Title of Invention	“A PROCESS FOR THE PREPARATION OF ETHANOLIC EXTRACT FROM ALTERNANTHERA BETTZICHIANA"
Abstract	The present invention provides physicochemical, phyto-chemical and pharmacological evaluation of the plant Aliernanthera bettzichiana. The present invention also relates to novel Aliernanthera bettzichiana extracts characterized by the presence of steroids, glycosides, proteins, carbohydrate, and alkaloids. The present invention further relates to processes for preparing AUernanthera bettzichiana extracts.

Title of Invention

“A PROCESS FOR THE PREPARATION OF ETHANOLIC EXTRACT FROM ALTERNANTHERA BETTZICHIANA"

Abstract

The present invention provides physicochemical, phyto-chemical and pharmacological evaluation of the plant Aliernanthera bettzichiana. The present invention also relates to novel Aliernanthera bettzichiana extracts characterized by the presence of steroids, glycosides, proteins, carbohydrate, and alkaloids. The present invention further relates to processes for preparing AUernanthera bettzichiana extracts.

Full Text	FORM-2 THE PATENTS ACT, 1970 (39 of 1970) & THE PATENTS RULES, 2003 COMPLETE SPECIFICATION (See Section 10; rule 13) ALTERNANTHERA 'BETTZICHJANA EXTRACTS AND PROCESSES FOR PREPARING THE SAME HUNDIWALE JOGENDRA CHANDRAKANT an Indian National of House No. 1167, Hundiwale Lane, Chopda, Dist. Jalgaon- 425107; Maharashtra, India THE FOLLOWING SPECIFICATION DESCRIBES THE NATURE OF THE INVENTION AND THE MANNER IN WHICH IT JS TO BE PERFORMED FIELD OF THE INVENTION The present invention relates to Alternanthera bettzichiana extracts and processes for preparing the same. BACKGROUND OF THE INVENTION AL TERNANTHERA BETTZICHIANA (REGEL) Phylum - Magnoliophyta Class - Magnoliopsida Order - Caryophyllales Family - Amaranthaceae Genus - Alternanthera There are about eighty varieties of genus Alternanthera Spread among tropical and subtropical regions of the world. The Ayurveda has already recognized Alternanthera pungens, Alternanthera sessilis, and Alternanthera philoxeroides as landmarks. About seven to eight species from this genus are located in Indian soil. Provided herein below are the species of the genus Alternanthera found in india: (Geeta C, et at. 1994.) 1. Alternanthera sessilis (L.) R. Br. ex DC. 2. Alternanthera pungens Kunth in H.B. & K. 3. Alternanthera paronychioides St. Hil. 4. Alternanthera philoxeroides (Mart.) Gris. 5. Alternanthera tenella colla 6. Alternanthera bettzichiana (Regel) Nicolson 7. Alternanthera caracasana Kunth in H.B. & K. Most of the species from the genus Alternanthera are considered as weeds and various species are utilized for the ornamental purpose. According to Janusz Lipecki, "A weed is defined to be a plant whose virtues have not been yet discovered and a plant in wrong place." This reflects negative nature of weeds (Janusz Lipecki, 2006). However, weeds also sometimes play positive role as natural components of the environment, for example Jimson weed {Datura stramonium). A. bettzichiana, a horticulture species, of many forms, is often found as an escape (Geeta C, et al.1994), reported as wild edible material (Arinathan V., et al. 2007) and as adsorbent for removal of Cr (VI) (Patil A.K., et al.2009). In order to make safe and sure use of the medicines, a necessary first step is the establishment of standards of safety and efficacy. Keeping this fact into consideration, in the present invention attempts are made to establish Pharmacognostical and pharmacological standards of the plant Altemanthera bettzichiana (Regel) Nicols which is a less exploited weed. OBJECTS OF THE INVENTION It is an object of the invention to provide Altemanthera bettzichiana extracts. It is another object of the present invention to provide processes for preparing the Altemanthera bettzichiana extract. It is still another object of the present invention to provide processes for preparing the Altemanthera bettzichiana extract which are simple, high yielding and economic. It is yet another object of the present invention to perform physicochemical, phyto-chemical and pharmacological evaluation of Altemanthera bettzichiana. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 illustrates HPTLC detection of cardiac glycosides wherein 1A is HPTLC chromatogram of ethanolic extract for cardiac glycosides; IB is HPTLC chromatogram of chloroform extract for cardiac glycosides; la is scanned HPTLC plate at 254 nm (before derivatization) for detection of cardiac glycosides; lb is scanned HPTLC plate at 366 nm (before derivatization) for detection of cardiac glycosides; and lc is scanned HPTLC plate at 366 nm(after derivatization) for detection of cardiac glycosides. Fig. 2 illustrates HPTLC detection of coumarin glycosides wherein 2A is HPTLC chromatogram of ethanolic extract for coumarin glycosides; 2 B is HPTLC chromatogram of chloroform extract for coumarin glycosides; 2a is scanned HPTLC plate at 254 nm (before derivatization) for detection of coumarin glycosides; 2b is scanned HPTLC plate at 366 nm (before derivatization) for detection of coumarin glycosides; and 2c is scanned HPTLC plate after derivatization in visible light for detection of coumarin glycosides. Fig. 3 illustrates HPTLC detection of steroids wherein 3A is HPTLC chromatogram of ethanolic extract for steroids; 3B is HPTLC chromatogram of chloroform extract for steroids. 3a is scanned HPTLC plate at 254 nm (before derivatization) for detection of steroids; 3b is scanned HPTLC plate at 366 nm (before derivatization) for detection of steroids; 3c is scanned HPTLC plate at 366 nm (after derivatization) for detection of steroids; and 3d is scanned HPTLC plate after derivatization in visible light for detection of steroids. Fig. 4 illustrates HPTLC detection of alkaloids wherein 4A is HPTLC chromatogram after scanning at wavelength 366nm for alkaloids; and 4B is scanned HPTLC plate at 366 nm (before derivatization) for detection of alkaloids. Fig.5 illustrates HPTLC detection of proteins wherein 5A is scanned HPTLC plate after derivatization in visible light for detection of proteins; 5B is HPTLC chromatogram after scanning at wavelength 254 nm for proteins; 5C is HPTLC chromatogram after scanning at wavelength 366 nm for proteins; and 5D is HPTLC chromatogram after scanning at wavelength 530 nm for proteins. Fig. 6 illustrates HPTLC detection of carbohydrates wherein 6A is HPTLC chromatogram after scanning at wavelength 254 nm for carbohydrates; 6B is HPTLC chromatogram after scanning at wavelength 366 nm for carbohydrates; 6C is HPTLC chromatogram after scanning at wavelength 530 nm for carbohydrates; 6a is scanned HPTLC plate at 254 nm (before derivatization) for detection of carbohydrates; 6b is scanned HPTLC plate at 366 nm (before derivatization) for detection of carbohydrates; 6c is scanned HPTLC plate at 366 nm (after derivatization) for detection of carbohydrates; and 6d is scanned HPTLC plate after derivatization in visible light for detection of carbohydrates. Figure 7A illustrates effect of different doses of ethanolic extract and diclofenac sodium on Carrageenan induced rat paw edema (% rise) at 1 hr. Figure 7B illustrates effect of different doses of ethanolic extract and diclofenac sodium on Carrageenan induced rat paw edema at 2 hrs. Figure 7C illustrates effect of different doses of ethanolic extract and diclofenac sodium on Carrageenan induced rat paw edema at 3 hrs. Figure 8A illustrates effect of different doses of ethanolic extract ofAlternanthera bettzichiana and diclofenac sodium on mean increase in cotton pallet weight. Figure 8B illustrates effect of different doses of ethanolic extract of Alternanthera bettzichiana and diclofenac sodium on percent inhibition of cotton pallet granuloma. Figure 8C illustrates cotton pellet with granuloma formation (Mouse model). Figure 9A illustrates effect of EEWPM and EEMPM on tumour volume at 5-10 days. Figure 9B illustrates effect of EEWPM and EEMPM on tumour volume at 11-15 days. Figure 9C illustrates effect of EEWPM and EEMPM on tumour volume at 16-20 days. Figure 9D illustrates effect of EEWPM and EEMPM on tumour volume at 21-25 days. Figure 10A illustrates effect of different concentration of EEWPM and EEMPM on cell viability. Figure 10B illustrates effect of different concentration of EEWPM and EEMPM on % cell viability SUMMARY OF THE INVENTION: In accordance with the first aspect of the present invention there is provided a process for the preparation of AIternanthera bettzichiana extract; said process comprising the following steps: a) selecting the Alternanthera bettzichiana plant material followed by washing and cleaning; b) drying the cleaned Alternanthera bettzichiana plant material; c) grinding the dried material to obtain powdered material followed by passing the powdered material through a sieve to obtain the Alternanthera bettzichiana powdered material ready for extraction; and d) subjecting the material to solvent extraction at a temperature of about 20 to 60 °C to obtain the Alternanthera bettzichiana extract. wherein said solvent extraction comprises extraction with at least one solvent or successive extraction or cold maceration. In accordance with one of the embodiments of the present invention the successive solvent extraction comprises extraction of the plant material using at least one solvent having low polarity followed by extraction with at least one solvent having high polarity. Typically, the solvent used for extraction is at least one selected from the group consisting of water, petroleum ether, chloroform, methanol and ethanol. In accordance with another embodiment of the present invention the solvent extraction further comprises i) filtering the extract in hot condition; ii) distilling on water bath; and iii) drying at low temperature under reduced pressure in a evaporator. In accordance with still another embodiment of the present invention the cold maceration comprises i) adding a solvent to the flask containing plant material under vigorous shaking; ii) plugging the flask with cotton plug and aluminium foil followed by keeping the flask aside for 12 hours; iii) shaking the flask vigorously and allowing to stand the flask for 7 days with frequent shaking; and iv) separating the fluid extract by decantation and filtration. In accordance with the second aspect of the present invention there is provided an Alternanthera bettzichiana extract; said extract comprising: i. steroids; it. glycosides; iii. carbohydrate; and iv. alkaloids. wherein said extract is alcoholic extract with extractive value of about 32% and is useful in the treatment of at least one disease selected from the group consisting of cancer, inflammatory diseases and bacterial infections. In accordance with the third aspect of the present invention there is provided an Alternanthera bettzichiana extract; said extract comprising: i. proteins; ii. glycosides; iii. carbohydrate; and iv. alkaloids. wherein said extract is aqueous extract with extractive value of about 19% and is useful in the treatment of at least one disease selected from the group consisting of cancer, inflammatory diseases and bacterial infections. In accordance with the fourth aspect of the present invention there is provided an Alternanthera bettzichiana extract; said extract comprising: i. steroids; and ii. glycosides. wherein said extract is chloroform extract with extractive Value of about 25% and is useful in the treatment of at least one disease selected from the group consisting of cancer, inflammatory diseases and bacterial infections. Typically, the glycoside comprises cardiac glycoside, anthraquinone glycoside, saponin glycoside and coumarin glycoside. In accordance with fifth aspect of the present invention there is provided a medicament for the treatment of cancer comprising administering in said subject an effective amount of Alternanthera bettzichiana extract. In accordance with sixth aspect of the present invention there is provided a medicament for the treatment of at least one disease selected from the group consisting of bacterial infections and inflammatory disease comprising administering in said subject an effective amount of Alternanthera bettzichiana extract. DETAILED DESCRIPTION OF THE INVENTION: In accordance with the first aspect of the present invention there is provided a process for the preparation of Alternanthera bettzichiana extract; said process comprising the following steps: First step is selecting the Alternanthera bettzichiana plant material. Fresh samples of the plant Alteanthera bettzichiana were collected from the different parts of Satpuda region of Chopda Tahsil, Jalgaon district in the month of September. The plant was authentificated from Botanical survey of India, Pune. Typically, the plant material used is wild plant material of A. bettzichiana or micro-propagated plant material of A. bettzichiana. The selected plant material is washed and cleaned properly. Second step is drying the cleaned Alternanthera bettzichiana plant material in shade. In next step the dried material is grinded using electric grinder to obtain the powdered material. The powdered material is then passed through a sieve no. 80 to obtain the Alternanthera bettzichiana powdered material ready for extraction. In the final step the obtained Altemanthera bettzichiana powdered material is subjected to solvent extraction at a temperature of about 20 to 60 °C to obtain an Altemanthera bettzichiana extract. In one of the embodiments of the present invention the solvent extraction comprises extraction with at least one solvent. In accordance with another embodiment of the present invention the solvent extraction is successive solvent extraction which comprises extraction of the plant material using at teast one solvent having low polarity followed by extraction with at least one solvent having high polarity. Typically, the solvent used for extraction is at least one selected from the group consisting of water, petroleum ether, chloroform, methanol and ethanol. In successive solvent extraction, a dried material was extracted with different solvents, starting from solvent of low polarity by using soxhlet extraction apparatus. After extraction by one solvent, material is removed from thimble, dried and recharged, extracted with solvent of successively high polarity. Typically, the glass Soxhlet apparatus consist of four parts • Round bottom flask (RBF), • Main body/Thimble, • Syphone glass tube, and • Condenser. 