1. INTRODUCTION:

Cancer is a disease described by uncontrolled engendering of cells that have changed from the typical cells of the body. The malignant growth cells can attack the neighbouring and distant tissues via the circulation. In advanced stages, a malignant growth patient may die because of either ill-advised finding or treatment disappointment. Malignancy is one of the push zones for which powerful medications at reasonable costs are not accessible until now presumably because of an absence of understanding the disease pathophysiology. For such a ghastly infection hostile to malignancy drugs have been created from an assortment of sources extending from normal items (plants and organisms) to synthetic particles.

The broadly utilized medications that are malignant growth chemotherapeutic specialists experience the ill effects of the downside of high danger, for example, bone marrow concealment, alopecia, queasiness and spewing and are not inside the compass of a typical man [1,2].

Medicines acquired from plants have assumed a central job in the social insurance of ahead of schedule and late societies. Ayurveda, the Indian arrangement of medication for the most part utilizes plant based medications or formulations to treat different sicknesses including malignancy. About 60% of medications allowed for cancer treatment are of natural source. Vincristine, Etoposide, Irinotecan, Taxanes and Camptothecines are instances of plant-derived anti-cancer compounds. [3,4]

A few malignancies inquire about investigations accompanied using traditional medicinal plants in solidarity to find new helpful specialists that do not have the dangerous reactions connected with current chemotherapeutic agents and the medications under clinical trials, phytomedicines have improved strongly in the previous two decades. [5,6]

Current treatment of malignant growth has significantly more side effects to defeat from that; natural remedies are helpful, adequate and demanding by the disease patients because of its less or no toxic effect.

Henceforth here is a significant need to create substitute restorative techniques against this deadly illness. There is consistently the expectation that the pursuit among the traditional therapeutic plants may furnish strong and safe prescriptions with few or no poisonous impacts.

Reactive oxygen species are thought to assume a significant job in numerous human maladies. Radical scavenging power is essential because of the lethal job of free radicals in biological systems. Numerous secondary metabolites like phenols, polyphenols, and flavonoids fill in as wellsprings of antioxidants and perform scavenging activity. [7]

In spite of assuming a key job in cell forms, reactive oxygen species promptly join and oxidize biomolecules, for example, sugars, proteins, and lipids and in this manner making them inactive with sub-sequent harm to cells, tissues, and organs prompting beginning of numerous maladies including malignancy. In this manner, by diminishing free radicals and oxidative pressure, antioxidants assume a job in improving DNA harm, decreasing the rate of abnormal cell division, and lessening mutagenesis. Hence, numerous antioxidant-rich plants possess cancer prevention activity. [8,9]

Ceratophyllum demersum L., having a place with the family Ceratophyllaceae [10], is one of the major appealing aquatic therapeutic plant that has been utilized as pain relieving, antipyretic, anti-inflammatory and in the treatment of ulcer, looseness of the bowels, wounds, burning sensation, hemorrhoids or piles, inherent hemorrhages, hyperdipsia, epistaxis, hematemesis [11,12]. Furthermore, it was accounted for that the aqueous, chloroform, ethanol and methanol extracts of C. demersum have an antimicrobial impact against confined strains of bacteria and fungi [13]. Moreover, other chemical compounds, for example, tricin-7-O-β-D-glycoside, naringenin-7-O-β-D-glycoside, esculetin, β-sitosterol, 7α-hydroxyl-β-sitosterol,7α-methoxyl-β-sitosterol, and palmitic acid have likewise been isolated from C. demersum [14].

The assessment of the range of biological activities (antineoplastic and anti-inflammatory) with a prediction of activity spectra for substances (PASS) for the significant parts of essential oil of C. demersum alongside different plants extracted with hexane was studied. The anticipated estimation of anti-inflammatory and antineoplastic activities with probability above 0.8 was detected for 12 compounds (2Z,4Z)-Hepta-2,4-dienal; 2-Phenylacetaldehyde; (3E,5E)- Octa-3,5-dien-2-one; 2,6-Dimethylcyclohexan-1-ol; geranylacetone; 𝛼-muurolene; 𝛽-ionone; 𝛽-eudesmol; α- eudesmol; biformen; kaurene and manool [15].

