Anti-Proliferative activity of Chlorophyllin from Phyllanthus Emblica L. against MCF-7 and Vero Cell line

 

Durga Devi M¹* , Banu N²

¹Assistant Professor, Department of Biotechnology, Mohamed Sathak College of Arts and Science, Sholinganallur, Chennai- 119.

²Associate Professor, Department of Biotechnology, Vels Institute of Science, Technology and Advanced Studies (VISTAS), Vels University, Pallavaram, Chennai.

 

ABSTRACT:

Chlorophyllin refers to any one of a group of closely related water-soluble salts that are semi-synthetic derivatives of chlorophyll. It is most common form is sodium copper derivative used as a food additive and in alternative medicine. In this study sodium copper chlorophyllin was extracted from Phyllanthus emblica, a medicinal plant and estimated by visible spectrophotometer and further characterized by TLC, IR, NMR and GC-Mass Chromatographic techniques and studied the anticancer activity against MCF-7 cell line and Vero cell line. Result shows that Chlorophyllin of Phyllanthus emblica has good anticancer activity for breast cancer cell line by using MTT assay.

 

 

 

1.INTRODUCTION:

Medicinal plants are sources of important therapeutic aids for alleviating human ailments. The number of Medicinal and Aromatic Plants (MAPs), which constitute the viable component of human health care at one time or another, is above 80,000.¹  India being one of the richest countries in the world for medicinal and aromatic plants. Plants have the ability to synthesize a wide variety of chemical compounds that are used to perform important biological functions, and to defend against attack from predators such as insects, fungi and herbivorous mammals. Normally medicinal plants have bioactive compounds which are used for curing various human diseases and also play an important role in healing.² Phytochemicals have two categories i.e., primary and secondary constituents.

 

Primary constituents have chlorophyll, proteins sugar and amino acids.³ Secondary constituents contain terpenoids and alkaloids. Medicinal plants have anti-fungal, anti-bacterial and anti-inflammation activities. Terpenoids exhibit various important pharmacological activities i.e., anti-inflammatory, anti-cancer, anti-malarial, inhibition of cholesterol synthesis, anti-viral and anti-bacterial activities.⁴ Phyllanthus emblica L. is one of the most extensively studied plants and reports suggest that it contains tannins, alkaloids, and phenolic compounds. It has been reported that fruits of P. emblica contains elevated amount of vitamin C and significantly high concentrations of the majority minerals, protein and amino acids like glutamic acid, proline, aspartic acid, alanine, cystine and lysine.⁵ Vitamin C levels are more than those in oranges, tangerines, or lemons. The fresh pericarp of P. emblica contains higher amount of hydrolysable tannins like emblicanin A and B, punigluconin, pedunculagin. Activity directed fractionation and purification process identified phytochemicals present in P. emblica including gallic acid, methyl gallate, corilagin, furosin and geraniin by a chromatographic and spectroscopic method. Phytochemical investigations showed that P.emblica contains higher amount of flavonoid like quercetin. Fruits were also analyzed for their alkaloidal content.⁶ Cancer is a class of diseases characterized by out-of-control cell growth. There are over 100 different types of cancer, and each is classified by the type of cell that is initially affected. Cancer harms the body when damaged cells divide uncontrollably to form lumps or masses of tissue called tumors (except in the case of leukemia where cancer prohibits normal blood function by abnormal cell division in the blood stream). Tumors can grow and interfere with the digestive, nervous, and circulatory systems and they can release hormones that alter body function. Tumors that stay in one spot and demonstrate limited growth are generally considered to be benign. Breast cancer is a type of cancer that develops from breast tissue. Signs of breast cancer may include a lump in the breast, a change in breast shape, dimpling of the skin, fluid coming from the nipple, or a red scaly patch of skin.  In those with distant spread of the disease, there may be bone pain, swollen lymph nodes, shortness of breath, or yellow skin. Breast cancer is a complex and heterogeneous disease. The first human cell line was established in a Baltimore laboratory over 50 years.⁷ The first breast cancer cell line to be established was BT-20 in 1958.⁸ The most commonly used breast cancer cell line in this world is MCF-7 established in 1973 at the Michigan Cancer Foundation.⁹ The popularity of MCF-7 is largely due to its exquisite hormone sensitivity through expression of oestrogen receptor (ER) making it an ideal model to study hormone response.¹⁰

 

2. MATERIALS AND METHODS:

2.1. Collection of Plant:

Phyllanthus emblica L. was collected from Unani Hospital, Royapettah, Chennai.