150 gm of dried, powered crude drug was kept in a filter paper cover which was already placed in thimble. Then the solvent was slowly poured onto it. The solvent from thimble goes to lower round bottom flask via siphon tube due to the siphoning or siphon cycle. Such 2-3 cycles of solvent were performed and then the drug powder was kept for 12 hrs with solvent for imbibitions. After 12 hours imbibitions, solvent from flask heated to form vapors. Due to heat the solvent from round bottom flask gets converted into its vapors and then these vapors pass .via side tube into the condenser where it gets condensed. This solvent was dripped again on to drug material which was placed in thimble. This process was continued till thimble gets filled with solvent and when level of solvent reaches to siphon tube, pulling of whole solvent into the flask is taken place. All this events repeated several times and drug material was extracted continuously with fresh solvent. This process was performed for 3 days and when siphon solution showed negative test for phytoconstituents, extraction was completed. Then the extracts were filtered in hot condition and distilled on water bath. All the extract was finally dried at low temperature under reduced pressure in a rotary evaporator. Typically, the dried powdered plant material was extracted successively with petroleum ether followed by chloroform, 90% ethanoi and water. In accordance with another embodiment of the present invention there is provided an aqueous extract which is prepared by cold maceration. 100 gm of powered crude drug was taken into 2 liters of conical flask. To this, one liter of solvent was added with vigorous shaking. Then the flask was plugged with cotton plug and aluminum foil and kept aside for 12 hours. After 12 hours flask was again shacked vigorously and then flask was allowed to stand for 7 days with frequent shaking. After completion of 7 days the fluid extract was separated by decantation and filtration. Typically, the filtration was carried out by using vacuum filtration assembly. Typically, the extract is in the form selected from the group consisting of powder, semisolid and liquid. In accordance with the second aspect of the present invention there is provided an Alternanthera bettzichiana extract; said extract comprising: i. steroids; ii. glycosides; i i i. carbohyd rate; and iv. alkaloids. wherein said extract is alcoholic extract with extractive value of about 32% and is useful in the treatment of at least one disease selected from the group consisting of cancer, inflammatory diseases and bacterial infections. In accordance with the third aspect of the present invention there is provided an Alternanthera bettzichiana extract; said extract comprising: i. proteins; ii. glycosides; iii. carbohydrate; and iv. alkaloids. wherein said extract is aqueous extract with extractive value of about 19% and is useful in the treatment of at least one disease selected from the group consisting of cancer, inflammatory diseases and bacterial infections. In accordance with the fourth aspect of the present invention there is provided an Alternanthera bettzichiana extract; said extract comprising: i. steroids; and ii. glycosides. wherein said extract is chloroform extract with extractive value of about 25% and is useful in the treatment of at least one disease selected from the group consisting of cancer, inflammatory diseases and bacterial infections. In accordance with one of the embodiments of the present invention the glycosides comprises cardiac glycoside, anthraquinone glycoside, saponin glycoside and coumarin glycoside. Physicochemical and Phy to-chemical investigation of the extract: a) Total Ash: 1g powdered drug was taken in a tarred silica dish previously dried and weighed. It was ignited in a furnace until free from carbon. The ash obtained was weighed. b) Acid Insoluble ash: To the crucible containing the total ash, 25 ml of dilute hydrochloric acid was added, covered with a watch glass boiled gently for 5 minutes. The insoluble matter was collected on an ash less filter paper, washed with hot water until the filtrate is neutral. It was allowed to cool in a suitable desiccator for 30 minutes, and then weighed without delay (World Health Organization, Geneva, 1998 and Khandelwal K.R., 2001). c) Water-soluble ash: To the crucible containing the total ash, 25ml of water was added and boiled for 5 minutes. The insoluble matter was collected on an ash less filter paper, washed with hot water, and ignited in a crucible for 5 minutes. The weight of this residue was subtracted from the weight of total ash. d) Extractive Value: This is the most useful method for the evaluation of a crude drug, which gives the idea about the active constituents in the given amount of medicinal plant material when extracted with solvents. About 5 g of the powdered drug was weighed in a weighing bottle and transferred to a dry 250 ml conical flask. 100 ml graduated flask was filled to the delivery mark with the solvent. The weighing bottle was washed out and the washings were poured, together with the rest of the solvent into the conical flask. Then the flask was corked with cotton plug and aluminum foils and was set aside for 24 hours with vigorous shaking. After completion of period, it was filtered into a 50 ml measuring cylinder up to mark. 25 ml of this filtrate was transferred to a weighed, thin porcelain dish and evaporated to dryness on a water-bath. The drying was complete in an oven at 100°C followed by cooling in a desiccator and weighing. The percentage w/w of extractive with reference to the air-dried drug was calculated. Ash Value Total ash 14% Acid-insoluble ash 1.6%- Water soluble ash 3.0 % Extractive values Petroleum ether 6.4 % Chloroform 25.6 % Ethano! 32.0 % Water 19.2% The above table depicts the physicochemical characters in which the total ash value was higher than that of acid insoluble and water soluble ash value and a decrease in the acid insoluble ash value may be due to presence of siliceous matters. The ethanolic extractive value was higher than petroleum ether, chloroform and aqueous extractive value revealing the presence of larger amounts of alcohol soluble constituents in the plant material. The yield of petroleum ether extract was dull greenish semisolid mass with a yield of 6.4% w/w, 25.6% w/w chloroform extract in the form of brilliant green-brown semisolid, 32% w/w for ethanolic extract in the form of dark brown semisolid mass, while aqueous extract yield found to be 19.2% w/w in the form of black-brown semisolid mass. A) HPTLC detection of cardiac glycosides: Ethanolic extract (10 mg/ml) and chloroform extract (10 mg/ml) were dissolved in ethanol and chloroform respectively. An appropriate volume30 pi\, 20 \i\, 10 u! and 05 uj for ethanolic extract and 20 μl, 12 μl, 07 μl and 03 ul for chloroform extract were applied on HPTLC plate, developed using Ethyl Acetate- Methanol- Ethanol- Water (16ion. Derivatizing Reagent: Anisaldehyde -.2: 2.2: 0.8: 1.6) as mobile phase and scanned at 254 nm and 366 nm before and after derivatizat Sulphuric acid reagent (0.5 ml Anisaldehyde is mixed with 10 ml Glacial Acetic Acid, followed by 85 ml methanol and 5 ml of concentrated sulphuric acid in that order). After spraying with anisaldehyde-sulphuric acid reagent, the ethanolic extract showed nine well resolved peaks at Rf 0.12, 0.32, 0.41, 0.51, 0.55, 0.68, 0.83, 0.88 and 0.90 (Table 1). Rf values with 0.12, 0.32, 0.83 were found to be the major one's with about 23.51, 26.69 and 31.59 peak areas (Figure 1A), respectively (Tablel). While the chloroform extract showed seven peaks at Rf 0.48, 0.64, 0.69, 0.77, 0.84, 0.87, and 0.92 (Table 2). Rf values with 0.48 and 0.77 were found to be the major one with about 29.85, 21.33 peak areas (Figure IB), respectively (Table 2).Very week fluorescence quenching at 254 nm (Figure la) without chemical treatment, presence of light blue and dark blue fluorescence (Figure lb) in ethanoiic extract and orange, pah brown fluorescence (Figure Ic) in chloroform extract at 366 nm were the confirmatory mark for the presence of cardiac glycosides (Wagner H., et al. 1984) in A. bettzichiana. TabSe 1: HPTLC profile of crude ethanolic extract of A. bettzichiana for detection of cardiac glycosides λmax Peak Start Rf MaxRf EndRf Area % 366 1 0.06 0.12 0.15 23.51 2 0.20 0.32 0.37 26.69 3 0.40 0.41 0.45 2.32 4 0.47 0.51 0.53 2.68 5 0.54 0.55 0.57 1.13 6 0.59 0.68 0.68 5.08 7 0.74 0.83 0.87 31.59 8 0.87 0.88 0.89 4.39 9 0.89 0.90 0.92 2.61 λm&x Peak Start Rf Max Rf End Rf Area % 366 1 0.35 0.48 0.57 29.85 2 0.57 0.64 0.65 12.99 3 0.65 0.69 0.71 11.25 4 0.72 0.77 0.80 21.33 5 0.81 0.84 0.85 12.65 6 0.85 0.87 .0.91 11.71 7 0.91 0.92 0.92 0.23 B) KPTLC detection of Coumarin Glycosides; Ethanolic extract (10 mg/ml) and chloroform extract (10 mg/ml) were dissolved in ethanol and chroform respectively. An appropriate volume30 ul, 20 ul, 10 JJ.1 and 05 ul for ethanolic extract and 20 ul, 12 u.1, 07 ui and 03 u.1 for chloroform extract were applied on HPTLC plate, developed using Toluene- Diethyl Ether (1:1) Saturated with 10 % Acetic acid (Separately prepared 10 % acetic acid solution and toiuene-Diethyl ether (1:1) and both the contents were mixed. The contents were transferred in separating funnel and aqueous layer was discarded. The resulting ether: toluene layer was utilized as mobile phase) as mobile phase and scanned at 254 nm and 366 nm before and after derivatization. Derivatizing Reagent: Vanillin - Phosphoric acid Reagent (Dipping solution): Dissolve 1 gm of Vanillin (4-hydroxy-3-methoxy benzaldehyde) in 25 ml ethanol and add 25 ml water and 35 ml O-phosphoric acid. In HPTLC screening for coumarms, distinct fluorescence quenching at 254 nm (Wagner H., et al. 1984) in ethanolic and chloroform extract (Figure 2a), intense blue fluorescence (may be of simple coumarins), blue fluorescence (may be of furano and pyranocoumarins) in ethanolic extract and brown fluorescence (Wagner H., et al. 1984) in chloroform extract on scanning the plate at 366 nm were observed. (Figure 2b). After development and spraying with vanillin-phosphoric acid reagent, ten spots were observed at Rf 0.11, 0.17, 0.20, 0.24, 0.30, 0.43, 0.56, 0.68, 0.77, 0.89 in chloroform extract (Table 3) and four spots at Rf 0.18, 0.30, 0.32, 0.55 were observed in ethanolic extract (Table 4, Figure 2A) under 366 nm. Rf values with 0.30, 0.56, 0.68 were found to be the major one's with about 17.76, 33.51 and 31.12 peak areas (Figure 2B), respectively in chloroform extract (Table 3). Table 3: HPTLC data for presence of coumarin glycosides in chloroform extract of A. bettzichiana λtnax Peak Start Rf Max Rf End Rf Area % 366 1 0.06 0.11 0.12 5.45 2 0.13 0.17 0.17 1.17 3 0.17 0.20 0.23 2.93 4 0.23 0.24 0.25 0.59 5 0.25 0.30 0.37 17.76 6 0.39 0.43 0.46 1.72 7 0.47 0.56 0.61 33.51 8 0.61 0.68 0.76 31.12 9 0.76 0.77 0.84 3.91 10 0.85 0.89 0.92 1.85 Table 4: HPTLC data for presence of coumarin glycosides in ethanolic extract oA. bettzichiana λmax Peak Start Rf Max Rf End Rf Area % 366 1 0.13 0.18 0.21 23.42 2 0.25 0.30 0.31 30.18 3 0.32 0.32 0.38 25.72 4 0.50 0.55 0.58 20.67 C) HPTLC detection of Steroids: Ethanolic extract (10 mg/ml) and chloroform extract (10 mg/ml) were dissolved in ethanol and chroform respectively. An appropriate volume30 pi, 20 (if, 10 μl and 05 pi for ethanolic extract and 20 μl, 12 μl, 07 pi and 03 μl for chloroform extract were applied on HPTLC plate, developed using Toluene- Ethyl Acetate- Formic acid (10: 03: 0.5). The chromatogram was evaluated densitometrically using win CAT software and the steroids were traced after treatment with dertvatizing reagent. Derivatizing Reagent: 10 % Sulphuric acid In present HPTLC screening for steroids, distinct fluorescence at 254 nm (Figure 3a), blue and violet fluorescence (Figure 3b) at 366 nm before derivatization were observed. The ethanolic extract shows blue and slightly green colored bands (Figure 3c) at 366 nm after derivatization (D.Pradhan, et ai 2009). The HPTLC chromatogram of chloroform extract and ethanolic extract showed the presence of seven components at Rf values 0.06, 0.08, 0.19, 0.39, 0.53, 0.65, 0.84 (Table 5) and 0.02, 0.08, 0.19, 0.44, 0.53, 0.81, 0.83 (Table 6) respectively after development and spraying with 10% sulphuric acid, indicating the confirmation of steroidal components in A. bettzichiana. Rf values with 0.39, 0.53, 084 in chloroform extract (Table 5) and 0.02, 0.53, 0.81, 0.83 in ethanolic extract (Table 6) were found to be major one's with about their respective peak areas (Figure 3B and 3A). Table 5: HPTLC profile for detection of steroids in chloroform extract of A, bettzichiana λmax Peak Start Rf Max Rf End Rf Area % 366 1 0.03 0.06 0.07 3.69 2 0.07 0.08 0.10 4.52 3 0.16 0.19 0.24 3.77 4 0.34 0.39 0.46 20.42 5 0.46 0.53 0.57 14.94 6 0.58 0.65 0.68 10.05 7 0.74 0.84 0.91 42.60 Table 6: HPTLC profile for detection of steroids in ethanolic extract of A. bettzichiana Xrnax Peak Start Rf MaxRf EndRf Area % 366 i—i 0.00 0.02 0.03 44.86 2 0.07 0.08 0.09 2.23 3 0.16 0.19 0.20 4.37 4 0.40 0.44 0.48 7.86 5 0.49 0.53 0.60 13.77 6 0.74 0.81 0.81 11.33 7 0.82 0.83 0.91 15.55 D) HPTLC detection of Alkaloids: 01 g of powdered drug was moistened with about 1 ml 10 % ammonia solution. To it 5 ml of methanol was added and the drug was extracted for 10 min on a water bath. 20 ul, 15 ul, 10 ul and 05 u! of above solvent was applied on HPTLC plate, developed using Toluene-Ethyl acetate- Diethylamine (7:2:1) as mobile phase and scanned at 254 nm and 366 nm before and after derivatization. Derivatizing Reagent: Dragendorffs reagent. {Preparation of Dragendorffs reagent: Solution A- 0.85 g of basic bismuth nitrate was dissolved in 10 mi glacial acetic acid and 40 mi water with heating. Solution B- 08 g of potassium iodide was dissolved separately in 30 ml of water. Solution A and B were mixed in the ratio of 1 :1 and labeled as stock solution. For spraying purpose 1 ml of stock solution is mixed with 2 ml glacial acetic acid and 10 ml water.) During HPTLC screening for alkaloids blue and violet fluorescence was observed at UV 366 nm (Figure 4B) before derivatization (Wagner H., et al. 1984) and the fluorescence intensity increases with increase in concentration. The HPTLC finger print at 366 nm showed the presence of two compounds with Rf 0.28 and 0.84 (Table 7; Figure 4A) after derivatization with Dragendorffs reagent. Table 7: HPTLC profile for detection of alkaloids in A. bettzichiana λmax Peak Start Rf Max Rf End Rf Area % 366 1 0.26 0.28 0.30 17.76 2 0.80 0.84 