C. demersum has been generally utilized as bioindicators of heavy metals in air contamination, radioactivity indicators [16,17], biomonitoring [18] in the aquatic condition, genetic engineering [19]. It is likewise one of the well-known plants in the aquatic industry due to its resistance in a wide range of aquatic conditions [20,21]. It additionally gives a phenomenal living condition for shelter to fish and aquatic organisms. It is likewise utilized as a wellspring of food for some livestock, poultry [22]and fish [23]. C. demersum is astringent, bitter, sweet, oleaginous, fragrant and purgative [24]

However, this plant has not been studied for anticancer action on colon cancer and there is a just a single accessible examination study on antioxidant properties of this plant. Therefore, we have made an endeavour to utilize herbal plant to check adequacy against colon malignant growth cell lines. The present investigation was aimed meant to assess preliminary phytochemical evaluation, in vitro antioxidant potential, and in vitro cytotoxicity against HT-29 colon malignant growth cell lines.

2. MATERIAL AND METHOD:

2.1 Chemicals:

All solvents were analytical grade. Pet Ether, Chloroform, ethanol, Ascorbic acid, ferric chloride, aluminium chloride, potassium acetate, DPPH reagent, sodium carbonate, was obtained from Loba Chemie Pvt. Ltd. Mumbai. Shimadzu 1800 UV-Vis Spectrophotometer was utilized in all spectrophotometric estimations.

2.2 Cell culture:

The human colon cancer cell line (HT-29) was obtained from National Centre for Cell Sciences (NCCS), Pune, India. The cells were kept up at 37°C in a humidified atmosphere (90%) containing 5% CO₂ and afterword, cultured in (Dulbecco's Modified Eagle Medium) with low glucose, Thermo fisher scientific (Cat No-11965-092) with 10% (v/v) FBS, 100 units/mL penicillin, and 100 μg/ml streptomycin.

2.3. Strategy for Collection of plant:

The plant Ceratophyllum demersum Linn. chosen for the investigation was collected from the Kolhapur district of Maharashtra. The plant was authenticated and a Voucher specimen was kept at the herbarium of the Department of Botany, Shivaji University Kolhapur, Maharashtra, India. (Voucher reference numbers: SSA-01).

2.4. Phytochemical Evaluation:

The whole plant of Ceratophyllum demersum Linn. were collected, washed with water, shade dried, and powdered. The powdered material was exposed to successive solvent extraction with a selected order of polarity i.e. pet ether, chloroform, ethanol and water maceration. The resulted successive extracts i.e. Pet ether Extract [PECD], chloroform Extract [CECD], Ethanol Extract [EECD], Aqueous Extract [AECD], were exposed to phytochemical investigation for the identification of phytoconstituents present.

2.4.1 Tests for carbohydrates:

The carbohydrates were tried by using Benedict’s test, Fehling’s test and Molisch test.

2.4.2 Test for proteins:

Various extracts were dissolved in a few ml of water and treated with Million’s reagent.

2.4.3 Tests for Fats and Oils:

Fats and oils were tested with Translucent Spot test, Acrolein test,

2.4.4 Tests for sterols:

The sterols were tested by using the Libermann-Burchard test and the Salkowski test.

2.4.5 Tests for glycosides:

Keller Kiliani Test, Borntrager’s test were utilized for the analysis of glycosides.

2.4.6 Test for saponins:

Foam test was performed for the presence of saponins.

2.4.7 Test for Flavonoids:

The flavonoids were tested by the Shinoda test and Ferric chloride test.

2.4.8 Tests for alkaloids:

The alkaloids have been tested by using the Dragendroff ’s test and Wagner’s test.

2.4.9 Tests for tannins:

Test for tannins, ferric chloride test, and lead acetate test were performed. [25, 26, 27]

2.5. Pharmacological Evaluation:

2.5.1. In vitro Antioxidant Activity:

The in-vitro antioxidant activity of different extracts of Ceratophyllum demersum Linn. was finished by utilizing Ferric reducing antioxidant power (FRAP) assay, Phosphomolybdenum assay and 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability assay. [28]

2.5.1.1 Ferric ion reducing antioxidant power assay (FRAP):

Ferric reducing antioxidant power (FRAP) assay is a broadly utilized technique that utilizes antioxidants as reductants in a redox-connected colorimetric response, Ferric iron (Fe³⁺) is initially reduced by electron-giving antioxidants present inside the sample to its ferrous form (Fe²⁺). The iron colorimetric test complex builds a dark blue color product upon reduction which can be measured at 700nm. Antioxidants are molecules that go about as reducing agents by giving electrons to free radicals to stabilize them and limit the harm brought about by free radicals to DNA, cells and organ systems. Antioxidant incorporates substances such as polyphenols; flavonoids; vitamins and enzymes like glutathione peroxidase and superoxide dismutase. [29]

