Title of Invention	A COMPOSITION USEFUL FOR TREATING ACUTE AND CHRONIC MYELOID LEUKEMIA
Abstract	This invention relates to a herbal based composition for treatment of CD33+ acute and chronic myeloid leukemia by Piper betel leaf extracts, fractions of Piper betel leaf extracts and 3-O-p-coumaryl quinic acid purified from Piper betel leaves extract. 12

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Title: An herbal based composition for treating acute and chronic myeloid leukemia. Field of invention This invention relates to a herbal based composition for treatment of CD33+ acute and chronic myeloid leukemia by Piper betel leaf extracts, fractions of Piper betel leaf extracts and 3-O-//-coumaryl quinic acid purified from Piper betel leaves extract. Background and prior art references Myeloid leukemia, both acute (AML) and chronic (CML) are lethal, there is no drug directing towards the destruction of the myeloid cells and these cells poorly respond to chemotherapy, which is always non-specific, thus adversely affecting normal cells. Unique property of the therapy with Piper betel components is the killing of myeloid cancer cells by recognizing CD33+ marker on the cell membrane, leaving normal cells without getting affected. Myeloid leukemia is usually subdivided into two groups: Acute Myeloid Leukemia (AML) and Chronic Myeloid Leukemia (CML). AML is characterized by an increase in the number of myeloid cells in the bone marrow and an arrest in their maturation. In the United States, the annual incidence of AML is approximately 2.4 per 100,000 and it increases progressively with age, to a peak of 12.6 per 100,000 in adults of 65 years age or older. The CML is a malignant clonal disorder of hematopoietic stem cells. The median age at presentation is 53 years, but it occurs at all age groups, including children. The natural history of CML is progression from a benign chronic phase to a rapidly fatal blast crisis within three to five years or even earlier. The prognosis of CML is also poor inspite of vast advancement of clinical medicine (1). CD33 represents a specific and useful marker in the process of myeloid cell differentiation (2). Recent reports suggest that engagement of CD33 by monoclonal antibody induced apoptosis leading to growth inhibition of proliferation of AML and CML cells in vitro (2,3). Exploiting the myeloid specific expression of CD33, humanized anti-CD33 monoclonal antibody conjugated with anti-cancer drug has been tried in AML patients with significant success (4). With the extract from Piper betel leaves anti-myeloid activity was claimed earlier (Patent filed no. PCT/INOO/00118 dated December 12, 2000). Hence, applicant's earlier finding is in directed consonance with the present patent filing on Piper betel leaf extract, fractionated leaf extracts and purified compound 3-O-p-coumaryl quinic acid (Fig. 1) obtained from leaf extract for treating CD33+ acute and chronic myeloid leukemia. Piper betel leaves have a strong pungent aromatic flavour and are widely used in India as a masticatory. Generally, mature or overmature leaves, which have ceased growing but not yet become brittle are used for chewing. The basic preparation for chewing purposes consists of betel leaf smeared with hydrated lime and catechu to which scrapings of arecanut are added; flavourings such as coconut shavings, clove, cardamom, fennel, powdered liquorice, nutmeg and also tobacco are used according to one's taste. In some places prepared Piper betel leaf preparation is covered with silver or gold film. As a masticatory, it is credited with many properties: it is aromatic, digestive, stimulant and carminative. Medicinally it is useful in catarrhal and pulmonary infections; it is also used for poultices. The effects of chewing of betel leaves with arecanut and other adjuncts are the excitation of the salivary glands and the irritation of the mucous membrane of the mouth. The red coloration produced is due to a pigment in the arecanut, which manifests itself under the action of alkali in lime and catechu. A mild degree of stimulation is produced, resulting in a sensation of warmth and well-being, besides imparting a pleasant odour. The most important factor determining the aromatic value of the leaf is the amount and particularly the nature of the essential oil present. Betel leaves from different regions vary in smell and taste. The most pungent is the Sanchi type, while the most mild and sweet ones are from Varanasi. The betal leaves contain