 

2.2. Plant Extract and Estimation:

Chlorophyllin extract were done by using Schertz, 1928¹¹ method. Ten grams of fresh leaves were taken and 1gm of sodium carbonate was added to neutralize the acidity. The plant material was ground with 50 – 100ml acetone and filtered using filter paper. This procedure is repeated until the residue becomes colorless. It was then washed with 50 – 150ml of diethyl ether to wash off acetone. The mixture was poured into a separating funnel and acetone was washed off using distilled water. This was repeated until a yellow color separates off which consists of flavones. The solution was poured into a bottle and 10 – 25ml of methanol saturated with potassium hydroxide pellets was added. The solution was shaken thoroughly and kept in icebox for overnight. The alkaline solution of chlorophyllin was poured into a separating funnel and 100ml diethyl ether was added and left for 30mins. Chlorophyllin separates off greenish layer which was removed. The ether layer was washed off with dilute potassium hydroxide and distilled water, to remove traces of chlorophyllin salts. The filtrate was evaporated to dryness in rotary evaporator and the extract was stored in ice box.

 

2.3. Visible Spectroscopic Analysis:

The partially purified chlorophyllin was analysed by VIS absorption by dissolving in diethyl ether and red at 405nm in a Beckman DU-40 Spectrophotometer and compared with authentic chlorophyllin.

 

2.4. Column Chromatography:

Weigh 20g silica gel 40 in a 50ml erlenmeyer flask and some amount of petroleum ether acetone solution slowly while mixing the slurry with a glass rod. Place the glass wool at the bottom of the column. Load the sample and pour 10ml 7:3 of petroleum ether and acetone solution into the column. Place the slurry in the column with a pipette continuously, then the purified sample was eluted from the column and it was used for further studies.

 

2.5. Thin Layer Chromatography:

 Pre-coated silica gel plates (Merck, Germany) were cut with ordinary household scissors. Plate markings were made with soft pencil. Glass capillaries were used to spot the sample for TLC applied sample volume 5-μl sample by using capillary at distance of 1 cm. In the twin trough chamber with acetone- water (8:2) after pre-saturation with mobile phase for 20 min for development were used. After development the plate was visualized under white light, UV 254, UV 365 nm and photographed.

 

               Distance travelled by solute

R_f  =      ------------------------------------

                Distance travelled by solvent

 

Standard R_f values are: 0.16-Xanthophyll; 0.32-chlorophyll b; 0.44-chlorophyll a; 0.95-β-carotene; 0.97-Standard Chlorophyllin.

 

2.6. FT-IR Spectroscopy Analysis:

The purified chlorophyllin was ground with IR grade potassium bromide (KBr) (1:10) pressed into discs under vacuum using spectra lab Pelleteir. The Fourier-Transform Infrared Spectra were recorded in a region 450-4000cm^-1 using Shimadzu FT-IR 8000 series instrument.

 

2.7. Nuclear Magnetic Resonance:

The carbon – 13 NMR spectral analyses was performed by taking the chlorophyllin sample in NMR tubes dissolved in D₂O. The NMR was recorded at 25.15MHz on a Burker AV III series instrument.

 

2.8. Gas Chromatography-Mass Spectroscopy:

Chlorophyllin extract of the plant sample was injected into the Gas chromatography unit Shimadzu GC-MS QP2010 was the instrument used for GC-MS analysis. It is separated into various constituents with different retention time which are detected by mass spectrophotometer.