FRAP assay was utilized to quantify the total antioxidant potential of the extracts. Antioxidant activity assays were performed by the technique portrayed by Benzie and Strain with slight drugs. All extract of Ceratophyllum demersum Linn. and Ascorbic acid in various concentrations ranging from 12.5µg/ml to 200µg/ml, and were blended with 2.5ml of 0.2mM phosphate buffer (pH 7.4) and 2.5ml of potassium ferricyanide [1% weight/volume (W/V)]. The resulting mixture is incubated at 50^oC for 20 minutes, followed by the addition of 2.5mL of trichloroacetic acid (10% W/V) and centrifuged at 3000 rpm for 10 minutes. At that point, 2.5ml of distilled water was included and later 0.5 ml of ferrous chloride (0.1% W/V). At long last, the absorbance was estimated at 700nm. Ascorbic acid was utilized as a positive reference standard. [30]

2.5.1.2. Phosphomolybdenum assay (PM):

The antioxidant assay depends on the reduction of Phosphate-Molybdenum (VI) to Phosphate-Molybdenum (V). The incubation of extracts with the Molybdenum (VI) will communicate the presence of antioxidant constituents in the extract, which can be assessed by recording the absorbance at 695nm (to distinguish the reduced green molybdenum complex). In this way, this assay is very helpful to conceive the antioxidant capability of crude extracts.

The total antioxidant action was evaluated by Phosphomolybdenum (PM) assay utilizing the standard procedure of Prieto et al. All extracts of Ceratophyllum demersum Linn. and Ascorbic acid in different concentrations ranging from 12.5µg/ml to 200µg/ml were added to each test tube individually containing 3ml of distilled water and 1 ml of molybdate reagent. These tubes were kept incubated at 95^oC for an hour and a half. After incubation, they are kept at room temperature for 20–30 minutes and the absorbance was estimated at 695 nm. The positive reference standard was utilized in this assay was Ascorbic acid. [30,31]

2.5.1.3. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability assay:

The anti-oxidant capability of any compound can be resolved based on its scavenging activity of the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical. DPPH is a steady free radical containing an odd electron in its structure and generally used for the detection of the radical scavenging activity in chemical analysis. [32] The lambda max of a stable DPPH radical in methanol was at 517nm. The decline in absorbance of DPPH radical is brought about by antioxidants, in view of the reaction between antioxidant particles and radical progresses, which brings about the scavenging of the radical by hydrogen donation. [33,34]

Free radical scavenging effect of plant extract was resolved utilizing the 2-diphenyl-1picrylhydrazyl (DPPH) with slight meds of the technique portrayed by Brand-Williams et al. Brieﬂy; the concentrations of extracts ranging from 12.5µg/ml to 400µg/ml were set up in ethanol. DPPH solution (0.004%) was prepared in ethanol and 1ml of this solution was blended in with a similar volume of extracts and standard ascorbic acid solution separately. The mixture was incubated for half an hour in the dark at room temperature and the absorbance was measured at 517nm. The level of DPPH purple decolourization to DPPH yellow showed the scavenging effectiveness of the extract. Lower absorbance of the reaction mixture showed higher free radical-scavenging activity. The scavenging activity against DPPH was determined utilizing the equation:

DPPH scavenging activity (%) = AC-AT/AC× 100

AC: Absorbance of Control, AT: Absorbance of sample

The results were analyzed in triplicate. The IC50 value is the concentration of the sample required to inhibit 50% of the DPPH free radical. [30,35]

2.5.2. In-vitro Anticancer Activity:

The viability assay most as often as possible utilized all through the world is the MTT assay. This colorimetric assay utilizes a reduction of a yellow tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, or MTT) to measure cellular metabolic activity as a proxy for cell viability. Viable cells contain NAD(P)H- dependent oxidoreductase enzymes which reduce the MTT reagent to formazan, an insoluble crystalline product with a deep purple shading. Formazan crystals are then dissolved utilizing a solubilizing solution and absorbance is estimated at 500-600 nm utilizing a platereader. The darker the solution, the more the quantity of viable and metabolically active cells. [30, 36, 37]

The in-vitro anticancer activity of different extracts of Ceratophyllum demersum was was finished by utilizing HT-29 human colon malignant growth cell lines.[38]

