TECHINICAL FIELD

The invention is related to a herbal synergistic composition for managing cancer. The synergistic composition comprises extracts of Daucus carota, Cucumis sativus, Piper nigrum , and Curcuma longa. The invention also relates to the method of preparation of synergistic composition.

BACKGROUND AND PRIOR ART

Cancer accounts for one of the leading causes of death amongst humans. Therapy comprises both palliative and curative measures including surgery, which may be in combination with radiation therapy and/or chemotherapy. Side effects of surgery, radiation and chemotherapy include fatigue, appetite loss, nausea, vomiting, constipation, skin problems. However, the treatments do not guarantee the cure of the cancer. Hence emphasis is on natural ways which can help in preventing and/or management of the cancer. Consistent research is going on to understand the effects of commonly available plants and parts thereof on cancer.

Many plants and parts thereof help the body to fight free radicals, maintain healthy cells, and prevent inflammation. The antioxidant property helps in decrease of cancer cell growth and increase of cancer cell death in cells.

There is subtle information on the knowledge of anti-cancer properties of plants and parts thereof in public domain, however there is no candid study which shows the clear effect of a single plant or composition of plants or parts thereof on the retardation of cancer cells. The present study focus is to provide a synergistic composition comprising parts/extracts of four plants Daucus carota, Cucumis sativus, Piper nigrum, and Curcuma longa or extract thereof or both, as a potential cytotoxic composition for managing cancer. The invention also provides a method of preparation of the synergistic composition.

OBJECTS OF INVENTION

An object of the invention is to identify plants and parts or extract thereof including vegetable, spices for adoption in a synergistic composition which can manage cancer.

Another object of present invention is to provide a synergistic composition of plants and parts or extract thereof for managing cancer.

SUMMARY OF INVENTION

Accordingly, the present invention provides a synergistic composition comprising vegetables, spices of different plants for managing cancer. The vegetables are Carrot ( Daucus carota ), Cucumber ( Cucumis sativus ), and spices are Marica ( Piper nigrum ), Curcumin ( Curcuma longa).

The present invention provides a synergistic composition comprising extract of root of Daucus carota 25%wt/wt, extract of fruit of Cucumis sativus 25%wt/wt, extract of seeds of Piper nigrum 25%wt/wt, and extract of root of Curcuma longa 25% wt/wt, optionally with excipients.

The present invention also provides a method of preparation of synergistic composition comprising extract of root of Daucus carota 25%wt/wt, extract of fruit of Cucumis sativus 25%wt/wt, extract of seeds of Piper nigrum 25% wt/wt, and extract of root of Curcuma longa 25% wt/wt; said method comprising acts of- a) obtaining the extract of root of Daucus carota wherein the extracts are water soluble ranging from 78% to 82% and drying, b) obtaining the extract of fruit of Cucumis sativus wherein the extracts are water soluble ranging from 84% to 86%and drying, c) obtaining extract of seeds of Piper nigrum wherein the extract are water soluble ranging from 15% to 17% and drying , d) obtaining extract of root of Curcuma longa wherein the extract comprises curcumin ranging from 94% to 96% and drying, and e) blending the dried extracts obtained in steps (a)-(d) and optionally adding excipients to obtain a synergistic composition comprising extract of root of Daucus carota , extract of fruit of Cucumis sativus , extract of seeds of Piper nigrum, and extract of root of Curcuma longa.

A method of managing cancer, said method comprising act of oral adoption of the synergistic composition comprising extract of root of Daucus carota 25%wt/wt, extract of fruit of Cucumis sativus 25%wt/wt, extract of seeds of Piper nigrum 25%wt/wt, and extract of root of Curcuma longa 25% wt/wt, optionally with excipients.

BRIEF DESCRIPTION OF FIGURES

The features of the present invention can be understood in detail with the aid of appended figures. It is to be noted however, that the appended figures illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope for the invention.

