|Year : 2020 | Volume
| Issue : 1 | Page : 24-28
Cytotoxic and antimigratory effects on michigan cancer foundation-7 cells of Morinda citrifolia L. leaf extract and formulation of tablets from extract
Supavadee Boontha1, Benjaporn Buranrat2, Tasana Pitaksuteepong3
1 Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences, University of Phayao, Phayao, Thailand
2 Faculty of Medicine, Mahasarakham University, Maha Sarakham, Thailand
3 Department of Pharmaceutical Technology, Faculty of Pharmaceutical Sciences, Naresuan University, Phitsanulok, Thailand
|Date of Web Publication||3-Feb-2020|
Dr. Supavadee Boontha
Department of Pharmaceutical Sciences, School of Pharmaceutical Sciences, University of Phayao, 19 Moo 2, Tambon Maeka, Ampur Mueng, Phayao 56000
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Cancer is one of the deadliest diseases known to man. Efforts to combat the disease remain ongoing. Morinda citrifolia L. is a medicinal plant which is gaining interest as a natural chemotherapeutic agent for breast cancer treatment. Objective: This study aimed to determine the anticancer activity of M. citrifolia leaf extract on Michigan cancer foundation 7 (MCF-7) breast cancer cells as well as formulate tablets containing the extract to assess their viability. Materials and Methods: M. citrifolia leaf extract was prepared using the maceration method. Cytotoxicity and cell migration suppression, representing anticancer activity, were also assessed in MCF-7 cells by the sulforhodamine B assay and wound-healing assay. Tablets containing the extract were formulated using the wet granulation method. The prepared tablets were then evaluated regarding their physical properties and scopoletin content, a marker in the extract, before and after stability testing. Results: The extract showed cytotoxicity on MCF-7 cells in a dose- and time-dependent manner with 50% inhibitory concentration values of 39.9 ± 3.5 μg/mL for 48 h. In addition, it showed an antimigratory effect on MCF-7 cells with a significant effect at 25 μg/mL. The prepared tablets had good characteristics and were found to meet the requirements of the United States Pharmacopeia. Moreover, they could maintain their physical properties and scopoletin content over a 2-month period. Conclusion: This study showed that M. citrifolia leaf extract exhibited potential anti-breast cancer activity. The prepared tablets containing the extract could be a potential formulation for breast cancer treatment. However, the underlying mechanism of the extract as an antibreast cancer agent requires further investigation in subsequent study.
Keywords: Antibreast cancer, antimigratory effect, Morinda citrifolia L. leaf, scopoletin content, tablets
|How to cite this article:|
Boontha S, Buranrat B, Pitaksuteepong T. Cytotoxic and antimigratory effects on michigan cancer foundation-7 cells of Morinda citrifolia L. leaf extract and formulation of tablets from extract. Phcog Res 2020;12:24-8
|How to cite this URL:|
Boontha S, Buranrat B, Pitaksuteepong T. Cytotoxic and antimigratory effects on michigan cancer foundation-7 cells of Morinda citrifolia L. leaf extract and formulation of tablets from extract. Phcog Res [serial online] 2020 [cited 2020 Dec 5];12:24-8. Available from: http://www.phcogres.com/text.asp?2020/12/1/24/277476
- Morinda citrifolia leaf extract had scopoletin content and could also inhibit Michigan cancer foundation 7 human breast cancer cell viability and cell migration
- Tablets containing the extract were formulated and consisted of 0.4% w/w M. citrifolia extract, polyvinylpyrrolidone K-30, corn starch (F-1) or sodium starch glycolate (F-2), magnesium stearate, talcum and microcrystalline cellulose
- The tablets prepared as formulation F-2 had good physical properties within the acceptable range of the United States Pharmacopeia requirements through a stability period. Thus, it could be a promising formulation to develop for the treatment of breast cancer.
Abbreviations Used: DT: Disintegration time; HPLC: High performance liquid chromatography; IC50:50% inhibitory concentration; MCF-7: Michigan cancer foundation 7; SRB: Sulforhodamine B assay; USP: The United State pharmacopeia.
| Introduction|| |
Medicinal plants in the fight against cancer are currently a topic of focus because treatment with anticancer drugs have shown high toxicity and high drug resistance rates, frequently leading to cancer treatment failure. Among cancers, breast cancer causes the greatest proportion of morbidity and mortality among women worldwide., Thus, novel chemotherapeutic agents with greater effects and less toxicity are needed for breast cancer treatment.
