Breast and cervical cancers are the leading cause of death in women, and chemotherapy with cytotoxins is the usual treatment. This study evaluated the cytotoxicity of guava leaf (Psidium guajava L.) extracts as an alternative chemotherapeutic drug. Although many studies related to the cytotoxic effects of guava leaf (Psidium guajava L.) on cancer cells have been reported, the effects of guava leaf fractions on human breast and cervical cancer cells (T47D, MCF-7, and HeLa) have never been evaluated. Herein, we researched candidate activities of ethanol, ethyl acetate, and n-extracts from guava leaf fractions and their effect on various human cancer cell lines (T47D, MCF-7, and HeLa cells). The cytotoxicity test was carried out using the microtetrazolium assay for all fractions. We confirmed and showed the in vitro antitumor activity of guava leaf (Psidium guajava L.) fractions in human breast and cervical cancer cells. We found that the effectiveness of anticancer activity increased from ethanol to ethyl acetate to n-hexane fraction. This work underlines the potential of n-hexane fraction as a chemotherapeutic drug. These novel results have important implications for further isolation, identification, and characterization of Psidium guajava L.-based anti-cancer extracts.
Cancer is ranked as the third leading cause of death in the world. Estimates from the IARC (International Agency for Research on Cancer) indicate that, by 2,040, the number of new cancer cases could reach 30.2 million cases with a death rate of up to 16.3 million cases. The most common cancers are breast cancer (11.7%), lung cancer (11.4%), colon or rectum cancer (10%), prostate cancer (7.3%), stomach cancer (5.6%), liver cancer (4.7%), cervical cancer (3.1%), and other cancers (46.2%) [1,2]. However, this situation has been an object of interest for many researchers and they have carried out impressive studies on the isolation and synthesis field to end the harms of cancer [3,4,5,6].
Breast and cervical cancer is malignant cancer, which is most commonly found in women and is one of the causes of death, especially in developing countries . Because of the high prevalence of cancer, complementary and alternative medicines are needed in cancer treatment . Several strategies in the discovery of new anticancer compounds have been performed such as isolating active compounds from natural products, exploring secondary metabolites to specifically inhibit cancer cells, and synthesizing potential organic compounds with anticancer activity [9,10,11,12,13].
It is known that 30,000–40,000 species of medicinal plants in the world are available in Indonesia. However, only a portion of these plants has been developed for therapeutic drugs , particularly cancer therapy . Several plants have been investigated as therapeutic agents for leukemia, lung cancer, breast cancer, liver cancer, and prostate cancer [16,17,18,19,20,21,22,23]. Several anticancer compounds have also been isolated from other sources such as microbes (endophytic bacteria/yeast and fungi). For example, Pseudomonas putida can produce secondary metabolites that function as anticancer .
Guava (Psidium guajava L.) is a member of the Myrtaceae family and many investigations have reported that the leaf has antimicrobial [25,26,27], antidiabetic , antimalarial, , and antitumor activities [30,31]. In addition, it has been used to treat acne , coughs , and dental diseases . Guava leaf contains mostly phenolic compounds, flavonoids (morin-3-O-glycoside, morin-3-O-arabinoside, quercetin, and quercetin-3-O-arabinoside), essential oils, lipids, gallic acid, catechin, epicatechin, rutin, tannin, saponin, alkaloids, and sesquiterpenes . In particular, the flavonoids in guava leaf are reported to have strong antibacterial  and anticancer activities . However, research on the activity of guava leaf as an anticancer drug is still very limited.
To the best of our knowledge, there are several studies related to examining the anticancer activity of guava Psidium guajava L. using cell cultures. The cancer cells that have been used to test the anticancer activity of guava are prostate cancer cells , cervical cancer , colon cancer , gastric cancer , and blood cell cancer . Based on the results of the anticancer activity, we hypothesize that guava leaf has potential as an anticancer drug because it can prevent or inhibit the growth of cancer cells . The anticancer potential of the extracts is evaluated using the microtetrazolium (MTT), MTS, SRB, thymidine incorporation, and trypan blue exclusion assay to observe their cytotoxicity to cancer cells [37,38,39,40]. Although some studies related to guava leaf (Psidium guajava L.) as an anticancer were reported to exhibit cytotoxic effects in human cancer cells [41,42,43,44,45,46,47], the effects of guava leaf fraction on human breast and cervical cancer cells (T47D, MCF-7, and HeLa) have never been evaluated.