Figure 1 shows photomicrographs representing the cytotoxic effect on MCF-7 breast cancer cells when cancer cells are only present with solvent control. Figure 2 shows photomicrographs representing the cytotoxic effect on MCF-7 breast cancer cell when the dosage is (a) 3.906 ug/ml (b) 7.8125 ug/ml, (c) 15.625 ug/ml and (d) 31.25 ug/ml, (e) 62.5 ug/ml, (f) 125 ug/ml, (g) 250 ug/ml and (h) 500 ug/ml.

Figure 3 shows photomicrographs representing cytotoxic effect on colon cancer cells when dosage is 7.81 pg/ml Curcumin; 7.81 pg/ml Piper nigrum; 7.81 pg/ml Gajor( carrot);

7.81 pg/ml Cucumber; 31.25pg/ml of synergistic composition.

Figure 4 shows photomicrographs representing cytotoxic effect on colon cancer cells when dosage is 15.62 pg/ml Curcumin; 15.62 pg/ml Piper nigrum; 15.62 pg/ml Gajor(carrot); 15.62 pg/ml Cucumber; 62.5 pg/ml of synergistic composition. Figure 5 shows the cell viability assay of 7.81 pg/ml Curcumin; 7.81 pg/ml Piper nigrum;

7.81 pg/ml Gajor( carrot); 7.81 pg/ml Cucumber; 31.25pg/ml of synergistic composition.

Figure 6: shows the cell viability assay of 15.62 pg/ml Curcumin; 15.62 pg/ml Piper nigrum; 15.62 pg/ml Gajor( carrot); 15.62 pg/ml Cucumber; 62.5 pg/ml of synergistic composition. Figure 7: shows photomicrographs representing cytotoxic effect on lung cancer cells when dosage is 7.81 pg/ml Curcumin; 7.81 pg/ml Piper nigrum; 7.81 pg/ml Gajor; 7.81 pg/ml Cucumber; 31.25pg/ml synergistic composition.

Figure 8: shows photomicrographs representing cytotoxic effect on lung cancer cells when dosage is 15.62 pg/ml Curcumin; 15.62 pg/ml Piper nigrum; 15.62 pg/ml Gajor( carrot); 15.62 pg/ml Cucumber; 62.5 pg/ml synergistic composition.

Figure 9: shows the cell viability assay wherein the dosage is 7.81 pg/ml Curcumin;

7.81 pg/ml Piper nigrum; 7.81 pg/ml Gajor; 7.81 pg/ml Cucumber; 31.25pg/ml synergistic composition. Figure 10: shows the cell viability assay when dosage is 15.62 pg/ml Curcumin; 15.62 pg/ml Piper nigrum; 15.62 pg/ml Gajor( carrot); 15.62 pg/ml Cucumber; 62.5 pg/ml synergistic composition. DETAILED DESCRIPTION OF DISCLOSURE

The foregoing description of the embodiments of the invention has been presented for the purpose of illustration. It should not be construed that the scope of the invention is limited to the disclosure herein.

The various embodiments of the synergistic composition of the present invention along with its method of preparation are described below with reference to the figures.

It may further be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by persons skilled in the art. Abbreviations:

DMEM: DULBECCO’S MODIFIED EAGLE’S MEDIUM FBS: FETAL BOVINE SERUM

MTT dye: 3(4,5 Dimethylthiazol -2)2,5 Diphenyl tetrazolium bromide OD: OPTICAL DENSITY

The present invention focuses on formulating a synergistic composition comprising plants or parts of plants or extract thereof. The parts of plants may be vegetables, root, seed, stem, fruit and the like. In an embodiment of the invention, the synergistic composition comprises Carrot/ Gajor ( Daucus carotd) Cucumber ( Cucumis sativus), Marica ( Piper nigrum ), and Curcumin ( Curcuma longa).

In at least one embodiment of the present invention comprises Carrot/Gajor ( Daucus carotd) about 25 % w/w, Cucumber ( Cucumis sativus) comprises about 25 % w/w,

Marica {Piper nigrum) comprises about 25 % w/w and Curcumin {Curcuma longa) about 25 % w/w of total composition.