 

2.9. Collection of Cell Line:

Breast Cancer Cell Line (MCF-7) and African Green Monkey Kidney (VERO) cell lines were obtained from National centre for cell sciences, Pune (NCCS). The cells were maintained in Minimal Essential Media supplemented with 10% FBS, penicillin (100U/ml), and streptomycin (100μg/ml) in a humidified atmosphere of 50μg/ml CO₂ at 37 °C.

 

2.10. Sub culturing and Maintenance of Cell Line:

The medium was brought to room temperature for thawing. The tissue culture bottles were observed for the growth, cell degeneration, pH and turbidity using an inverted microscope. If the cells (MCF-7 & VERO) attained 80% confluent it was taken for sub culturing process. The mouth of the bottle was wiped with cotton soaked with spirit to remove the adhering particles. The growth medium was discarded in a discarding jar. Then 4-5 ml of Minimum essential medium (MEM) without Foetal Calf Serum (FCS) was added and gently rinsed with titling. The dead cells and excess FCS was washed out and medium was discarded. TPVG was added over the cells and incubated a 37ºC for 5 min. for disaggregation. The cells disaggregate and becomes individual cells and is present as suspension. 5 ml of 10% MEM with FCS was added using a serological pipette. The process was repeated if any clumps were present.

 

2.11. Seeding of Cells:

The cell suspension was taken and poured into 24 well plate. In each well 1ml of the suspension was added and kept in a desiccators in 5% CO₂ atmosphere.

 

2.12. Cell Observation:

After 24hrs of incubation the cells were observed in an inverted microscope and photographs were taken (Olympus, Japan).

 

2.13. Cytotoxicity Assay:

In order to study the anti- tumor activity of a new drug, it is important to determine the cytotoxicity concentration of the drug. Cytotoxicity test defines the upper limit of extract concentration, which is non-toxic to the cell line. The concentration at which the drug is non toxic to the cells is chosen for anti- cancer assay. After the addition of drugs, cell death and cell viability was estimated. The result was confirmed by additional metabolic intervention experiment such as MTT assay.

 

 

2.14. Stock Drug Concentration-100mg/ml:

10µl of drug from stock was dissolved in 990µl of DMSO giving a working concentration of 1 mg/ml. The working concentration was prepared fresh and filtered through 0.5µl filter before each assay. 500µl of serum free MEM was taken in 9 eppendroff tubes. Then 500µl of this volume was transferred from first to last tube by serial dilution to obtain the desired concentration of drug. As a result the volume remains constant but there is a change in concentration.

 

2.15. Sampling:

Forty eight hour monolayer culture of MCF-7 and VERO cells at a concentration of 1lakh cells/well were seeded in 24 well titer plates. The plates were microscopically examined for confluent monolayer, turbidity and toxicity if the cells become confluent. The growth medium (MEM) was removed using a micropipette. Care was taken so that the tip of the pipette did not touch the cell sheet. The monolayer of cells was washed twice with serum free MEM to remove the dead cells and excess FCS. To the washed cell sheet, 1 ml of serum free medium containing defined concentration of the drug was added in the perspective wells. To the cell control wells 1ml of serum free MEM was added. Every experiment included a set of negative control and positive control. The plates were incubated at 37˚C in 5% CO₂ environment and observed for cytotoxicity using inverted microscope.

 

2.16. MTT Assay:

The tetrazolium salt 3, (4,5-dimethyl tiazol-2yl)-2, 5-diphenyl tetrazolium bromide is commonly known as MTT. It is a dye and is widely used in cytotoxicity assays. MTT assay was first proposed by Mossman, 1983.

 

2.17. Principle:

MTT is cleaved by mitochondrial dehydrogenase in viable cells, yielding a measurable purple colour product formazan. This formazan production is directly proportional to the viable cell number and inversely proportional to the degree of cytotoxicity (Mosmann, 1983).