In this study, we are interested in finding alternative medicine to chemotherapy based on guava leaves (Psidium guajava L.). Our research group explored and carried out the fractionation process to separate the active compounds in guava leaves based on their polarity levels using ethanol (polar), ethyl acetate (semi-polar), and n-hexane (non-polar) as solvents to find which fractions of the compounds are active as anti-cancer agents. This will be very useful for further exploration processes in the future, which include isolation, identification, and characterization of active anti-cancer compounds. The fractionation process in this study refers to several previous studies, where fractionation was carried out with a fractionation column using non-polar, semi-polar, and ending with polar solvents continuously [35,36]. We also reported that different fractions from guava leaves act as anticancer agents characteristically and selectively against three cancer cells. Then, we focused on anticancer tests that were carried out in vitro using the MTT assay. Furthermore, we confirmed the in vitro antitumor activity of the guava leaf (Psidium guajava L.) fraction in human breast and cervical cancer cells (T47D, MCF-7, and HeLa). By understanding the selectivity of each fraction, it can be developed into an anticancer agent. The fractions used were ethanol, ethyl acetate, and n-hexane fractions of guava leaves.
2 Materials and methods
All chemicals used were of analytical grade from JT Baker (n-hexane, ethyl acetate, ethanol, and chloroform), Merck and Co. Inc (methanol, acetic acid, acetonitrile, DMSO, silica gel 60 [0.063–0.200 mm], silica gel 60 PF254, Na2SO4, H2SO4, NaOH), Sigma-Aldrich (acetone, Mg metal, HCl 37%, Dragendroff reagent, Mayer’s reagent, FeCl3, phosphate buffer saline, RPMI culture media, trypsin-EDTA, dimethyl sulfoxide [DMSO], sodium dodecyl sulfate (SDS), Dulbecco's modified Eagle’s medium [DMEM], formaldehyde, MTT). The Psidium guajava L. leaves were obtained from a tree cultivated in the Sleman District and have passed the determination and identification test by our student at the Laboratory of the Faculty of Biology, Gadjah Mada University Indonesia, Yogyakarta Province. T47D, MCF-7, and HeLa cancer cells were obtained from the collection of Gadjah Mada University.
All analysis instruments used in this study are LC-MS/MS UPLC-QToF-MS/MS System (Waters), hemositometer, laminar air flow (Innotech V-800), ELISA reader (Bio Rad Benchmark), incubator (Memmert), centrifuge (Hettich Rotina 22R), analytical measure (Fujitsu), Rotary evaporator (Heidolph Laborata 4000 efficient), shaker (SCILOGEX SK-O330-Pro), micropipette (Eppendorf Research), microtube, and 96-well microplate.
2.2 Preparation of Psidium guajava L. leaves as raw materials
The Psidium guajava L. leaves were dried in an open space without direct sunlight until dry. A total of 700 g of dried samples were blended to powder. The powder was stored in a bottle to be used in the next step.
2.3 Preparation of ethanol, ethyl acetate, and n-hexane fraction of guava leaves
The extraction was carried out using the maceration technique. About 100 g of dry Simplicia powder was immersed in ethanol in an airtight container. After 1 day, the ethanol extract was separated from the macerate using filter paper. Then, the extract was concentrated using a rotary evaporator to obtain the ethanol crude extract. This crude extract was impregnated using silica gel and put in a glass chromatography column. The process was continued by fractionating the crude extract using a fractionation column chromatography using non-polar (n-hexane), semi-polar (ethyl acetate), and finally with polar (ethanol) solvents continuously. Then, the extract was concentrated to get n-hexane, ethyl acetate, and ethanol fractions of the guava leaf as non-polar, semi-polar, and polar fractions, respectively.
