FIELD OF THE INVENTION
The present invention deals with the use of bioactive of the botanical plant extract for acute myloid leukaemia. The botanical plant extract containing the different bioactives, mostly secondary metabolites are well effective on acute myloid leukaemia cells even at lower concentration with minimal effect on the healthy normal peripheral blood mononuclear cells (PBMCs)
BACKGROUND OF THE INVENTION
Many important plant bioactive compounds have been discovered from natural sources using bioactivity directed fractionation and isolation (Balunas and Kinghom 2005), Continuing discovery has also been facilitated by the recent development of new bioassay methods. These bioactive compounds are mostly plant secondary metabolites, and many naturally occurring pure compounds have become medicines, dietary supplements, and other useful commercial products. Active lead compounds can also be further modified to enhance the biological profiles and has been developed as clinical trial candidates.
AML is a malignant disease of the bone marrow in which precursors of blood cells are arrested in an early stage of development. Most AML subtypes show more than 30% 'blasts' in the blood, bone marrow, or both. There is maturational arrest of bone marrow cells in the first stages of development. The mechanism involves the activation of abnormal genes through chromosomal translocations and other genetic abnormalities. This reduces the number of normal blood cells. Any therapeutic intervention that kills these undifferentiated 'blast' cells without showing cytotoxic effect on normal peripheral blood mononuclear cells (PBMC) is the need of the hour. In this discovery, we have enlisted a plant bioactive, which has shown this effect on the AML cells.
OBJECTIVE OF THE INVENTION
The current drugs available for AML therapy eradicate 'blast' cells but has severe side effects on the normal heamatopoeitic cells. The major objective of this invention is to disclose a plant-based compound that is effective against AML blast cells with minimal effect on normal haematopoeitic cells.

SUMMARY OF PRESENT INVENTION
The invention embodies the isolation of a plant bioactive with cytotoxic and anti¬proliferative effect on acute myeloid leukemia (AML) cells. The results show that the bioactive is effective even at low concentrations (0.1 -1 µg/ml) on the AML cells with minimal effects on the healthy normal peripheral blood mononuclear cells (PBMCs). Cell cycle analysis using propidium-iodide (PI) and flow cytometry showed that the plant bioactive extract causes gradual G2/M arrest of the AML cells from 14.3% to 17.90% over a dose ranging from 0.1-1 µg/ml. To elucidate the mode of action of the plant bioactive on AML cells, apoptotic assays using Annexin V-PI was carried out. It was found that the bioactive extract caused apoptosis of the AML cells ranging from 31.2% -39.5% over a concentration range of 0.1-10 µg/ml.
DESCRIPTION OF DIAGRAMS
Figure 1: TLC Fingerprinting profile
Figure 2 : Anatomical Studies
Figure 3 : Powder Analysis
Figure 4 : Chromatographic profile of Ca3.
Figure 5 : Comparative chromatograph overlay of three runs
Figure 6 : TIC of+EMS of Ca3
Figure 7 : TIC of-EMS of Ca3
Figure 8 : TIC of+EMS of Ca3 ethyl acetate farction
Figure 9 : TIC of-EMS of Ca3 ethyl acetate fraction:
Figure 10 : TIC of+EMS of Ca3 chloroform farction
Figure 11: TIC of-EMS of Ca3 chloroform fraction
Figure 12: TIC of+EMS of Ca3 butanol farction
Figure 13 : TIC of-EMS of Ca3 butanol fraction:
Figure 14 : TIC of+EMS of Ca3 water farction
Figure 15: TIC of-EMS of Ca3 water fraction
Figure 16 : Average of two independent experiments of Ca3 on KGI at 24h.
Figure 17 : Average of two independent experiments of Ca3 water fraction on KGI at 24h.

