{"id":56476,"date":"2024-03-20T11:12:03","date_gmt":"2024-03-20T11:12:03","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=56476"},"modified":"2024-04-01T19:25:12","modified_gmt":"2024-04-01T19:25:12","slug":"cytotoxic-and-antiproliferative-testing-of-hela-cervical-cancer-cells-using-seagrass-ethanolic-extraction-cymodocea-rotundata-and-enhalus-acoroides","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no1\/cytotoxic-and-antiproliferative-testing-of-hela-cervical-cancer-cells-using-seagrass-ethanolic-extraction-cymodocea-rotundata-and-enhalus-acoroides\/","title":{"rendered":"Cytotoxic and Antiproliferative Testing of HeLa Cervical Cancer Cells Using Seagrass Ethanolic Extraction (Cymodocea rotundata and Enhalus acoroides )"},"content":{"rendered":"\n

Introduction <\/strong><\/p>\n\n\n\n

Occurrence of cancer cases tends to increase\nthroughout the years. The highest incidence of cancer\ncases is in Asia countries, from which in Indonesia itself has a total of 398,914 cases with the highest category is breast cancer at 16.6% and followed by cervical\ncancer at 9.2%1<\/sup>. From Ministry of Health of Indonesian\nRepublic, Cervical cancer is a type of cancer that\ndevelops in the cervix which is in the lower third of the uterus and is\nconnected to the vagina through the external uterine ostium2<\/sup>. This happens because of the presence of the\nHPV virus (Human Papilloma Virus<\/em>).\nHPV 16 and 18 are oncogenes which are responsible for 70-80% of cervical cancer\ncases in the world3<\/sup>.<\/p>\n\n\n\n

Currently there are several ways of treating cervical cancer such as removal of localized cancer tissue (surgery), irradiation of radiation, and chemotherapy. Each of these treatments is carried out according to the type and stage of cancer when diagnosed4<\/sup>. However, this therapy has side effects such as scarring after surgery, experiencing alopecia, nausea, emesis, anemia, fatigue, infection, infertility, menopause, weight changes, hepatotoxicity and triggers cancer in other organs5<\/sup>.  Therefore, searching other alternative treatments must be conducted, one of which is by utilizing natural ingredients such as seagrass Cymodecea rotundata<\/em> and Enhalus acoroides<\/em> which have to be known contain some bioactive compounds such as alkaloids, flavonoids, saponins and steroids6<\/sup> as drugs, antioxidants, antitumor and anticancer7<\/sup>. Previous study8<\/sup> indicated stated that methanolic extraction of Enhalus acoroides<\/em> had potential as an anticancer by performing cytotoxic anti-proliferative activity on HeLa<\/em> cell line. Based on that previous study, the aim of this study was to further analyze the action of cytotoxic and antiproliferative activity of other sea grasses, namely Cymodecea rotundata<\/em> as well as Enhalus acoroides<\/em> against HeLa<\/em> cell line. But at this study ethanol was used to extract some bioactive from two different sea grasses, since ethanol is the most commonly compound used for bioactive extraction of many plants. <\/p>\n\n\n\n

Material and methods <\/strong><\/p>\n\n\n\n

This\nresearch was conducted from September to June 2023. Cymodecea rotundata<\/em> was obtained from\nthe Tegal Perak beach, Pesawaran Regency of Lampung\nIndonesia and Enhalus acoroides<\/em> was obtained from Dollar Beach Padada beach,\nSouth Lampung Regency of Lampung Indonesia. The seagrass obtained was cleaned with running water, dried,\nextracted by maceration method using 96% ethanol with a ratio of 1:10 for 24\nhours. The obtaining extract then were\nevaporated using a rotary evaporatory with a temperature of 50\u00b0C until a thick\nextract was obtained9<\/sup>.\nThen the phytochemical determination was carried out with the following\nprocedure:<\/p>\n\n\n\n

Table 1: Procedure for Determining Secondary Metabolites10<\/sup><\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
\n