Morinda citrifolia L., commonly called Noni or Yor in Thai, is an edible and medicinal tropical plant in the Rubiaceae family. The plant grows in many tropical regions of the world, including Thailand and other countries in Southeast Asia., The leaves and fruits of M. citrifolia are used around the world as internal and external treatments in traditional medicine. Additionally, they have been used extensively as a dietary supplement for many diseases including hypertension, diabetes, arthritis, headaches, cancer, and many more.,,,, In Thailand, the leaves of M. citrifolia are usually used for cooking in a popular Thai dish called “Hor Mhok Bai Yor.” Moreover, it has been used in traditional herbal remedies for both single remedies and combination remedies with other ingredients, since the last century. Thus, the leaves of M. citrifolia can be used in either food or herbal medicine.
Thani et al. reported that the methanolic M. citrifolia leaf extract exhibited cytotoxic effects on many cancer cell lines. It also inhibited Michigan cancer foundation 7 (MCF-7) cell viability with an 50% inhibitory concentration (IC50) value of 245.00 ± 8.37 μg/mL. This study revealed that the ethanolic M. citrifolia leaf extract had low cytotoxic effect on MCF-7 cells with an IC50 value higher than 600 μg/mL. Meli et al. reported that the ethanolic M. citrifolia shoot extract exhibited highly cytotoxic effects on breast cancer (MDA-MB-231) and colorectal cancer (HT-29) cell lines with an IC50 value of 49.72 μg/mL and 65.43 μg/mL, respectively. Therefore, M. citrifolia leaf extract will be investigated as an alternative natural source and safer chemotherapeutic agent for breast cancer treatment. Besides cytotoxic effect, the antimigratory effect on breast cancer cell, indicating cancer cell metastasis, is necessary to study the antibreast cancer effect of the medicinal plant extract. Wound scratch-healing assay is generally used to determine the ability of cancer cells to migrate. There are only a few studies concerning the anti-migratory effect of the ethanolic M. citrifolia leaf extract on MCF-7 cells. Therefore, the cytotoxic and anti-migratory effects of the extract on MCF-7 breast cancer cells were evaluated in this study.
It has been reported that M. citrifolia contains a number of major components including scopoletin, octanoic acid, potassium, Vitamin C, terpenoids, alkaloids, anthraquinones, β-sitosterol, carotene, flavone glycosides, rutin, and many more. Scopoletin, a coumarin compound, can be found in M. citrifolia and various medicinal plants, including Artemisia, Brunfelsia, Mallotius and Solanum species. Scopoletin has been reported to have antioxidant and anti-inflammatory activities as well as exhibiting a cytotoxic effect on cancer cells., Moreover, it has been used as a reference compound for the cytotoxic activity of M. citrifolia leaf extract. Hence, scopoletin content can represent the cytotoxic activity of the extract on the MCF-7 cancer cells in this study. Herbal dietary supplement products are currently available in many different dosage forms, including powders, capsules, tablets or liquids., When comparing all the formulations, tablets are more stable and preferable, while giving an accurate amount of the drug and being in a convenient dosage form.,
Thus, this study aimed to determine the anticancer effect of M. citrifolia L. leaf extract on MCF-7 cancer cells as well as formulate tablets containing the extract.
| Materials and Methods|| |
Chemicals and reagents
Ethanol was purchased from RCI Labscan (Bangkok, Thailand). Acetic acid, acetonitrile, and methanol (high-performance liquid chromatography [HPLC] grade) were purchased from Prolabo (Briare, France). Scopoletin were purchased from Sigma-Aldrich (Steinheim, Germany). MCF-7, a human breast cancer cell line, was purchased from the American Type Culture Collection (ATCC) (ATCC #HTB-22; Manassas, Virginia, USA). Dulbecco's modified Eagle's medium, fetal bovine serum, and other reagents for cell culture were purchased from Gibco-Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Trichloroacetic acid, sulforhodamine B (SRB) and crystal violet were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Corn starch, sodium starch glycolate, magnesium stearate, and talcum were obtained from Union Science Co. Ltd. (Chiang Mai, Thailand). Polyvinylpyrrolidone K-30 (PVP K-30) and microcrystalline cellulose were obtained from TTK science (Bangkok, Thailand).