0.90 82.54 E) HPTLC detection of Proteins: Hydrolysis of protein a. 50 mg of aqueous extract was weighed in to a 10 mi ampoule, to this 8 ml of 6N HCL was added followed by degassing the ampoule with nitrogen and sealing; the sealed ampoule was stored in an oven at 110°C for 16 h; the content of ampoule was transferred in to beaker containing barium hydroxide followed by manually shaking for 10 min and filtering using Whatmann filter paper; and the filterate was evaporated to dryness. b. The residue was transferred to 25 ml volumetric flask containing 10 ml of 10 % isopropanol and volume was adjusted to the mark using same solvent. The same solution was used for chromatographic separation. a) 25 ul, 20 ul, 15 ul and 10 ul of above filtrate was applied on HPTLC plate, developed using Toluene: n-butanol-acetic acid-water (3:1:1) as mobile phase and scanned at 254 nm, 366 nm and 530 nm before and after derivatization. Derivatizing Reagent: Ninhydrin reagent (5% ninhydrin, 5% acetic acid, and 1% cadmium acetate in 96% ethanol) (Bernard Fried, et al. 1996) In HPTLC screening for proteins light violet, yellow and intense violet colored bands were observed after spraying the plate with ninhydrin reagent in visible light (Figure 5A). After derivatization the plate was scanned at 254 nm, 366 nm, and 530 nm. Seven spots with Rf 0.29, 0.33, 0.47, 0.64, 0.76, 0.84, 0.91, were observed under 254 nm. Only one compound with Rf 0.69, was observed under 366 nm. While six spots with Rf 0.20, 0.30, 0.35, 0.48, 0.64, 0.76 were observed under 530 nm (Table 8a). Rf values with 0.64, 0.76 at 254 nm (Figure 5B), 0.69 at 366 nm (Figure 5C) and 0.30, 0.35, 0.48 at 530 nm (Figure 5D) were found to major one's with about their respective peak areas. Table 8a: HPTLC data for presence of proteins in A. bettzichiana λmax Peak Start Rf Max Rf End Rf Area % 254 1 0.26 0.29 0.31 6.64 2 0.32 0.33 0.36 2.85 3 0.43 • 0.47 0.50 7.94 4 0.56 0.64 0.72 50.15 5 0.73 0.76 0.82 23.03 6 0.82 0.84 0.87 5.32 7 0.88 0.91 0.94 4.07 366 1 0.61 0.69 0.77 100.00 530 1 0.11 0.20 0.25 15.85 2 0.25 0.30 0.32 18.51 3 0.33 0.35 0.42 26.44 4 0.43 0.48 0.54 18.29 5 0.59 0.64 0.72 15.62 6 0.75 0.76 0.87 5.29 F: HPTLC detection of Carbohydrates: 05 g of powdered plant material was boiled with 25 ml hot 80% alcohol with stirring. Then the solution was decanted in to a suitable flask, and the solids were again re-extracted with 80% alcohol. Then the mixture was transferred to the flask, and allowed it to cool. The combined extracts were filtered and evaporated until alcohol odor disappears. The resulting extract was filtered through a thin mat of Celite in a Buchner funnel. Then the Celite layer was washed with small amount of distilled water. The washings and filtrate were diluted to 50 ml with distilled water. Then the purification was done with the addition of lead acetate to the solution, till formation of precipitate takes place. Shake the contents and allow it to stand for few minutes. Filter the contents in a beaker containing an excess amount of sodium acetate crystals. Formation of precipitate takes place, which was then filtered and washed with small amount of distilled water. The filtrate and washings were finally diluted up to 50 ml with distilled water. A precoated HPTLC plate was soaked in 0.3NNaH2PO4 solution for one minute. Immediately removed after one minute and dried at 100°C for 30 min. 25 ul, 20 ul, 15 ul and 10 ul of above filtrate was applied on HPTLC plate, developed using Toluene: n-butanol-acetone- 0.3N.NaH2PO4 (4:5:1) as mobile phase and scanned at 254 nm, 366 nm and 530 nm before and after derivatization. Derivatizing Reagent: ADP reagent (2 ml aniline + 2 g diphenylamine + 10 ml H3P04 + 88 ml methanol), The HPTLC screening for confirmation of sugars in A. bettzichiana revealed presence of fluorescence at 254 nm (Figure 6a) and light blue, blue and violet colored fluorescence at 366 nm (Figure 6b) before derivatization. The fluorescence quenching was increased with increase in concentration. After derivatization, a clear separation of light brown to dark brown colored bands was observed on scanning the plate in visible light (Figure 6d). Five spots with Rf 0.41, 0.52, 0.63, 0.67, 0.83 under 254 nm (Figure 6A), 0.04, 0.50, 0.62, 0.77, 0.88 under 366 nm (Figure 6B) and eleven spots with Rf 0.06, 0.10, 0.18, 0.33, Q.40, 0.57, 0.66, 0.70, 0.73, 0.81, 0.85 were observed under 530 nm (Table 8b, Figure 6C) after derivatization. Rf values with 0.67, 0.83 at 254 nm, 0.04, 0.50, 0.62 at 366 nm and 0.33, 0.57 at 530 nm were found to major one's with about their respective peak areas (Table 8b). Table 8b: HPTLC data for presence of sugars in A. bettzichiana λmax Peak Start Rf Max Rf End Rf Area % 254 1 0.39 0.41 0.46 5.55 2 049 0.52 0.54 4.14 3 0.60 0.63 0.65 9.53 1 ■7 4 0.65 0.67 0.70 19.05 5 0.72 0.83 0.88 61.74 366 1 0.02 0.04 0.06 19.69 2 0.42 0.50 0.53 27.57 3 0.57 0.62 0.64 29.69 4 0.76 0.77 0;82 11.66 5 0.84 0.88 0.94 11.40 530 1 0.05 0.06 0.07 1.54 2 0.07 0.10 0.11 4.98 3 0.13 0.18 0.22 8.39 4 0.26 0.33 0.37 12.45 5 0.37 0.40 0.40 1.10 6 0.48 0.57 0.64 66.95 7 0.64 0.66 0.69 2.15 8 0.69 0.70 0.71 0.56 9 0.73 0.73 0.76 0.65 10 0.79 0.81 0.82 0.66 11 0.84 0.85 0.87 0.56 In accordance with fifth aspect of the present invention there is provided a medicament for the treatment of cancer comprising administering in said subject an effective amount of Alternanthera beltzichiana extract. In accordance with sixth aspect of the present invention there is provided a medicament for the treatment of at least one disease selected from the group consisting of bacterial infections and inflammatory disease comprising administering in said subject an effective amount of Alternanthera beltzichiana extract. Following examples illustrate the invention, but are not intended to limit the scope of the present invention. Example 1: Antibacterial Studies: The antimicrobial screening of various extracts of A. beltzichiana was performed by using agar disc diffusion method and was tested against gram positive bacteria viz. Bacillus subtilis, Staphylococcus aureus and gram negative strains of E. coli and Salmonella typhi. Table 9 depicts the results of antimicrobial activity of the various extracts of A. beltzichiana. The results of zone of inhibition revealed that the petroleum ether, chloroform, ethanol and aqueous extract posses antimicrobial activity in a concentration dependant manner against the test organisms and was also comparable with the standard drugs streptomycin (10 ug/ml) included in the study. As shown in table 9, the extracts of A. beltzichiana showed antimicrobial activity with the diameter of zone of inhibition (Figure 4.6.1) ranging from 4.9 mm to 13.8 mm. The petroleum ether extract exhibited zone of inhibition (mm) ranging between 7.0 ± 0.6 to 9.5 ± 0.9 against the tested organisms. The chloroform extract exhibited zone of inhibition (mm) ranging between 7.0 ± 0.7 to 11.0 ± 1.0 against the tested organisms. The ethanolic extract showed zone of inhibition (mm) ranging between 8.0 ± 0.7 to 13.8 • 0.9 and the aqueous extract revealed 4.9 ± 0.5 to 7.4 ± 0.6 mm zone of inhibition against the tested organisms. No zone of inhibition was observed in E. coli with aqueous extract. Ethanolic extract of A. bettzichiana exhibited significant antibacterial activity. In case of all these extracts MIC 80 to 120 jig/ml was observed against the test organisms and was determined by using broth dilution method. Table 9: Antibacterial activity of various extracts of/4, bettzichiana Type of Extract Cone. ug/ml Zone of inhibition (mm) Bacteria Bacillus subtillis S. aureus E. coli S. typhi Petroleum ether extract 100 300 500 7.5 ± 0.6 8.7 ±0.7 9.0 ±0.6 . 7.0 ±0.6 8.2 ±0.8 9.5 ± 0.9 7.5 ±0.5 7.9 ±0.6 8.0 ± 0.7 7.9 ±0.6 8.0 ±0.8 8.4 ±0.8 Chloroform extract 100 300 500 9.0 ±0.8 10.4 ± 0.9 11.0 db 1.0 7.0 ± 0.7 7.8 ± 0.6 10.5 ±0.9 7.5 ± 0.6 8.2 ±0.8 8.5 ±0.7 8.5 ±0.7 8.9 ±0.9 9.5 ± 0.9 Ethanolic extract 100 300 500 11.0±1.1 12.0 ±1.0 13.8 ±0.9 9.5 ± 0.8 10.6 ±0.9 11.9± 1.1 8.0 ± 0.7 9.0 ±0.8 9.7 ±0.9 10.5 ±1.0 11.5±l.l 12.0 ±1.1 Aqueous extract 100 300 500 5.5 ±0.5 6.3 ±0.5 7.4 ±0.6 5.1 ±0.4 6.0 ± 0.6 7.0 ± 0.7 ~ 4.9 ±0.5 5.3 ±0.5 6.0 ±0.6 ■ Standard streptomycin 10 15.0 ±1.2 14.5 ±1.3 15.0 ±1.3 15.0± 1.4 Example 2: Anti-inflammatory activity of A. bettzichiana extracts: A) Animals utilized for Anti-inflammatory Studies Albino wistar rat within weight range of 200-250 g were used for the anti-inflammatory studies. They were housed in polyethylene cages under standard conditions of 12 h light and dark cycle at 25 ± 2°C. Animals were supplemented with standard pelletized feed (Amrut Rat Feed, Pune, India) and water was made available ad libitum. The ethical clearance was approved by IAEC of R.C. Pate! Institute of Pharmaceutical Education and Research, Shirpur (M.S.) with resolution number RCCOP/IAEC/2007-08/42. Anti-inflammatory activity of the ethanolic extract of A. bettzichiana was evaluated using acute and chronic inflammatory models. Acute activity was evaluated by Carrageenan induced rat paw edema method (Winter C.A., et al. 1962) and the chronic activity was evaluated by cotton pellet granuloma model (D, Arcy P.F., et al. 1960). I) Carrageenan Induced Paw Edema Model: Wistar albino rats were randomly divided in five groups (n=6) and 12 h prior to the experiments, the animals were deprived of food and water was made available during experiments. The first group (negative control) received normal saline (0.9% Nacl), second group (positive control) received 10 mg/kg p.o., diclofenac sodium and rest of the three groups received ethanolic extract of A. bettzichiana at the dose of 100 mg/kg, 200 mg/kg, and 400 mg/kg p.o., concentrations. In animals of all the groups acute inflammation was produced by sub plantar injection of 0.1 ml of 1% Carrageenan (A.4) in normal saline in right hind paw of rat. Animals were pre-medicated with drug/extracts 1 h before Carrageenan injection. Paw volumes (in ml) was measured by dislocation of the water Column in a plethysmometer (Ugo Basile 7140, Italy) immediately after Carrageenan application at 1, 2, and 3 h intervals after the stimulus (Winter C.A., et al. 1962). The volume of paw recorded just before Carrageenan injection was recorded as initial volume (Vo) in each case. The mean percent rise in the paw volume was determined by the following formula (Chandragouda R. Patil, et al. 2009) Where Vt = Volume at different intervals after Carrageenan injection Different doses of ethanolic extract of A. bettzichiana showed dose dependant significant antiinflammatory activity. The dose concentration of 100 mg/kg did not exhibit significant activity in the early phase of I h (Table 10). But in the later phase (2 h and 3 h), the dose of 200 mg/kg and 400 mg/kg of ethanolic extract of A. bettzichiana significantly decreased rise in paw volume as compared to control (Table 11). It was observed that the dose 200 mg/kg and 400 mg/kg inhibited increase of paw volume (1.1 ± 0.096 and 1.1 ± 0.031 respectively) as compared with control (1.3 ± 0.021). The diclofenac sodium (Standard drug) also exhibited significant inhibition in rise of paw volume (Table 10). The test drug showed 52%, 39%, and 32% inhibition in rise of paw edema at the doses of 100, 200, and 400 mg/kg respectively at the end of 3 hrs. The percent rise of paw volume of diclofenac sodium, a reference standard drug, was found to be 23% (Table 11). The Carrageenan inflammation is a useful model to detect oral action of anti-inflammatory agents (Di Rosa M., et al. 1971). The development of Carrageenan induced edema is biphasic, the early phase (1-2 h) is mainly mediated by histamine, serotonin and prostaglandins. After which increased vascular permeability is maintained by the release of kinins up to 2.30 hrs. The later phase (from 2.30 up to 6 hsr.) is sustained by prostaglandin release and mediated by bradykinin, leukotriens, polymorphonuclear cells and prostaglandins produced by tissue macrophages (Britto A.R.M.S., et al. 1998). The later phase is reported to be sensitive to most of the clinically effective antiinflammatory agents (Smucker E., et al. 1967). The ethanolic extract of A. bettzichiana revealed significant acute anti-inflammatory activity in early phase (Figure 7A and 7B) as well as later phase (Figure 7C). Table 10: Effect of different doses of ethanolic extract and diclofenac sodium as reference standard on Carrageenan induced rat paw edema. Group Dose (mg/kg) Mear i increase in paw volume l hr. 2 hrs. 3 hrs. Negative Control — 0.96 ±0.012 1.1 ±0.017 1.3 ±0.021 Diclofenac Sodium 10 0.92 ±0.013 1.0± 0.017 1.0 ±0.046 Ethanolic Extract 100 0.96 ±0.016 1.1 ±0.019 1.3 ±0.020 Ethanolic Extract 200 0.95 ±0.019 1.1 ±0.020 1.1 ±0.016* Ethanolic Extract 400 0.96 ±0.020 1.1 ±0.022 1.1 ±0.031 P Table 11: Effect of different doses of ethanolic extract on % rise in paw volume of Carrageenan induced rat paw edema. Group Dose (mg/kg) Percent Rise in Paw Edema 1 hr. 2 hrs. 3 hrs. Negative Control — 20 ±0.84 38 ±2.3 60 ± 1.2 Diclofenac Sodium 10 11 ± 0.35.