Test type<\/strong><\/p>\n<\/td>\n

\n

Treatment<\/strong><\/p>\n<\/td>\n

\n

Indicator<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Alkaloids<\/p>\n<\/td>\n

\n

0.5 ml sample was used then 5 drops of chloroform was added followed by 5 drops of Mayer’s reagent (1 g of KI dissolved in 20 ml dH2<\/sub>O and added by 0.271 g HgCl2<\/sub>)<\/p>\n<\/td>\n

\n

The color of the solution is brownish white<\/p>\n<\/td>\n<\/tr>\n

\n

Flavonoids<\/p>\n<\/td>\n

\n

0.5 ml sample was added with 0.5 g Mg and 5 ml concentrated HCl (added drop by drop)<\/p>\n<\/td>\n

\n

The color of the solution is red or yellow in the form of foam<\/p>\n<\/td>\n<\/tr>\n

\n

Saponins<\/p>\n<\/td>\n

\n

0.5 ml of sample was added with 5 ml of dH2<\/sub>O, shaken well for 30 seconds<\/p>\n<\/td>\n

\n

Formed foam<\/p>\n<\/td>\n<\/tr>\n

\n

Steroids<\/p>\n<\/td>\n

\n

0.5 ml sample was added with 0.5 ml glacial CH3<\/sub>COOH and 0.5 ml H2<\/sub>SO4<\/sub><\/p>\n<\/td>\n

\n

The color of the sample changes to blue or purple<\/p>\n<\/td>\n<\/tr>\n

\n

tannins<\/p>\n<\/td>\n

\n

1 ml of sample was added with 3 drops of FeCl3<\/sub> solution<\/p>\n<\/td>\n

\n

The color of the solution becomes black<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

HeLa cell culture media was prepared according to procedure of CCRC12<\/sup>.  At room temperature, 5 ml of 10% Fetal Bovine Serum (FBS) and 0.5 ml Penicillin Streptomycin (Pensterp) was added and blended in a sterilized bottle (to thaw cell line), then followed by adding DMEM (Dulbecco’s Modified Eagle’s Medium) up to 50 ml.<\/p>\n\n\n\n

HeLa cell cultures were taken from liquid nitrogen tanks and then thawed in a water bath at 37\u00b0C for 3 minutes. By using a sterile conical tube containing 10 ml of DMEM culture medium, cells were placed and incubated for 4 hours at 36\u00b0C. The cancer cells then were centrifuged for 5 minutes at 1500 rpm to separate from the medium. The obtaining supernatant was removed and HeLa cells were grown in 4 tissue culture flasks containing 10% FBS dissolved in DMEM. The flasks were kept at 36\u00b0C with 5% CO2<\/sub> flow and lid flask loosened to optimize aeration for cell growth. The media was replaced after 3 days and the cells were grown again until 80% concentration was sufficiently for treatments11<\/sup>.<\/p>\n\n\n\n

HeLa cell harvesting was carried out after the cells reached 80% confluence as indicated by the cells filling the tissue culture flask, then the cells were released from the flask wall by aspirating the media using a sterile Pasteur pipette. Five (5) ml of PBS was used to wash cultured cells and it was repeated twice. Cells then were added with 0.25% trypsin EDTA solution to release cells and again they were incubated in a CO2<\/sub> incubator at 36\u00b0C for 5 minutes. The cells then were added by 5 ml of media which had been mixed with 10% FBS and Pensterp, and then re-suspended again with a pipette until it did not show any clotting. The re-suspended cells were then put into a sterile conical tube11<\/sup>.<\/p>\n\n\n\n

Cell count. The cells in the conical tube were then filled with 3 ml of media. Then the cells were centrifuged for 5 minutes at 1500 rpm. The supernatant was discarded and 10 ml of fresh media was added to the precipitated formed nathan.  Cells as much 10 \u03bcl was pipetted into the well plate and was added by 10 \u03bcl of trypan blue. The number of cells was calculated, 10 \u03bcl of the mixed cells was pipetted into a hemocytometer. Live cells were colorless or clear, while non-living cells were blue. Cell count with a hemocytometer was carried out by selecting 4 counting chambers under a microscope. A series of calculations for determining the number of cells to be cultured was followed12,13<\/sup>.<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