The cells were seeded at a density of around 5×10³cells/well in a 96-well flat-bottom microplate and kept at 37⁰C in 95% humidity and 5% CO₂ overnight. Distinctive concentration (800, 400, 200, 100, 50, 25 µg/ml) of samples were treated. The cells were incubated for extra 48 hours. The cells in well were washed twice with phosphate buffer solution, and 20µl of the MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide), staining solution (5mg/ml in phosphate buffer solution) was added to each well and plate was incubated at 37⁰C. After 4 hours, 100µl of Dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals, and the absorbance at 570 nm was measured with UV- Spectrophotometer using DMSO as the blank. Measurements were performed and the concentration required for a 50% inhibition (IC50) was determined. The % cell viability was determined utilizing the following formula:

% Cell viability = A₅₇₀ of treated cells / A₅₇₀of control cells × 100

A₅₇₀: Mean absorbance at 570nm. [30, 39, 40]

3. STATISTICAL ANALYSIS:

All the determinations of the experimental work were carried out in triplicates and the acquired information were classified and analyzed by using Microsoft Office Excel 2013. The results are expressed as mean values and standard Error Mean (SEM).

4. RESULTS:

4.1 Phytochemical evaluation:

The existences of numerous secondary metabolites such as sterols, flavonoids, saponins, glycosides were detected in ethanol and aqueous extracts. These significant metabolites were identified based on color changes, the formation of precipitation or formation of persistent forms qualitatively. On the other hand, a lower amount of carbohydrate, alkaloids and tannins were identified. Traces of fats and oils were found in Pet ether extract. (Table 1)

Table 1: Phytochemical evaluation of Ceratophyllum demersum L. extracts

Compounds	PECD	CECD	EECD	AECD
Carbohydrates	-	-	+	+
Proteins	-	-	-	+
Fats and Oil	+	-	-	-
Sterols	-	++	+++	++
Glycosides	-	-	+	++
Saponins	-	-	+	++
Flavonoids	-	-	++	++
Alkaloids	-	+	+	+
Tannins	-	-	+	+

+++: Strongly present, ++: Moderately present, +: Present in traces, -: Absent

4.2 In vitro antioxidant activity:

Antioxidant capacities were shown highest in ethanol extracts based on the FRAP assay, PM assay and DPPH radical scavenging ability assay.

4.2.1. Ferric ion reducing antioxidant power assay:

In the present investigation, extracts of Ceratophyllum demersum L. were exposed to FRAP assay alongside standard ascorbic acid. In the results obtained, Ethanol extract showed higher activity than other extracts which was comparable to standard Ascorbic acid. (Table 2)

4.2.2 Phosphomolybdenum assay:

The Pet Ether (PECD), Chloroform (CECD), Ethanol (EECD) and Aqueous (AECD) extracts of Ceratophyllum demersum L. were subjected to PM assay along with standard ascorbic acid. In the results obtained, Ethanol extract showed higher activity than other extracts which was comparable to standard Ascorbic acid. (Table 3)

4.2.3. 2, 2-Diphenyl-1-picrylhydrazyl radical scavenging ability assay:

In the present study, Pet Ether (PECD), Chloroform (CECD), Ethanol (EECD) and Aqueous (AECD) extracts of Ceratophyllum demersum L. were subjected to DPPH free radical scavenging assay. The antioxidant potential of the extract was compared with ascorbic acid as the standard antioxidant. Ethanol extract showed higher activity than other extracts which was comparable to standard Ascorbic acid. (Table 4) IC50 values of DPPH scavenging activity of extracts of Ceratophyllum demersum L. for antioxidant activity are represented in Figure 1.

Table 2: Mean absorbance of Ceratophyllum demersum L. extracts for FRAP assay.

Concentration (µg/ml)	PECD	CECD	EECD	AECD	AS
200	0.354±0.025	0.412±0.021	0.643±0.017	0.467±0.015	0.962±0.009
100	0.275±0.017	0.265±0.018	0.444±0.028	0.312±0.020	0.612±0.001
50	0.161±0.025	0.138±0.026	0.295±0.039	0.217±0.027	0.42±0.002
25	0.094±0.023	0.077±0.007	0.18±0.035	0.079±0.009	0.266±0.007
12.5	0.052±0.014	0.06±0.008	0.086±0.017	0.054±0.011	0.164±0.009

Data expressed as Mean ± Standard error of the mean.