Preparation of Morinda citrifolia extract
The M. citrifolia sample was identified and deposited in the Applied Thai Traditional Medicine Department, Faculty of Medicine, Mahasarakham University (specimen voucher no. MSUT_7225) and the Faculty of Science, Mahasarakham University, Thailand. The fresh leaves of M. citrifolia were washed, cut into small pieces and then dried in the air. They were also dried at 50°C using a hot-air oven (Memmert, Schwabach, Germany) for 48 h. Subsequently, they were macerated using 95% ethanol, followed by the leaf pieces being ground into a powder. After filtration, the filtrate was further concentrated at a controlled temperature of 50°C by a rotary evaporator (Heidolph, Germany). The percentage yield of the obtained extract was determined by comparing the crude extract dried weight (g) to the M. citrifolia leaf powder dried weight (g).
Determination of scopoletin content in the extract
Scopoletin content was determined to be a marker for the obtained extract using HPLC (CBM-20Alite®, Shimadzu, Japan). The assay was performed on a phenomenex luna C18 column (150 mm × 4.60 mm, 5 μm). The mobile phase consisted of 0.01M acetic acid, acetonitrile and methanol at a ratio of 60:20:20. The flow rate was run at 1.0 mL/min. The sample (20 μl) was added for ultraviolet detection at 254 nm. Scopoletin content was determined according to the peak area of the chromatogram.
Cell culture and cell viability method
The effects of the extract on cancer cell viability were examined using the SRB method, as previously described., The MCF-7 cells were exposed to various concentrations of the extract (0–250 μg/mL) for 24–48 h. After that, the MCF-7 cells had their viability determined in terms of the percentage of cell viability by comparing with the untreated control groups. The IC50 was then calculated from the dose-response curve.
Cell migration method
The effects of the extract on cancer cell migration was examined using the wound healing method, as previously described., Briefly, MCF-7 cells were scratched and then the complete medium containing various concentrations (0–50 μg/mL) of the extract was added. The wound distance (%) was determined by taking a photo of the uncovered area of the wound and then comparing with the control group.
Preparation and characterization of tablets containing extract
The tablets containing M. citrifolia extract were prepared by the wet granulation method. The ingredients of the tablets consisted of PVP K-30 as a binder, corn starch or sodium starch glycolate as a disintegrant with magnesium stearate, and talcum as glidants, while microcrystalline cellulose served as a filler. The tablet formulations are shown in [Table 1]. The composition of tablet formulations are shown in [Table 1]. The ingredients were weighed and then mixed together in a mortar using the geometric dilution technique. The extract was dissolved in the binder solution before being mixed with other ingredients. Using a hydraulic press (PerkinElmer, IL, USA), the tablets were compressed with a compaction force of 2 kN and a round flat-faced punch of diameter 10 mm was used. The obtained tablets were then evaluated for their physical properties. The nonofficial evaluation (i.e., thickness and hardness) and official evaluation (i.e., % weight variation, friability test and disintegration test [DT]) were evaluated based on the standard United States Pharmacopeia (USP) guidelines.
Thickness and hardness
Ten tablets were measured using a hardness and thickness tester (Erweka, Germany). Data was reported in terms of mm units for thickness and kilopond (kP) units for hardness.
Twenty tablets were weighed individually and the weight of each tablet was recorded. It was considered acceptable if the average percentage weight variation was in the range of ± 5%.
Ten tablets were accurately weighed together and friability was tested using a friability tester (Erweka, Germany) with rotation at 25 rpm for 4 min. The friability of the prepared tablets was determined by comparing the weight of the tablets before rotation (g) to the weight of the tablets after rotation (g). In this test, any free dust was removed from the tablets before weighing, with reweighing carried out following rotation. It was considered acceptable if the friability was not more than 1%.
Six tablets were tested using a disintegration tester (Erweka, Germany). The DT of the tablet was determined in distilled water at a controlled temperature of 37°C. It was considered acceptable if the DT was not more than 15 min.
Physicochemical stability was evaluated at 45°C and relative humidity of 75% for a 2-month period. The prepared tablets and their physical properties were evaluated as previously described, while scopoletin content was determined as follows.