2.4 Phytochemical screening
Phytochemical screening in research is an initial stage that aims to obtain an overview of the class of compounds contained in a sample. The phytochemical screening method was performed by visualizing the color testing reaction. The screening included the content of tannin/polyphenol compounds, alkaloids, flavonoids, and terpenoids according to Singh et al. .
2.5 In vitro anticancer activity of guava leaf fractions on T47D, MCF-7 cells (breast cancer), and HeLa cells (cervical cancer) with MTT assay
The in vitro anticancer activity of guava leaf fractions (n-hexane, ethyl acetate, and ethanol fraction) on T47D, MCF-7, and HeLa cells was performed with the preparation of the RewI Park 1640 culture media for T47D, HeLa cells, and DMEM for MCF-7 cells, followed by cell activation, harvesting, and calculation as initial processes . Afterward, each fraction (ethanol, ethyl acetate, and n-hexane fractions) was diluted into a solution using DMSO at concentrations of 200, 100, 50, 25, 12.5, 6.25, and 3.125 μg mL. Then, 100 μL of the fraction was added to a cell suspension in each well and incubated at 37°C. The test was replicated three times. After 24 h, culture media and the MTT reagent (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were added in a ratio of 9:1 and then incubated. After 4 h, 100 μL of SDS as stopper solution was added to the well and then placed in a dark room for 24 h. The final stage was carried out by absorbance reading using an ELISA reader (wavelength 595 nm). Cytotoxicity tests were carried out by the same method, namely in vitro on Vero cells. After IC50 values were obtained for T47D, MCF-7, HeLa, and Vero cells, then the selectivity index (SI) was calculated (see the Supporting information) by the following formula:
where A is the absorbance of the treatment (cell + culture media + fraction), B is the absorbance of the media control (culture media), and C is the absorbance of the negative control (cell + culture media).
3 Results and discussion
3.1 Phytochemical screening
Phytochemical screening results on ethanol, ethyl acetate, and n-hexane fractions of guava leaves are shown in Table 1.
|Ethanol fraction||Alkaloids||Dragendorff||Greenish-yellow solution (fixed)||−|
|Wagner||Greenish-yellow solution (fixed)||−|
|Flavonoids||HCl + Mg||Orange solution||+|
|Tannins/polyphenols||FeCl3||deposits (bluish-black solution)||+|
|Terpenoids||Anhydrous acetic acid + H2SO4||Purplish-red solution||+|
|Ethyl acetate fraction||Alkaloids||Dragendorff||Brown solution (fixed)||−|
|Wagner||Greenish-yellow solution (fixed)||−|
|Flavonoids||HCl + Mg||Precipitate (green solution)||+|
|Terpenoids||Anhydrous acetic acid + H2SO4||Dark-green solution||+|
|n-Hexane fraction||Alkaloids||Dragendorff||Brown solution||−|
|Wagner||Blackish red solution||−|
|Flavonoids||HCl + Mg||Precipitate (red solution)||+|
|Terpenoids||Anhydrous acetic acid + H2SO4||Purplish-red solution||+|
Note: (+), positive; (–), negative.
Phytochemical screenings are carried out to identify the class of active compounds in the sample, i.e., tannin/polyphenols, alkaloid, flavonoid, and terpenoid compounds. Based on visual observation, it was concluded that the n-hexane fraction contained flavonoids and terpenoids; the ethyl acetate fraction contained flavonoids, tannins/polyphenols, and terpenoids; the ethanol fraction contained terpenoids, flavonoids, and tannins/polyphenols, and these results are in line with previous studies from other researchers .