* 22 ±0.41 23 ±3.8 Ethanolic Extract 100 17 ±0.50* 30 ±0.53 52 »1.6 Ethanolic Extract 200 15 ±0.28 27 ±0.23 39 ±2.8** ■Ethanolic Extract 400 13 0.30* 24 ±0.31 32 ±4.6 *P II) Cotton Pellet Induced Granuloma Model: Chronic or proliferative phase of inflammation was assessed by cotton pellet granuloma in rat (D' Arcy, et at I960). The model was based on the foreign body granuloma formed in rats by subcutaneous implantation of compressed cotton pellets. After several days, histologically giant cells and undifferentiated connective tissue can be measured by weighing the dried pellets after removal. The cotton pellets weighing exactly 15 ± 1 mg each, were made from cotton rolls. Then the pellets were sterilized in an autoclave for 30 min at 121°C and 15 lb pressure. Then the pellets were soaked in distilled water containing penicillin and streptomycin and implanted subcutaneously in to both sides of the groin region of each rat under light ether anesthesia, after shaving the fur of the animals. In this model, the animals were divided in to five groups as described in Carrageenan induced rat paw edema model. The drug and extracts were administered orally to the respective group of animals for seven consecutive days, from the day of cotton pellet implantation. On the 8lh day, the animals were anaesthetized again; the cotton pellets were removed surgically and made free from extragenous tissues. The pellets were incubated at 37°C for 24 h and dried at 60°C to constant weight. The weight of the cotton pellet before implantation was subtracted from the weight of the dried granuloma pellets. The increase in dry weight of the pellets was taken as a measure of granuloma formation. The cotton pellet granuloma bioassay is considered a chronic inflammation study model for studies on chronic inflammation and considered as a typical feature of chronic inflammatory reaction (Spector W.G., 1969). During the study with reference to chronic model, pellet caused a significant increase in granuloma weight in control group. The study of ethanolic extract of A. beiizichiana on chronic and proliferative phase of inflammation indicated that ethanolic extract (200 mg/kg and 400 mg/kg, p.o.) slightly but significantly (P The cotton pellet method is widely used to evaluate the transudative and proliferative components of the chronic inflammation. In this model, inflammation and, granuloma develops during the period of several days (D. Sheshadrishekar, et al 2009). Inflammation involves proliferation of macrophages, neutrophils and fibroblasts, which are basic sources of granuloma formation (Figure 8C). The wet weight of the cotton pellets correlates with the transuda; the dry weight of the pellets correlates with the amount of granulomatous tissue (Olajide O.A., et ah, 2000). Hence, the degree in the weight of granuloma indicates that the proliferative phase was effectively suppressed by the ethanolic extract of A. bettzichiana. The present anti-inflammatory protocol showed that the ethanolic extract of A. bettzichiana elicited a significant anti-inflammatory activity in Carrageenan induced paw edema (acute) and cotton pellet granuloma (chronic) rat models. This effect may be due to presence of secondary metabolites, probable coumarins, steroids and alkaloids present in the plant. This study indicates that the ethanolic extract of A. bettzichiana possess significant anti-inflammatory activity. Table 12: Effect of different doses of ethanolic extract of A, bettzichiana on % inhibition of Cotton Pellet Induced Granuloma. Treatment Groups Dose (mg/kg) Mean increase % Inhibition Control — 75 ± 2.5 — Diclofenac Sodium 10 42±1 8 45.70 Ethanolic Extract 100 71 ± 1.2 6.34 Ethanolic Extract 200 62 ± 2.7* 17.37 Ethanolic Extract 400 56 ±1.6** 26.35 P Example 3: Anti-tumour activity of EEWPM and EEMPM of A. bettzichiana'. A) Animals used for Anti-tumour Studies: The anti-tumour activity was performed with 6-7 week old Fj hybrid Swiss Albino mice. Animals with both sex and between weighing range of 27 ± 3 gm were utilized for the study. Animal care and research protocol were in accordance with the principles and guidelines of CPCSEA norms and approved by ethical committee (Registration No.500/la/CPCSEA72002) of Jawaharlal Nehru cancer Hospital and Research, Bhopai (MP.) with resolution number 500/1 a/CPCSEA/2002. B) Division of Animals in Different Groups for in yivo Anti-tumour Studies: The animals were divided in the following groups and in each animal the melanoma cell line (B16F10) was injected subcutaneously (S.C.) Table 13: Types of groups Sr. No. Group Treatment No. of animals/ group 1. Control Group Tumour only 04 2. Test Group A1 Tumour + EEWPM (250 mg/kg) 04 Test Group A2 Tumour + EEWPM (500 mg/kg) 04 4. Test Group A3 Tumour + EEWPM (750 mg/kg) 04 5. Test Group B1 Tumour + EEMPM (250 mg/kg) 04 6. Test Group B2 Tumour + EEMPM (500 mg/kg) 04 7. Test Group B3 Tumour + EEMPM (750 mg/kg) 04 8. Radiation Group Tumour + Radiation (4 Gy) 04 Where £EWPM= ethanolic extract of wild plant material of A. bettzichiana and EEMPM= ethanolic extract of micro-propagated plant material of A. bettzichiana. B16F10 melanoma cell line is a highly metastatic melanoma cell line (G.Kuttan, et ai 2005) which gives rise to a tumour nodule in most mouse lines. Ex-vivo melanoma tumour was -induced by B16F10 melanoma cell lines. Five lakh cells were injected subcutaneously and after 8-10 days tumour started growing- When the tumour was palpable, the tumour volume was measured at a given interval of days. When the tumour was observed as palpable, the tumour volume measurement was started from the 5th day and calculated the mean of every 5th day till 25th day. During first few days of drug treatment (5th-l0lh days) slow and steady tumour growth was observed in all groups (Figure 9 A). During 1 l'h-15th day tumour volume was increased rapidly in all the cases (Figure 9B). No tumour regression was noticed in test as well as radiation group. But from 15th-20th days and onward, a drastic regression in tumour size was noticed in test groups as compared to radiation group (Figure 9C). On the other hand, on the 25th day, the tumour volume in control group was doubled by 10 folds (Figure 9D). From the observed values it was cleared that, both the extract revealed statistically significant (P The most remarkable feature observed during in vivo antitumour activity was ethanolic extract of micropropagated plant material (EEMPM) of A. bettzichiana revealed significant dose dependant regression in tumour volume between 16th-25th day (Figure 9C and Figure 9D) of studies as compared to ethanolic extract of wild plant material (EEWPM). From the overall results, it was well deared that A bettzichiana has shown the tumour inhibition potential. C] In vitro Anti-tumour Activity: I) Cell Culture: The B16F10 melanoma cell lines were provided by Department of Research, Jawaharlal Nehru Cancer Hospital and Research, Bhopal (M.P.). Cells were cultured in 5 ml Eagle's minimum essential media (EMBM) in Petri plates containing NaHCO3 (220 mg/100 ml). Media containing NaHCO3 was supplemented with 10% Fetal Calf Serum (FES) and streptomycin plus penicillin (100 μg/ml and 100 IU/ml, respectively). Cells were cultured in a 5% CO2 humidified atmosphere at 37°C until near confluence. A confluent monolayer was detached with 0.25% trypsin in PBS (pH 7.4, 0.01 M) and suspension was utilized for subculturing. Serially cultured cell lines at third stages were utilized for in vitro antitumour activity (Rodrigueg J., 2002). All the processes were carried out strictly under aseptic condition in a vertical laminar flow chamber. The in vitro screening was performed by inoculating l x l05 cells/ml of suspension in culture media and incubated for 3 h at 37°C in 5% CO2. After 3 h, the cells were exposed to different concentrations of EEWPM and EEMPM of A. bettzichiana. Another set of parallel cultures was also run throughout the study. Duplicate plates of each culture were incubated for further 24 h. After incubation for 24 h at 37°C, the cells were trypsinized (0.25% in PBS) and then centrifuged at 1000 rpm for 5 min. Supernant was discarded and the cells were re-suspended in PBS to make protein free suspension. Then the cell viability was measured by trypan blue exclusion test and counted using neuber's WBC counting chamber. II) Drug Treatment: Ethanolic extract of wild plant material (EEWPM) and micropropagated plant material (EEMPM) of A. bettzichiana were prepared at a concentration of 250 mg/ml, 500 mg/ml, and 750 mg/ml in DDW. The drug extracts were treated with plates containing B16F10 melanoma cell lines. While a plate containing B16F10 melanoma cell lines only was maintained as control during the study. III) Trypan Blue Exclusion Test: The percentage of viable and non-viable cells was determined by using trypan blue exclusion test (James Kumi-Diaka, et al. 1999). It is based on the principle that live cells posses intact cell membrane that exclude certain dyes such as trypan blue, eosin or propidium. Cell growth and was measured by adding I part of 0.4% trypan blue with 1 part of cell suspension. The mixture was then allowed to incubate in room temperature for 3. min. and cells were immediately counted using hemocytometer. Percent cell viability was calculated by the formula: Number of viable cells (Unstained cells) Cell Viability= X 100 Total number of cells (Stained and unstained cells) The in vitro cytotoxic effect of different concentrations of ethanolic extract of wild plant material (EEWPM) and ethanolic extract of micropropagated plant material (EEMPM) of A bettzichiana species on cell viability was determined by using B16FI0 melanoma cell lines. Treatment of extracts against B16FI0 melanoma cell lines, at all concentrations showed decrease in percent cell viability as compared to that control when examined by trypan blue exclusion test. In overall variations of test samples, 750 mg/ml concentration of both types of extracts (EEWPM and EEMPM) showed its best activity (Figure 10A). Both the extracts revealed significant dose dependant activity with decrease in the total number of viable cells with increasing number non-viable cells (Table 15). Below 45% of ceil viability at 750 mg/ml concentration as compared with control as 100% was observed in case of both the extracts (Figure 10B). Table 14: Effect of different doses of ethanolic extract (Wild and Micropropagated plant) on tumour volume for anti-tumour studies Groups Tumour vo j————™ lume(cm ) 5th-l0th,Day 11th-15th Day 16,th-20thDay 2th-25th Day Control 0.38 ±0.01 0.87 ±0.018 2 ± 0.0085 3.7 ±0.029 Radiation 0.23 ±0.01 0.79 ± 0.01 0.9 ± 0.036 • 1.2 ±0.029 EEWPM 250 mg/kg 0.015 ±0.00041 0.074 ± 0.00041 0.29 ± 0.00063 0.36 ± 0.00075 EEWPM 500 mg/kg 0.01 ±0.00065 0.053 ± 0.00065 0.091 ±0.00063 0.83 ± 0.0063 EEWPIM 750 mg/kg 0.015 ±0.00065 0.072 ± 0.00065 0.033 ± 0.00065 0.025 ± 0.00063 EEMPM 250 mg/kg 0.016 ±0.00085 0.11 ±0.00085 0.3 ± 0.00087 0.55 ±0.0022 EEMPM 500 mg/kg 0.014 ±0.00063 0.12 ±0.00063 0.23.± 0.00065 0.085 ±0.0019 EEMPM 750 mg/kg 0.014±0.014 0.074 ±0.0013 0.062 ±0.001 0.014 ±0.00048 Where EEWPM= ethanolic extract of wild plant material of A. bettzichiana and EEMPM= ethanolic extract of micropropagated plant material of A. bettzichiana Values are expressed as Mean ± SEM. Statistical analysis was performed by one way ANOVA followed by Bonferroni's multiple comparison test. P Table 15: In vitro cytotoxic effect of different concentration of A. bettzichiana extracts on B16F10 cell lines. Different Cone, of Extract Total No. of viable cells Total No. of non-viable cells Control 125000 — EEWPM 250 mg 108750 16250 EEWPM 500 mg 80500 46250 EEWPM 750 mg 53000 71250 EEWPM 250 mg 112500 18000 EEWPM 500 mg 80250 43000 EEWPM 750 mg 54250 70750 TECHNICAL ADVANCEMENT AND ECONOMIC SIGNIFICANCE: o The present invention provides physicochemical, phyto-chemical and pharmacological evaluation of the plant Alternanthera betlzichiana. o Further, the present invention provides novel Alternanthera betlzichiana extracts characterized by the presence of steroids; glycosides; proteins; carbohydrate; and alkaloids. o Still further, the present invention provides simple, high yielding and economic processes for preparing the Alternanthera bettzichiana extract. o Furthermore, the present invention provides Alternanthera bettzichiana extracts which exhibit significant anti-bacterial, anti-inflammatory and anti-cancer activity. While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the invention. These and other changes in the preferred embodiment of the invention will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation. I CLAIM: 1. A process for the preparation of Alternanthera bettzichiana extract; said process comprising the following steps: a. selecting the Alternanthera bettzichiana plant material followed by washing and cleaning; b. drying the cleaned Alternanthera bettzichiana plant material; c. grinding the dried material to obtain powdered material followed by passing the powdered material through a sieve to obtain the Alternanthera bettzichiana powdered material ready for extraction; and d. subjecting the material to solvent extraction at a temperature of about 20 to 60 °C to obtain the Alternanthera bettzichiana extract. wherein said solvent extraction comprises extraction with at least one solvent or successive extraction or cold maceration. 2. The process as claimed in claim 1, wherein the successive solvent extraction comprises extraction of the plant material using at least one solvent having low polarity followed by extraction with at least one solvent having high polarity. 3. The process as claimed in claim 1, wherein the solvent used for extraction is at least one selected from the group consisting of water, petroleum ether, chloroform, methanol and ethanol. 4. The process as claimed in claim 1, wherein the solvent extraction further comprises i) filtering the extract in hot condition; it) distilling on water bath; and iii) drying at low temperature under reduced pressure in a evaporator. 5. The process as claimed in claim 1, wherein the cold maceration comprises i) adding a solvent to the flask containing plant material under vigorous shaking; ii) plugging the flask with cotton plug and aluminium foil followed by keeping the flask aside for 12 hours; iii) shaking the flask vigorously and allowing to stand the flask for 7 days with frequent shaking; and iv) separating the fluid extract by decantation and filtration. 6. Alternanthera bettzichiana extract; said extract comprising: i. steroids; ii. glycosides; iii. carbohydrate; and iv. alkaloids. wherein said extract is alcoholic extract with extractive value of about 32% and is useful in the treatment of at least one disease selected from the group consisting of cancer, inflammatory diseases and bacterial infections. 7. Alternanthera bettzichiana extract; said extract comprising: i. proteins; ii. glycosides; iii. carbohydrate; and iv. alkaloids. wherein said extract is aqueous extract with extractive value of about 19% and is useful in the treatment of at least one disease .selected from the group consisting of cancer, inflammatory diseases and bacterial infections. 8. Alternanthera bettzichiana extract; said extract comprising: i. steroids; and ii. glycosides. wherein said extract is chloroform extract with extractive value of about 25% and is useful in the treatment of at least one disease selected from the group consisting of cancer, inflammatory diseases and bacterial infections. 9. The Alternanthera bettzichiana extract as claimed in claim 6,7 and 8 wherein the glycosides comprises cardiac glycoside, anthraquinone glycoside, saponin glycoside and coumarin glycoside. 10. A medicament for the treatment of at least one disease selected from the group consisting of cancer, inflammatory diseases and bacterial infections comprising administering in said subject an effective amount of Alternanthera bettzichiana extract.