Another embodiment of present invention relates to a synergistic composition comprising extract of root of Daucus carota 25%wt/wt, extract of fruit of Cucumis sativus 25%wt/wt, extract of seeds of Piper nigrum 25%wt/wt, and extract of root of

Curcuma longa 25% wt/wt, optionally with excipients.

Another embodiment of present invention comprises synergistic composition of the extract of root of Daucus carota with water soluble extracts ranging from 78% to 82%, extract of fruit of Cucumis sativus with water soluble extracts ranging from 84% to 86%, extract of seeds of Piper nigrum with water soluble extracts ranging from 15% to 17% and extract of root of Curcuma longa with curcumin ranging from 94% to 96%.

In still another embodiment of present invention, the synergistic composition comprises the extract of root of Daucus carota with water soluble extracts of 80.92%, extract of fruit of Cucumis sativus with water soluble extracts of 85.63%, extract of seeds of Piper nigrum with water soluble extracts of 15.33% and extract of root of Curcuma longa with 95% of curcumin.

In another embodiment of present invention, the synergistic composition may be in the form of solid, liquid, gel or combination thereof. In still another embodiment of present invention, the synergistic composition may be combined with excipients selected from a group comprising but not limiting to colouring agent, gelling agent, binders, preservative, flavours, bioenhancers and sweetners.

In still another embodiment of present invention, the bioenhancer is Piperine.

In another embodiment of present invention, a method of preparation of synergistic composition comprising extract of root of Daucus carota 25%wt/wt, extract of fruit of Cucumis sativus 25%wt/wt, extract of seeds of Piper nigrum 25%wt/wt, and extract of root of Curcuma longa 25% wt/wt; said method comprising acts of- a) obtaining the extract of root of Daucus carota wherein the extract comprises water soluble extracts ranging from 78% to 82% and drying, b) obtaining the extract of fruit of Cucumis sativus wherein the extract comprises water soluble extracts ranging from 84% to 86%and drying, c) obtaining extract of seeds of Piper nigrum wherein the extract comprises water soluble extracts from 15% to 17% and drying , d) obtaining extract of root of Curcuma longa wherein the extract comprises curcumin ranging from 94% to 96% and drying, and e) blending the dried extracts obtained in steps (a)-(d) and optionally adding excipients to obtain a synergistic composition comprising extract of root of Daucus carota , extract of fruit of Cucumis sativus , extract of seeds of Piper nigrum, and extract of root of Curcuma longa.

In an embodiment of the present invention, the extract is obtained by a method comprising but not limiting to solvent extraction, maceration, infusion, percolation, decoction, soxhlet extraction, microwave assisted extraction, ultrasound-assisted extraction, accelerated solvent extraction, and supercritical fluid extraction.

In still another embodiment of present invention, the extracts are obtained from solvent extraction. In still another embodiment of present invention, the solvent is selected from a group comprising Water, Methanol, Ethanol, Butanol, Iso butanol, Carbon tetrachloride, Chloroform, Dichloromethane and combination thereof.

In another embodiment of present invention, synergistic composition is used in managing cancer, comprising but not limiting to breast cancer, lung cancer, colon cancer. In another embodiment synergistic composition is used for prevention of cancers. In another embodiment synergistic composition can be used for overcoming chemoresistance during the treatment of cancer. In another embodiment synergistic composition can be used as drug bioenhancer.

EXPERIMENTATION The Breast cancer cell lines (MCF-7), Colon cancer cells(HCT116) and Lung cancer cells (A549) cell lines are obtained from the National Centre for Cell Science (NCCS), Pune, India.

The extracts of Carrot (Daucus carota), Cucumber (Cucumis sativus), and Marica (Piper nigrum) are commercially obtained from Tulsi Amrit Private Limited, India and Curcumin (Curcuma longa) is commercially obtained from Himrishi Herbal,

India.