4.3. In vitro anticancer activity:

The result of MTT assays revealed that the ethanol extract was found to be selectively cytotoxic to HT-29 human colon cancer cell line as compared to other extracts compared with mostly used Paclitaxel as cytotoxic and normal control. (Table 5). IC50 values of extracts of Ceratophyllum demersum L. against HT-29 Colon cancer cell lines are represented in Figure 2

5. DISCUSSION:

These days, the pharmaceutical ventures were concentrating in the therapeutic plants as a wellspring of lead bioactive specialists to deliver novel medications. Around 25-50% of the modern drugs were acquired from therapeutic plants. Numerous medicinal plants were interesting in their biological activities however it has been utilized by various tribes or nations for various ailments, this shows plants have a wide scope of healing powers which are recognized to their synthetic composition. [41]

In recent years, the use of herbal medicines in cancer treatment has received increasing attention due to their varied Phyto-metabolic contents with multiple biological activities [42]. The plant collected from the Western Ghats was recognized by their taxonomical characters as Ceratophyllum demersum L. and analyzed for the presence of phytochemicals with four solvent extracts. Preliminary phytochemical analysis revealed the presence of secondary metabolites in the selected extracts of the plant (Table 1). These secondary metabolites are reported to have many biological and therapeutic properties [43]. Among the various phytochemicals, sterols and flavonoids have picked up the regard for various zones of utilizations such as pharmaceutical, health, food, and cosmetic industries. These composites basic in the plant space as piece of our everyday eating routine and are appealing as attractive natural antioxidants. [44,45]

Reactive oxygen species (ROS) are thought to assume a significant job in numerous human illnesses. Free Radical scavenging activities are exceptionally vital because of the poisonous job of free radicals in biological systems. Numerous secondary metabolites like flavonoids fill in as origins of antioxidants and do scavenging activity. [46] ROS unreservedly consolidates and oxidizes biomolecules, for example, proteins, sugars, and lipids in this manner making them lethargic with consequent harm to cells, tissues, and organs prompting disease progression. [47,48]

In the present work, FRAP, PM and DPPH strategies were utilized to assess the entire antioxidant capacity of pet ether, chloroform, ethanol, and aqueous extracts.

The Ferric ion reducing capability of the extract may help as a significant presentation of its antioxidant activity. The presence of antioxidants, which have been shown to be an impart antioxidant action by breaking the free radical chain by donating a hydrogen molecule [49, 50].

The presence of antioxidants in the extract would bring about the reduction of ferricyanide Fe³⁺ to ferrocyanide Fe²⁺ by giving an electron which was estimated spectrophotometrically at 700nm. In this assay, the yellow shade of the test solution changes to various shades of green and blue, depending on the reducing power of plant extract. The reducing power decreased with the decrease in the extract concentrations. This might be filled in as a critical marker of its antioxidant potential. The ethanol extract indicated more absorbance at 700nm than other extracts (0.643±0.017); subsequently this investigation assumed that ethanol extract of Ceratophyllum demersum L. may have a high measure of antioxidant property than other extracts which was comparable to that of the synthetic antioxidant standard used Ascorbic acid (Table 2).

By utilizing phosphomolybdenum technique, the absolute antioxidant activity of the sample was investigated. It is a colorimetric quantitative technique that estimates the reduction of Phosphate-Mo (VI) to Phosphate-Mo (V) by the sample and resulting development of a pale blue green shaded Phosphate-Mo (V) complex [30]. It helps to watch the reduction rate among antioxidant and molybdenum ligand. In the present investigation, ethanol extract displayed higher absorbance at 695nm than other extracts (0.923±0.011) (Table 3). Henceforth this examination perceived that ethanol extract of Ceratophyllum demersum L. may have a high measure of antioxidant property than other extracts which was practically comparable to that of synthetic antioxidant standard used Ascorbic acid

DPPH is steady nitrogen focused free radical which is ordinarily used to decide free radical scavenging activities of antioxidants present in plant extracts or synthetic compound. [51,52,53] The reduction capacity of DPPH radical is dictated by the decline in absorbance at 517nm prompted by antioxidants. Greater absorbance of the reaction mixture specified lesser free radical scavenging activity. [54] In the present examination, ethanol extract displayed higher free radical scavenging activity than other extracts (79.58±1.15) (Table 4). Thus this examination accepted that ethanol extract of Ceratophyllum demersum L. may have a high measure of antioxidant property than other extracts which was practically comparable to that of the standard synthetic antioxidant agent Ascorbic acid. The outcome indicated that ethanol extract significantly scavenges the free radical and was the most potent extract with an IC50 value of 71.52μg/ml (Figure 1).