Full Text

FORM-2
THE PATENTS ACT, 1970 (39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See Section 10; rule 13)
ALTERNANTHERA 'BETTZICHJANA EXTRACTS AND PROCESSES FOR PREPARING THE SAME
HUNDIWALE JOGENDRA CHANDRAKANT
an Indian National
of House No. 1167, Hundiwale Lane, Chopda, Dist. Jalgaon- 425107; Maharashtra, India
THE FOLLOWING SPECIFICATION DESCRIBES THE NATURE OF THE INVENTION AND THE MANNER IN WHICH IT JS TO BE PERFORMED

FIELD OF THE INVENTION
The present invention relates to Alternanthera bettzichiana extracts and processes for preparing the same.
BACKGROUND OF THE INVENTION
AL TERNANTHERA BETTZICHIANA (REGEL)
Phylum - Magnoliophyta
Class - Magnoliopsida
Order - Caryophyllales
Family - Amaranthaceae
Genus - Alternanthera
There are about eighty varieties of genus Alternanthera Spread among tropical and subtropical regions of the world. The Ayurveda has already recognized Alternanthera pungens, Alternanthera sessilis, and Alternanthera philoxeroides as landmarks. About seven to eight species from this genus are located in Indian soil. Provided herein below are the species of the genus Alternanthera found in india: (Geeta C, et at. 1994.)
1. Alternanthera sessilis (L.) R. Br. ex DC.
2. Alternanthera pungens Kunth in H.B. & K.
3. Alternanthera paronychioides St. Hil.
4. Alternanthera philoxeroides (Mart.) Gris.
5. Alternanthera tenella colla
6. Alternanthera bettzichiana (Regel) Nicolson
7. Alternanthera caracasana Kunth in H.B. & K.
Most of the species from the genus Alternanthera are considered as weeds and various species are utilized for the ornamental purpose. According to Janusz Lipecki, "A weed is defined to be a plant whose virtues have not been yet discovered and a plant in wrong place." This reflects negative nature of weeds (Janusz Lipecki, 2006). However, weeds also sometimes play positive role as natural components of the environment, for example Jimson weed {Datura stramonium).
A. bettzichiana, a horticulture species, of many forms, is often found as an escape (Geeta C, et al.1994), reported as wild edible material (Arinathan V., et al. 2007) and as adsorbent for removal of Cr (VI) (Patil A.K., et al.2009).