essential oils, the content of oil varies from 0.7 to 2.6 per cent depending upon the varieties of leaves. The oil consists of phenols and terpens. The higher the proportion of phenol the better the quality. An isomer of eugenol named chavibetol (betel phenol; 4-alkyl-2-hydroxy-lmethoxy benzene) is considered to be characteristic constituent of betel oil. Betel oil of Indian types contains a predominant phenolic constituent and used in the treatment of various respiratory problems, either as a local application or by gargle. It has carminative properties. It exhibits in different actions on the central nervous system of mammals. The essential oil and extracts of the leaves possess activity against several Gram-positive and Gram-negative bacteria such as Micrococcus pyogenes var. Albus, Bacillus subtilis and B. Megaterium, Diplococcus pneumoniae, Streptococcus pyogenes, Eschenchia coli, Salmonella typhosa, Vibrio comma, Shigella dysenteriae, Proteus vulgaris, Pdseudomonas solanacaerum, Sarcina lutea and Erwinia carotorora. The essential oil and leaf extracts also showed anti-fungal activity against Asperigillus niger and A. Ory:ae. Curvularia lunata and Fusarium oxysporum. The oil is found to be lethal in about 5 minutes to the protozoa, Paramaeceum caudatum (5). Steam-distillate of the leaves showed activity against Mycobacterium tyberculosis. References 1. Sawyers CL, The New England Journal of Medicine, 340 (17), 1330-1340, 1999. 2. Vitaie, C; Romagnani, C, et al., Proc. Natl. Acd. Sci. USA, 96 (26), 15091-15096, 1999. 3. Vitaie, C et al., Proc. Natl. Acd. Sci, USA., 98 (10), 5764-5769, 2001. 4. Sievers EL, Appelbaum, FR et al., Blood, 93, 3678-3684, 1999. Object of the invention The main object of the invention is to provide a new herbal-based composition for the treatment of CD33+ acute and chronic myeloid leukemia by Piper betel leaf extracts. Another object of the invention is to provide a process for the isolation of active fractions from leaves or any other plant parts of Piper betel to treat CD33+ AML and CML. Still another object of the invention is to provide a simplified method of isolation of active components from all plant parts of Piper betel possessing biological activities relevant to the treatment of CD33+ AML and CML. Yet another object of the invention is to provide a new herbal product from active fraction from leaves or any other plant parts of Piper betel for the treatment of CD33+ AML and CML. Yet another object of the invention is to provide a herbal compound 3-O-p-coumaryl quinic acid purified from leaves of Piper betel for the treatment of CD33+ AML and CML. Yet another object of the invention is to provide a process for the preparation of extract from leaves or any other plant parts of Piper betel for the treatment of CD33+ AML and CML. Yet another object of the invention is to provide a simplified method of extract preparation from leaves or any other plant parts of Piper betel for the treatment of CD33+ AML and CML. Yet another object of the invention is to provide a process for the preparation of 3-O-p-coumaryl quinic acid from leaves of Piper betel for the treatment of CD33+ AML and CML. Yet another object of the invention is to provide a simplified method of preparation of 3-O-p-coumaryl quinic acid from all plant parts of Piper betel for the treatment of CD33+ AML and CML. Description of the Invention Accordingly, the present invention provides a composition useful for treating acute and chronic myeloid leukemia said composition comprising water, methanol fraction nos. 1 and 9 obtained from polar extract of plant piper betel by HPLC, in a ratio ranging between 1:1 (by wt) with retention time of 3.6 and 24.0 minutes respectively. Doses for myeloid leukemia patient depends on the in-vitro efficiency of the drug from piper betel either individually or in combination, the doses of in-vitro application for human myeloid cancer patients may range from 200 mg to 500 mg per day per patient if given as individual fraction (fraction 1 or fraction 9) or as a combination of fraction 1 and fraction 9 in the ratio between 0.1:5 to 5.0.1 preferably in the ration 1 :1 totaling 125 mg to 370 mg per day per patient through intravenous route. Accordingly the present invention provides a method of isolating fraction from piper betel, said method comprising steps of: a. cutting arial parts of piper betel into small pieces, b. homogenizing the said pieces with polar solvent selected from water, alcohol or their mixture