Figure 18 : Average of two independent experiments of Ca3 on PBMC at 24h.
Figure 19 : Cell cycle analysis: Effect of Ca3 on KGl-AML Cell line, control without
treatment
Figure 20 : Cell cycle analysis: Effect of Ca3 on KGl-AML Cell line, Treatment with
cisplatin-Positiive control (8µM).
Figure 21 : Cell cycle analysis: Effect of Ca3 on KGl-AML Cell line,Treatment with
parthenolide- Positive control (5.8 µM).
Figure 22 : Cell cycle analysis: Effect of Ca3 on KGl-AML Cell line, Treatment with Ca3
(O.µg/ml)
Figure 23 : Cell cycle analysis: Effect of Ca3 on KGl-AML Cell line. Treatment with Ca3
(lµg/ml)
Figure 24 : Cell cycle analysis: Effect of Ca3 on KGl-AML Cell line. Treatment with Ca3
(10µg/ml)
Figure 25 : Apoptosis assay: Effect of Ca3 on KGl cells: Control cells (KGl) unstained.
Figure 26 : Apoptosis assay: Effect of Ca3 on KGl cells: Control KGl cells stained with
Annexin.
Figure 27: Apoptosis assay: Effect of Ca3 on KGl cells: Control KGl cells stained with 7-
AAD
Figure 28 : Apoptosis assay: Effect of Ca3 on KGl cells: Control KGl cells stained with
Annexin and 7-AAD
Figure 29 : Apoptosis assay: Effect of Ca3 on KGl cells: KGl cells treated with cisplatin
8µM followed by staining with Annexin and 7-AAD
Figure 30 : Apoptosis assay: Effect of Ca3 on KGl cells: KGl cells treated with
Parthenolide 8µM followed by staining with Annexin and 7-AAD
Figure 31: Apoptosis assay: Effect of Ca3 on KGl cells: KGl cells treated with Ca3
O.lµg/ml followed by staining with Annexin and 7-AAD
Figure 32 : Apoptosis assay: Effect of Ca3 on KGl cells: KGl cells treated with Ca3
lµg/ml followed by staining with Annexin and 7-AAD
Figure 33 : Apoptosis assay: Effect of Ca3 on KGl cells: KGl cells treated with Ca3 10
µg/ml followed by staining with Annexin and 7-AAD
Figure 34 : Flow chart of Solvent Fractionation of Ca3

DETAILED DESCRIPTION OF THE INVENTION
Composition/ Pharmacognosy Of The Bioactive:
The in-house bioactive Ca3 mentioned through out this document was derived from Camellia sinensis or Green tea. Green tea is rich in catechins that include gailocatechin, epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (EGC) and epigallocatechin gallate (EGCG). One of the anti-tumor properties of green tea polyphenols and Epigallocatechin Gallate was studied by Thangapazham RL et.al (2007). In this study, Thangapazham RL et.al (2007), explained the effectiveness of green tea polyphenols (GTP) and its constituent Epigallocatechin Gallate (EGCG) in tumor regression was seen both in-vitro cell culture models and in vivo athymic nude mice models of breast cancer. The anti-proliferative effect of GTP and EGCG on the growth of human breast cancer MDA-MB-231 cell was studied using a tetrazolium dye-based assay, which showed that both had the ability to arrest the cell cycle at Gl phase as assessed by flow cytometry. Nude mice inoculated with human breast cancer MDA-MB-231 cells and treated with GTP and EGCG were effective in delaying the tumor incidence, reducing the tumor burden, induce apoptosis and inhibit the proliferation.
Hsuuw YD and Chan WH (2007) demonstrated that the catechins could evoke various responses, including cell death of human MCF-7 cells by Epigallocatechin-3-gallate (EGCG), a catechin that is highly abundant in green tea, which could induce apoptotic changes, including mitochondrial membrane potential changes and activation of c-Jun N-terminal kinase (JNK), caspase-9, and caspase-3. In contrast, higher concentrations of EGCG do not induce apoptosis, but rather trigger necrotic cell death in MCF-7 cells. Immunoblotting revealed that treatment of MCF-7 cells with 10-50 microM EGCG caused increases in Bax protein levels and decreases in Bcl-2 protein levels, shifting the Bax-Bcl-2 ratio to favor apoptosis. Reactive oxygen species (ROS) and ATP also play an important role to switch cell death types with apoptosis or necrosis. Collectively, these results indicate that EGCG supports apoptosis or necrosis. The most abundant and biologically active green tea catechin, epigallocatechin-3-gallate (EGCG), has been shown to act as a proteasome inhibitor and tumor cell death inducer. When MDA-MB-231 tumors were induced in nude

mice, followed by treatment with Pro-EGCG or EGCG, in vivo study showed a significant inhibition of breast tumor growth by Pro-EGCG, compared with EGCG, associated with increased proteasome inhibition and apoptosis induction in tumor tissues. In conclusion, it has shown that Pro-EGCG increases the bioavailability, stability, and proteasome-inhibitory and anticancer activities of EGCG in human breast cancer cells and tumors, suggesting its potential use for cancer prevention and treatment (Landis-Piwowar KR et.al, 2007).
Administration of Polyphenon E (a standardized preparation of green tea extract) in drinking water delayed tumor onset and suppressed tumor growth by 40%, with no adverse side effects. Histological analysis of mammary glands showed that green tea slowed the progression of ductal lesions to advanced mammary intraepithelial neoplasias and suppressed tumor invasiveness. Green tea also inhibited the proliferation of ductal epithelial cells and tumors and, overall, disrupted post-pubertal ductal growth. Immunohistochemical analyses also demonstrated that green tea inhibited angiogenesis through a decrease in both ductal epithelial and stromal VEGF expression and a decrease in intratumoral microvascular density Leong H et.al, (2007). Telomerase, an enzyme, is found to be elevated in >90% of breast carcinomas and therefore has received much attention as a target for breast cancer therapy and cancer diagnostic research. EGCG down-regulated telomerase in human breast carcinoma MCF-7 cells, leading to the suppression of cell viability and induction of apoptosis, thus providing the molecular basis for the development of EGCG as a novel chemopreventive and pharmacologically safe agent against breast cancer Mittal A et.al, (2004).
Tealeaves contain many compounds, such as polysaccharides, volatile oils, vitamins, minerals, purines, alkaloids (eg.caffeine) and polyphenols (catechins and flavonoids). Although all three-tea types have antibacterial and free radical capturing (antioxidising) activities, the efficacy decreases substantially the darker the variety of tea is. This is due to lower contents of anti-oxidising polyphenols remaining in the leaves (Chopra 2000), Polyphenols including flavonoids, tea tannins called catechin that includes gallocatechin, epicatechin (EC), epigallocatechin (EGC), epicatechingallate (ECG), and epigallocatechin gallate (EGCG) (Chopra 2000) are