3.2 In vitro anticancer activity of guava leaf fractions on T47D, MCF-7 cells (breast cancer), and HeLa cells (cervical cancer) with MTT assay
The in vitro anticancer activity test is required to pass through several preliminary stages, i.e., preparation and harvesting of cancer cells, cytotoxicity test, MTT assay, and absorbance reading. Cell preparation was carried out by activating the cells and regenerating them until they were confluent (where the cell growth was homogeneously or equally distributed as monolayer cells covering the cover glass). In this study, our research group uses T47D and MCF-7 cells for breast cancer cells and HeLa cells for cervical cancer cells. T47D breast cancer cells are obtained from the breast tissue of adolescent and adult women who are affected by ductal carcinoma and grown in RPMI media (Roswell Park Memorial Institute). Different from T47D cells, MCF-7 cells (Michigan Cancer Foundation-7) are obtained from Caucasian female patients with DMEM. These cells were obtained from breast epithelial tissue with a pleural effusion breast adenocarcinoma metastatic point from a 69-year-old woman of Caucasian ethnicity with blood group O with Rh+. On the other hand, HeLa cells were derived from human cervical cancer epithelial cells. These cells have been isolated since 1951 from the uterus of the 31-year-old first woman, Henrietta Lack, with cervical cancer, from Baltimore, USA. Last, but not least, research related to the discovery of cancer drugs needs to be done with cytotoxic tests on normal cells to find out whether compounds that inhibit cancer cells are selective on cancer cells or attack normal body cells as well. The Vero cell is a cell line derived from the kidney of the African green monkey and used as a representative of normal cells .
In the harvesting step, cells are grown, multiplied using RewI Park 1640 culture media for T47D and HeLa cells and DMEM for MCF-7 cells and ended with the addition of trypsin to release the interaction between the cell to the matrix (cell–matrix) and cell to cell (cell–cell) to prevent cell damage. Cancer cell counting was carried out using a hemocytometer and observed using an inverted microscope to determine the number of cells in the initial step. The number of T47D and MCF-7 cells was 46 × 104/mL and 144.25 × 104/mL, respectively, and HeLa cells were 453.75 × 104/mL. The initial morphology of T47D, MCF-7, and HeLa cells are shown in Figure 1.
The cell subculture, sample preparation, and cell treatment were carried out as part of the cytotoxicity test. At the cell subculture, cells were transferred from confluent conditions to an empty place for growing optimally and generated 2.17 × 104 µL for T47D; 0.22 × 104 µL for MCF-7; and 0.69 × 104 µL for HeLa. These cells were used again in the harvesting step and divided into three groups, namely cell control, media control, and test group. The test group consisted of ethanol, ethyl acetate, and n-hexane fractions. About 100 µL of cell suspension was pipetted into plate 96, except for media control. Then, the plate was incubated in a 5% CO2 incubator at 37°C for 24 h. This enabled the cells to reach the logarithmic phase, where cells are in optimum growth conditions. The logarithmic phase is characterized by the condition of cells that are 80% confluent, covering the surface of the medium container.
The next step is the sample preparation including sample (fraction) dissolution and preparation of concentration series solutions with DMSO as a solvent. First, the sample solution was prepared by adding 10 mg of each fraction and dissolving it with 100 µL of DMSO. Then, the multilevel concentration of the solution was made (200, 100, 50, 25, 12.5, 6.25, and 3.125 µg/mL) with RPMI for T47D and HeLa cells and DMEM for MCF-7 cells. Later, 100 µL of the test solution was added into the wells and replicated three times for each concentration. For control cells, only 100 µL of the medium was added without the fraction solution. Then, the plates were incubated again in a 5% CO2 incubator at 37°C for 24 h.
After the treatment, an MTT assay was carried out by adding the MTT reagent into each well and then incubating for 4 h. The reaction was stopped using 10% SDS as a stopper reagent. The stopper reagent dissolves the formazan crystals which are reduced by the cell reductase system. Then, the plates were incubated at room temperature for 24 h. Furthermore, the absorbance was measured using an ELISA reader with a wavelength of 595 nm. From Figure 2, for T47D cells, it can be seen that the n-hexane fraction of guava leaves at 200, 100, and 50 µg/mL shows yellow color and purple color at 25, 12.5, 6.25, and 3.125 µg/mL. A change in the color from purple to yellow indicates that the cancer cells have died. There is no reductase enzyme produced in the mitochondria of the cell that reduces tetrazolium salt into purple formazan crystals. However, in the ethyl acetate fraction, the yellow color was only observed at a concentration of 200 µg/mL and purple at a lower concentration. Meanwhile, in the ethanol fraction, the observed color intensity was purple from high to low concentrations. This indicated that the number of living cells is still quite a lot.