The synergistic composition used in managing cancer is described herein and further illustrated in the following examples. The effect of synergistic composition in managing cancer cells is studied on MCF-7 breast cancer cells, HCT116 colon cancer cells and A549 lung cancer cells.

Cell line culture expansion: The HCT116 colon cancer cells and A549 lung cancer cells are adherent cells and are culture expanded as per previous methods reported from our laboratory. The cells are culture expanded in standard DMEM (low glucose, Glutamax supplement) (Cat. No. 10567014, GIBCO, USA) supplemented with 10% FBS (Cat. No. 10270106, GIBCO, Brazil), 1% Antibiotic -Antimycotic (Cat. No. 15240062, GIBCO, USA) at 37°C under normoxic conditions.

A. Preparation of Synergistic composition:

General Procedure:

Carrot (Daucus carota), Cucumber (Cucumis sativus), Marica (Piper nigrum), and Curcumin (Curcuma longa) as a whole or extract of a part are dried and ground to get a uniform particle size powder. 25 % (w/w) of each ingredient is weighed and mixed thoroughly to get a uniform composition to get a synergistic composition.

The synergistic composition is constituted with root extracts of carrot, fruit extract of cucumber and a bioactive component Curcumin (95% purity) from the rhizome of turmeric, pepper seeds optionally with a bioenhancer, Piperine. All the constituents are devoid of microbial contamination and heavy metal content.

Example 1:

25 g of dried extract of Carrot (Daucus carota), 25 g of dried extract of Cucumber (Cucumis sativus), 25 g of dried extract of pepper (Piper nigrum), and 25 g of Curcumin (Curcuma longa) 95% are pulverized and mixed in a blender for lhr to obtain synergistic composition. The synergistic composition is stored in air tight container below 25°C and used for experimentation. Example 2:

10 g of extract of Curcumin longa with curcumin 95%, is mixed with a composition comprising lOg of dried extract of Carrot (Daucus carota), 10 g of dried extract of Cucumber (Cucumis sativus), 10 g of dried extract of pepper (Piper nigrum) and blended for 20 min to obtain synergistic composition, stored below 25°C and used for experimentation .

The synergistic composition and its various constituents are diluted to a concentration of lmg/ml. lmg of the formulation and also its various constituents individually are dissolved in 300ul Dimethyl sulphoxide (DMSO) and made up to 1 ml with Dulbecco's Modified Eagle Medium (DMEM) and filter sterilized. Further dilutions are made from this stock as required and stored in cold temperature, preferably below 25°C for further use. The cells are treated for 48Hours with the diluted synergistic composition and its combination of constituents with the working concentrations mentioned below.

B. Results of experimentation on MCF-7 breast cancer cells

The said synergistic composition is tested on MCF-7 breast cancer cells by varying concentration.

At first MCF-7 breast cancer cells are cultured for 24 hours before treatment in DMEM and 10% FBS. After which the cells are treated for a period of 24 hours at the various doses as mentioned below. Following the treatment period, cell viability /death is assessed visually and by MTT assay. The untreated control and solvent control during the experiment is maintained all through the experimentation. The results of the MTT assay are presented in table 1.

Table 1: MTT assay

Figures 1 and 2 and table 1 shows the effect of synergistic compositions on MCF-7 breast cancer cells. It can be seen from table 1 that as the dose of the synergistic composition increases, percentage viability of cancer cells also decreases i.e., death of cancer cells increases. This is supported by figures 1 and 2. Figure 1 shows the breast cancer cell in just solvent control i.e. Cancer cell was not treated with synergistic composition. Figure 2 (a-b) shows the cytotoxic effect of synergistic composition with dosage 3.906 ug/ml and 7.8125 ug/ml respectively. It can be seen that percentage viability of the cancer cell decreases. The dosage of synergistic composition is increase step wise and photomicrographs representing of the effect on MCF-7 breast cancer cells is studied and can be seen in Figure 2 (c-h). It is seen from the figures that as the dosage increases the percentage viability of the cancer cell decreases i.e. as the dosage of the synergistic composition increased - there is increase in cell death. C. Assessment of cytotoxicity activity in Colon Cancer cell line:

1. Morphological assessment

Colon cancer cell line (HCT116) is cultured in the 10% FBS supplemented DMEM with 1% antibiotic and antimycotic solution, later is trypsinized after attaining 80% confluency. The HCT116 cells are seeded at a seeding density of 50x104 cells/well in a 6 well plate with 10% FBS supplemented DMEM and incubated at 37°C under normoxic conditions for 24 hours for cell attachment. After 24 hours of incubation, the spent medium is removed and the serially diluted various concentrations of drug supplemented DMEM (7.81 pg/ml of curcumin, 7.81 pg/ml pepper, 7.81 pg/ml

Carrot, 7.81 pg/ml cucumber; 31.25 pg/ml of synergistic composition for first set and 15.62 pg/ml of curcumin, 15.62 pg/ml pepper, 15.62 pg/ml Carrot and cucumber; 62.5 pg/ml of synergistic composition are added to the cells and incubated at 37°C under normoxic conditions for 48 hours. The images are captured with Leica Optica inverted microscope.

2.Cell viability assessment

The HCT116 cells are seeded at a seeding density of 4000 cells per well of a 96 well plate with 10% FBS supplemented DMEM and incubated at 37°C under normoxic conditions for 24 hours for cell attachment. After 24 hours of incubation, the spent medium was removed and the serially diluted various concentrations of drug supplemented DMEM (7.81 pg/ml of curcumin, pepper, carrot and cucumber; 31.25 pg/ml of synergistic composition; 15.62 pg/ml of curcumin, pepper, carrot and cucumber; 62.5 pg/ml of synergistic composition and a combinational treatment of each 15.62 pg/ml of curcumin and pepper; pepper, carrot and cucumber; and cucumber and curcumin; 31.25 mg/ml of curcumin and pepper; pepper and carrot, carrot and cucumber; and cucumber and curcumin respectively) are added to cells and incubated at 37°C under normoxic conditions for 48 hours. After 48 hours of incubation, IOmI of CCK8 reagent is added to the cells containing medium and incubated for 4 hours. The optical density is measured at 450nm and the percentage of cell viability is calculated.

D. Assessment of cytotoxicity activity on LUNG CANCER cell line

1. Morphological assessment

Lung cancer cell line (A549) has epithelial, adherent lung carcinoma cell line. Cells are cultured in the 10% FBS supplemented DMEM with 1% antibiotic and antimycotic solution and later was trypsinized once they attained 80% confluency. For experiments, the A549 cells are seeded at a seeding density of 50x104 cells/well in a 6 well plate with 10% FBS supplemented DMEM and incubated at 37°C under normoxic conditions for 24 hours for cell attachment. After 24 hours of incubation, the spent medium is removed and the serially diluted various concentrations of drug supplemented DMEM (7.81 pg/ml of curcumin, 7.81 pg/ml pepper, 7.81 pg/ml Carrot and 7.81 pg/ml cucumber; 31.25 pg/ml of synergistic composition for the first set and 15.62 pg/ml of curcumin, 15.62 pg/ml pepper, 15.62 pg/ml carrot and 15.62 pg/ml cucumber; 62.5 pg/ml of synergistic composition for the second set) are added to the cells and incubated at 37°C under normoxic conditions for 48 hours. The images are captured with Leica Optica inverted microscope.