 

2.18. Procedure:

After incubation the medium was removed from the wells carefully for MTT assay. Each well was washed with serum free MEM for 2-3 times. And 200µl of MTT was added and incubated for 6-7 hours in 5% CO₂ incubator for cytotoxicity_. After incubation 1ml of DMSO was added to each well and mixed using a pipette and left for 45 seconds. If any viable cells were present the formazan crystals after adding solibilizing reagent (DMSO) showed purple colour formation. The suspension was then transferred to the cuvette of spectrophotometer and the OD values were red at 595 nm by taking DMSO as a blank. A graph was plotted by taking concentration of a drug on X-axis and relative cell viability on Y-axis. The percentage of cell viability was calculated using the formula

 

Cell viability (%) = Mean OD×100/control OD.

Toxicity (%) = 100 – Cell Viability (%)

 

3. RESULTS:

Chlorophyllin from Phyllanthus emblica L. were extracted and estimated under the visible spectroscopic analysis by dissolving in diethyl ether and red at 405nm in Beckman DU-40 Spectrophotometer. The Chlorophyllin of P.emblica was estimated about 12.54μg/ml when compared to authentic chlorophyllin (20.2μg/ml). Further it is purified by column chromatography, after the elution progress, the yellow and green pigments collected from the column were concentrated by removing the solvents using a rotary evaporator. The pigments left behind in the Erlenmeyer flask after rotary evaporation are transferred into which glasses using spatula.

 

3.1. Thin Layer Chromatrography:

The TLC chromatogram showing the single spot at the R_f value of 0.97 at the tested solvent system of acetone – water (8:2). Both standard and test sample showed similar band at same R_f value of 0.97, indicated the selective isolation of chlorophyllin.

 

3.2. Infra Red Spectroscopic Analysis:

The presence of chlorophyllin was proved by FTIR spectrum at 450-4000 cm^-1 range. With reference to the sample from the spectra obtained, the following interpretation could be made for each respective plant.

 

3.3. Infra Red Analysis of Standard Chlorophyllin:

The peak at 3392cm^-1 confirms the presence of –OH and NH group. The peak at 1151cm^-1 was due to COO^- which is for the wet sample. The aromatic ring system was confirmed by the presence of peaks at 1141cm^-1 and 1640cm^-1. A small peak at 2081cm^-1 was due to C-N stretch. The peak at 761cm^-1 was due to OOP (Out of Plane) bending vibration arise due to aromatic ring system or C=C system. The peak at 1077cm^-1 was due to the C-H bending vibration. The peak at 2927cm^-1 confirms the presence of sp³ hybridized bending vibration (Fig. 1).

 

Figure 1: IR Spectrum of Standard Chlorophyllin

3.4. Infra Red Analysis of Phyllanthus emblica L. Chlorophyllin:

The peaks at 3861cm^-1, 3722cm^-1 and 3305cm^-1 confirm the presence of –OH and NH group. The aromatic ring system was confirmed by the presence of the peaks at 1558cm^-1 and 1455cm^-1. The peak at 1189cm^-1 was due to COO^- stretch which was due to the presence of wet sample. The sp³ hybridization was confirmed by the peaks at 2921cm^-1 and 2850cm^-1. The C-H bending vibration was due to the presence of the peak at 1082cm^-1 (Fig. 2).

 

Figure 2: IR Spectrum of Phyllanthus emblica L. Chlorophyllin.

 

3.5. NMR Spectroscopic Analysis:

The presence of chlorophyllin structure in the extract of Phyllanthus emblica L. was further confirmed by the NMR analysis. The sample and the standard chlorophyllin were analyzed and compared. Predominant peaks are seen both in the standard as well as the sample. The C₁₃ NMR Spectra of the sample chlorophyllin shows clear NMR signals at 60.5Δ, 63Δ, 69.5Δ, 71Δ -72Δ, 73Δ -74Δ and 76Δ indicates the existence of magnetically different various carbon and the spectral data nicely resembles the spectral data of the standard chlorophyllin (Figs. 3 and 4).