the important phytochemical constituents in Green tea among other regular phytochemicals.
Pharmacognosy Data: Camellia sinensis (L.) O, Kuntze.
Batch No. AGT/COG/LF/9/1/05/030
Family Theaceae
Parts Used Leaf
[Note: AGT/COG/LF/9/1/05/030: Plant Raw Material Batch Number]
Description: An evergreen shrub or tree, 9-15 m in height (60-150 cm in height owing to pruning in cultivation); leaves simple, alternate, elliptic-ovate or lanceolate with serrate margins, usually glabrous, leathery; flowers white, fragrant, solitary or 2-4 together; fruits depressed capsules, 3-comered, 3-seeded.
Physicochemical And Organoleptic Parameters:
Table 1: physicochemical constants and organoleptic characters data

(TA - Total Ash; AIA - Acid Insoluble Ash; ASE - Alcohol Soluble Extractive; WSE - Water Soluble Extractive; Limit as per Ayurvedic Pharmacopoeia of India)
Characters In Anatomical Studies (Shown in figure 2)
1. Single layer epidermis both upper and lower epidermis,
2. Large vascular bundle at the center of the midrib,
3. Sclerenchymatous cells are present covered by the phloem cells, xylem
present in the mid rib region protoxylem cells present towards the lower
epidermis and metaxylem cells present towards upper epidermis.

4. Stomats are seen both upper and lower epidermal region with guard ceils,
5. Starch grains are present hypodermal region of the midrib region.
Characters In Powder Analysis:
Powder Colour: Green: (Shown in figure 3)
1. Epidermal cells with thin cuticle layer, palisade parenchymatous cells are present,
2. Stomats are seen with guard cells phloem fibers present.
3. Lignified vessels and calcium oxalate crystals.
Microbial Limit Test:
Total Aerobic Bacterial Count (TABC): 55.2 x 103 cfu g'
Total Yeast and Mould Count (TYMC): 0.6 x 103 cfu g"'
(Microbial contamination limit for raw herbs- TABC: <1 x l07cfu g'',TYMC: <1 x lO5cfu g-')
EXTRACTION OF PLANT MATERIAL DETAILS:
Green tea leaves collected from chikmangalore district were shade dried and powdered before extraction.
REFLUX EXTRACTION: Powdered Camellia sinensis leaves weighed into a round-bottomed flask. Two volumes of distilled water were added to the flask, few ceramic chips or glass beads were taken in the flask and kept on a heating mantle. A condenser with cold water circulating through it was placed on the flask before setting the temperature to the boiling point of water. The water vapor from the flask passes through the inlet of the extractor and condenses. The condensed (distilled) water extracts the polar compound from the plant material. This process is continuous as long as there is stable heat and water circulation to condense the vapors. The extraction was continued for 2 hours at room temperature and extracted at least for two times. After 2 hours the mantle was switched off and the water flow was stopped. After cooling the extract was collected separately and centrifuged. The extract was concentrated by drying under vacuum.