The evaluation of the cytotoxicity of plant extracts is important for innocuous treatment. It empowers the identification of the inborn toxicity quality of the plant. [55,56] The MTT assay is utilized in screening the crude extracts to assess the toxicity. It could likewise give a sign of conceivable cytotoxic properties of the tested plant extracts. MTT assay depends on the reduction of MTT by mitochondrial dehydrogenase by purple formazan product. It is often utilized as an in vitro model framework to measurement of cytotoxic impacts of plant extracts against malignant growth cell lines. [57,58,59]

In vitro cytotoxicity test using HT-29 colon malignant cell lines was performed to screen potentially toxic compounds that affect basic cellular functions and morphology. The four extracts (Pet ether, Chloroform, Ethanol and Aqueous) of Ceratophyllum demersum L. appeared in vitro development hindrance consequences for the malignant growth cell lines (HT-29), while there was no impact on the development of normal cells. Such specific impacts were focus just as, incubation period subordinate, regarding concentration (25, 50, 100, 200, 400, 800μg/ml) of each extract were assessed in triplicates by sequential dilutions. Among these six concentrations, 800μg/ml of ethanol extract was the best in creating percentage growth inhibition (Cell viability: 46.18±0.61) trailed by aqueous extract (Cell viability: 57.5±0.60) as compared to other extracts. However, the standard paclitaxel drug showed significant inhibition (Cell viability: 21.87±0.67) on the cancer cell lines (Table 5).

The outcome indicated that ethanol extract is most potent extract with an IC50 value of 571.3μg/ml (Figure 2).

6. CONCLUSION:

It was seen that the plant Ceratophyllum demersum L. contains a wide assortment of secondary metabolites that hold strong antioxidants and anticancer capability dependent on the analyses performed, which gives a logical proof to direct further examinations and research the lead compounds present in the plant and assess its anticancer potential on other cancer cell lines and on in vivo animal models and set forward an endeavour to carry out trials on human beings.

7. ACKNOWLEDGMENT:

We would like to thank Prof. D. D. Chougule, Principal, Dr. Shivajirao Kadam College of Pharmacy, Kasabe Digraj, Sangli, Maharashtra, and Mr. Vijay Kumbhar, Maratha Mandal's N.G.H. Institute of Dental Sciences and Research Centre, Belgaum, Karnataka, India for provided necessary research facilities to carry out the research work.

8. CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 28.02.2020 Modified on 21.03.2020

Research J. Pharm. and Tech. 2021; 14(1):28-36.

DOI: 10.5958/0974-360X.2021.00006.8

Concentration (µg/ml)	PECD	CECD	EECD	AECD	AS
200	0.645±0.033	0.721±0.021	0.923±0.011	0.732±0.020	1.199±0.011
100	0.447±0.011	0.541±0.017	0.696±0.007	0.413±0.011	0.903±0.009
50	0.208±0.014	0.274±0.016	0.412±0.009	0.321±0.004	0.658±0.014
25	0.121±0.019	0.133±0.009	0.196±0.006	0.164±0.008	0.442±0.009
12.5	0.08±0.012	0.098±0.004	0.164±0.008	0.114±0.005	0.237±0.004

Concentration (µg/ml)	PECD	CECD	EECD	AECD	AS
400	59.52±0.83	66.62±1.20	79.58±1.15	72.91±1.17	96.71±0.85
200	47.95±1.42	57.83±0.07	68.6±1.03	62.39±2.39	87.57±1.76
100	37.06±1.40	33.28±0.24	53.08±1.34	42.17±0.43	77.86±2.06
50	27.84±3.46	21.79±1.18	43.78±1.00	32.83±1.09	63.95±1.38
25	14.55±1.10	12.01±1.53	33.94±1.71	23.61±1.00	46.83±1.91
12.5	8.75±0.52	5.98±0.45	20.39±0.96	14.37±1.50	27.14±3.41

Conc. µg/ml	PECD	CECD	EECD	AECD	Paclitaxel
25	96.18±0.66	95±0.26	95.74±0.78	94.71±0.79	92.6±0.47
50	90.35±0.77	91.77±0.46	88.47±1.11	86.76±0.35	82.01±0.83
100	84.56±0.34	86.03±0.64	80.26±0.57	76.62±1.26	68.83±0.41
200	79.79±1.57	79.44±0.34	63.68±0.45	66.32±0.39	50.53±0.50
400	74.29±0.032	73.65±0.70	60±0.64	55.44±0.52	37.39±0.43
800	64.53±0.75	61.21±0.58	46.18±0.61	57.5±0.60	21.87±0.67
NC	100