 

Figure 3:  NMR Spectroscopic Analysis of Chlorophyllin.

 

Figure 4:  NMR Spectroscopic Analysis of Phyllanthus emblica L. Chlorophyllin.

 

3.6. Gas Chromatography – Mass Spectroscopy Analysis:

The GC-Mass spectra of the sample chlorophyllin gives the fragmentation pattern in such a way that it highly correlates with the standard chlorophyllin. The molecular ion peak at m/e at 724.1689 ascertains the existence of chlorophyllin molecular samples in accordance with standard chlorophyllin. Other fragmentations are also observed in the GC-Mass of the sample as that of the sample chlorophyllin. Thus the GC-Mass spectra of the sample chlorophyllin authentically indicate the existence of chlorophyllin (Figs. 5 and 6).

 

Figure 5: GC-Mass Spectroscopic Analysis of Standard Chlorophyllin.

 

Figure 6: GC-Mass Spectroscopic Analysis of Phyllanthus emblica L. Chlorophyllin.

3.7. Anti- Proliferative Activity of Phyllanthus emblica L.

3.7.1. Standard Chlorphyllin on Vero Cell Line:

Screening of Chlorophyllin standard on Vero cell line which is treated with increasing concentrations (25µg-125µg/ml) for 48Hrs and the IC₅₀ value was absorbed at 125µg/ml. Cyclophosphamide is used as a Positive control. The cells were examined by phase contrast microscopy for the morphological apoptosis which showed typical polygonal intact appearance. The Chlorophyllin treated cells exhibited morphological characters like Cellular shrinkage (low toxicity), rounding (medium toxicity) and poor adherence (high toxicity) as well as round floating shapes (Table 1).

 

Table 1: Anti-cancer effect of Standard Chlorophyllin on VERO cell line

 

3.7.2. Standard Chlorophyllin On MCF-7 Cell Lines:

The effect of standard chlorophyllin was evaluated on MCF-7 cell line. The cells were treated with increasing concentrations of standard chlorophyllin (25µg-125µg/ml) for 48Hrs and IC₅₀ value was 125µg/ml. Cyclophosphamide is used as a Positive control. The cells were examined by phase contrast microscopy for the morphological apoptosis. The cells showed typical polygonal intact appearance. The Chlorophyllin treated cells exhibited morphological characters like Cellular shrinkage (low toxicity), rounding (medium toxicity) and poor adherence (high toxicity) as well as round floating shapes (Table 2).

 

Table 2: Anti-cancer effect of Standard Chlorophyllin on MCF-7 cell line

 

3.7.3. Phyllanthus emblica L. Chlorophyllin on Vero Cell Line:

The effect of Phyllanthus emblica L. chlorophyllin was evaluated on Vero cell line. The cells were treated with increasing concentrations of Phyllanthus emblica L. chlorophyllin (25µg-125µg/ml) for 48Hrs and IC₅₀ value was 125µg/ml. Cyclophosphamide is used as a Positive control. The cells were examined by phase contrast microscopy for the morphological apoptosis. The cells showed typical polygonal intact appearance. The Chlorophyllin treated cells exhibited morphological characters like Cellular shrinkage (low toxicity), rounding (medium toxicity) and poor adherence (high toxicity) as well as round floating shapes (Table 3).

 

Table 3: Anti-cancer effect of Phyllanthus emblica L.  Chlorophyllin on Vero cell line

 

3.7.4. Phyllanthus emblica L. Chlorophyllin on MCF-7 Cell Line:

The effect of Phyllanthus emblica L. chlorophyllin was evaluated on MCF-7 cell line. The cells were treated with increasing concentrations of Phyllanthus emblica L. chlorophyllin (25µg-125µg/ml) for 48Hrs and IC₅₀ value was 125µg/ml. Cyclophosphamide is used as a Positive control. The cells were examined by phase contrast microscopy for the morphological apoptosis. The cells showed typical polygonal intact appearance. The Chlorophyllin treated cells exhibited morphological characters like Cellular shrinkage (low toxicity), rounding (medium toxicity) and poor adherence (high toxicity) as well as round floating shapes (Table 4).

