Cytotoxicity of Ethanolic Extract of Plumeria rubra L. Stem bark to Cancer Cells and Lymphocytes

 

Nur Kuswanti^{1, 2}, Sri Widyarti¹, Widodo Widodo¹, Muhaimin Rifa’i^1*

¹Jurusan Biologi, Fakultas Matematika dan Ilmu Pengetahuan Alam, Universitas Brawijaya, Malang, Jawa Timur, Indonesia

²Jurusan Biologi, Fakultas Matematika dan Ilmu Pengetahuan Alam, Universitas Negeri Surabaya, Surabaya, Jawa Timur,  Indonesia

 

ABSTRACT:

This study aimed to examine the cytotoxicity of a stem bark ethanolic extract of Plumeria rubra L. from East Java, Balongbendo District of Sidoarjo, Indonesia on a model cancer cell line and lymphocytes in vitro. The cancer cells (i.e., T47D cells) and lymphocytes were treated using various concentrations, i.e. 0.3, 0.6, 1.2, 2.4, 4.9, 9.8, 19.5, 39.1, 78.1, 156.1, 312.5, 625, 1,250, 2,500, 5,000 and 10,000 µg/mL of the stem bark P. rubra extract. The number of dead and live cells were determined using direct blue staining after 24 hours of incubation in the extract. The data were analyzed by the Kolmogorov-Smirnov test and probit analysis using SPSS 16.0. The result showed that after administration of the extract of P. rubra, the T47D cells had an LC50 of 275.744 µg/mL with a linear R² = 0.802. Its LC50 to lymphocytes was more than 10,000 µg/mL. It can be concluded that the extract was toxic to the T47D cells, but it was not toxic to the lymphocytes. Thus, the extract of P. rubra seemed to have a high capability of preventing the growth of cancer cells, i.e., T47D cells. The higher toxicity of the extract to T47D cells than to lymphocytes indicates the potential to use the extract as an anticancer agent that is safe for normal cells.

 

 

 

INTRODUCTION:

Some plant extracts can be toxic to certain animals cells, due to various secondary metabolites¹. Even in a single individual plant, various parts contain different compositions of secondary compounds².

 

Cancer incidence is getting higher every year, with breast cancer having the highest incidence rate³. The conventional treatments, i.e., radiation, chemotherapy, and surgery, have side effects, e.g., causing inflammation⁴ and even promoting cancer growth⁵. Immune cells are natural killers of the cancer cells without any side effects under standard conditions⁴. It is important to investigate cancer treatments without or having a low side effect, such as inducing an immunomodulatory effect. Thus, such low side-effect treatments should not kill normal cells, especially immune cells.

 

 

Plumeria rubra L., a small tree in the Dogbane family, is one of many plant species that contain secondary metabolites and has been widely used as a drug for curing some disease^6,7. Its organs have different compositions of metabolites that relate to their different functions. Based on metabolic identification in several studies, the highest number of secondary metabolites was in the stem bark of the plant^2,6,8-10. Some of these have been shown to have at least some toxicity to various cancers^11-17. Using plant crude extracts for curing disease can have a synergic effect of various metabolites of the plant¹. Some P. rubra metabolites have indeed been shown to have a synergistic effect, such as plumericin and isoplumericin^18,19.

 

Some regional/varietal variation in metabolite composition may be seen as well. Based on LCMS analysis, the stem bark of P. rubra specifically found in Balongbendo District of Sidoarjo, East Java, Indonesia, showed different types and composition of secondary metabolites, compared with samples from other areas (data not shown). The latter contained 42 types of secondary metabolites that consisted of terpenoids, phenolics, and vitamins. Based on some partial studies, most of these metabolites were capable of being cytotoxic, apoptosis inducers and proliferation inhibitors of various cancer cells^20-22. Furthermore, some of the compounds also acted as anti-inflammatory agents, antioxidants and radical scavengers and anti-angiogenesis agents. The last three capabilities can be related to inhibition of the initiation, promotion and progression of various tumor and cancers^13,16.23,24. This study was performed to determine the toxicity of stem bark ethanolic extract of P. rubra harvested from the Balongbendo district of Sidoarjo, East Java, Indonesia, on breast cancer cells (T47D) and lymphocytes.

 

MATERIAL AND METHODS:

Preparation of Secondary Metabolites:

To extract the secondary metabolites, first, the stem bark was sun-dried, then crushed to be powder. The metabolites were extracted using ethanol. The result was filtered and evaporated for drying; It was done until semisolid brown mass resulted. The extract was stored at -20^oC for further use.