Number of cells counted\/ml = mean\ncells x dilution factor x 10 4<\/sup><\/p>\n\n\n\n

Total number required cells = number of wells x number of cells per well<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

The\ncell harvest volume calculations then were\ntransferred into the conical tube and\nmedia was added.<\/p>\n\n\n\n

Stock solution preparation<\/strong><\/p>\n\n\n\n

The ethanol extracts of Cymodocea rotundata<\/em> and Enhalus acoroides<\/em> were performed prior to the cytotoxicity and antiproliferation tests. The stock solution was prepared by dissolving 10 mg of the extract with 1 ml of 5% dimethyl sulfoxide (DMSO). Then, it was put in a bath at 30\u00b0C until the extract dissolves. The stock solution was put into closed sterile microtubes and stored in the refrigerator prior used.  For the study treatments, the stock solution then was diluted again to concentrations of 2000 ppm, 1000 ppm, 5000 ppm, 250 ppm, 125 ppm and 62.5 ppm, either for Cymodocea rotundata<\/em> and Enhalus acoroides<\/em>. As for Doxorubicin (used as control treatment), we used concentrations of 20 ppm, 10 ppm, 5 ppm, 2.5 ppm, 1.25 ppm, and 0.625 ppm. The prepared Doxorubicin and extract solutions in various concentrations were then tested on HeLa<\/em> cells in a Laminar Air Flow Cabinet12<\/sup>.<\/p>\n\n\n\n

Cytotoxic activity<\/strong><\/p>\n\n\n\n

For the cytotoxic evaluation, the final harvested volume of cells was used and affixed to the medium.  They were loaded into each well plate as much as 100 \u03bcl followed by incubation in 5% CO2<\/sub> incubator for 24 hours at 36\u00b0C aiming that the cells would stick to the wall of the flask12<\/sup>. The 24 hour- cultured cells were removed from the incubator and then perceived under a microscope. Prior rinsing with PBS solution, the culture medium was discarded. Each well plates then was given sea grass extract of 50 \u00b5l of each treatment  concentrations (62.5 ppm, 125 ppm, 250 ppm, 500 ppm, 1000 ppm and 2000 ppm for both sea grasses extract) as well as for Doxorubicin at concentration of 0.625 ppm, 1.25 ppm, 2.5 ppm, 5 ppm, 10 ppm and 20  ppm.  Meanwhile for the control cells, they were given DMSO which used as solvent. They were then incubated again in a 5% CO2<\/sub> incubator at 36\u00b0C for 24 hours. After incubation time, medium was removed from each well plates and each well plates was rinsed using PBS and given 50 \u00b5l of CCK-8 reagent and reared again in a CO2<\/sub> incubator at 36\u00b0C for 2 hours. After incubation for 2 hours, viability of the cells of each well plates were determined by using ELISA reader at a wavelength of 450 nm13,14<\/sup>. To obtain HeLa cell viability (in percent), the following formula was used13<\/sup>.<\/p>\n\n\n\n

\"\"<\/figure>\n\n\n\n

The percentage value of cell viability was\nthen converted into a probit value to determine the IC50<\/sub> value using\nthe Microsoft Excel program.<\/em><\/p>\n\n\n\n

Antiproliferation activity determination<\/strong><\/p>\n\n\n\n

A 100 \u03bcl of cell suspension was filled to each well plates followed by incubation in a CO2<\/sub> incubator 24 hours at 37\u00b0C. After 24 hours of incubation, the cells were removed from the incubator and then perceived under a microscope. After discarding culture media, cells were washed with PBS solution. With the same procedure as those of cytotoxic activity determination, each well plates were incubated a 5% CO2<\/sub> incubator at 36\u00b0C within different time treatments, namely 24, 48 and 72 hours. After the incubation time was attained, the test solution was removed, and the well plates were washed again with PBS solution and given with 50 \u03bcl of CCK-8 reagent and again incubated in a CO2<\/sub> incubator at 36\u00b0C for 2 hours. After incubation for 2 hours, viability of the cells of each well plates were determined by using ELISA reader at a wavelength of 450 nm13,14,15<\/sup>. In the anti-proliferation test, data processing was carried out to determine the difference in the number of living cells from extract treatment with numerous concentrations.   Meanwhile the different incubation times was used to determine the doubling time value for each treatment using the following formula16<\/sup>:<\/p>\n\n\n\n

