Anti-Mitotic and Cytotoxic Effect of Mussaenda queensirkit

MATERIALS AND METHODS

Chemicals
Acetocarmine, Quercetin, 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) and DMEM (Dulbecco’s Modified Eagle Medium) were purchased from M/s Sigma Laboratories, Monsanto, U.S.A. Brine Shrimp (Artemia salina) Eggs were purchased from Tuticorin local market and the Nauplii were hatched in artificial sea water before experiment. Allium cepa bulb was purchased from the local market and stored throughout the study. All other chemicals used were of analytical grade and were procured from E.Merck, Mumbai.

Plant Material
Fresh flowers of Mussaenda queensirkit have been collected in and around Thanjavur during the month of December 2005. The plant was taxonomically authenticated by the Botanists at Rabinat Herbarium, St. Joseph’s College (Autonomous), Tiruchirapalli and a voucher specimen has been deposited in the CARISM herbarium. The extract was prepared by soaking 100 g of the flowers (pink variety) in 500 mL of 70% methanol and concentrated in vacuuo. The yield obtained was found to be 0.48% (w/w). The concentrated extract was dried and stored in a dessicator.

HPLC Profile
The extract was qualitatively assessed for the presence of flavonoids (Shinoda Test). The profile was developed at λ max 280 nm. Ten milligram of methanolic extract of MQF was dissolved in methanol and was defatted using petroleum ether. The profile was obtained using RP-C18 (Waters) column monitored by PDA detector. The chromatogram was obtained using the gradient, methanol: water (30:70, 15 min) → methanol: water (50:50, 20 min) → methanol: water (80:20, 25 min).

Brine Shrimp Lethality Bioassay
The test was performed according to the method of Meyer et al. (1993). The extract was tested at concentrations of 1000, 100 and 10 μg mL^-1 dispensing 100 μL in six replicates into wells of a 24 well microplate.

Anti-Mitotic Activity
This activity was evaluated using A. cepa root meristamatic cells (Williams and Omoh, 1996). A. cepa bulbs were sprouted in tap water for 48 h at room temperature. The bulbs that developed uniform roots were used for the experiment. These roots (Three roots per concentration) were treated with the extracts at concentrations of 2000, 1000, 500 and 250 μg mL^-1. Water was used as medium/vehicle for dilution. A control was set with three roots from the same bulb and water was used as medium. After 3 h of treatment, the root tips were fixed in a fixing solution of acetic acid and alcohol. Squash preparations were made by staining with acetocarmine stain. The mitotic index was calculated as-

Cytotoxicity by MTT Assay
The fibroblasts cultured from skin were seeded on to the 48 well plate with a known count of cells (2500 cells/well) suspended in DMEM (Dulbecco’s Modified Eagle Medium) medium containing 10% Fetal Bovine Serum (FBS), Penicillin G (100 U mL^-1) and Streptomycin (100 μg mL^-1). The cells were maintained at 37°C in (5:95; CO₂: Air) incubator. The MTT assay was performed according to the method of Skehan et al. (1990) Cells (100 cells/well) were incubated for 24 h with the extract at the concentrations of (100, 250 and 500 μg mL^-1) and the standard, quercetin at concentrations of 10, 25 and 50 μg mL^-1). At various concentrations 20 μL of MTT (5 mg mL^-1) were added to each well and incubated for 4 h at 37°C. The formazan product was dissolved by adding 100 μL dimethylsulfoxide to each well and the plates were read at 550 nm. All measurements were performed in triplicate and each experiment was repeated at least three times.

Statistics
The values obtained for cytotoxic and anti-mitotic activities were expressed as mean of triplicate analyses as earlier studies. The LC₅₀ value for Brine Shrimp Lethality Bioassay was calculated using Graph Pad Prism Software and Student t-test was performed for MTT assay using SPSS software.

RESULTS

HPLC Profile
The HPLC profile shows two distinct peaks with Rt at 9.65 and 44.54 min. The UV spectrum of compound at Rt 44.54 showed bands similar to that of a flavonol glycoside.

Brine Shrimp Lethality
The results from brine shrimp lethality test were analysed using mortality data. The LC₅₀ value was found to be 83.33 μg mL^-1 (Table 1). The results were expressed as mean of six replicates.

Anti-Mitotic Activity
Anti mitotic activity of the MQF was comparable to that of Quercetin. The extract showed highest activity at the concentration of 2000 μg mL^-1, with the lowest mitotic index (Table 2). The maximal inhibition produced by MQF was considerably lower than that exhibited by quercetin.

Cytotoxicity
The results showed that 70% methanolic extract of MQF exhibited 71.44% activity at the concentration of 500 μg mL^-1. The reference compound (Quercetin) displayed maximum activity at the concentration of 10 μg mL^-1 (Table 3). The results were mean of three replicates.

Table 1:	Brine shrimp lethality bioassay of MQF (n = 6)
* An average of six replicates

Table 2:	Anti-mitotic activity of MQF (n = 3)
Values are expressed as mean±SD; P-Prophase; M-Metaphase; A-Anaphase; T-Telophase

Table 3:	Cytotoxicity of MQF against fibroblast culture (MTT assay)
*MQF/Quercetin when compared with respective controls (p<0.5)

DISCUSSION

The results from brine shrimp lethality, antimitotic and cytotoxicity assay show that the 70% methanolic extract of fresh flowers of Mussaenda queensirkit (MQF) has considerable cytotoxic effect.

The preliminary phytochemical analysis suggested the flowers of MQ are found to be rich in flavonoids. The critical relationship of fruit and vegetable intake and cancer prevention has been thoroughly documented in a review (Block et al., 1992). Flavonoids of citrus fruits are known to have anticancer properties. Several flavonols, flavones and flavonones as well as the isoflavone biochanin A, were highly active; a carbonyl function at C-4 of the flavone nucleus was found to be essential for anti-mutagenic activity (Edenharder et al., 1999). Flavone -8 acetic acid was also shown to have antitumour effects (Thomsen et al., 1991).

The MQF seems to inhibit at prophase stage of mitoic cell division where the DNA duplication occurs. Generally, compounds inhibiting mitosis (M phase) affect microtubule configuration which results in an increased proportion of cells arrested in the metaphase (Armbruster et al., 1991; Lazareva et al., 2003). However in the extract treated cells, there was no increase in the number of cells in all mitotic phases except prophase when compared to control roots. This suggests that extract might not directly affect the process in mitosis but inhibit prophase of the cell cycle, resulting in the prevention of the cells entry into mitosis. The possible factors related to this action are the inhibition of DNA synthesis in the S phase, blocking the cell cycle progress in the G1/S or G2/M interface, fragmentation of DNA, malfunction of cyclin dependent kinase activity or proteolysis of cell cycle regulator (Planchais et al., 2000). The mitotic index does not increase considerably in the case of the extract when compared to the reference compound, Quercetin. This may be due to the fact that the extract may be mediating its effect through other mechanisms also. The extract may be binding with cell proteins responsible for cell division.

ACKNOWLEDGMENT

The authors wish to thank Prof. R. Sethuraman, The Vice Chancellor, SASTRA University, Thanjavur, for his constant support and encouragement for our research activities. Our thanks are due to Dr. G. Victor Rajamanickam Dean, CARISM, SASTRA University for providng the laboratory facilities of CARISM. Our sincere thanks are due to Mr. N. Shanmughasundaram, Department of Biotechnology, Central Leather Research Institute, Chennai for his assistance in the study. Financial support for the experiments from the Drugs and Pharmaceuticals Division, Department of Science and Technology, Govt. of India are gratefully acknowledged.