In order to make safe and sure use of the medicines, a necessary first step is the establishment of standards of safety and efficacy. Keeping this fact into consideration, in the present invention attempts are made to establish Pharmacognostical and pharmacological standards of the plant Altemanthera bettzichiana (Regel) Nicols which is a less exploited weed.
OBJECTS OF THE INVENTION
It is an object of the invention to provide Altemanthera bettzichiana extracts.
It is another object of the present invention to provide processes for preparing the Altemanthera bettzichiana extract.
It is still another object of the present invention to provide processes for preparing the Altemanthera bettzichiana extract which are simple, high yielding and economic.
It is yet another object of the present invention to perform physicochemical, phyto-chemical and pharmacological evaluation of Altemanthera bettzichiana.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 illustrates HPTLC detection of cardiac glycosides wherein
1A is HPTLC chromatogram of ethanolic extract for cardiac glycosides;
IB is HPTLC chromatogram of chloroform extract for cardiac glycosides;
la is scanned HPTLC plate at 254 nm (before derivatization) for detection of cardiac
glycosides;
lb is scanned HPTLC plate at 366 nm (before derivatization) for detection of cardiac
glycosides; and
lc is scanned HPTLC plate at 366 nm(after derivatization) for detection of cardiac glycosides. Fig. 2 illustrates HPTLC detection of coumarin glycosides wherein
2A is HPTLC chromatogram of ethanolic extract for coumarin glycosides;
2 B is HPTLC chromatogram of chloroform extract for coumarin glycosides;
2a is scanned HPTLC plate at 254 nm (before derivatization) for detection of coumarin
glycosides;
2b is scanned HPTLC plate at 366 nm (before derivatization) for detection of coumarin
glycosides; and
2c is scanned HPTLC plate after derivatization in visible light for detection of coumarin
glycosides.

Fig. 3 illustrates HPTLC detection of steroids wherein
3A is HPTLC chromatogram of ethanolic extract for steroids;
3B is HPTLC chromatogram of chloroform extract for steroids.
3a is scanned HPTLC plate at 254 nm (before derivatization) for detection of steroids;
3b is scanned HPTLC plate at 366 nm (before derivatization) for detection of steroids;
3c is scanned HPTLC plate at 366 nm (after derivatization) for detection of steroids; and
3d is scanned HPTLC plate after derivatization in visible light for detection of steroids. Fig. 4 illustrates HPTLC detection of alkaloids wherein
4A is HPTLC chromatogram after scanning at wavelength 366nm for alkaloids; and
4B is scanned HPTLC plate at 366 nm (before derivatization) for detection of alkaloids. Fig.5 illustrates HPTLC detection of proteins wherein
5A is scanned HPTLC plate after derivatization in visible light for detection of proteins;
5B is HPTLC chromatogram after scanning at wavelength 254 nm for proteins;
5C is HPTLC chromatogram after scanning at wavelength 366 nm for proteins; and
5D is HPTLC chromatogram after scanning at wavelength 530 nm for proteins. Fig. 6 illustrates HPTLC detection of carbohydrates wherein
6A is HPTLC chromatogram after scanning at wavelength 254 nm for carbohydrates;
6B is HPTLC chromatogram after scanning at wavelength 366 nm for carbohydrates;
6C is HPTLC chromatogram after scanning at wavelength 530 nm for carbohydrates;
6a is scanned HPTLC plate at 254 nm (before derivatization) for detection of carbohydrates;
6b is scanned HPTLC plate at 366 nm (before derivatization) for detection of carbohydrates;
6c is scanned HPTLC plate at 366 nm (after derivatization) for detection of carbohydrates; and
6d is scanned HPTLC plate after derivatization in visible light for detection of carbohydrates. Figure 7A illustrates effect of different doses of ethanolic extract and diclofenac sodium on
Carrageenan induced rat paw edema (% rise) at 1 hr. Figure 7B illustrates effect of different doses of ethanolic extract and diclofenac sodium on
Carrageenan induced rat paw edema at 2 hrs. Figure 7C illustrates effect of different doses of ethanolic extract and diclofenac sodium on
Carrageenan induced rat paw edema at 3 hrs. Figure 8A illustrates effect of different doses of ethanolic extract ofAlternanthera bettzichiana and
diclofenac sodium on mean increase in cotton pallet weight. Figure 8B illustrates effect of different doses of ethanolic extract of Alternanthera bettzichiana and
diclofenac sodium on percent inhibition of cotton pallet granuloma. Figure 8C illustrates cotton pellet with granuloma formation (Mouse model). Figure 9A illustrates effect of EEWPM and EEMPM on tumour volume at 5-10 days.

Figure 9B illustrates effect of EEWPM and EEMPM on tumour volume at 11-15 days.
Figure 9C illustrates effect of EEWPM and EEMPM on tumour volume at 16-20 days.
Figure 9D illustrates effect of EEWPM and EEMPM on tumour volume at 21-25 days.
Figure 10A illustrates effect of different concentration of EEWPM and EEMPM on cell viability.
Figure 10B illustrates effect of different concentration of EEWPM and EEMPM on % cell viability
SUMMARY OF THE INVENTION:
In accordance with the first aspect of the present invention there is provided a process for the preparation of AIternanthera bettzichiana extract; said process comprising the following steps:
a) selecting the Alternanthera bettzichiana plant material followed by washing and cleaning;
b) drying the cleaned Alternanthera bettzichiana plant material;
c) grinding the dried material to obtain powdered material followed by passing the powdered material through a sieve to obtain the Alternanthera bettzichiana powdered material ready for extraction; and
d) subjecting the material to solvent extraction at a temperature of about 20 to 60 °C to obtain the Alternanthera bettzichiana extract.
wherein said solvent extraction comprises extraction with at least one solvent or successive extraction or cold maceration.
In accordance with one of the embodiments of the present invention the successive solvent extraction comprises extraction of the plant material using at least one solvent having low polarity followed by extraction with at least one solvent having high polarity.
Typically, the solvent used for extraction is at least one selected from the group consisting of water, petroleum ether, chloroform, methanol and ethanol.
In accordance with another embodiment of the present invention the solvent extraction further comprises i) filtering the extract in hot condition; ii) distilling on water bath; and iii) drying at low temperature under reduced pressure in a evaporator.
In accordance with still another embodiment of the present invention the cold maceration comprises i) adding a solvent to the flask containing plant material under vigorous shaking; ii) plugging the flask with cotton plug and aluminium foil followed by keeping the flask aside for 12 hours; iii) shaking the flask vigorously and allowing to stand the flask for 7 days with frequent shaking; and iv) separating the fluid extract by decantation and filtration.

In accordance with the second aspect of the present invention there is provided an Alternanthera bettzichiana extract; said extract comprising: i. steroids; it. glycosides; iii. carbohydrate; and iv. alkaloids. wherein said extract is alcoholic extract with extractive value of about 32% and is useful in the treatment of at least one disease selected from the group consisting of cancer, inflammatory diseases and bacterial infections.
In accordance with the third aspect of the present invention there is provided an Alternanthera bettzichiana extract; said extract comprising: i. proteins; ii. glycosides; iii. carbohydrate; and iv. alkaloids. wherein said extract is aqueous extract with extractive value of about 19% and is useful in the treatment of at least one disease selected from the group consisting of cancer, inflammatory diseases and bacterial infections.
In accordance with the fourth aspect of the present invention there is provided an Alternanthera bettzichiana extract; said extract comprising:
i. steroids; and
ii. glycosides. wherein said extract is chloroform extract with extractive Value of about 25% and is useful in the treatment of at least one disease selected from the group consisting of cancer, inflammatory diseases and bacterial infections.
Typically, the glycoside comprises cardiac glycoside, anthraquinone glycoside, saponin glycoside and coumarin glycoside.
In accordance with fifth aspect of the present invention there is provided a medicament for the treatment of cancer comprising administering in said subject an effective amount of Alternanthera bettzichiana extract.

In accordance with sixth aspect of the present invention there is provided a medicament for the treatment of at least one disease selected from the group consisting of bacterial infections and inflammatory disease comprising administering in said subject an effective amount of Alternanthera bettzichiana extract.
DETAILED DESCRIPTION OF THE INVENTION:
In accordance with the first aspect of the present invention there is provided a process for the
preparation of Alternanthera bettzichiana extract; said process comprising the following steps:
First step is selecting the Alternanthera bettzichiana plant material.
Fresh samples of the plant Alteanthera bettzichiana were collected from the different parts of
Satpuda region of Chopda Tahsil, Jalgaon district in the month of September. The plant was
authentificated from Botanical survey of India, Pune. Typically, the plant material used is wild
plant material of A. bettzichiana or micro-propagated plant material of A. bettzichiana.
The selected plant material is washed and cleaned properly.
Second step is drying the cleaned Alternanthera bettzichiana plant material in shade.
In next step the dried material is grinded using electric grinder to obtain the powdered material. The
powdered material is then passed through a sieve no. 80 to obtain the Alternanthera bettzichiana
powdered material ready for extraction.
In the final step the obtained Altemanthera bettzichiana powdered material is subjected to solvent
extraction at a temperature of about 20 to 60 °C to obtain an Altemanthera bettzichiana extract.
In one of the embodiments of the present invention the solvent extraction comprises extraction with at least one solvent.
In accordance with another embodiment of the present invention the solvent extraction is successive solvent extraction which comprises extraction of the plant material using at teast one solvent having low polarity followed by extraction with at least one solvent having high polarity.
Typically, the solvent used for extraction is at least one selected from the group consisting of water, petroleum ether, chloroform, methanol and ethanol.
In successive solvent extraction, a dried material was extracted with different solvents, starting
from solvent of low polarity by using soxhlet extraction apparatus. After extraction by one solvent,
material is removed from thimble, dried and recharged, extracted with solvent of successively high
polarity.
Typically, the glass Soxhlet apparatus consist of four parts