Double time =\n \n<\/p>\n\n\n\n

In which  Y = Log (2 x Number of initial living cells); A = Intercept; B = Slopes<\/p>\n\n\n\n

Results and Discussion <\/strong><\/p>\n\n\n\n

The\nweight of simplisia and the pasta of the sea grasses, Cymodocea rotundata<\/em> and Enhalus acoroides<\/em>,<\/em> extraction could be seen in Table 2. This result was\nneeded to show how much percentage that we could gain from 100 gram of\nsimplisia (dry mass of both sea grasses) which seemingly that both had similar\npercentage of less than 1 %. <\/p>\n\n\n\n

Table 2: The mass extract of Cymodocea rotundata <\/em>and Enhalus acoroides<\/em><\/strong><\/p>\n\n\n\n\n\n\n\n\n
\n

No<\/strong><\/p>\n<\/td>\n

\n

Seagrass<\/strong><\/p>\n<\/td>\n

\n

Sample Mass (grams)<\/strong><\/p>\n<\/td>\n

\n

Percent<\/strong><\/p>\n

(%)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Simplisia<\/strong><\/p>\n<\/td>\n

\n

Pasta<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

1<\/p>\n<\/td>\n

\n

Cymodocea rotundata<\/p>\n<\/td>\n

\n

100<\/p>\n<\/td>\n

\n

4.586<\/p>\n<\/td>\n

\n

0.046<\/p>\n<\/td>\n<\/tr>\n

\n

2<\/p>\n<\/td>\n

\n

Enhalus acoroides<\/p>\n<\/td>\n

\n

100<\/p>\n<\/td>\n

\n

4.952<\/p>\n<\/td>\n

\n

0.049<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

While the phytochemical\ntests (qualitatively, just to indicate that some\nbioactive was visible) on the extracts of Cymodocea rotundata <\/em>and Enhalus acoroides <\/em>waspresented in table 3 as follows:<\/p>\n\n\n\n

Table 3: Phytochemical content of Cymodocea rotundata and Enhalus acoroides extracts<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n
\n

No.<\/strong><\/p>\n<\/td>\n

\n

Phytochemical Test<\/strong><\/p>\n<\/td>\n

\n

Cymodocea rotundata<\/strong><\/p>\n<\/td>\n

\n

Enhalus acoroides<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

1<\/p>\n<\/td>\n

\n

Alkaloids<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n<\/tr>\n

\n

2<\/p>\n<\/td>\n

\n

Flavonoids<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n<\/tr>\n

\n

3<\/p>\n<\/td>\n

\n

Saponins<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n<\/tr>\n

\n

4<\/p>\n<\/td>\n

\n

Steroids<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n<\/tr>\n

\n

5<\/p>\n<\/td>\n

\n

tannins<\/p>\n<\/td>\n

\n

+<\/p>\n<\/td>\n

\n

–<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

Note: +\/-: Indicating of such bioactive compound exist or not<\/p>\n\n\n

The cytotoxic activity of the ethanolic extraction of sea grasses, Cymodocea rotundata<\/em> and Enhalus rotundata<\/em> by using CCK-8 assay could be seen in Fig 1 and 2 as follows. <\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1<\/strong>:<\/strong> Cytotoxic of <\/strong>Cymodocea rotundata <\/em><\/strong>ethanol extract <\/strong>on HeLa<\/em> Cell line <\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Percentage of HeLa<\/em> cell viability was reduced in treatment using the ethanol extract of Cymodocea rotundata<\/em> compared to control cells.  Meanwhile, Cymodocea rotundata<\/em> at concentrations of 1000 ppm and 2000 ppm, was able to sharply reduce the viability of HeLa<\/em> cells, even compared to the Doxorubicin at the highest concentration in this study (5 \u2013 10 ppm). The decrease in cell viability at these concentrations was 97.61% and 99.02%.  This indicated that ethanolic extraction of Cymodocea rotundata<\/em> contained of bioactive which presumably could be elaborated to be one of potential anticancer drug from one of marine plants. Yet, further much deeper study is needed.  <\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 2<\/strong>:<\/strong> Cytotoxic of Enhalus acoroides<\/em><\/strong> ethanol extract <\/strong>on HeLa<\/em> Cell line<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