2. Cell viability assessment:

The A549 cells are seeded at a seeding density of 4000 cells per well of a 96 well plate with 10% FBS supplemented DMEM and incubated at 37°C under normoxic conditions for 24 hours for cell attachment. After 24 hours of incubation, the spent medium was removed and the serially diluted various concentrations of drug supplemented DMEM (7.81 pg/ml of curcumin, 7.81 pg/ml pepper, 7.81 pg/ml carrot and 7.81 pg/ml cucumber individually; 31.25 pg/ml of synergistic composition for the first set and 15.62 pg/ml of curcumin, 15.62 pg/ml pepper, 15.62 pg/ml carrot and

15.62 pg/ml cucumber; 62.5 pg/ml of synergistic and a combinational treatment of each 15.62 pg/ml of curcumin and pepper, 15.62 pg/ml pepper and carrot; 15.62 pg/ml carrot and cucumber; and 15.62 pg/ml cucumber and curcumin and; 31.25 pg/ml of curcumin and pepper; 31.25 pg/ml pepper and carrot; 31.25 pg/ml carrot and cucumber; and 31.25 pg/ml cucumber and curcumin respectively) are added to cells and incubated at 37°C under normoxic conditions for 48 hours. After 48 hours of incubation, lOpl of CCK8 reagent is added to the cells containing medium and incubated for 4 hours. The optical density is measured at 450nm and the percentage of cell viability is calculated. 3. Statistical analysis:

The optical density is measured at 450nm and the percentage of cell viability is calculated. Data obtained from experiments performed in triplicate manner are presented as mean ± SEM. The unpaired t test (two-tailed) is used to determine significant differences between two groups of data (control and activator or inhibitor treated). P-values of less than 0.05, less than 0.01 and less thatn 0.001 are considered as statistically significant and are indicated by asterisks (*,**, and *** respectively). All data are analysed by using graph pad V5.0software.

Assessment of cytotoxicity activity on colon cancer cell line:

Morphological assessment -First set Doses 7.81 mg/ml Curcumin; 7.81 mg/ml Piper nigrum; 7.81 mg/ml Carrot; 7.81 mg/ml Cucumber; 31.25mg/ml of synergistic composition.

After 48 hours of incubation with various concentrations as mentioned previously the components of synergistic composition individually and the whole formulation, cells have attained the spherical morphology in both Curcumin (b) and synergistic composition (e) and have started to detach from the culture plate which is evident from the figure 3. Comparatively a large number of cells have detached in synergistic composition rather than the Curcumin. Whereas, Piper nigrum (c), Carrot (d), and Cucumber (e) doesn’t show any significant morphological difference compared with solvent control (a) which remained in the epithelial morphology.

Second set:

The doses adopted are 15.62 pg/ml Curcumin; 15.62 pg/ml Piper nigrum; 15.62 pg/rnl carrot; 15.62 pg/ml Cucumber; 62.5 pg/ml synergistic composition.

In the second set with higher dose of treatment, the cells have appeared as a spherical morphology in both Curcumin (b) and synergistic composition (e) and all the cells have detached from the plate. After the treatment with synergistic composition for 48 hours, the cells have become more shrunk and completely dissociated from the plate. Similar to 31.25 pg/ml concentration, Piper nigrum (c), carrot(d), and Cucumber (e) doesn’t show any significant morphological difference compared with solvent control (a) (figure 4).

Cell viability assessment

The CCK-8 results showed that treatment with 7.81 pg/ml curcumin and 31.25 pg/ml of synergistic composition inhibited the colon cancer cell proliferation with a significant p-value of 0.006 and 0.001 respectively. The synergistic composition reduces the cell viability more than curcumin which is evident from figure 5. Whereas, the other natural components in the synergistic composition like Piper nigram, Carrot and Cucumber at a dose of 7.81 pg/ml did not show any significant reduction in the cell viability which correlates to our previous findings in morphological assessment. However, the synergistic effect amongst the constituents together in the composition inhibited the colon cancer cell proliferation more significantly than the individual components. With 15.62 pg/ml of the Curcumin treatment, has greatly reduced the cell proliferation to cell viability of 28.4% with a significant p-value of 0.004. Whereas the treatment with 61.5 pg/ml synergistic composition has cell viability of 16.3% with a very high significant p-value of 0.001. The combinational treatment of