Determination of scopoletin content in tablets
To determine the scopoletin content in the tablets, five obtained tablets were crushed using a mortar and pestle, after which 25 mL of methanol was added for extraction. After extract filtration, filtrate with a volume of 20 μL was analyzed by the HPLC technique.
The data values were expressed in terms of mean ± standard deviation and assessed by one-way ANOVA with post hoc least significant difference test. The data values were analyzed using Sigma Stat software version 3.5 (Systat Software Inc., San Jose, CA, USA). The data were considered statistically different when P < 0.05.
| Results|| |
Physical appearance, percentage yield and scopoletin content of Morinda citrifolia extract
The extract was a viscous greenish-brown paste with pH of 6.7. The percentage yield of the extract was approximately 30% (w/w). The peaks of the scopoletin had retention times of 4.3 min and it was well-resolved under isocratic elution using the HPLC technique. The scopoletin amount in the extract was 0.58% (w/w).
Effect of extract on cell viability and migration
The effect of M. citrifolia leaf extract on breast cancer MCF-7 cell viability was explored by the SRB method. This study indicated that the extract could decrease the cell viability of cancer cells in a dose and time-dependent manner when compared to the control group [Figure 1], with IC50 values of 156.9 ± 10.4 μg/mL for 24 h and 39.9 ± 3.5 μg/mL for 48 h. It was found that the extract had a significant role in inhibiting MCF-7 cell viability at a concentration of 25 μg/mL for 24 h and concentration of 100 μg/ml for 48 h. These results revealed that the extract showed four times higher inhibition effect on MCF-7 cell viability with increased incubation time.
|Figure 1: Effect of Morinda citrifolia leaf extract on cancer cell, Michigan cancer foundation-7, viability. Cells were exposed to extract (0–250 μg/mL) for 24 and 48 h. All results are percentages of control groups with three independent experiments and represent mean ± standard error of mean values. *P < 0.05 as compared with day 0 (cell treated with the extract for 24 h) and #P < 0.05 as compared with day 0 (cell treated with the extract for 48 h)|
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The effect of the extract on MCF-7 cell migration was determined by the wound healing assay. The results revealed that the extract inhibited MCF-7 cancer cell migration and was significant at 25–50 μg/mL [Figure 2]a and [Figure 2]b with an IC50 value of 39.8 ± 16.8 μg/mL. As shown in [Figure 2]a, cell death seemed to be observed when the MCF-7 cells were treated with 50 μg/mL of the extract for 48 h. This was due to the extract exhibiting a cytotoxic effect on MCF-7 cells with IC50 values of 39.9 ± 3.5 μg/mL at 48 h. From the experiments, it was found that the extract had a significant effect on inhibiting MCF-7 cell viability and cell migration.
|Figure 2: Effect of Morinda citrifolia leaf extract on cancer cell, Michigan cancer foundation-7, migration. Cells were exposed to extract (0–50 μg/mL) for 48 h and migration was captured using inverted microscopy (Magnification ×40) (a). All results are percentage of control groups with three independent experiments and represent mean ± standard error of mean values. *P < 0.05 when compared with control (b)|
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Properties of prepared tablets
The prepared tablets from both formulations had a diameter of 10 ± 0.43 mm with a round shape and flat surface. As shown in [Figure 3], the prepared tablets were light brown in color when compared with the tablets without the extract (data not shown). The two prepared tablet formulations had good characteristics. Their properties are shown in [Table 2]. The prepared tablets showed a narrow weight variation range with friability of <1%. In addition, the prepared tablets were completely disintegrated within 5 min. Therefore, they were found to be acceptable under the USP standards. The thickness of the prepared tablets was in the range of 3.03–3.15 mm, depending on the size of the die. Tablet hardness was in the range of 8.76–11.02 kP, indicating that the tablets should be strong enough to resist the packing and shipping process for the finished product, as revealed by the data from tablet tests.
|Figure 3: The physical appearance of the tablets prepared (F-1) before (a) and after stability test (b)|
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|Table 2: Physical properties of the tablet prepared before and after stability test|
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After the stability test, the tablets prepared from the two formulations were a dark brown color. The thickness and hardness of the prepared tablets were decreased, whereas weight variation and % friability were increased [Table 2]. However, the % weight variations and % friability were within the acceptable working range of the USP requirements. The hardness of the tablets prepared using corn starch (F-1) was reduced, resulting in faster DT, while the results of the tablets prepared using sodium starch glycolate (F-2) were different, though not significantly. The tablet formulation F-2 showed better physical properties than tablet formulation F-1 after the stability test. The scopoletin amount in both prepared tablet formulations were reduced after the stability test, as shown in [Figure 4]. However, they were not significantly different in terms of the decrease in the scopoletin content of the prepared tablets.