The percentage yield of the extract is calculated with respect to the initial weight of the plant material taken before extraction.
Percent Yield = wt. of lyophilized extract (after drying) * 100
Wt. of dry Plant material (initial)
Result:
There was 18,3% yield of Camellia sinensis by reflux extraction and this was labelled as Ca3 AVG BC023_Le_DrRf (95) 08(00) 05/02/08 as in-house labelling system and quoted as Ca3 throughout this document to keep it simple and easy to write.
Polyphenol Estimation:
Polyphenol assay is carried out using Floin-Ciocalteu reagent as performed by Using singleton, V., Rossi, J.A.Jr, method (1965), In brief to a 200µl of 50% Methanol / Standard / test sample with various concentrations, add l000 µl of FC reagent, mixed and incubate at RT for 5min. add 800 µ1 7.5% sodium carbonate, mix and incubate at RT for 30min. Read the absorbance at 750nm against blank by spectrophotometer. Gallic acid was used as a standard and the total polyphenol content in Ca3 was found to be 25.2 % Gallic acid equivalent (GAE).
Free Radical Scavenging activity:
DPPH is a free radical, has got free electron, very unstable generally. When this free radical reacts with antioxidant, antioxidant donates electron to free radical and makes it stabilize. This reduced DPPH gives change in colour from black to yellow and the change in absorbance at 517nm is followed which can be measured spectrophotometrically. Ca3 had high antioxidant potency in DPPH scavenging activity with an EC50 (the concentration of the extract required to bring about 50% of the scavenging effect as compared to control) of 5.2 µg.
ALKALOID ESTIMATION:
CaS was subjected for alkaloid estimation to test for the presence of alkaloid by Dragendorff s reagent method as reported by Sreevidya N 2003. In this method the alkaloid dragendorff reagent precipitates present in the plant extract and there is a

formation of yellow bismuth complex formed with thiourea. There was a 0.22% of alkaloid content in Ca3 extract as estimated by this method.
Hplc Fingerprinting Of AVGBC023_LE_DRRF(95)08(00)/ Ca3:
Metabolic fingerprinting of AVGBC023_Le_DrRf(95)08(00) and also to establish its authenticity by providing reproducibility .
Sample preparation
l0mg of the extract was weighed out into sterile eppendorf and dissolved in 1ml of HPLC grade water and sonicated for 15 minutes to ensure that the extract dissolves completely. The extract was centrifuged at 13,000rpm for 15 minutes at room temperature and the supernatant was filtered through 0.2u filters and collected into sterile recovery vials before analysis was performed.
Instrument initiation
Instrument was switched on and connected to the data station through communication
bus module. Each line was purged with the respective solvents for five minutes.
Further the column was washed with 100% methanol for 30 minutes and then
equilibrated with the initial run conditions of the corresponding LC time program.
Instrument parameters: Shimadzu LC20AT
Data acquisition: LC stops time: 65 mins, Acquisition time (PDA), Sampling: 3.125
Hz, Start time: Omins, End time: 50mins, Time constant: 0.64sec, Column used:
Ascentis ^^ RP -Amide,5nm 25x4.6mm
Pump: Pump Model: LC-20AT, Mode: Low Pressure Gradient Total Pump flow: 1
ml/min,
Total pump A flow: 1 ml/min, Solvent B Cone: 1, Solvent C cone: 0, Solvent D
Cone: 0, Max pressure limits: 5405
Column Oven Temperature: 33°C
Column used: Ascentis "^ RP -Amide,5µm 25x4.6mm
Controller: Model: CBM-20A, Power: On
Mobile phase:
Mobile phase A: HPLC grade water with 0.1 %TFA Mobile phase B: 0.1% TFA acidified acetonitrile


Auto sampler (SIL 20A): Sample rack: 1.5ml standard, Rinsing volume: 200µ1, Needle stroke: 52mm, Control vial needle stroke: 52mm, Rinsing speed: 35µl/sec, Sampling speed: 15µl/sec, Purge time: 25 min. Rinse mode: Before and after aspiration, Rinse dip time: lOSec,
Auto purge: Purge order. Mobile phase A: 5 mins. Mobile phase B: 5 mins, Mobile phase C: 5 mins, Mobile phase D: 5 mins. Auto sampler: 5 mins. Total pump A flow: Iml/min. After checking all the above mentioned parameters the inlets from every solvent system were purged with the respective solvents for five minutes to clear the tubes of any air bubbles. Further the column was washed with HPLC grade water (100%) for 30 min and then equilibrated with the initial run conditions of the corresponding LC time program.
Run conditions
No of runs performed for each sample: 5
Injection volume: 25)4,1
LC time program used: Ca_MD 1

Results
The extract was injected at a constant volume of 25µ1 and its metabolite profiling monitored at 254nm, shown in figure 4. A comparative profiling was performed between the runs/ extract to check the reproducibility of the results, shown in the figure 5.