to obtain an extract, c. filtering the extract to collect fi Iterate, d. lyophilizing the clear extract solution to obtain a semi-solid mass, e. fractionating the said semi-solid mass using column chromatography with only water, water-methanol with ratio ranging between 1:5 to 5:1, and only methanol, as eluents, f. selecting water : methanol fraction from the column chromatography, g. running HPLC with flow rate of 1.0 ml/min, using solvent system of methanol : water : acetic acid of ratio about 23:76:1, h. detecting about 12 peaks at about 280 nm, with varied retention time ranging between 3.6 to 36 minutes, i. separating the said peaks in a preparative HPLC with flow rate of 12ml/min, and obtaining fraction nos. 1 and 9 having desired biological activity In an embodiment of the present invention the ratio of water and methanol is ranging between 1:5 to 5:1. In another embodiment of the present invention the fraction 9 contains compound 3-O-p-coumaryl quinic acid. In yet another embodiment of the present invention dosage of each fraction individually ranging between 200-500mg/day. In still another embodiment of the present invention dosage of fraction nos. 1, and 9 in combination is ranging between 125 to 370mg/day. In still another embodiment of the present invention ratio of dosage of fraction nos. 1 and 9 is ranging between O.I: 5 to 5: 0.1, preferably, 1:1. In still another embodiment of the present invention the compound 3-O-p-coumaryl quinic acid shows 100% efficacy against acute myeloid leukemia at concentration of about 600ug/ml. In still another embodiment of the present invention compound 3-O-p-coumaryl quinic acid shows about 85% efficacy against chronic myeloid leukemia at concentration of about 600ug/ml. In still another embodiment of the present invention compound 3-O-p-coumaryl quinic acid shows about 60% efficacy against acute myeloid leukemia at concentration of about lOOug/ml. In an embodiment of the invention relates to evaluation of bioactivity of fractions 1 to 9 isolated from piper betel leaf against cancer cells. In an embodiment of the invention relates to evaluation of bioactivity of fractions 1 to 9 isolated from piper betel leaf for the treatment of CD33+ acute and chronic myeloid leukemia. The invention also provides a process for isolation of 3-O-p-coumaryl quinic acid from piper betel, which is reported for the first time. Another embodiment of the invention relates to a process for the isolation of active fractions from leaves or any other plant parts of Piper betel to treat CD33+ AML and CML. Still another embodiment of the invention relates to a simplified method of isolation of active components from all plant parts of Piper betel possessing biological activities relevant to the treatment of CD33+ AML and CML. Yet another embodiment of the invention relates to a new herbal composition/ fraction from leaves or any other plant parts of Piper betel for the treatment of CD33+ AML and CML. Yet another embodiment of the invention relates to new activity of compound 3-O-p-coumaryl quinic acid purified from leaves of Piper betel for the treatment of CD33+ AML and CML. Yet another embodiment of the invention relates to a process for the preparation of extract from leaves or any other plant parts of Piper betel for the treatment of CD33+ AML and CML. Yet another embodiment of the invention relates to simplified method of extract preparation h from leaves or any other plant parts of Piper betel for the treatment of CD33+ AML and CML. Yet another embodiment of the invention relates to a process for the preparation of 3-O-p- coumaryl quinic acid from leaves of Piper betel for the treatment of CD33+ AML and CML. Yet another embodiment of the invention relates to a simplified method of preparation of 3- O-p-coumaryl quinic acid from all plant parts of Piper betel for the treatment of CD33 -f AML and CML. Brief description of accompanying drawings Figure 1 represents HPLC analysis of fraction E. The first peak having retention time 3.6 min is designated as fraction 1, retention time 24.0 is designated as fraction 9. Figure 2 represents structure of fraction 9 deduced as 3-O-p-coumaryl quinic acid. Figure 3 represents destruction of CD33+ myeloid leukemia cells in vitro by P.Betel leaf extract as demonstrated by flow cytometry. Data is presented as histograms. In upper panels, Forward Scattering of light (X-axis) is plotted against the cell numbers (counts Y-axis). In the lower panels, side scattering of light (X-axis) is