|Figure 4: Percentage remaining of scopoletin content of the extract formulated in F-1 and F-2 formulations following stability study. The results are presented as mean ± standard deviation values (n = 3)|
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| Discussion|| |
Presently, medicinal plants are considered to be effective sources of anticancer agents based on their lower toxicity. Many anticancer agents comprise plant-derived compounds including vinblastine, vincristine, paclitaxel, topotecan, and irinotecan. Moreover, herbal dietary supplements are used in traditional cancer treatment. This investigation revealed that the extract from M. citrifolia leaves had scopoletin content of 0.58% (w/w) and inhibited viability and migration in MCF-7 cells. The SRB method and wound scratch healing method are usually used to evaluate cell viability and cell metastasis, respectively. Consequently, this study indicated that the extract had anticancer activity on MCF-7 breast cancer cells, as based on its cytotoxic activity on the MCF-7 cells in a dose-and time-dependent manner. This study confirmed that M. citrifolia leaf extract exhibited a cytotoxic effect on breast cancer cell lines, as reported by previous studies., In this study, the ethanolic M. citrifolia leaf extract showed higher potential cytotoxic effect on human MCF-7 cancer cells with lower IC50 value when compared with the results from previous research. This might be due to a different in the percentage of extraction solvent. Also, M. citrifolia leaf extract showed higher potential cytotoxic effect on breast cancer (MDA-MB-231) cell lines with lower IC50 value and shorter incubation time, as previously reported by Meli et al. The M. citrifolia leaf extract seemed to be selective and safer because previous studies reported that M. citrifolia extract did not show any cytotoxic effect on normal cells, including mice fibroblast (L929) and mouse fibroblast (BALB/c 3T3) cell lines after incubation with the extract., The suppression effect on MCF-7 cell migration of the extract was observed, with the results showing that the extract had an anti-migratory effect, indicating inhibited metastasis on MCF-7 cells. The mechanism of M. citrifolia extract for growth inhibition on MCF-7 cancer cells will be identified by using cell cycle analysis, colony formation, caspase-three activity, and reactive oxygen species formation.
When preparing the tablet formulation containing the extract, the finished product is light brown, flat and round with satisfactory characteristics according to USP guidelines. The tablet hardness and % friability represent the tablet strength and resistance to friability for the packing and shipping process of the finished product, as described above, whereas the DT represented the initial prominent step of the tablets to disintegrate into smaller granules in solution, which are then soluted in the medium. Generally, the faster the DT of the tablets, the faster the onset of action will be. In this study, corn starch or sodium starch glycolate was used as a disintegrant and added to give a faster DT. After the stability study, the physical properties of the M. citrifolia tablets exhibited no significant differences. This result indicated that the tablets could remain stable throughout the duration of the stability test, with no significant decrease in scopoletin content. However, stability over a longer time period and the dissolution test of the prepared tablets will be determined in future studies.
| Conclusion|| |
This study revealed that M. citrifolia leaf extract had a scopoletin content of 0.58% (w/w) and could inhibit viability and migration in the MCF-7 cell. Therefore, the extract has the potential for development as an anticancer agent for breast cancer. Further, the tablet formulation consisting of 0.4% w/w M. citrifolia extract, PVP K-30, sodium starch glycolate, magnesium stearate, talcum, and microcrystalline cellulose could potentially be developed for the treatment of breast cancer. However, further study is needed to understand the underlying mechanism of the extract as a chemotherapeutic agent for breast cancer and the possibility of development as a dietary supplement formulation.
The authors would like to express our thanks to Assistant Prof. Dr. Saengrawee Sutthiparinyanont for HPLC technical assistance and Dr. Jolyon Dodgson for his editing assistance and advice on English expression in the manuscript.
Financial support and sponsorship
This study was financially supported by the School of Pharmaceutical Sciences, University of Phayao, Thailand and Thailand Research Fund (MRG6080071).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]