• Round bottom flask (RBF),
• Main body/Thimble,
• Syphone glass tube, and
• Condenser.
150 gm of dried, powered crude drug was kept in a filter paper cover which was already placed in thimble. Then the solvent was slowly poured onto it. The solvent from thimble goes to lower round bottom flask via siphon tube due to the siphoning or siphon cycle. Such 2-3 cycles of solvent were performed and then the drug powder was kept for 12 hrs with solvent for imbibitions. After 12 hours imbibitions, solvent from flask heated to form vapors. Due to heat the solvent from round bottom flask gets converted into its vapors and then these vapors pass .via side tube into the condenser where it gets condensed. This solvent was dripped again on to drug material which was placed in thimble. This process was continued till thimble gets filled with solvent and when level of solvent reaches to siphon tube, pulling of whole solvent into the flask is taken place. All this events repeated several times and drug material was extracted continuously with fresh solvent. This process was performed for 3 days and when siphon solution showed negative test for phytoconstituents, extraction was completed. Then the extracts were filtered in hot condition and distilled on water bath. All the extract was finally dried at low temperature under reduced pressure in a rotary evaporator.
Typically, the dried powdered plant material was extracted successively with petroleum ether followed by chloroform, 90% ethanoi and water.
In accordance with another embodiment of the present invention there is provided an aqueous
extract which is prepared by cold maceration.
100 gm of powered crude drug was taken into 2 liters of conical flask. To this, one liter of solvent
was added with vigorous shaking. Then the flask was plugged with cotton plug and aluminum foil
and kept aside for 12 hours. After 12 hours flask was again shacked vigorously and then flask was
allowed to stand for 7 days with frequent shaking. After completion of 7 days the fluid extract was
separated by decantation and filtration. Typically, the filtration was carried out by using vacuum
filtration assembly.
Typically, the extract is in the form selected from the group consisting of powder, semisolid and
liquid.
In accordance with the second aspect of the present invention there is provided an Alternanthera bettzichiana extract; said extract comprising:
i. steroids;

ii. glycosides;
i i i. carbohyd rate; and
iv. alkaloids. wherein said extract is alcoholic extract with extractive value of about 32% and is useful in the treatment of at least one disease selected from the group consisting of cancer, inflammatory diseases and bacterial infections.
In accordance with the third aspect of the present invention there is provided an Alternanthera bettzichiana extract; said extract comprising: i. proteins; ii. glycosides; iii. carbohydrate; and iv. alkaloids. wherein said extract is aqueous extract with extractive value of about 19% and is useful in the treatment of at least one disease selected from the group consisting of cancer, inflammatory diseases and bacterial infections.
In accordance with the fourth aspect of the present invention there is provided an Alternanthera bettzichiana extract; said extract comprising:
i. steroids; and
ii. glycosides. wherein said extract is chloroform extract with extractive value of about 25% and is useful in the treatment of at least one disease selected from the group consisting of cancer, inflammatory diseases and bacterial infections.
In accordance with one of the embodiments of the present invention the glycosides comprises cardiac glycoside, anthraquinone glycoside, saponin glycoside and coumarin glycoside.
Physicochemical and Phy to-chemical investigation of the extract:
a) Total Ash:
1g powdered drug was taken in a tarred silica dish previously dried and weighed. It was ignited in a furnace until free from carbon. The ash obtained was weighed.
b) Acid Insoluble ash:
To the crucible containing the total ash, 25 ml of dilute hydrochloric acid was added, covered with a watch glass boiled gently for 5 minutes. The insoluble matter was collected on an ash less filter paper, washed with hot water until the filtrate is neutral. It was allowed to cool in a suitable

desiccator for 30 minutes, and then weighed without delay (World Health Organization, Geneva, 1998 and Khandelwal K.R., 2001).
c) Water-soluble ash:
To the crucible containing the total ash, 25ml of water was added and boiled for 5 minutes. The insoluble matter was collected on an ash less filter paper, washed with hot water, and ignited in a crucible for 5 minutes. The weight of this residue was subtracted from the weight of total ash.
d) Extractive Value:
This is the most useful method for the evaluation of a crude drug, which gives the idea about the active constituents in the given amount of medicinal plant material when extracted with solvents. About 5 g of the powdered drug was weighed in a weighing bottle and transferred to a dry 250 ml conical flask. 100 ml graduated flask was filled to the delivery mark with the solvent. The weighing bottle was washed out and the washings were poured, together with the rest of the solvent into the conical flask. Then the flask was corked with cotton plug and aluminum foils and was set aside for 24 hours with vigorous shaking. After completion of period, it was filtered into a 50 ml measuring cylinder up to mark. 25 ml of this filtrate was transferred to a weighed, thin porcelain dish and evaporated to dryness on a water-bath. The drying was complete in an oven at 100°C followed by cooling in a desiccator and weighing. The percentage w/w of extractive with reference to the air-dried drug was calculated.

Ash Value Total ash 14%
Acid-insoluble ash 1.6%-
Water soluble ash 3.0 %
Extractive values Petroleum ether 6.4 %
Chloroform 25.6 %
Ethano! 32.0 %
Water 19.2%
The above table depicts the physicochemical characters in which the total ash value was higher than that of acid insoluble and water soluble ash value and a decrease in the acid insoluble ash value may be due to presence of siliceous matters. The ethanolic extractive value was higher than petroleum ether, chloroform and aqueous extractive value revealing the presence of larger amounts of alcohol soluble constituents in the plant material. The yield of petroleum ether extract was dull greenish semisolid mass with a yield of 6.4% w/w, 25.6% w/w chloroform extract in the form of brilliant green-brown semisolid, 32% w/w for ethanolic extract in the form of dark brown semisolid mass, while aqueous extract yield found to be 19.2% w/w in the form of black-brown semisolid mass.
A) HPTLC detection of cardiac glycosides:

Ethanolic extract (10 mg/ml) and chloroform extract (10 mg/ml) were dissolved in ethanol and chloroform respectively. An appropriate volume30 pi\, 20 \i\, 10 u! and 05 uj for ethanolic extract and 20 μl, 12 μl, 07 μl and 03 ul for chloroform extract were applied on HPTLC plate, developed using Ethyl Acetate- Methanol- Ethanol- Water (16ion.
Derivatizing Reagent: Anisaldehyde -.2: 2.2: 0.8: 1.6) as mobile phase and scanned at 254 nm and 366 nm before and after derivatizat Sulphuric acid reagent (0.5 ml Anisaldehyde is mixed with 10 ml Glacial Acetic Acid, followed by 85 ml methanol and 5 ml of concentrated sulphuric acid in that order).
After spraying with anisaldehyde-sulphuric acid reagent, the ethanolic extract showed nine well resolved peaks at Rf 0.12, 0.32, 0.41, 0.51, 0.55, 0.68, 0.83, 0.88 and 0.90 (Table 1). Rf values with 0.12, 0.32, 0.83 were found to be the major one's with about 23.51, 26.69 and 31.59 peak areas (Figure 1A), respectively (Tablel). While the chloroform extract showed seven peaks at Rf 0.48, 0.64, 0.69, 0.77, 0.84, 0.87, and 0.92 (Table 2). Rf values with 0.48 and 0.77 were found to be the major one with about 29.85, 21.33 peak areas (Figure IB), respectively (Table 2).Very week fluorescence quenching at 254 nm (Figure la) without chemical treatment, presence of light blue and dark blue fluorescence (Figure lb) in ethanoiic extract and orange, pah brown fluorescence (Figure Ic) in chloroform extract at 366 nm were the confirmatory mark for the presence of cardiac glycosides (Wagner H., et al. 1984) in A. bettzichiana.
TabSe 1: HPTLC profile of crude ethanolic extract of A. bettzichiana for detection of cardiac glycosides

λmax Peak Start Rf MaxRf EndRf Area %
366 1 0.06 0.12 0.15 23.51

2 0.20 0.32 0.37 26.69

3 0.40 0.41 0.45 2.32

4 0.47 0.51 0.53 2.68

5 0.54 0.55 0.57 1.13

6 0.59 0.68 0.68 5.08

7 0.74 0.83 0.87 31.59

8 0.87 0.88 0.89 4.39

9 0.89 0.90 0.92 2.61

λm&x Peak Start Rf Max Rf End Rf Area %
366 1 0.35 0.48 0.57 29.85

2 0.57 0.64 0.65 12.99

3 0.65 0.69 0.71 11.25

4 0.72 0.77 0.80 21.33

5 0.81 0.84 0.85 12.65

6 0.85 0.87 .0.91 11.71

7 0.91 0.92 0.92 0.23
B) KPTLC detection of Coumarin Glycosides;
Ethanolic extract (10 mg/ml) and chloroform extract (10 mg/ml) were dissolved in ethanol and
chroform respectively. An appropriate volume30 ul, 20 ul, 10 JJ.1 and 05 ul for ethanolic extract and
20 ul, 12 u.1, 07 ui and 03 u.1 for chloroform extract were applied on HPTLC plate, developed using
Toluene- Diethyl Ether (1:1) Saturated with 10 % Acetic acid (Separately prepared 10 % acetic
acid solution and toiuene-Diethyl ether (1:1) and both the contents were mixed. The contents were
transferred in separating funnel and aqueous layer was discarded. The resulting ether: toluene layer
was utilized as mobile phase) as mobile phase and scanned at 254 nm and 366 nm before and after
derivatization.
Derivatizing Reagent: Vanillin - Phosphoric acid Reagent (Dipping solution): Dissolve 1 gm of Vanillin (4-hydroxy-3-methoxy benzaldehyde) in 25 ml ethanol and add 25 ml water and 35 ml O-phosphoric acid.
In HPTLC screening for coumarms, distinct fluorescence quenching at 254 nm (Wagner H., et al. 1984) in ethanolic and chloroform extract (Figure 2a), intense blue fluorescence (may be of simple coumarins), blue fluorescence (may be of furano and pyranocoumarins) in ethanolic extract and brown fluorescence (Wagner H., et al. 1984) in chloroform extract on scanning the plate at 366 nm were observed. (Figure 2b). After development and spraying with vanillin-phosphoric acid reagent, ten spots were observed at Rf 0.11, 0.17, 0.20, 0.24, 0.30, 0.43, 0.56, 0.68, 0.77, 0.89 in chloroform extract (Table 3) and four spots at Rf 0.18, 0.30, 0.32, 0.55 were observed in ethanolic extract (Table 4, Figure 2A) under 366 nm. Rf values with 0.30, 0.56, 0.68 were found to be the major one's with about 17.76, 33.51 and 31.12 peak areas (Figure 2B), respectively in chloroform extract (Table 3).

Table 3: HPTLC data for presence of coumarin glycosides in chloroform extract of A. bettzichiana

λtnax Peak Start Rf Max Rf End Rf Area %
366 1 0.06 0.11 0.12 5.45

2 0.13 0.17 0.17 1.17

3 0.17 0.20 0.23 2.93

4 0.23 0.24 0.25 0.59

5 0.25 0.30 0.37 17.76

6 0.39 0.43 0.46 1.72

7 0.47 0.56 0.61 33.51

8 0.61 0.68 0.76 31.12

9 0.76 0.77 0.84 3.91

10 0.85 0.89 0.92 1.85
Table 4: HPTLC data for presence of coumarin glycosides in ethanolic extract oA. bettzichiana

λmax Peak Start Rf Max Rf End Rf Area %
366 1 0.13 0.18 0.21 23.42

2 0.25 0.30 0.31 30.18

3 0.32 0.32 0.38 25.72

4 0.50 0.55 0.58 20.67
C) HPTLC detection of Steroids:
Ethanolic extract (10 mg/ml) and chloroform extract (10 mg/ml) were dissolved in ethanol and chroform respectively. An appropriate volume30 pi, 20 (if, 10 μl and 05 pi for ethanolic extract and 20 μl, 12 μl, 07 pi and 03 μl for chloroform extract were applied on HPTLC plate, developed using Toluene- Ethyl Acetate- Formic acid (10: 03: 0.5). The chromatogram was evaluated densitometrically using win CAT software and the steroids were traced after treatment with dertvatizing reagent. Derivatizing Reagent: 10 % Sulphuric acid

In present HPTLC screening for steroids, distinct fluorescence at 254 nm (Figure 3a), blue and violet fluorescence (Figure 3b) at 366 nm before derivatization were observed. The ethanolic extract shows blue and slightly green colored bands (Figure 3c) at 366 nm after derivatization (D.Pradhan, et ai 2009). The HPTLC chromatogram of chloroform extract and ethanolic extract showed the presence of seven components at Rf values 0.06, 0.08, 0.19, 0.39, 0.53, 0.65, 0.84 (Table 5) and 0.02, 0.08, 0.19, 0.44, 0.53, 0.81, 0.83 (Table 6) respectively after development and spraying with 10% sulphuric acid, indicating the confirmation of steroidal components in A. bettzichiana. Rf values with 0.39, 0.53, 084 in chloroform extract (Table 5) and 0.02, 0.53, 0.81, 0.83 in ethanolic extract (Table 6) were found to be major one's with about their respective peak areas (Figure 3B and 3A).
Table 5: HPTLC profile for detection of steroids in chloroform extract of A, bettzichiana

λmax Peak Start Rf Max Rf End Rf Area %
366 1 0.03 0.06 0.07 3.69

2 0.07 0.08 0.10 4.52

3 0.16 0.19 0.24 3.77

4 0.34 0.39 0.46 20.42

5 0.46 0.53 0.57 14.94

6 0.58 0.65 0.68 10.05

7 0.74 0.84 0.91 42.60
Table 6: HPTLC profile for detection of steroids in ethanolic extract of A. bettzichiana