plotted against the cell numbers. Solid lines represent data with myeloid leukemia cells before treatment with P.betel leaf extract and the dotted lines represent data with myeloid leukemiccells after treatment with P.betel leaf extract (10.0 mg/ml) for48hours. Figure 4 represents destruction of CD33" myeloid leukemic cells in vitro by fraction E as demonstrated by flow cytometry. Dot plots of Forward Scatter vs. Side Scatter with CD33+ myeloid leukemic cells before and after treatment with fraction E (100.0 u.g/ml) for 48 hours are shown in left panels where gated region (Rl) represents myeloid leukemic cells. Two colour dot plots are also shown after staining with fluorescein isothiocyanate (FITC) conjugated anti-CD33 and phycocrytbrin (PE) conjugated anti-CD13 monoclonal antibodies. Isotype matched control antibodies (FITC and PE conjugated) were used as negative controls. Upper left quadrants represented CD13, CD33+ cells and upper right quadrants represented CD13+,CD33+ cells. Figure 5 represents CD33" myeloid leukemiccells are unaffected by fraction E as demonstrated by flow cytometry. Two colour dot plots after staining CD33" myeloid leukemic cells with anti-CD33-FITC and anti-CD13-PE monoclonal antibodies before and after treatment with fraction E (100.0 ug/ml) for 48 hours are shown. Isotype matched control monoclonal antibodies (FITC and PE labelled) were used as negative controls. Upper left quadrants represented CD134, CD33 cells and upper right quadrants represented CD13+, CD33+ cells. Figure 6 represents flow diagram for example 2 Figure 7 represents flow diagram for example 3 Exam pie-1 Collection of plant material The leaves and all other plant parts of Piper betel were collected from the climber from different areas and West Bengal, India. A voucher specimen was deposited at the Deptt. of Medicinal Chemistry at the Indian Inst. of Chemical Biology, 4 Raja S.C. Mullick Road, Kolkata-700 032. Example 2 Fresh leaves 5.3 kgs of Piper betel is thoroughly washed with distilled water. Leaves are cut into small pieces. It is then homogenized with distilled water 1.5 liter in a mixture-blender. The extract was filtered through a tine cheese-cloth and the filtrate was collected. The plant material inside the cloth was again extracted with water (1000 ml). The extract was filtered in a similar way. This process of extraction was repeated three times. The combined extract was centrifuged to obtain a clear solution. The clear extract was lyophilized yielding a semi-solid mass 150 gms. The extract was then tested for biological activity. A potion (10.02 gm) of the extract was fractionated on Sephadex LH-20 column chromatography. The column was eluted with water, water-methanol (1:1) and methanol as eluentto give three fractions, fractions 1-3, in the order of elution. The three fractions were then tested for biological activity. The fraction-2 (designated as Fraction E) showed biological activity, i.e., destruction of myeloid cells in human patients' ex vivo blood. Thin layer chromatography of this fraction showed two spots having Rf. 0.50 and 0.41 in the solvent system n-butanol: acetic acid : water (13:3:5). The HPLC analysis of this fraction showed several peaks as in Fig.l using Intersil ODS-3 (4.6x250 mm) analytical column, with a solvent system methanol: water: acetic acid (23:76:1), having flow rate of 1.0 ml/min and detection at 280nm. With the retention time 3.60, 8.60, 9.30, 11.50, 12.70, 16.40, 19.20,20.00, 24.00 and 36.60 mins respectively. Each peak of this fraction E was separated in a preparative HPLC using u.-Bonda pak column (19x300 mm), with a flow rate 12ml/min and the detection at 280nm. Compounds isolated from individual peak were tested for biological activity as described above. A purified compound was isolated from the peak having retention time 24.00 min. The structure of the compound was established as 3-O-p-coumaryl quinic acid (Fig.2), mp. 245 to 247° C, [oc]D -75°(methanol). IRYKBrma*crrV1 :3379(OH), 1691 (CO), 1514, 1383, 1269, 1173 and 830 1H-NMR (CD3OD) 7.64 (1H,DJ=15.9, H-7), 7.46 (2H,d, J=8.4, H-2 and 6), 6.81 (2H,d,J=8.4, H-3 and 5), 6.35 (1H,d,J=15.9,H-8), 5.38 (1H,m,H-3), 4.19 (1H, br, H-4) 3.84 (1H,m,H-5), 2.2 (2H, br., H-2) and 2.0 (2H, br., H-6) 13C-NMR (CD3OD) 176.00 (COOH), 167.65 (C-9), 160.24 (C-4), 145.68 (C-7), 130.17 (C-2 and 6), 126.25 (C-1), 115.81 (C-3 and 5), 75.16 (C-11), 72.48, 70.98, 70.57, 37.79 and 37.22 FAB MS m/z 329 (M++H), 361 (M++Na), 377 (M++K) Example-3 The fresh