As well as those seen in Cymodocea rutundata<\/em>, the ability of Enhalus acoroides<\/em> ethanolic extract\nto suppress cell viability at concentrations of 1000 ppm and 2000 ppm was higher than those of the control\ngroup and\nDoxorubicin treated groups. The\npercentage of decreasing in cell\nviability at these concentrations was 97.44% and 85.48%.  Again,\nthis indicated that ethanol extract of Enhalus acoroides<\/em> at these two concentrationa were more toxic than those of control drug using Doxorubicin.<\/p>\n\n\n\n

In\norder to determine the IC50<\/sub> value of both sea-grasses ethanolic extract, linear\nregression was plotted for both sea-grasses ethanolic extraction, which could be seen in Figures 3 and 4 as follows.  From\nthese figures (Fig. 3,\n4), it\ncould be seen that the\nextracts of Cymodocea rotundata<\/em> and Enhalus acoroides<\/em> were\nboth able to decrease cell\nviability of HeLa<\/em> with each IC50<\/sub> values less than 1000 ppm.  Beside, the IC50<\/sub> value of the Enhalus acoroides<\/em> extract was also\nlower than\nthat of Cymodocea rotundata<\/em> <\/em>extract.<\/p>\n\n\n\n

Cytotoxic activity of HeLa<\/em>\ncells using Cymodocea rotundata<\/em> and Enhalus acoroides<\/em> extracts were carried\nout to test the toxic potential of the extracts used using the test parameter,\nnamely IC50<\/sub> (50% inhibitory concentration)17<\/sup>. IC50<\/sub>\nis the concentration used to inhibit or inhibit the activity of cancer cells.\nThe smaller the IC50<\/sub> value obtained, the better some compound to\nhave alternative potential as an anticancer drug by inhibiting as much as 50%\nof the proliferative activity of the cancer cells themselves14<\/sup>. It\nis known that the ethanolic extract of Enhalus\nacoroides<\/em> had an IC50<\/sub> value of 645.96 ppm, which was lower than Cymodocea rotundata<\/em> of 856.65 ppm.<\/p>\n\n\n\n

According to the NCI (National Cancer Institute) it is known\nthat there are categories of compounds that are considered to be toxic. The\ncategories are seen from their IC50<\/sub> values based on U.S. National Cancer Institute (NCI) and Geran protocol,\nwhich are as follows: IC50<\/sub> \u2264 20 \u03bcg\/ml = high, IC50<\/sub> 21-200\n\u03bcg\/ml = moderate, IC50<\/sub> 201-500 \u03bcg\/ml = weak and IC50<\/sub> >\n501 \u03bcg\/ml = no toxic18<\/sup>. Based on these categories, the ethanol\nextracts of Cymodocea rotundata<\/em> and Enhalus acoroides<\/em> of this study was included in\ncompounds that was not toxic but both does have the potential as\nanticancer agents to inhibit HeLa<\/em>\ncell growth. This is in accordance with the finding of other study19<\/sup>\nwhich states that if a compound had an IC50<\/sub> value of <1000 \u03bcg\/ml\nthen the compound has the potential as an anticancer.<\/p>\n\n\n\n

