0. On day 1, the cells will be treated with varying concentrations of extracts in triplicate. On day 3, live cell number will be counted under a microscope.

Objective 2: To identify the anti-proliferative effects from Streblus asper root extract on osteosarcoma cells.

Total number of cells will be seeded in 6-well plates on Day 0. After 24 hours, the cells will be treated with extracts at the IC₅₀ concentration. The total live cell numbers will be quantified under microscope or the MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolim bromide assay) will be conducted on Day 1, Day 2, Day 3, Day 4, Day 6 and Day 8. The cell proliferation graph will be plotted. The cell growth medium will be changed for every 3 days.

Objective 3: To identify of the effectiveness of Streblus asper root extract on apoptosis of osteosarcoma cells.

Total of cells will be seeded in 6-well plates. After 24 hour, the cells will be treated with extracts at the IC50 concentration for 72 hours. Treated cells will be trypinised and washed three times with 1 ml of Nexin buffer. They will be then centrifuged for collection and resuspended in 50 µl of Nexin buffer. Aliquots of 5 µl of Annexin V-PE and 5 µl of 7-amino-acitinomycin D (7-AAD) will be added to 40µl of cells. The mixtures will be incubated on ice for 20 min under shielding from light. The cells will be examined by Flow cytometry (BD FACSCanto II).

1.4.3 Research tools and variables

Research tools:

• Cell lines - Human osteosarcoma cells (ATCC code: CRL1543)

• Hemocytometer

• Biosafety cabinet Class II

• Centrifuge machine

• Waterbath

• Micropipetts

• Glass pipetts

• CO₂ incubators

• Inverted microscopes

Research variables:

• Independent variable

- Human osteosarcoma cells

- IC₅₀ concentration

• Dependent variable

- Different concentrations of Streblus asper root extract

1.4.4 Statistical method

• ANOVA on SPSS version 12 is used for data analysis.

1.4.5 Ethical issue consideration

• There is no ethical issue involved as the oral bacteria in the study will be inoculated from ATCC culture medium.

1.5 Expected limitation of the study

• No circulation, no dynamism of cells or proteins

• Mechanical loading factors are not considered

• Inadequate for studying systemic reactions

1.9 References

• Rastogi et al., (2006). Streblus asper Lour. (Shakhotaka): A Review of its Chemical, Pharmacological and Ethnomedicinal Properties. eCAM. 3(2):217-222
• Kirtikar KR, Basu BD (1933). Indian Medicinal Plants, Allahabad: Lalit Mohan Basu Publications. 3:2291
• Bhakuni DS, Dhar ML, Dhar MM, Dhawan BN, Mehrotra BN (1969). Screening of Indian plants for biological activity. Indian J Exp Biol. 7:250-62
• The Ayurvedic Pharmacopoeia of India (2001), Vol. III, Part I, Delhi: Department of ISM and Homoeopathy, Ministry of Health and Family Welfare. 460.
• Gupta AK, Tandon N, Sharma M (2005). Quality Standards of Indian Medicinal Plants, Vol. II, New Delhi: Indian Council of Medical Research. 227-34
• Saxena VK, Chaturvedi SK (1985). Cardiac glycosides from the roots of Streblus asper. Planta Med. 4:343.
• Pandey PN, Das UK (1990). Therapeutic assessment of Shakhotaka Ghana Vati on Slipada (Filariasis). J Res Ayur Siddha. 11:31-37
• Rastogi RP, Dhawan BN (1990). Anticancer and antiviral activities in Indian medicinal plants: a review. Drug Dev Res. 19:1-12