 

Table 4: Anti-cancer effect of Phyllanthus emblica L. Chlorophyllin on MCF-7 Cell Line

Concentration (µg/ml)	OD at 570nm	% of Toxicity	% of Viability
25	0.56	10.32	86.25
50	0.53	15.45	78.25
75	0.48	20.0	65.45
100	0.42	24.39	55.36
125	0.36	22.12	50.42
Control	0.70	0	100
Positive Control (Cyclophosphamide)	0.51	26.65	73.34

 

4. DISCUSSION:

Chlorophyll and its derivatives are believed to be among the family of phytochemical compounds. Water soluble derivatives of chlorophyll including chlorophyllides, chlorophyllin are known to cure cancer. Although, most research has focused on commercial grade sodium copper chlorophyllin, the extent to which natural chlorophyll and its derivative chlorophyllin modulate biomarkers of cancer is also being exploded. MTT proliferation assay was carried out to determine the parameters like cell viability, growth inhibition and morphological changes and compared with untreated (Control). The chlorophyll has shown broad spectrum cytotoxicity and it had most active cytotoxic activity on breast carcinoma cells. The IC₅₀ of extract in cell line less than 100μg/ml is categorized as a potential cytotoxic substances. A linear relationship between the formazen generated and the number of viable cells were demonstrated together with time-dependent manner for MCF-7 cells by Ferrari et al., 1990.¹² In the present study, the percentage of toxicity also varies on dose dependent manner, ranges from 43.80–48.78. The percentage of non-viable cells on cell lines increased with the increasing period of treatment with Goniothalamin towards human breast cancer cells. The criteria of cytotoxicity activity for the crude extracts as established by American National Cancer Institute (NCI) is an IC₅₀ <30μg/ml in the preliminary assay. Chlorophyllin has been used successfully as a cancer chemopreventive agent in human population residing in certain parts of China who are at high risk of eating aflatoxin-contaminated food.¹³ In the past, most of the work with chlorophyllin was done with analytical grade chlorophyllin. In the present study, Sodium-copper chlorophyllin (SCC) was purified and subjected to further study. The chlorophyllin was extracted from Phyllanthus emblica and it was estimated about 12.54μg/ml and further purified using column chromatography. Thin layer chromatogram showed R_f value at 0.97, which was compared with authentic chlorophyllin. The functional groups present in the chlorophyllin was further confirmed by FT-IR, compared to that of Standard Chlorophyllin (Figs. 1 and 2). From the analysis of standard chlorophyllin, it was found out that the peak at 3392cm^-1 confirms the presence of -OH and NH groups. The peak at 1151cm^-1 was due to COO- which is for the wet sample. The aromatic ring system was confirmed by the presence of peaks at 1141cm^-1 and 1640cm^-1. A small peak at 2081cm^-1 was due to C-N stretch. The peak at 761cm^-1 was due to OOP (Out of Plane) bending vibration arise due to aromatic ring system (or) C=C system. The peak at 1077cm^-1 was due to the C-H bending vibration. It clearly indictes that the replacement of Mg⁺ with Na⁺ (or) K⁺ (or) Cu⁺ on the central ion in the porphyrin ring structure. Hence the IR spectrum clearly indictes the existence of monovalent substituted carboxyl group, Keto group, nitrogen substituted heterocyclic ring may be porphyrin ring system (Figs. 1 and 2). The NMR spectra of chlorophyllin from Phyllanthus emblica showed NMR signals at 60.5Δ, 63Δ, 69.5Δ, 71Δ, 72Δ, 73Δ, 74Δ and 76Δ indicates the existence of magnetically different carbon and it resembles the data of standard chlorophyllin (Figs. 3 and 4). The GC-Mass spectra also proven the presence of similarity in fragmentation pattern of Chlorophyllin from Phyllanthus emblica  with that of standard chlorophyllin (Figs. 5 and 6). In the present study, the Sodium-copper chlorophyllin, generally called chlorophyllin was extracted and purified from the medicinal plant Phyllanthus emblica was tested for its anti-cancerous activity against Vero cell line and breast cancer cell line (MCF-7). MTT assay was performed at different concentrations of standard chlorophyllin and chlorophyllin of Phyllanthus emblica on these two cell lines. The parameters like cell viability, growth inhibition and morphological changes were compared with untreated (Control) cells. The assay detects the reduction of MTT by mitochondrial dehydrogenase to blue formazan product, which refelects the normal function of mitochondria and cell viability.