 

Getting T47D and Lymphocytes:

The T47D cells were obtained from cell lines preserved at -80 ˚C in The Animal Physiology Laboratory, Biology Department of Brawijaya University, Malang. The preserved cells were thawed. Then, as much as 200 µl of the suspension was put into a T-25 culture flask to which complete media (89% RMPI, 1% penicillin and streptomycin and 10 % Fetal Bovine Serum) was added. The flask was put in an incubator at 37 ˚C and 5% CO₂. After confluence, the cells were trypsinated and centrifuged for 5 minutes at 2,500 rpm at 10 ˚C. To the resulting pellet was added complete media to 1 mL. Cell number was counted using a haemocytometer (Neubauer Improved). The cells were plated in 96 culture wells at a concentration of 8,000 cells per well for further use.

 

The lymphocytes were processed using density gradient configuration method. About 3 mL blood was put into a 15 ml tube after adding 1:1 3 ml of ficoll solution (Lympho Spin Medium, cat 60-0092-10). The solution was centrifuged at 1600 rpm, 25 ˚C for 30 minutes. The resulting lymphocyte pellet was pipetted out and put into a 15 ml tube. The pellet was washed using 10 ml of PBS, then centrifuged at 2500 rpm and 10 ˚C for 5 minutes. The supernatant was removed, and the lymphocytes remained at the bottom of the tube were added with the complete media until the scale of 1 ml. The number of leucocytes was counted using haemacytometer. The cells were filled with about 50,000 cells and plated into each well of a 96 culture well plate for further treatment.

 

Extract Dilution:

The extract was diluted using water. It was put into a small bottle. A volume of water is suitable for specific concentration was poured into the bottle. The solution was stirred at 1250 rpm and room temperature. The result was centrifuged at 10 ˚C and 2,500 rpm for 5 minutes. The supernatant was obtained and easily filtered using a 0.02 µM Millipore filter.

 

Treatment:

T47D treatment:

Next, 24 hours after plating in the 96-well cell plate, the T47D cells were treated with the following extract concentrations: 0 (as control sample), 0.3, 0.6, 1.2, 2.4, 4.9, 9.8, 19.5, 39.1, 78.1, 156.1, 312.5, 625, 1,250, 2,500, 5,000 and 10,000 µg/mL. The treatments were done in triplicate. After a 24-hour incubation, the live and dead cells were counted using a hemacytometer (Neubauer Improved) under Direct Blue 1 (TCI, Cat no. B0782) staining up to 1 minute. The dead cells were determined based on their capability to absorb the direct blue solution, shown by blue color. On the other hand, the live ones saw no change in color.

 

Lymphocyte treatment:

After putting the lymphocytes in the wells, the cells were directly treated using the stem bark ethanolic extract, using the same concentrations listed above for the T47D cells. The cells were also treated in triplicate; then toxicity was examined using direct blue as described above.

 

Data Analysis:

The obtained data were organized and corrected using Abbots Formula then their normality was tested using the Kolmogorov Smirnov test. Furthermore, they were plotted using probit analysis. The Kolmogorov Smirnov and probit analysis were done using SPSS 16.0.

 

RESULTS:

Extract Dilution:

After dilution in sterile water and centrifugation, the extract was not completely dissolved. This was shown by the material settled at the base of the centrifugation tube. The settled extract was weighed for the determination of soluble and insoluble parts. The resulting weights are shown in Table 1.

 

Table 1. The weight of an ethanolic extract of Plumeria rubra L. before and after dilution

Extract weight (%)
Insoluble	soluble
47.5	52.5

 

The stem bark of ethanolic extract of P. rubra L. could not be easily diluted. The result showed that the soluble part portion 52.5% of the original weight.

 

Figure 1. Mortalities of T47D cells and lymphocytes after treatment of stem bark ethanolic extract of Plumeria rubra L.

 

LC50 analysis:

The data showed that the stem bark ethanolic extract of P. rubra L. was toxic to T47D cell line. It showed a positive linear correlation between extract concentration and cell mortality: the higher the concentration, the higher the mortality (Figure 1.). Plotting the data resulted in a linear line with a linear R² = 0.802 (Figure 2). Its toxicity was also shown by the 275.7 µl/mL LC50 (Table 2).

 

Table 2. LC50 of the stem bark ethanolic extract of Plumeria rubra L. on T47D and lymphocytes

Name of Cells	LC50 (µg/mL)
T47D	275.7
Lymphocyte	more than 10,000

 

Also, lymphocyte mortalities were lower than those of T47D cells (Figure 1). At concentrations up to 10,000 µg/mL of extract concentration, the lymphocyte mortality remained under 50% (Table 2). This means that the stem bark ethanolic extract of P. rubra was toxic to T47D cells, but not toxic to lymphocytes (Figure 1).