Based on the analysis using ELISA reader at 595 nm, the IC50 values were obtained and are presented in Table 2 (see the Supporting information for calculation).
*It means data from reference.
The IC50 values for potential anticancer is below 30 μg/mL, 30 < IC50 < 100 μg/mL for moderately active, and more than 100 μg/mL for less active [54,55]. Based on Table 2, it can be concluded that the ethanol fraction is inactive on T47D cells, active on MCF-7 cells, and moderately active on HeLa cells. The ethyl acetate fraction was active against T47D cells, moderately active against MCF-7 cells, and less active on HeLa cells. The n-hexane fraction has the best activity based on IC50 values, very active against T47D and MCF-7 cells but moderately active against HeLa cells.
A cytotoxicity test was also carried out on normal cells (Vero cells) to determine the selectivity of the fractions. The purpose of this selectivity value is to determine the level of safety of an anticancer compound against normal cells so that it can be further developed as a chemopreventive agent. The steps carried out were the same as the activity test on cancer cells for each fraction with seven concentration variations: 200, 100, 50, 25, 12.5, 6.25, and 3.125 µg/mL. Tables 2 and 3 show that the guava leaf fraction is not harmful to normal cells (Vero cells). This can be shown from the IC50 value, which is >100 μg/mL. Moreover, the selectivity value is very important. It was obtained from the IC50 values of normal cells divided by the IC50 values of the cancer cells for each fraction. For SI values of >10, it can be categorized to be good selectivity.
Based on our analysis, the n-hexane fraction has high activity and selectivity to cancer cells. The cell morphology after n-hexane treatment as the best fraction for three cancer cells was observed under an inverted microscope for (a) T47D, (b) MCF-7, and (c) Hela cells, and shown in Figure 3. The morphology of Hela cells after interacting with the n-hexane fraction became long and spiky wrinkled. It looks different after the interaction with T47D where the cells look like little dots. Dramatically, the interaction with MCF-7 cells shrunk the structure into small pieces causing cell damage. This shows that there are differences in interactions between the fraction and breast cancer cells.
The inhibition analysis (IC50) values as a reference for the effectiveness of the fraction as a chemopreventive agent were strengthened by the cell viability tests, where cell viability indicates the number of living cells. The test results are presented in Tables 4–6.
|Fraction||Concentration (μg/mL)||Viability cells (%)|
|Fraction||Concentration (μg/mL)||Viability cells (%)|
|Concentration (μg/mL)||Viability cells (%)|
As seen from Table 4, at the same concentration (25 μg/mL) of ethanol fraction, the cell viability is around 77.18%, which means that more than 50% of the cells are alive, whereas 26.19% for ethyl acetate and n-hexane had lowest cell viability about 4.38%. The phenomena showed that at this concentration the n-hexane fraction had greater inhibition than the ethanol and ethyl acetate fraction. Therefore, it can be concluded that the n-hexane fraction is more effective in inhibiting T47D cancer cells than others.
As shown in Table 5, at the same concentration (25 μg/mL) of ethanol fraction, the viability or the number of living cells was 46.82% (less than 50%), and ethyl acetate fraction was not much different (59.45%), while the n-hexane fraction had much lower viability (15.87%). The phenomena also showed that the n-hexane fraction has a better inhibition ability to MCF-7 than the other two fractions at fairly low concentrations.
Based on Table 6, at the same concentration (25 μg/mL) of ethanol fraction, the viability was 60.77, 71.39% for ethyl acetate, and 62.10% n-hexane. This shows that the three fractions cannot inhibit the growth of HeLa cells at low enough concentrations so that cell viability is above 50%. In addition, when compared to T47D and MCF-7 cells, the three fractions showed better activity and effectiveness than in HeLa cells.