leaves and all other plant parts of Piper betel (1.2 kg) was homogenized with methanol (800 ml) in a mixture-blender and then sonicated in an ultrasonic bath with 3 burst each for 15 min and allowed to be extracted over night for 16 hrs. Filtering through Whatman No.l filter paper separated the methanol-extracted material. The process of extraction was repeated for three times. The combined extract was evaporated to dryness in a flash evaporator under reduced pressure at 40" C. The residual substance was then dried under high vacuum and the semi-solid mass (35.22 gm) was tested for biological activity (as mentioned above). The methanol extract (14 gm) was chromatographed on Diaion HP-20 (380 gm), elution being carried out with water, water-methanol (1:1) and methanol. Eluted three fractions were tested for biological activity. HPTLC of water-methanol fraction (fraction E) showed similar Rf values (0.5 and 0.41) as described in example. Active compound, 3-O-p-coumaryl quinic acid was isolated from the fraction E by using preparative HPLC as described in example-2. Example 4 Preparation of peripheral blood mononuclear cells (PBMC) from CD33+ myeloid leukemia patients ex vivo blood. Whole blood (10 ml) was drawn from a previously diagnosed CD33+ myeioid leukemic patient and mononuclear cells were separated by Ficoll/hypaqal density gradient centrifugation. Example 5 PBMC from CD33" myeioid leukemia patients. Whole blood (10 ml) was drawn from a previously diagnosed CD33" myeioid leukemia patient. Mononuclear cells were separated by Ficoll/hypaque density gradient centrifugation. ExampIe-6 Incubation of PBMC of myeioid leukemia patients with crude Piper betel extract PBMC (2xl06 /ml) of myeioid leukemia patients were incubated with Piper betel extract (10.0 mg/ml) for 48 hours and then washed and counted for viability. Example -7 Incubation of PBMC of myeioid leukemia patients with fraction E in vitro PBMC (2xl06/ml) of myeioid leukemia patients were incubated with varied concentrations of fraction E for 48 hours. Cells were then washed and counted for viability. Example -8 Incubation of PBMC of myeioid leukemia patients with purified compound 3-O-/?-coumaryl quinic acid in vitro PBMC (2x106 /ml) of myeioid leukemia patients were incubated with varied concentrations of purified compound for 48 h. Cells were then washed, and counted for viability. Example 9 Flow cytometry of myeioid leukemia cells Myeioid leukemia cells before and after in vitro incubation with crude Piper betel extract, purified fraction E or purified 3-O-p-coumaryl quinic acid were marked with anti-CD33-FITC and anti-CD13-PE monoclonal antibodies. Cells were then analysed in a flow cytometer (FACS Calibur, Section Dickinson, USA) Example 10 Incubation of myeioid leukemic cells with fractions having retention time 3.60 (fraction-1) and 24.00 min (fraction 9) PBMC of myeioid leukemic patients were incubated with above-mentioned fractions, alone or in combination of the two for 48 hrs. Cells were then washed and counted for viability. Results of the experiments Results of Example-6 As shown in Fig.3, CD33+ myeioid leukemia cells were killed in the presence of crude Piper betel extract. On the other hand, crude Piper betel extract has no effect on the CD33" myeioid leukemic cells. Results of example 7,8 and 9 As shown in Fig.4, CD33+ myeloid leukemia cells are destroyed by fraction E. On the other hand, CD33" myeloid leukemia cells remained unaffected.(Fig.5). PBMC of CD33+ myeloid leukemia patients (both AML and CML) are destroyed by purified compound 3-O-p-coumaryl quinic acid (Table -1). Results of Example 10 As shown in Table 2 combining fractions of 1 and 9 produced greater effects as compared to their individual activity at lower concentration. Table 1 Growth inhibition of AML and CML cells in vitro by purified compound (Table Remove) We Claim: 1. A composition useful for treating acute and chronic myeloid leukemia said composition comprising water: methanol fraction nos. 1 and 9 obtained from polar extract of plant piper betel by HPLC, in a ratio ranging between 1 : 1 (by wt) with retention time of 3.6 and 24.0 minutes respectively. 2. A composition as claimed in claims 1 to 2, wherein the fraction 9 contains compound 3-O-p-coumaryl quinic acid. 3. A composition useful for treating acute and chronic myeloid leukemia as herein described with reference to the examples 1 to 8 and drawings accompanying the specification.

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