31.25 pg/ml of curcumin and pepper, 31.25 pg/ml of curcumin and cucumber has significant palpable changes whereas 31.25 pg/ml of Piper nigram and carrot, and 31.25 pg/ml of carrot and cucumber do not show any significant reduction in the cell viability (Figure 6) which correlates with findings in morphological assessment. This suggests that the synergistic composition acts as a better anti-cancer agent compared when treated with curcumin and pepper, cucumber, curcumin and curcumin alone and the other natural compounds in a synergistic manner do hinder the cell proliferation of colon cancer cells significantly and exhibits a greater anti-cancer activity of synergistic composition on colon cancer cells. Assessment of cytotoxicity activity on lung cancer cell line

Morphological assessment- First set

Doses 7.81 pg/ml Curcumin; 7.81 pg/ml Piper nigrum; 7.81 pg/ml Carrot; 7.81 pg/ml Cucumber; 31.25pg/ml synergistic composition. After 48 hours of incubation with various concentrations as mentioned previously mentioned of various the components of synergistic composition individually and the whole formulation, the cells have reduced its proliferation in both Curcumin (b) and synergistic composition (e) with lesser colonies which is evident from the figure 7. Comparatively fewer colonies of cells are observed in synergistic composition (f) rather than the Curcumin (b). Whereas, Piper nigrum (c), carrot (d), and Cucumber (e) did not display any significant chronological difference compared with solvent control (a) which remained in the epithelial morphology.

Second set Doses 15.62 pg/ml Curcumin; 15.62 pg/ml Piper nigrum; 15.62 pg/ml Carrot; 15.62 pg/ml Cucumber; 62.5 pg/ml synergistic composition.

In the second set of treatment with higher dose of treatment like 62.5 pg/ml treatment, the cells have greatly reduced its proliferation and started to appear a spherical morphology in both Curcumin (b) and synergistic composition (e). After the treatment with synergistic composition for 48 hours, the cells are more shrunk and no colonies are formed which is evident from the figure 8. Similar to 31.25 pg/ml concentration, Piper nigrum (c) and Cucumber (e) doesn’t show any significant morphological difference compared with solvent control (a). However, the cells treated with carrot (d) also had reduced proliferation rate. Cell viability assessment

The CCK-8 results showed that treatment with 7.81 pg/ml curcumin inhibited the Lung cancer cell proliferation with a percentage of 91% and 31.25 pg/ml synergistic composition treatment with 64% with a significant p-value of 0.04 and 0.03 respectively. The synergistic composition has greatly reduced the cell viability more than curcumin which is evident from figure 9. Whereas, the other natural components in the synergistic composition like pepper, Carrot, and Cucumber and its combinations treatment did not show any significant reduction in the cell viability which correlates to our previous findings in morphological assessment. Although some visible morphological differences are evident in the components when compared to the solvent treated cells. However, the synergistic effect of all the components together in the formulation of synergistic composition inhibited the lung cancer cell proliferation more significantly than the individual components. When treatment with 15.62 pg/ml of Curcumin treatment has greatly reduced the cell proliferation to cell viability of 47.7% with a significant p-value of 0.005. Whereas when treated with 61.5 pg/ml of synergistic composition, it caused reduction in cell viability of 38.7% with a very high significant p-value of 0.002. The combinational treatment of unlike curcumin alone and synergistic composition; Piper nigram, Carrot, and Cucumber and their combination do not show any significant reduction in the cell viability (figure 10) which correlates with findings in morphological assessment. This suggests that the synergistic composition acts as a better anti-cancer agent compared when treated with curcumin alone and the other natural compounds do not hinder the cell proliferation significantly and provides a greater anticancer activity of synergistic composition on lung cancer cells.

The synergistic composition of Curcumin, Pepper, Carrot, and Cucumber extracts enhanced cancer cell apoptosis. The composition has a higher apoptotic activity on cancer cells. Thus, the synergistic composition of present invention is a useful option in supplementation therapy for Cancer treatment including but limiting to breast cancer, colon cancer and lung cancer.