Table 3: Summary of percentage conservation of AVGBCO 23_Le_DrRf(95)08(00)/Ca3 extract scanned from 200 to 700 nm using MetaGridTM

SOLVENT FRACTIONATION OF Ca3
Procedure:
Solvent fractionation of Ca3 - crude water extract was subjected to solvent-based fractionation where there is a phase formation of two immiscible solvents. 5 grams of

Ca3 dry extract powder was dissolved in 500 ml of distilled water and taken in a 2-
liter separating funnel. 1000ml of Ethyl acetate was added to the funnel containing
Ca3 followed by through mixing of the two solutions repeatedly and allowing it to
separate in to two phases. Compounds that are soluble into ethyl acetate will be in the
Ethyl acetae phase that is collected from the separating funnel. Again 1 liter of Ethyl
acetate is added and the same procedure followed until the ethyl acetae phase is
colorless.
The remaining aqueous phase is again fractionated with Chloroform following the
same procedure as given above. Once all the Chloroform phase is removed the
aqueous phase is again fractionated with n-Butanol. The same procedure of shaking
the two immiscible solvents, facilitating the fractionation of compounds those are
soluble in the organic phase from aqueous phase is followed throughout sequentially
from Ethyl acetate, Chloroform and Butanol.
The collected fractions were concentrated by rotary evaporator under vacuum to
remove the solvents. Finally four fractions were obtained Ethyl acetate, Chloroform,
Butanol and water.
Table 4 : Fraction of Ca3 - Solvent/phase fractionation


Liquid chromatography and Mass spectrometry of AVGBC023_LE_DRRF(95) 08(00)/ Ca3 and ethylacetate, chloroform, butanol and water fractions :
Identification and characterization of various phytochemicals present in Ca3 by LC-
MS/MS
i) Acquisition of + EMS in full scan mode from m/z 50 amu to 1000 amu
ii) Acquisition of- EMS in full scan mode from m/z 50 amu to 1000 amu
iii) Acquistion MS/ MS of selected ions
iv) Analysis of the mass peaks and characterization of the metabolites
Procedure:
5mg of Ca3 extarct in water, ethyl acetate fraction in 50% methanol, chloroform fraction in 50% methanol , butanolfraction in 50% methanol and water fraction of Ca3 extract in water were prepared for LC-Ms analysi.sThe contents of the vials were mixed thoroughly for 5 min. by a vortex following this; the vials were placed in a sonicator bath for 1 hour. The vials were then centrifuged for 15 min at 14000 rpm and 4°C to remove any suspended particles. The extracts were filtered through a 0,2-//-syringe filter, the clarified extracts were carefully transferred into respective autosampler vials (1.5 mL capacity, Shimadzu, Prominence). The Ca3 extract and fractions were subjected to an autosampler (SIL20AC) attached to HPLC (Shimadzu, Prominence). The temperature of the autosampler was maintained at 8°C throughout the experiment. The samples were eluted from HPLC by a binary gradient through a 5 ^ particle size RP-18 column, (4.6 mm D x 250 mm xL) held at 40°C in a temperature controlled column oven (CTO 20AC) at a flow rate of 0.5ml/min over 30.01 min. The gradient system consisted of 0.1% aqueous formic acid (A) and 0.1% formic acid in acetonitrile (B). The gradient was programmed to attain 95% (B) over 24 min, remains same till 26 min and decreases instantly to 5% at the end of 27 min. The 5% (B) remains till 30 min and the HPLC stops at 31.01 min.The HPLC eluent was further directed into mass spectrometer (Applied Biosystems MDS SCIEX 4000 Q Trap MS/MS) by applying a splitter.
The Mass spectrometer was operated in an EMS positive and negative polarity mode with ion spray voltage 2750, source temperature 350°C, vacuum 4.6"' Torr, curtain gas 20, Collision Energy (CE) 5, GSl 40, GS2 60, collision energy 5 and declustering

potential of 35. The scan rate was set at 1000 amu/ s with the interface heater 'on', 967 scans in a period and LIT fill time 20 m sec and dynamic LIT fill time on. The acquisition of EPI by LC-MS/MS- The enhanced product ion and MS/MS was performed at LC flow rate of 0.5 mL min"' over a period of 30.01 min,. The MS was operated both in positive and negative polarity mode. For positive polarity mode the curtain gas was set to 20, Collision Energy 30, CES 10, ion spray voltage was set at 4000.00 GSl 40, GS2 60 with interface heater and the dynamic fill time on. For negative polarity mode the curtain gas was set to 20, Collision Energy -30, CES 10, ion spray voltage was set at -3000.00, temp 300.00, GSl 40, GS2 60 with interface heater and the dynamic fill time on.
Data Processing:
For the processing, the total ion chromatogram (TIC) of blank (solvent) and test sample were Gaussian smooth, base line subtracted and noise to be set to 1%. The TIC of blank was subtracted fi-om that of the TIC of test and the spectrum was generated using Analyst Software 1.4.2. The noise level of spectrum was set to 1%. The processed spectrum is also manually verified. The data list is then generated to check the number of ions present with their m/z, centroid m/z, peak intensities, resolution, peak area and their charge specification. Next level of processing involves the elimination of the multiple charge ions by checking their singly charged ions. The low intense ions are further extracted to obtain Extracted ion chromatogram (XIC) or amplified.
Result:
The number of ions obtained in positive and negative node was 817, 862 in Ca3 extract, 74 and 205 firom ethyl acetate fraction of Ca3 extract , 93 and 275 from chloroform fraction of Ca3 extract, 167and 398 from butanol fraction of Ca3 extract and 184 and 187 water fraction of Ca3 extract respectively, (Fig 6-15)