Differences\nin IC50<\/sub> cytotoxic values in sea-grasses extract of Cymodocea<\/em> rotundata<\/em>\nand Enhalus acoroides<\/em> could be\naccounted from the differences in bioactivity produced from both sea-grasses,\nfrom  which can be also influenced by their environment (chemical and\nphysical condition) where they grow. These differences were thought to\naffect differences in chemical content so that the bioactivity could also be\ndifferent20<\/sup>. Sea-grasses Cymodocea\nrotundata<\/em> has a special feature where the leaf edges are not serrated, and\nthe leaf sheaths are closed. The rhizomes of this sea-grass are smooth, and\nhave irregularly branched roots. This sea-grass habitat is on a muddy sand\nsubstrate. Whereas the Enhalus acoroides<\/em>\nsea-grass has a feature where its largest size can reach 1 meter and there are\nhairs on its rhizomes. Enhalus acoroides<\/em>\nusually grows and lives in tidal areas21<\/sup>.<\/p>\n\n\n\n

Besides being influenced by their environment, the cytotoxic\nability of Cymodocea rotundata<\/em> and Enhalus acoroides<\/em> is also influenced by\nthe secondary metabolites contained therein. Cymodocea rotundata<\/em> and Enhalus\nacoroides<\/em> are known to contain active compounds such as alkaloids,\nflavonoids, saponins and steroids. In addition, Cymodocea rotundata<\/em> is also known to contain tannins as those also indicated from its\nphytochemical tests. Tannins contained in Cymodocea rotundata<\/em> are thought to have anticancer activity by\ninhibiting tyrosine kinase and also as antioxidants. Tannins also have\nmechanisms that can inhibit enzymes such as transcriptase and DNA topoisomerase22<\/sup>.\nOther study also indicated\nthat Cymodocae rotundata<\/em>\ncontained coumarins, flavonoids, phenols, proteins, free amino acids, quinones,\nsaponins, sterols, sugars, terpenoid bioactive compounds with antibacterial,\ncytotoxic and hemolytic activity23<\/sup>.<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 3<\/strong>:<\/strong> Cytotoxic Activity of Cymodocea rotundata <\/em>extract
 <\/strong>on HeLa<\/em> cell line <\/strong>(IC50<\/sub> 856.65 ppm).<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n
\"\"<\/td>\n\n

Figure 4<\/strong>:<\/strong> Cytotoxic Activity of Enhalus acoroides <\/em><\/strong>on HeLa <\/em><\/strong>cell line <\/strong>(IC50<\/sub> 645.96 ppm).<\/strong><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The antiproliferation activity of both sea-grasses could be seen in Figure 5 (a, b) as follows:<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 5<\/strong>:<\/strong> C<\/strong>ell viability at 24, 48, and 72 hours of incubation of ethanol extract <\/strong>(a) <\/strong>Cymodocea rotundata <\/em><\/strong>(b) Enhalus acoroides<\/em><\/strong> <\/em><\/p>\n

<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

The highest average number of living cells in Cymodocea rotundata<\/em> extract was found at 62.5 ppm group at 24 hours of incubation. The lowest\nnumber of live cells was found at a concentration of 500 ppm at 48 hours. This\nshowed that given of Cymodocea rotundata<\/em> had a significant\neffect on HeLa<\/em> cell death because the\nhigher the concentration and time, the lower the living cell average. In the treatment of\nEnhalus acoroides<\/em> extract there was a\nsignificant difference among\ntreatment groups with the highest number of\nliving cells was\nobtained from 2000 ppm at 24 hours of incubation. The lowest cell count was at\na concentration of 500 ppm and within 24 hours. <\/p>\n\n\n\n

Furthermore, the calculation of the doubling time value was carried out to determine each treatments on the proliferative ability of HeLa<\/em> cells. The results of the doubling time values obtained after being treated with the ethanolic extracts of Cymodocea rotundata<\/em> and Enhalus acoroides<\/em> could be seen in Table 4 as follows:<\/p>\n\n\n\n

Table 4: Doubling time of HeLa<\/em> Cell line  given extracts of C. rotundata <\/em>andE. acoroides<\/em><\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

Sea-grass<\/strong><\/p>\n<\/td>\n

\n

Concentration (ppm)<\/strong><\/p>\n<\/td>\n

\n

Incubation Time Equations with Logs of Cell Count<\/strong><\/p>\n<\/td>\n

\n

Slope<\/strong><\/p>\n<\/td>\n

\n

Value of doubling time (hours)<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

