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In-vitro Cytotoxicity and Antioxidant Property Evaluation from Methanolic Extract of Cuscuta Reflexa Flowers

Md. Hossan Sakib
Department of Pharmacy, University of Science and Technology Chittagong, Bangladesh.

Mohammad Shahadat Hossain
Department of Pharmacy, University of Science and Technology Chittagong, Bangladesh.

Muhammad Sazzad Hossain
Department of Pharmacy, University of Science and Technology Chittagong, Bangladesh.

Asif Al Mahmood
Department of Pharmacy, University of Science and Technology Chittagong, Bangladesh.

Md. Yasin Sarkar
Department of Pharmacy, International Islamic University Chittagong, Bangladesh.

Sadequr Rahman
Department of Pharmacy, International Islamic University Chittagong, Bangladesh.

Limon Kanti Shill
Department of Pharmacy, International Islamic University Chittagong, Bangladesh.

Abstract/ Executive Summary:

This investigation is made upon the plant Cuscuta reflexa, the flowers of it, to find out its Cytotoxicity property. The anti-oxidant property of this plant part was investigated using methanol extraction. Methanolic extract of Cuscuta reflexa was used to evaluate its cytotoxicity in Brine shrimp lethality bioassay where vincristine sulphate was used as standard drug. In Brine shrimp lethality bioassay, LC50 value of the extract was 36.72μg/ml and vincristine sulphate served as the positive control showed LC50 value 10.51μg/ml. So, compared to vincristine sulphate, it is evident that the methanol extract of flowers of Cuscuta reflexa was cytotoxic. In case of anti-oxidant the scavenging power (IC₅₀₎ of DPPH radical was 29.26, 17.07, 18.29, 19.55 and 54.87µg/ml respectively.

Keywords: Cuscuta reflexa; Cytotoxicity; Scavenging; Reducing power; Anti-oxidant

Location: Chittagong

Start Date:

End Date:

Program Area: Pest Management

Commodity: Organic fertilizer

Objectives:

1. To find out the plant Cuscuta reflexa Cytotoxicity property and

2. To evaluate the plant Cuscuta reflexa cytotoxicity in Brine shrimp lethality bioassay where vincristine sulphate was used as standard drug.

Methodology:

The flowers of Cuscuta reflexa were collected from Chittagong local forest area; the leaves of Cuscuta reflexa were collected at their fully mature form. After cleaning, the leaves were taken and splitting the peal, then air dried for 8 days, and then kept in an oven at 45°C at 72 hours. 250 gm of dried powder was cold extracted with Methanol. Dried powder soaked with methanol for 7 days. Then filtered to take the concentrated extract, extract containing beaker was placed on the water bath (at 40°C-45°C) to evaporate the solvent from the extract. The extract is prepared by cold extraction process. In this process the coarse powder was submerged in ethanol (95%) since ethanol is the most common solvent for extracting most of the constituents present in herbal materials. Amber glass bottle were used for this purpose, which were kept at room temperature and allowed to stand for 7 days with occasional shaking and stirring. When the solvent became concentrated the contents were first decanted by using cotton and then filtered through Whatmann No.1 filter paper. The filtrate so obtained was then concentrated to dryness through the evaporation of solvent using rotary evaporator. Finally we got the concentrated semi-solid extract. The concentrated were then used as crude extract of respective test experiments. In our present investigation, we used methanol extract for cytotoxic and antioxidant activity. Brine shrimp lethality bioassay is widely used in the bioassay for the bioactive compounds. Here simple zoological organism (Artemia salina) was used as a convenient monitor for the screening. The dried cyst of the brine shrimp were collected from an aquarium shop (Chittagong, Bangladesh) and hatched in artificial seawater (3.8% NaCl solution) with strong aeration for 48 hours day/dark cycles to mature shrimp called nauplii. The cytotoxicity assay was performed on brine shrimp nauplii using Meyer method. Artemia salina Leach (brine shrimp eggs), Sea salt non ionized NaCl, Small tank with perforated dividing dam to hatch the shrimp, Lamp to attract the nauplii, Pipette (1 ml and 5 ml), Micropipette (1-10 micro liter), Glass vials (5ml), Magnifying glass, Test sample for experimental plants. Artemia salina Leach (brine shrimp eggs) collected from the pet shop was used as the test organism. Simulated sea water was taken in the small tank and the shrimp eggs (1.5gm/L) were added to one side of the tank and this side was covered. The shrimps were allowed to one side of tank and this side was covered. The shrimp were allowed for two days to hatch and mature as nauplii (larvae). Constant oxygen supply was carried out during the hatching time. The hatched shrimps were attracted to the lamp on the other side of the divided tank through the perforated dam. These nauplii were taken for this bioassay. In the present study, vincristine sulphate was used as the positive control. 3 mg of vincristine sulphate was dissolved in 1.8 ml of distilled water to get a concentration of 5 mg/ml. This was used as stock solution of vincristine sulphate. With the help of a micropipette 80, 70, 50, 30 and 10 μl of the stock solution were transferred in 6 different vials. NaCl solution (brine water) was added to each vial making the volume up to 5 ml. The final concentration of vincristine sulphate in the vials became 80µg/ml, 70µg/ml, 60µg/ml, 50µg/ml and 10μg/ml respectively. 0.1ml of each concentration of extract (0, 5, 25, 50, 100 and 200µg/ml) was added to 3ml of 0.004% methanol solution of DPPH. The mixture was kept in dark place for proper reaction, as the reaction is light sensitive. After 30 min, absorbance of the resulting solution was measured at specified wavelength. The percentage was calculated using formula following, where A₀ is absorbance of control and A₁ is absorbance of test.

Source of Information: Asian J. Med. Biol. Res. 2015, 1 (2), 285-291; ISSN 2411-4472 (Print) 2412-5571 (Online).

Article Link (Online Journal): www.ebupress.com/journal/ajmbr

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

From the above study it can be concluded that the methanolic extract of cuscuta reflexa may be a potential candidate for future cytotoxic agent. Furthermore study and isolation is needed to obtain site specific and more potent agent that causing this effect. The cytotoxic result obtained 36.72µg/ml (LC₅₀) it so good, but proper isolation can make it more potent and useful. So, it could be suggested to modify for site specific use. From the experimental literature these compounds have record of broad spectrum action as anti-oxidant and they are originating for such reason from long period of time. Anti-oxidant activities of methanolics are caused by their donation of hydrogen atoms to free radical scavenging action. They possess an ideal structure for scavenging property.

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