CYTOTOXICITY ASSAY OF THE BIOACTIVE Ca3:
A) Screening Of Bioactives On Acute Myeloid Leukemic Cell Line KGl
It is also a non-radioactive assay, allows sensitive colorimetric assays for determination of the number of viable cells in cell proliferation and cytotoxicity assays. It uses highly water-soluble tetrazolium salt- WST-8 that is reduced by dehydrogenases in cells to give an orange colored product (Formazan) in the presence of an electron mediator. The formazan is soluble in the tissue culture media. The amoimt of the formazan dye generated by dehydrogenases in cells is directly proportional to the number of living cells. lOµl of CCK-8 is added to 100µ1 of the sample and incubated for l-4h in the incubator. The absorbance is read at 450nm.
Procedure: The assay was carried out in 96 well plates. The optimized seeding density for KGl was 1x10^ cells were seeded in each of the 96 well. The lOmg of bioactive Ca3 was weighed and dissolved in 1ml of sterile water and filtered through syringe filter 0.22 micron. The stock solution of bioactive (lOmg/ml) was further serially diluted with respective media (Ix IMDB + 20%FBS) to Img/ml, O.lmg/ml, 0,01mg/ml and O.OOlmg/ml. The range of bioactive concentration used ranged fi-om O.lµg/ml, lµg/ml, lOµg/ml, 25µg/ml, 50µg/ml, 75µg/ml and 100µg/ml. Each concentration of bioactive Ca3 was carried out in triplicates. The KGl cells, 105 density was seeded in 96 well plates and after bioactive treatment they were incubated for 24h at 37°C, 5% CO2 incubator. After the respective incubation period, cell proliferation assay (CCK-8) was carried out and O.D taken at 450nm.
CCK-8- (Cell Counting Kit-8)- measures the cytotoxicity / viable cells in proliferation, via colorimetrically.

Table 4: Average of two independent experiments of Ca3 on KGl at 24 hours:

Results: The results of the cytotoxicity assay is the average of two independent assays and shows that Ca3 bioactive shows inhibitory effect (IC5Ovalue) on KGl cells at almost all the concentrations as represented in the graph above and mentioned in the figure 16.
Cytotoxicity Assay of the Bioactive Ca3 Fractions
The assay was carried out in 96 well plates. 1x105 cells were seeded in each of the 96 well. 10mg of four Ca3 bioactive fractions namely Ea, Ch, Bu and Wa were weighed and dissolved in 1ml of sterile water and filtered through syringe filter 0.22 micron. Each of the stock solution of bioactive (10mg/ml) was further serially diluted with respective media (Ix IMDB + 20%FBS) to Img/ml, O.lmg/ml, O.Olmg/ml and O.OOlmg/ml. The concentration of each of the bioactive fractions used ranged from 0.1µg/ml, l|Ag/ml, lOµg/ml and 25µg/m\. Each concentration of Ca3 bioactive fractions were plated in triplicates. The treated cells were incubated for 24h at 37°C, 5% CO2 incubator. After the incubation period, cell proliferation assay (CCK-8) was carried out and O.D taken at 450nm.
Results

The results of the cytotoxicity assay is based on two independent experiments which show that among the four Ca3 fractions, Ca3 bioactive fraction 'Wa' follows a dose-dependent inhibitory effect on KGl cells. The cytotoxicity for concentration of Wa fraction (0.1 - 25 µg/ml) ranged from 13.7 to 25.5% as shown in the graph (Fig. 17)
B) SCREENING OF Ca3 ON NORMAL HEALTHY PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMCS)
It is also a non-radioactive assay, allows sensitive colorimetric assays for determination of the number of viable cells in cell proliferation and cytotoxicity assays. It uses highly water-soluble tetrazolium salt- WST-8 that is reduced by dehydrogenases in cells to give an orange colored product (Formazan) in the presence of an electron mediator. The formazan is soluble in the tissue culture media. The amount of the formazan dye generated by dehydrogenases in cells is directly proportional to the number of living cells. lOjxl of CCK-8 is added to 100^1 of the sample and incubated for l-4h in the incubator. The absorbance is read at 450nm.
Procedure: The assay was carried out in 96 well plates. The optimized seeding density for PBMCs was 0.5x106 cells were seeded in each of the 96 well. The 10mg of bioactive Ca3 was weighed and dissolved in 1ml of sterile water and filtered through syringe filter 0.22 micron. The stock solution of bioactive (lOmg/ml) was further serially diluted with respective media (Ix RPMI + 10% FBS) to Img/ml, O.lmg/ml, O.Olmg/ml and O.OOlmg/ml. The range of bioactive concentration used ranged from O.lµg/ml, Ijig/ml, lOµg/ml, 25µg/ml, 50µg/ml, 75µg/ml and lOOµg/ml. Each concentration of bioactive Ca3 was carried out in triplicates. The PBMCs 0.5x10^ density was seeded in 96 well plates and after bioactive treatment they were incubated for 24h at 37°C, 5% CO2 incubator. After the respective incubation period, cell proliferation assay (CCK-8) was carried out and CD taken at 450nm.
CCK-8- (Cell Counting Kit-8)- measures the cytotoxicity / viable cells in
proliferation, via colorimetrically. [Note: - Ca3 - AVG BC023_Le_DrRf
(95) 08(00)]