Cymodocea rotundata<\/em><\/p>\n<\/td>\n

\n

2000<\/p>\n<\/td>\n

\n

Y= 6973.2 + -18741<\/p>\n<\/td>\n

\n

6973,2<\/p>\n<\/td>\n

\n

269<\/p>\n<\/td>\n<\/tr>\n

\n

1000<\/p>\n<\/td>\n

\n

Y= 11232 + -33386<\/p>\n<\/td>\n

\n

11232<\/p>\n<\/td>\n

\n

297<\/p>\n<\/td>\n<\/tr>\n

\n

500<\/p>\n<\/td>\n

\n

Y = 2499 + -522.5<\/p>\n<\/td>\n

\n

2499<\/p>\n<\/td>\n

\n

21<\/p>\n<\/td>\n<\/tr>\n

\n

250<\/p>\n<\/td>\n

\n

Y= 24515 + -83222<\/p>\n<\/td>\n

\n

24515<\/p>\n<\/td>\n

\n

339<\/p>\n<\/td>\n<\/tr>\n

\n

125<\/p>\n<\/td>\n

\n

Y= 50867 + -197726<\/p>\n<\/td>\n

\n

50867<\/p>\n<\/td>\n

\n

389<\/p>\n<\/td>\n<\/tr>\n

\n

62.5<\/p>\n<\/td>\n

\n

Y= 65162 + -261294<\/p>\n<\/td>\n

\n

65162<\/p>\n<\/td>\n

\n

401<\/p>\n<\/td>\n<\/tr>\n

\n

Enhalus acoroides<\/p>\n<\/td>\n

\n

2000<\/p>\n<\/td>\n

\n

Y= 44314 + -167948<\/p>\n<\/td>\n

\n

44314<\/p>\n<\/td>\n

\n

379<\/p>\n<\/td>\n<\/tr>\n

\n

1000<\/p>\n<\/td>\n

\n

Y= 7039.7 + -21094<\/p>\n<\/td>\n

\n

7039.7<\/p>\n<\/td>\n

\n

300<\/p>\n<\/td>\n<\/tr>\n

\n

500<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

250<\/p>\n<\/td>\n

\n

Y= 33696 + -124783<\/p>\n<\/td>\n

\n

33696<\/p>\n<\/td>\n

\n

370<\/p>\n<\/td>\n<\/tr>\n

\n

125<\/p>\n<\/td>\n

\n

Y= 46270 + -177917<\/p>\n<\/td>\n

\n

46270<\/p>\n<\/td>\n

\n

385<\/p>\n<\/td>\n<\/tr>\n

\n

62.5<\/p>\n<\/td>\n

\n

Y= 57926 + -229609<\/p>\n<\/td>\n

\n

57926<\/p>\n<\/td>\n

\n

396<\/p>\n<\/td>\n<\/tr>\n

\n

Cell Control<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

Y= 42842x – 16422<\/p>\n<\/td>\n

\n

42842<\/p>\n<\/td>\n

\n

38<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n\n\n

It was seen that the doubling times obtained in the treatment of Cymodocea rotundata<\/em> and Enhalus acoroides<\/em> ethanolic extracts were different. The slope value of Cymodocea rotundata<\/em> ethanol extract at concentrations of 62.5 ppm and 125 ppm were greater than the control cells with doubling time values of 401 hours and 389 hours. In the ethanol extract of Enhalus acoroides<\/em> it was found that the slope values at concentrations of 62.5 ppm, 125 ppm, and 2000 ppm had a greater slope value than the control group with doubling time values of 396 hours, 385 hours, and 379 hours. The smaller the slope value obtained the longer the time required for doubling the time and as well as the greater the slope value of the treatment groups compared to the control cell, the shorter the doubling time is.<\/p>\n\n\n\n