¹⁴  Decrease in cell viability and increase in growth inhibition by both standard chlorophyllin and chlorophyllin of Phyllanthus emblica was observed on all the cell lines including Vero cell line and MCF-7 cell line in dose dependent manner (Tables 1–4). From MTT, morphological characters were confirmed after 24h of incubation with various concentration of standard chlorophyllin and chlorophyllin of Phyllanthus emblica, under phase contrast, inverted microscope, showed the cytoplasmic shrinkage, loss of normal nuclear architecture, detached cells and free floating cells in the medium showed the indication of apoptosis. As a result, the number of cytotoxic cells, increased with Chlorophyllin concentrations, with the highest having the most pronounced inhibitory effect on cell proliferation than control (Tables 1–4), this was substantiated by the work of Pavithra and Banu, 2015.¹⁵ The antigenotoxic and anticancer effects of Chlorophyllin are believed to be mediated through the porphyrin ring either by scavenging free radicals (or) by forming complexes with planar carcinogens.^16,17 Vesenick et al., 2012 studied that chlorophyllin concentration of 500μg/ml was necessary for cytotoxicity at 24h.¹⁸ This concentration reduced the cell survival rate by approximately 20%. Cytotoxicity was observed starting at 48h for the lower concentration. Currently, the Food and Drug Administration allows three 200mg chlorophyllin tablets to be ingested daily. This dose has been effective in preliminary studies of populations at risk for aflatoxin B1 exposure.¹⁹ Vesenick et al., 2012 analyzed two chlorophyllin concentrations (100 and 500μg/ml) through morphological analysis under fluorescent microscopy and through gene expression analysis of caspase-8 and -9. The experimental results from the analysis of morphological changes in cells, such as chromatin condensation, formation of apoptotic bodies, and increased caspase expression, show that none of the studied concentrations induces apoptosis. The cytotoxicity detected in the MTT assay is not due to cell death; instead, it might be attributed to decreased cell proliferation (cytostatic effect). Chlorophyllin has gained considerable attention in recent years owing to its high safety and efficacy without any adverse side effects. Numerous studies in a panel of human cancer cell lines in a variety of experimental animal models have revealed that CHL influences multiple molecules and pathways involved in the metabolism of carcinogens, antioxidant defenses, cell proliferation, apoptosis, invasion and angiogenesis to exert its chemopreventive effects. Thus, dietary phytochemcials such as Chlorophyllin that affect multiple signal transduction pathways involved in cancer initiation and progression hold promise as ideal candidates for cancer chemoprevention and therapy.²⁰ There are many anticancerous works available on various extracts of the following plants of Phyllanthus amarus, Adhatoda vasica, Tridax procumbens, Calotropis gigantea, Phyllanthus emblica, Catharathus roseus, Gymnema sylvestre and Psoralea corylifolio from different cell line. But this was the first report of natural chlorophyll and chlorophyllin (Sodium Copper Chlorophyllin) on breast cancer cell line (MCF-7). Further, it is necessary to investigate the underlying mechanism and active principle by which this activity was exhibited.

 

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Accepted on 28.12.2016           © RJPT All right reserved