Table 5: Average of two independent experiments of Ca3 on PBMC at 24 hours:

Results: The results of the cytotoxicity assay,shown in the figure1 8, is the average of two independent assays and shows that Ca3 bioactive shows negligible effect at lower concentrations however, at higher concentrations ranging from 25µg/ml-100µg/ml inhibitory effect (IC50 value) on PBMCs was seen , this clearly shows that Ca3 at lower concentrations can be a considered as a potential bioactive for acute myeloid leukemia.
CELL CYCLE ANALYSIS: EFFECT OF Ca3 ON KGl CELL LINE:
The assay was carried out in 6 well plate. The optimized seeding density for KGl was 105 cells were seeded in each of the 6 well plate. The different treatments used for the assay was control with untreated cells, positive control was KGl cells treated with cisplatin 8µM (IC50 value) and Parthenolide 5.8µM, the experimental cells KGl cells treated with potent bioactive Ca3 at effective concentration (IC50 value) 0.1µg/ml, lµg/ml and 10µg/ml. The treated and untreated (control) were incubated for 24h at 37°C, 5% CO2 incubator. After the respective incubation period the contents of the cells were transferred to 15ml falcons and spun at 1500rpm for 5min, the supernatant discarded and resuspended in fresh PBS and spun again at 1500rpm for 5min to

remove traces of the media. The supernatant discarded and pellet (cells) is resuspended in 200-500µ1 of PBS and further used for PI staining.
PI Staining: The cells were fixed in 70% ethanol. 500µl of the cell suspension was added to 4ml of ice-cold ethanol in a 15ml falcon, while slowly vortexing to suspend them. The cells were kept on ice or incubated at 4°C for Ih and then stored at -20°C upto 24h before PI staining. The cells suspended in 70% ethanol were centrifuged at 1500rpm for lOmin at 4°C. The supernatant was carefully removed and the cells were resuspended in the PI master mix (PBS + PI 40µg/ml final concentration + RNase A-lOO^g/ml) at 106 cells /ml. The falcon was covered with aluminium foil and incubated for 45min at room temperature and analysed by FACS.
Results: The cell cycle analysis profile of KGl untreated cells is shown in Fig 19. The results of the cell cycle analysis for the positive control cisplatin treated KGl cells showed 74.5%, shown in figure 20 were in sub-GO phase and 22.9% in Glphase 3% synthesis phase and 1.1% G2/Mphase indicating the clear shift in population towards the subGO phase comprising of dead cells and necrotic cells. In case of parthenolide (another positive control specifically for AML), shown in figure 21, treated cells showed 29.9% of cells were seen in sub GO phase. 51.1% in Glphase, 12.2% synthetic phase and 9.8% G2/M phase. In addition to the cells shifting towards the apoptotic phase, there is also an arrest seen in synthetic phase when compared to the control KGl cells.
In the Ca3 treated KGl cells with increase in the concentration there is an increase in the synthetic phase when compared to the control cells showing that there is an arrest at the synthetic phase. In KGl cells treated with 0.1µg/ml of Ca3, figure 22 there is an increase in the synthetic phase indicating an S-phase arrest. In KGl cells treated with lµg/ml of Ca3, figure23 showed an increase in G2/M phase showing that there is G2/M arrest. In the case of 10µg/ml of Ca3 concentration, figure 24 there was an increase in the sub-GO phase with 19.3%, which comprises of dead cells and necrotic cells. In addition to this there was an increase in the synthetic phase also showing an arrest in the S-phase. Thus the cell cycle analysis shows that Ca3 has an effect on the cell cycle of KGl cells.