The\nantiproliferative ability of the ethanol extracts of Cymodocea rotundata<\/em> and Enhalus\nacoroides<\/em> showed in longer doubling time than control cells. This means\nthat the ethanol extracts of Cymodocea\nrotundata<\/em> and Enhalus acoroides<\/em> were able to inhibit the growth speed\nat which HeLa<\/em> cervical cancer cells\nmultiply. If such compound could delay the doubling time of cells, then there\nwas possibility that the compound could inhibit the genes and proteins which regulate\nthe cell cycle. Presumably, the inhibition of cell proliferation was caused by\nthe mechanism of secondary metabolites contained in the ethanol extract of Cymodocea rotundata<\/em>, Enhalus acoroides<\/em> such as alkaloids,\nflavonoids, saponins, steroids and tannins. Alkaloids, which also found in seagrass Cymodocea rotundata<\/em> and Enhalus acoroides,<\/em> are known to have\nanticancer activity24<\/sup>. From which alkaloids could act as antiangiogenic,\nantiproliferative, inhibit topoisomerase activity, tubulin polymerization and\ninduce apoptosis24<\/sup>. Alkaloids also can increase apoptosis by\ninducing DNA damage25<\/sup>. Alkaloids also have potential as antioxidants\nby donating H atoms to free radicals, so that free radicals will be stable26<\/sup>.<\/p>\n\n\n\n

Flavonoid compounds are usually found in several plants and are used as disease prevention, one of which is as an anticancer by inhibiting cell proliferation through inhibiting oxidative processes which can initiate cancer cells in the body. Presumably the enzymes xanthin oxidase, Cyclooxygenase (COX) and Lipooxygenase (LOX) was dropped, causing slowing down in cell cycle27<\/sup>. Flavonoids affect the induction of apoptosis by increasing the activity of caspase 3 and cox 228<\/sup>. In addition, the enzyme expression for topoisomerase I and II was also inhibited. The topoisomerase complex will be alleviated then by topoisomerase enzyme inhibitor triggering DNA to be cut and damaged29<\/sup>. Tannins have activity as free radical scavengers and are able to inhibit lipoxygenase and lipid peroxidase. Tannins inhibit the S phase or cell cycle synthesis. The cell will carry out DNA synthesis and chromosome replication in the S phase30<\/sup>. Based on the compounds contained in the seagrass Cymodocea rotundata<\/em> and Enhalus acoroides<\/em>, the two seagrasses can be used as anticancer agents based on the IC50<\/sub> value for inducing apoptosis and the doubling time value on the proliferative ability of HeLa<\/em>, cervical cancer cells.<\/p>\n\n\n\n

Conclusion <\/strong><\/p>\n\n\n\n

From this study we can conclude that the activity of the ethanolic extraction of Cymodocea rotundata<\/em> and Enhalus acoroides<\/em> was cytotoxic against HeLa<\/em> cervical cancer cells, as evidenced by reducing viability cell percentage related to the control group with IC50<\/sub> values of 856.65 ppm and 645.96 ppm. The ethanol extracts of Cymodocea rotundata<\/em> and Enhalus acoroides<\/em> also have antiproliferative against HeLa<\/em> cells. This can be seen in the longer doubling time compared to the control cells. Based on this, the ethanol extracts of Cymodocea rotundata<\/em> and Enhalus acoroides<\/em> have potential as HeLa<\/em> cervical anticancer agents.<\/p>\n\n\n\n

Acknowledgement  <\/strong><\/p>\n\n\n\n

We deeply appreciated to the Research and Community Service Institute (LPPM) of University of Lampung and Graduate University of Lampung and to the CC&C Laboratory-Faculty of Pharmacy, UNPAD (Padjajaran University-Indonesia) for helping us carry out this research.<\/p>\n\n\n\n

Conflict of Interest <\/strong><\/p>\n\n\n\n

There is no conflict of interest on publishing this article. <\/p>\n\n\n\n

Funding Source<\/strong><\/p>\n\n\n\n

The source of funding this research was supported by the University of Lampung under the Research and Community Service Institute (LPPM \u2013 UNILA) through BLU DIPA with contract number 861\/UN26.21\/PN\/2023.   <\/p>\n\n\n\n

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