In vitro Evaluation of Seaweed Gracilaria verrucosa for Cytotoxic Activity against Cervical HeLa Cells

Background: Seaweed macroalgae of Gracilaria verrucosa has been known to have a potent anticancer activity, however the cytotoxicity against cervical cancer has not been explored further. Objective: This study aims to utilize Indonesia’s marine resource which is focused on seaweed macroalgae G. verrucosa as a future anti-cervical cancer agent. Materials and Method: Seaweed G. verrucosa originated from Labuan Aji beach, Nusa Tenggara Barat, Indonesia, extracted, macerated, and fractionated into four organic solvents of different polarity, consisting of hexane, ethyl acetate, chloroform and ethanol. Then, the macroalgae extracts are diluted into 8 different concentrations. Afterwards, in vitro anticancer activity evaluation of hexane, ethyl acetate, chloroform and ethanol extracts of G. verrucosa against cervical HeLa cells were conducted by MTT cell proliferation assay. Triplo mechanism is also applied in this study to increase the accuracy of the results. The anticancer activity is measured using IC50 value. Results: The four concentrated extracts G. verrucosa showed cytotoxicity against cervical HeLa cells. The greatest anticancer activity is depicted by hexane extract with an IC50 of 14.94 μg/mL, followed by chloroform (IC50 15.74 μg/mL), ethyl acetate (IC50 16.18 μg/mL), and ethanol (IC50 19.43 μg/mL). Conclusion: Our results clearly indicate that hexane, ethanol, chloroform, and ethyl acetate extracts of seaweed G. verrucosa can be further developed to be anti-cervical cancer agents, with hexane extract displaying the greatest cytotoxic effect.

acetic acid (VIA).This method screens for any abnormal changes on the superficial part of the cervix.This diagnostic method is especially useful to be used low-resource settings such as in primary health care centers in Indonesia.When pre-cancerous lesion is detected, immediate treatment should be done to prevent progression to cancer. 4However, the tools and skills to perform VIA or cryotherapy are not evenly distributed and this inspection is subjective as it requires practice and experience in order to correctly perform it.According to the Ministry of Health Indonesia, VIA and breast physical examination as screening efforts to eradicate cervical and breast cancer have been done in 1 623 913 (3.4%) women out of 37 500 000 of total target from 2008-2016. 5Moreover, late diagnosis is common as this disease is asymptomatic at its early phases. 4Hence, more aggressive treatments with its deleterious side effects need to be applied.In addition to that, these aggressive treatments are only available in some health care centers.Thus, due to these conditions, there is an urgent need to develop a new practical, more effective, and less invasive preventive and/or curative effort to beat cervical cancer.G. verrucosa is an edible and relatively cheap seaweed species that can be easily found in Indonesia (Figure 1).Studies have shown that this seaweed has shown promising evidence indicating that extract of G. verrucosa is able to induce apoptosis through mitochondrial pathway by involving cytochrome c release from mitochondria. 6Moreover, the species G.tenuistipitata is able to have apoptosis-based cytotoxicity against oral cancer cells by induction of ROS, DNA damage, and mitochondrial depolarization against oral squamous carcinoma displayed by their secondary metabolites.Most studies about Gracilaria seaweeds have been focused on its anti-hypercholesterolemic, anti-oxidative, anti-inflammatory, and antimicrobial properties but not its anti-cancer activities. 7ased on this information, we are inspired to evaluate further the anticancer activity of G. verrucosa against cervical cancer cells.In this study, we explore the cytotoxic activity of seaweed macro algae G. verrucosa collected from Labuan Aji beach, Lombok, Nusa Tenggara Barat Province, Indonesia.This research utilizes experimental study design to determine the effects of G. verrucosa extract against the cervical HeLa cells.This research will be conducted in several stages.The first stage is the collecting and identification of sample G. verrucosa.Next, the sample will be rinse, dry, and grind G. verrucosa to give out a smooth, dry powder form.The third stage is to macerate and extract sample G. verrucosa.Then, it will be fractioned into four organic solvents with different polarity which are hexane (non-polar), chloroform (non-polar), ethyl acetate (semi polar), and ethanol (polar).The extracts will undergo serial dilution to produce 8 different concentrations.The HeLa cervical cancer cell line will be nourished, tested for its viability, and incubated first before being treated with any G. verrucosa extract.The last stage is in vitro cytotoxic activity evaluation of hexane, chloroform, ethyl acetate, and ethanol extracts of G. verrucosa against HeLa cervical cancer cells by MTT assay.
In our previous research we have a completed the phytochemistry test and TLC analysis of the seaweed G. verrucosa.Phytochemistry test showed that G. verrucosa extract contains metabolite triterpenoid and saponin, whereas TLC analysis of G. verrucosa extract reveals that n-hexane, chloroform solvents showed 7 bands, ethyl acetate solvent showed 6 bands, and ethanol solvent showed 2 bands.This suggests that n-hexane, chloroform solvents are non-polar in nature, while ethyl acetate is semi-polar, and ethanol solvent is polar. 8

Extraction and Fractionation Samples of Seaweeds
About 1000g of dry powder sample of G. verrucosa will be macerated in 500 mL of separate four organic solvents, namely, n-hexane, chloroform, ethyacetate and ethanol for 7 days inside a sealed vase container while being stirred at some times, 2-3 times a day.Maceration will be done three times to get as many extract as possible.The result of maceration will be filtered and concentrated with rotary evaporator to get n-hexane extract (0.3 g), chloroform extract (0.2 g), ethylacetate extract (1 g), and ethanol extract (1.5 g), respectively.Next, the 4 extracts will be diluted into 5 different concentrations of 3.125; 6.25; 12.5; 25; 50 µg/mL by serial dilution method.