APOPTOSIS ASSAY: Effect Of Ca3 On KGl Cell Line
The assay was carried out in 24 well plate. The optimized seeding density for KGl was 105 cells were seeded in each of the 24 well plate. The different treatments used for the assay was control with untreated cells, positive control was KGl cells treated with cisplatin 8µM (IC50 value) and Parthenolide 5.8µM, the experimental cells KGl cells- treated cells with potent bioactive Ca3 at effective concentration (IC50 value) 0.1 µg/ml,1µg/ml and 10µg/ml. The treated and untreated (control) were incubated for 24h at 37°C, 5% CO2 incubator. After the respective incubation period the contents of the cells were transferred to 15ml falcons and spun at 1500rpm for 5min, the supernatant discarded and resuspended in fresh PBS and spun again at1500rpm for 5min to remove traces of the media. The pellet is washed in 1x Binding buffer. The supematant discarded and pellet (cells) is resuspended in 100µl of 1x binding buffer andused for the apoptosis assay.
Apoptosis Assay: The KGl cell pellet suspended in 100^1 of Ix binding buffer was transferred to FACS tubes. The tubes were wrapped in aluminium foil. Add 5µI of Annexin (FITC) and 5µl of 7-AAD. Gently vortex the cells and incubate for ISmins at room temperature (25°C) in dark. Add 400µ1 of Ix Binding buffer to each tube. Analyse by flow cytometer within 1hr.
Results: Fig 25 depicts KGl unstained cells showing live cell gating. Fig 26 shows
KGl cells stained with Annexin V-FITC alone while Fig 27 shows KGl cells stained
with 7AAD-APC alone and 7AAD and Annexin-V together Fig 28. The results of the
apoptosis assay showed the control cells comprises of 74% live cells, 24.5% are in
early apoptosis and 1.6% in late apoptosis (Figure 25-28). Cisplatin treated cells
showed 62.5% live cells, 18% cells in early apoptosis and 19.4% in late apoptosis and
0.1% dead cells (Figure 29).
Parthenolide treated cells showed 66.2% live cells and 23.1% in early apoptosis and
10.7% in late apoptosis (Figure30)
The KGl cells treated with Ca3 0.1µg/ml showed 67.4% live cells, 31.2% early
apoptosis and 1.4% late apoptosis (Figure31).
The percentage of cells undergoing apoptosis increased with increase in Ca3
concentration. In KGl cells treated with Ca3 lµg/ml (figure 32) showed 63.4% live

cells and 35.3% of cells undergoing early apoptosis and 1.3% cells in late apoptosis. In case of KGl cells treated with Ca3 10µg/ml (figure 33) showed 59.2% live cells, 39.5% were in early apoptosis and 1.3% in late apoptosis.
In comparison to the untreated control the results showed an increase in the percentage of KGl cells undergoing apoptosis with increase in Ca3 concentration. In case of Ca3 0.lµg/ml showed a 6.7% increase in early apoptosis, in Ca3 lug/ml showed 10.8% cells in early apoptosis and in Ca3 10µg/ml showed 15% of KGl cells in early apoptosis as compared to the untreated control. The results are quite significant, since it is in log scale. The bioactive Ca3 can be considered as a potential in-house natural plant extract candidate for considering for AML therapy. The bioactive Ca3 could be taken further for future studies with regard to purification into pure molecule, studying the mechanism of action (signalling pathway), pre-clinical and finally clinical studies.

1. A method of obtaining a plant extract (plant bioactive), capable of showing
antiproliferative activity, comprising the following steps:
(a) Obtaining plant material from one or more parts of the plants of claim 1.
(b) Obtaining an extract from the plant material by contacting the plant material with an aqueous, an ethanolic or an organic solvent, or a combination or the fractions of the extracts thereof, thereby providing one or more plant extracts
(c) Analyzing the plant extracts for toxicity and presence of antiproliferative activity
2. A method of obtaining a plant extract (plant bioactive) according to Claim 1
wherein the plant bioactive has anti-proliferative effect on cancer cell lines
3. A method of obtaining a plant extract (plant bioactive) according to Claim 1
and Claim 2 wherein the cell line is Acute Myeloid Leukemia (AML) cell
lines.
4. A method of obtaining a plant extract (plant bioactive) according to
claim 1 and claim 2 wherein the plant bioactive has anti-proliferative
effect on Acute Myeloid Leukemia (AML) cells at a dose ranging from 0.1-10
mg/ml with minimal effect on normal Peripheral Blood Mononuclear Cells
(PBMCs).
5. A method of obtaining a plant extract (plant bioactive) according to claim 1 and
claim 2 wherein the mode of action of the bioactive on AML cells is by induction
of apoptosis in AML cells.
6. A method of obtaining a plant extract (plant bioactive) according to Claims 1, 2
and 3 wherein the bioactive not only shows anti-proliferative effect on AML
cells, but also in other cancer cell lines like head and neck, breast and prostate
cancer cell lines
7. A method of obtaining a plant extract (plant bioactive) according to Claim 1
wherein the bioactive shows anti-proliferative effect on cell lines having a high proporation of stem cells,like KGla.