3.3 LCMS/MS analysis of dominant compounds in the n-hexane fraction
The n-hexane fraction, which is the most active fraction of three cancer cells, was analyzed using LC-MS/MS with positive and negative ionization modes. The chromatogram and mass analysis of the n-hexane fraction are given in the supporting information. This analysis showed that there are several high and sharp peaks with near retention times either in the negative mode or positive mode. These peaks were determined as the dominant compounds contained in the n-hexane fraction. They were analyzed and compared with some references from the previous research group to determine their masses and are presented in Tables 7 and 8 [56,57,58,59,60,61]. This analysis has provided valuable information regarding the content of secondary metabolites in the n-hexane fraction and is very useful for the development of further research to investigate which compounds have an important role as anticancer from the n-hexane fraction.
|Component name||[M + H]+ or [M + Na]+ (m/z)||Neutral mass (Da)||Observed RT (min)|
|Oleanolic acid *||203.19||201.19||5.70|
|Quercetin isomer *||257.13||256.13||6.36|
|Quercetin hexoside *||179.07||178.07||6.36|
*LC-MS/MS data obtained from authors.
|Component name||[M–H]− (m/z)||Neutral mass (Da)||Observed RT (min)|
|Madecassic acid (triterpenoid) ||503.31||504.31||4.54|
|Gallic acid *||169.98||170.98||4.54|
|Trihydroxycinnamoylquinic acid isomers *||369.01||370.01||6.51|
|Guavenoic acid *||471.27||472.27||6.91|
*LC-MS/MS data obtained from authors.
Based on the LC-MS/MS analysis using negative ionization (Table 8), the presence of ellagitannin compounds was shown in high concentration and it was suspected that these compounds had a role in preventing or inhibiting the growth of cancer cells. Ellagitannin is known as a chemoprevention agent for colon, skin, cervical, prostate, breast, and liver cancers . Ellagitannin has specificity in inhibiting the growth of cancer cells by attacking the ER + point, where T47D cells have that point; therefore, the presence of ellagitannin makes the n-hexane fraction more active than other fractions and is effective for inhibiting T47D cells. Also in the positive ionization mode, genistein was identified to play a role in preventing the growth of breast cancer . By identifying these compounds in the n-hexane fraction of guava leaves, it can be concluded that ellagitannin and genistein play a key role to inhibit the growth of cancer cells in this study.
The chemopreventive agent from guava leaves was successfully prepared via maceration and fractionation methods. Phytochemical screenings have demonstrated that the n-hexane fraction contained flavonoids and terpenoids; the ethyl acetate fraction contained flavonoids, tannins/polyphenols, and terpenoids; and the ethanol fraction contained terpenoids, flavonoids, and tannins/polyphenols. Based on the MTT assay, it was found that the n-hexane fraction has high activity and selectivity to cancer cells with the IC50 for T47D, MCF-7, and HeLa being 8.92, 4.28, and 85.98, respectively, and the selectivity value being>10, which was categorized to be good selectivity. Further development, isolating active compounds, and investigation of the n-hexane fraction to make it more suitable as a chemopreventive agent is still underway in our laboratory.
The authors thank to the Direktorat Penelitian dan Pengabdian Masyarakat (DPPM), Universitas Islam Indonesia, for the research funding support with research contract number 002/Dir/DPPM/70/Pen.Unggulan/PI/IV/2019.
Funding information: This research was financially supported by Universitas Islam Indonesia (project number: 002/Dir/DPPM/70/Pen.Unggulan/PI/IV/2019).
Author contributions: Nurcahyo Iman Prakoso: research design, identification, toxicity assay, and writing/reviewing/editing the draft. Mila Tria Nita: plant identification, separation, extraction, toxicity assay, and phytochemical screening.
Conflict of interest: The authors declare no conflict of interest.
Ethical approval: The conducted research is not related to either human or animal use.
Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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