In vitro Cytotoxicity Assay
Malignant HeLa cervical cancer cells taken from the Department of Pathology Anatomy Faculty of Medicine University of Indonesia will be supplemented with 10% of fetal bovine serum (Gibco, USA) using laminar flow hood technique to ensure sterility.Then it will be cultured into RPMI 1640 (Gibco, USA).The supplemented cultured cells will be incubated in temperature of 37 o C and 4% CO 2 in a humidified atmosphere.The viability of cells will be tested using trypan blue 0.1%.If the viability of the cells > 90%, then the cells are able to be used in MTT assay.This is done to avoid inaccuracy in the results of the assay. 9The passage cell used here will be passage 5 cell obtained from Department of Pathology Anatomy Faculty of Medicine University of Indonesia.The next step is to input the cultured cells into 96-microwell with each well containing 10 4 cells.The well will be left overnight to make the cells adhere to the base of the wells.On the next day, the medium will be changed and the cells will be treated with the four extracts of G. verrucosa of 5 different concentrations.The treated HeLa cell line will be incubated for 48 hours.Later, an addition of 20 μl 5 solution 5mg/mL MTT phosphate-buffered saline (PBS) will be added into the wells.The mixture inside the plates are incubated for 4 hours, then it will be centrifuged and separated from its medium.About 20 μl DMSO is added into every well so that it will dissolve a blue purple sediment.Absorbance is measured in 590 nm on a microplate reader (Model 550, Bio-Rad, USA). 9-10Inhibitory activity of the extracts are measured using the following formula.%Inhibition absorbance of treatment group IC 50 is calculated using Microsoft Excel 2013 by connecting the G. verrucosa extract concentration (x-axis) and the percent inhibition (y-axis), from which a linear equation was then created.

Cytotoxic Activity of Seaweeds G. verrucosa
Figure 2 shows the percentage inhibition of all G. verrucosa extracts ranges from 13.8 -71.4% against cervical HeLa cells.Based on linear equation IC 50 all extract has small IC 50 under 20 µg/mL.Hexane extract of G. verrucosa showed the greatest anticancer activity compared to others, they are shown on Table 3.

Cytotoxic Activity of Seaweeds G. verrucosa
Cytotoxic activity is depicted by IC 50 value.The lower the IC 50 value of an extract, the greater the anticancer exhibited, while, percentage inhibition tells how well the extract can inhibit the cancer cell growth.The higher percentage inhibition is; the greater inhibition is shown by the sample towards cell growth.Figure 2 displays the percentage inhibitory activity and the concentration of the extract towards cervical HeLa cells.The results indicate that percentage inhibition and relative concentration of the extract is directly proportional.Which means, all of the four extracts are concentration-dependent in terms of inhibiting the growth of HeLa cells.Anticancer activity of G. verrucosa is depicted by IC 50 value (µg/mL).anticancer activity level of the extracts are categorized according to the median inhibitory concentration (IC 50 ) into four groups: ≤20 μg/mL, active; >20-100 μg/mL, moderately active; >100-1000 μg/mL, weakly active; and >1000 μg/mL, inactive. 11As displayed in Table 2, the IC 50 value of all extracts are less than 20, this means that all of G. verrucosa extracts are active in inhibiting the growth of cervical HeLa cells.The most effective extract is hexane extract, as it has the smallest IC 50 compared to other extracts.This means that it only requires a small amount of hexane extract of G. verrucosa to be able to inhibit 50% of cervical HeLa cells.This result proves that G. verrucosa has a big potential on becoming anti-cervical cancer drug.The main objective of making 4 different extracts of G. verrucosa in different solvents of varying polarity is to determine at which polarity, the secondary metabolites of seaweed G. verrucosa work best in inhibiting the growth of HeLa cervical cancer cells.Secondary metabolites of seaweed vary in their degree of polarity, and the presence of those secondary metabolites can be evaluated through several methods such as phytochemical analysis and thin layer chromatography.The principle is, compounds are dissolved according to the polarity of their solvent.Polar compounds are more likely to be dissolved in polar solvents, while non polar compounds are more likely to be dissolved in non polar solvents. 12The polarity, concentration, and physiochemical characteristics of solvents will affect the activity of bioactive compounds present in the seaweed. 13A study done in Jakarta, Indonesia performed phytochemical analysis on seaweed G. verrucosa which also comes from the same habitat studied here.The phytochemical result was positive for saponin and triterpenoid. 8The presence of triterpenoid and saponin in chloroform, hexane, ethanol, and ethyl acetate extract might be the reason why seaweed G. verrucosa is able to inhibit the growth of HeLa cervical cancer cells. 8Triterpenoid has nonpolar characteristic and dissolved better in a nonpolar solvent such as hexane and chloroform, thus it can be concluded that the most dominant secondary metabolite in inhibiting the growth of HeLa cervical cancer cells is triterpenoid rather than saponin. 12owever, this does not mean that the secondary metabolite of saponin is inactive against HeLa cervical cancer cells, because the ethanol also shown anticancer activity towards HeLa cervical cell line. 14[20]

CONCLUSION
Our results clearly demonstrate that seaweed G. verrucosa can be further developed to be an anti-cervical cancer drug.

Figure 2 :
Figure 2: The relationship between relative concentrations of an extract and their percentage inhibition towards cervical HeLa cells.