 

 

Figure 2. Probit-transformed responses of Log of extract concentration of ethanolic extract of Plumeria rubra L.

DISCUSSION:

Much cancer research has been done both in vitro and in vivo, especially for cancer treatments. In vitro method use cell culture, as an initial drug screening before preclinical and clinical examinations²⁵.

 

Toxicity of an extract is generally examined using LC50. The benefit of a lethal dose (LD50) is that it can reduce the number of animals used significantly²⁶. Accordingly, it also can be applied to cells, which can reduce the number of cells used in vitro. Lethal concentration (LC50) has been used to examine the toxicity of substances on cells, including cancer cells, as part of the preclinical evaluation of a drug candidate²⁷. The LC50 of a drug on normal and modified cells, such as cancer, can be one of some aspects to determining the drug concentrations that will be examined, which is termed an up and down procedure²⁶. Such that, after understanding safe and effective drug concentrations in vitro, can help formulate in vivo concentrations²⁸.

 

Table 1 shows that not all part of the ethanolic extract of the stem bark of Plumeria rubra L. was soluble in water, because of its nonpolar metabolites; most of the terpenoids and phenolics were nonpolar²⁹. Based on this result, the initial concentrations were not the extract used by the cells influencing their mortality. In this case, the original concentration should be about 52.5% of the initial concentration.

 

The toxicity of the ethanolic extract stem bark of P. rubra L. on T47D cells was shown by its LC50, 275.7 µg/mL. Since the extract was highly toxic to the cells, the types of the compound contained have a high possibility to function alone or synergistically to treat cancer cells directly and indirectly. The direct effect was shown by the high mortality of the cells after treatment.

 

 

Indirectly, the extract could be an immunomodulatory agent. This was supported by its LC50 being more than 10,000 µg/mL. This high LC50 means that it was not toxic on the cells, so that it is possible to be applied for killing cancer cells in a wide range of concentrations, although the mechanism of cell death, e.g., apoptotic or necrotic³⁰, has yet to be identified.

 

The pathway of cancer death is very important. The safest way is through apoptosis. In cancer treatments, an anticancer agent is targeted to induce apoptotic death of the cells. In this case, the death of the cells should be affected by the drug administered³¹. On the other hand, necrotic death can induce cancer growth³². This can be caused by the existence of reactive oxygen species (ROS)³³, and extract overdosing³⁴. The LC50 of the extract on lymphocytes being more than 10,000 µg/mL is very important because it shows that the extract was not toxic for normal cells, thus low side effects on normal cells and lymphocytes. Based on the result, there is a possibility for the extract to be an immunomodulator and increase immunosurveillance³⁵.

 

Based on previous studies, the stem bark of P. rubra contained more than 50 secondary metabolites^{2,6,8,11,17,36-39}. Some of them were cytotoxic to cancer cells^11,12,17 and prevented proliferation¹⁶.

 

Recent studies noted P. rubra contained 42 secondary metabolites, consisting of terpenoids, i.e fulvoplumierin, allamcin, isoplumericine, plumericin, allamandin, stigmasterol, β-Sitosterol, plumieride, plumerubroside, protoplumericin A (data not shown), phenolics, i.e. scopoletin, stigmasterol and betulinic acid, cinnamyl alcohol, camphor, geraniol, linalool, fenchyl alcohol, α-terpineol, citronellol, farnesol, benzyl benzoate, caryophyllene oxide, nerolidol, α-amyrin, lupeol, 3-O-acetyl lupeol, taraxasterol acetate, benzoic acid, gallic acid, ferulic acid, phenyl benzoate, kaempferol, quercetin, kaempfero-3-O-glucoside, quercitrin, quercetin-3-glycoside and rutin (data not shown), and vitamins, L-Bornesitol. The types of metabolites were different from metabolites contained in the same plant species from other areas previously studied^14,17,38,40. The previous findings showed that around 16 of more than 50 metabolites could treat cancer. However, a more recent study showed that 30 of 42 of the metabolites could be used to treat cancer. In addition, some potential anticancer compounds, e.g., lignanliliodendrin¹¹, 4-Hydroxy acetophenone¹⁷ and ursolic acid¹³, were not be identified in the present extract.

 

Most metabolites of the plants from Sidoarjo were cytotoxic on various cancer cells by inducing apoptosis and inhibiting proliferation. Some terpenoids, such as α-terpineol^41,42, β-sitosterol⁴³, α- and β-amyrin⁴⁴, betulinic acid⁴⁵, plumericin^11,12 were toxic on various cancer cells. Some phenolics, e.g., quercetin and kaempferol also showed some toxic effect⁴⁶.

 

The extract’s cytotoxicity may be caused via inducing apoptosis or inhibiting cancer cell proliferation. It was shown the existence of some terpenoids, e.g., camphor²⁰, geraniol⁴⁷, linalool⁴⁸, β-sitosterol⁴⁹, β-Amyrin and α-amyrin⁴⁴, lupeol⁵⁰, betulinic acid⁵¹, plumericin¹⁶ that were capable to inducing cell death. Some phenolics, quercetin^52-54, p-coumaric acid, ferulic acid^55,56, gallic acid^54,57 and kaempferol also could be toxic on cancer cells through apoptosis or interfering with the proliferative pathways.

 

The function of the secondary metabolites in inhibiting cancer growth was also shown by their capability to be anti-inflammatory agents, antioxidants, free radical scavengers, and anti-angiogenesis agents. Some secondary metabolites of the stem bark ethanolic extract of P. rubra seem to have anti-inflammatory activity, for example, α-terpineol, geraniol and citronellol^58-61. They reduced inflammation in single or combination treatment⁶¹. Stigmasterol⁶², β-sitosterol^43,63, lupeol^62,64,65, betulinic acid⁶⁶ and plumericin²³ acted in different ways, including inhibiting NF-κB activation. Some phenolics, such as gallic acid^57,67, quercetin⁵³, scopoletin⁶⁸, kaempferol⁶⁹ were also anti-inflammatory agents. Some of them could act as anti-inflammatory agents by reducing the activity of NF-κB⁵³. Inflammation weakens the immune response to the existence of cancer cells, e.g., suppressing T cells and B⁷⁰. Accordingly, anti-inflammatory agents can reduce the suppression and restore the immune system immunosurveillance, the initial capability to cancer cells. It also works as an immunomodulator. Thus, compounds having inflammatory capability also can act as immunomodulatory agents and immunosurveillance enhancers.

 

Other secondary metabolites functions relating to the inhibition of cancer growth are antioxidant and free radical scavenging⁴¹. The metabolites contained in the stem bark of P. rubra may be capable of such functions. Geraniol²¹, stigmasterol⁶², lupeol⁷¹, β-sitosterol⁴³, scopoletin⁶⁸, ferulic acid⁷² and quercetin⁵³ seem to be antioxidants or radical scavengers.

 

Some terpenoids acted as antioxidants by scavenging free radicals^41,53 are geraniol⁷³, stigmasterol^62,74, lupeol^71,75 and β-sitosterol⁴³. Other phenolics having the same activity are scopoletin⁶⁸, ferulic acid⁷² and quercetin⁵³.

 

Angiogenesis supports the growth of cancer by facilitating transport to the cancer cells. Some secondary metabolites found in the stem bark of P. rubra L. have been shown to be capable of inhibiting this process: gallic acid⁵⁷ and kaempferol⁷⁶.

 

As noted above, the treatment of the extract on lymphocytes did not indicate an LC50 was reached up to 10,000 µgr/mL (Table 2). Thus, the ethanolic extract of P. rubra was not toxic to lymphocytes. The very low toxicity of the extract on lymphocytes may because of its normal expression of NF-κB⁷⁷, as opposed to cancer cells. In relation to NF-κB expression, the cancer cells exhibit higher NF-κB expression than in normal ones⁷⁷. Because of its overexpression, the inhibition of NF-κB overexpressed induced breast cancer cell line death through apoptosis^77,78. It seemed an overexpression of NF-κB in a cell caused the cancer cells was sensitive to inhibition of NF-κB activation. As a result, the cells were more accessible to kill⁵.

 

CONCLUSION:

The stem bark ethanolic extract of Plumeria rubra L. grown in Balongbendo district of Sidoarjo, Indonesia seemed to have a high capability to prevent the growth of cancer cells. Its higher toxicity to cancer than immune cells shows potential for the use of the extract as an anticancer agent which is safe for normal cells. Thus, it also gives a possibility to use the extract as an immunomodulator in cancer patients. Based on the finding, further more-specific studies relating to cancer growth inhibition should be done to determine the appropriate use of the extract in curing cancer in vivo.

 

ACKNOWLEDGMENTS:

We would like to thank the Indonesian Ministry of Research, Technology and Higher Education for financial support.

 

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Accepted on 27.09.2018        © RJPT All right reserved

Research J. Pharm. and Tech 2018; 11(12): 5545-5550.