Xrnax Peak Start Rf MaxRf EndRf Area %
366 i—i 0.00 0.02 0.03 44.86

2 0.07 0.08 0.09 2.23

3 0.16 0.19 0.20 4.37

4 0.40 0.44 0.48 7.86

5 0.49 0.53 0.60 13.77

6 0.74 0.81 0.81 11.33

7 0.82 0.83 0.91 15.55

D) HPTLC detection of Alkaloids:
01 g of powdered drug was moistened with about 1 ml 10 % ammonia solution. To it 5 ml of
methanol was added and the drug was extracted for 10 min on a water bath. 20 ul, 15 ul, 10 ul and
05 u! of above solvent was applied on HPTLC plate, developed using Toluene-Ethyl acetate-
Diethylamine (7:2:1) as mobile phase and scanned at 254 nm and 366 nm before and after
derivatization.
Derivatizing Reagent: Dragendorffs reagent.
{Preparation of Dragendorffs reagent: Solution A- 0.85 g of basic bismuth nitrate was dissolved in 10 mi glacial acetic acid and 40 mi water with heating.
Solution B- 08 g of potassium iodide was dissolved separately in 30 ml of water.
Solution A and B were mixed in the ratio of 1 :1 and labeled as stock solution. For spraying purpose 1 ml of stock solution is mixed with 2 ml glacial acetic acid and 10 ml water.)
During HPTLC screening for alkaloids blue and violet fluorescence was observed at UV 366 nm (Figure 4B) before derivatization (Wagner H., et al. 1984) and the fluorescence intensity increases with increase in concentration. The HPTLC finger print at 366 nm showed the presence of two compounds with Rf 0.28 and 0.84 (Table 7; Figure 4A) after derivatization with Dragendorffs reagent.
Table 7: HPTLC profile for detection of alkaloids in A. bettzichiana

λmax Peak Start Rf Max Rf End Rf Area %
366 1 0.26 0.28 0.30 17.76

2 0.80 0.84 0.90 82.54

E) HPTLC detection of Proteins:
Hydrolysis of protein
a. 50 mg of aqueous extract was weighed in to a 10 mi ampoule, to this 8 ml of 6N HCL was added followed by degassing the ampoule with nitrogen and sealing; the sealed ampoule was stored in an oven at 110°C for 16 h; the content of ampoule was transferred in to beaker containing barium hydroxide followed by manually shaking for 10 min and filtering using Whatmann filter paper; and the filterate was evaporated to dryness.

b. The residue was transferred to 25 ml volumetric flask containing 10 ml of 10 % isopropanol and volume was adjusted to the mark using same solvent. The same solution was used for chromatographic separation. a) 25 ul, 20 ul, 15 ul and 10 ul of above filtrate was applied on HPTLC plate, developed using Toluene: n-butanol-acetic acid-water (3:1:1) as mobile phase and scanned at 254 nm, 366 nm and 530 nm before and after derivatization. Derivatizing Reagent: Ninhydrin reagent (5% ninhydrin, 5% acetic acid, and 1% cadmium acetate in 96% ethanol) (Bernard Fried, et al. 1996)
In HPTLC screening for proteins light violet, yellow and intense violet colored bands were observed after spraying the plate with ninhydrin reagent in visible light (Figure 5A). After derivatization the plate was scanned at 254 nm, 366 nm, and 530 nm. Seven spots with Rf 0.29, 0.33, 0.47, 0.64, 0.76, 0.84, 0.91, were observed under 254 nm. Only one compound with Rf 0.69, was observed under 366 nm. While six spots with Rf 0.20, 0.30, 0.35, 0.48, 0.64, 0.76 were observed under 530 nm (Table 8a). Rf values with 0.64, 0.76 at 254 nm (Figure 5B), 0.69 at 366 nm (Figure 5C) and 0.30, 0.35, 0.48 at 530 nm (Figure 5D) were found to major one's with about their respective peak areas. Table 8a: HPTLC data for presence of proteins in A. bettzichiana
λmax Peak Start Rf Max Rf End Rf Area %
254 1 0.26 0.29 0.31 6.64

2 0.32 0.33 0.36 2.85

3 0.43 • 0.47 0.50 7.94

4 0.56 0.64 0.72 50.15

5 0.73 0.76 0.82 23.03

6 0.82 0.84 0.87 5.32

7 0.88 0.91 0.94 4.07
366 1 0.61 0.69 0.77 100.00
530 1 0.11 0.20 0.25 15.85

2 0.25 0.30 0.32 18.51

3 0.33 0.35 0.42 26.44

4 0.43 0.48 0.54 18.29

5 0.59 0.64 0.72 15.62

6 0.75 0.76 0.87 5.29

F: HPTLC detection of Carbohydrates:
05 g of powdered plant material was boiled with 25 ml hot 80% alcohol with stirring. Then the solution was decanted in to a suitable flask, and the solids were again re-extracted with 80% alcohol. Then the mixture was transferred to the flask, and allowed it to cool. The combined extracts were filtered and evaporated until alcohol odor disappears. The resulting extract was filtered through a thin mat of Celite in a Buchner funnel. Then the Celite layer was washed with small amount of distilled water. The washings and filtrate were diluted to 50 ml with distilled water. Then the purification was done with the addition of lead acetate to the solution, till formation of precipitate takes place. Shake the contents and allow it to stand for few minutes. Filter the contents in a beaker containing an excess amount of sodium acetate crystals. Formation of precipitate takes place, which was then filtered and washed with small amount of distilled water. The filtrate and washings were finally diluted up to 50 ml with distilled water. A precoated HPTLC plate was soaked in 0.3NNaH2PO4 solution for one minute. Immediately removed after one minute and dried at 100°C for 30 min. 25 ul, 20 ul, 15 ul and 10 ul of above filtrate was applied on HPTLC plate, developed using Toluene: n-butanol-acetone- 0.3N.NaH2PO4 (4:5:1) as mobile phase and scanned at 254 nm, 366 nm and 530 nm before and after derivatization.
Derivatizing Reagent: ADP reagent (2 ml aniline + 2 g diphenylamine + 10 ml H3P04 + 88 ml methanol),
The HPTLC screening for confirmation of sugars in A. bettzichiana revealed presence of fluorescence at 254 nm (Figure 6a) and light blue, blue and violet colored fluorescence at 366 nm (Figure 6b) before derivatization. The fluorescence quenching was increased with increase in concentration. After derivatization, a clear separation of light brown to dark brown colored bands was observed on scanning the plate in visible light (Figure 6d). Five spots with Rf 0.41, 0.52, 0.63, 0.67, 0.83 under 254 nm (Figure 6A), 0.04, 0.50, 0.62, 0.77, 0.88 under 366 nm (Figure 6B) and eleven spots with Rf 0.06, 0.10, 0.18, 0.33, Q.40, 0.57, 0.66, 0.70, 0.73, 0.81, 0.85 were observed under 530 nm (Table 8b, Figure 6C) after derivatization. Rf values with 0.67, 0.83 at 254 nm, 0.04, 0.50, 0.62 at 366 nm and 0.33, 0.57 at 530 nm were found to major one's with about their respective peak areas (Table 8b).
Table 8b: HPTLC data for presence of sugars in A. bettzichiana

λmax Peak Start Rf Max Rf End Rf Area %
254 1 0.39 0.41 0.46 5.55

2 049 0.52 0.54 4.14

3 0.60 0.63 0.65 9.53
1 ■7

4 0.65 0.67 0.70 19.05

5 0.72 0.83 0.88 61.74
366 1 0.02 0.04 0.06 19.69

2 0.42 0.50 0.53 27.57

3 0.57 0.62 0.64 29.69

4 0.76 0.77 0;82 11.66

5 0.84 0.88 0.94 11.40
530 1 0.05 0.06 0.07 1.54

2 0.07 0.10 0.11 4.98

3 0.13 0.18 0.22 8.39

4 0.26 0.33 0.37 12.45

5 0.37 0.40 0.40 1.10

6 0.48 0.57 0.64 66.95

7 0.64 0.66 0.69 2.15
8 0.69 0.70 0.71 0.56

9 0.73 0.73 0.76 0.65

10 0.79 0.81 0.82 0.66

11 0.84 0.85 0.87 0.56
In accordance with fifth aspect of the present invention there is provided a medicament for the treatment of cancer comprising administering in said subject an effective amount of Alternanthera beltzichiana extract.
In accordance with sixth aspect of the present invention there is provided a medicament for the treatment of at least one disease selected from the group consisting of bacterial infections and inflammatory disease comprising administering in said subject an effective amount of Alternanthera beltzichiana extract.
Following examples illustrate the invention, but are not intended to limit the scope of the present invention.
Example 1: Antibacterial Studies:
The antimicrobial screening of various extracts of A. beltzichiana was performed by using agar disc diffusion method and was tested against gram positive bacteria viz. Bacillus subtilis, Staphylococcus aureus and gram negative strains of E. coli and Salmonella typhi. Table 9 depicts the results of antimicrobial activity of the various extracts of A. beltzichiana. The results of zone of inhibition revealed that the petroleum ether, chloroform, ethanol and aqueous extract posses antimicrobial activity in a concentration dependant manner against the test organisms and was also comparable with the standard drugs streptomycin (10 ug/ml) included in the study. As shown in table 9, the extracts of A. beltzichiana showed antimicrobial activity with the diameter of zone of inhibition (Figure 4.6.1) ranging from 4.9 mm to 13.8 mm. The petroleum ether extract exhibited

zone of inhibition (mm) ranging between 7.0 ± 0.6 to 9.5 ± 0.9 against the tested organisms. The chloroform extract exhibited zone of inhibition (mm) ranging between 7.0 ± 0.7 to 11.0 ± 1.0 against the tested organisms. The ethanolic extract showed zone of inhibition (mm) ranging between 8.0 ± 0.7 to 13.8 • 0.9 and the aqueous extract revealed 4.9 ± 0.5 to 7.4 ± 0.6 mm zone of inhibition against the tested organisms. No zone of inhibition was observed in E. coli with aqueous extract. Ethanolic extract of A. bettzichiana exhibited significant antibacterial activity. In case of all these extracts MIC 80 to 120 jig/ml was observed against the test organisms and was determined by using broth dilution method.
Table 9: Antibacterial activity of various extracts of/4, bettzichiana

Type of Extract Cone. ug/ml Zone of inhibition (mm)

Bacteria

Bacillus subtillis S. aureus E. coli S. typhi
Petroleum ether extract 100 300 500 7.5 ± 0.6 8.7 ±0.7 9.0 ±0.6 . 7.0 ±0.6 8.2 ±0.8 9.5 ± 0.9 7.5 ±0.5 7.9 ±0.6 8.0 ± 0.7 7.9 ±0.6 8.0 ±0.8 8.4 ±0.8
Chloroform extract 100 300 500 9.0 ±0.8 10.4 ± 0.9 11.0 db 1.0 7.0 ± 0.7 7.8 ± 0.6 10.5 ±0.9 7.5 ± 0.6 8.2 ±0.8 8.5 ±0.7 8.5 ±0.7 8.9 ±0.9 9.5 ± 0.9
Ethanolic extract 100 300 500 11.0±1.1 12.0 ±1.0 13.8 ±0.9 9.5 ± 0.8 10.6 ±0.9 11.9± 1.1 8.0 ± 0.7 9.0 ±0.8 9.7 ±0.9 10.5 ±1.0 11.5±l.l 12.0 ±1.1
Aqueous extract 100
300 500 5.5 ±0.5
6.3 ±0.5
7.4 ±0.6 5.1 ±0.4 6.0 ± 0.6 7.0 ± 0.7 ~ 4.9 ±0.5 5.3 ±0.5 6.0 ±0.6
■ Standard streptomycin 10 15.0 ±1.2 14.5 ±1.3 15.0 ±1.3 15.0± 1.4
Example 2: Anti-inflammatory activity of A. bettzichiana extracts:
A) Animals utilized for Anti-inflammatory Studies
Albino wistar rat within weight range of 200-250 g were used for the anti-inflammatory studies. They were housed in polyethylene cages under standard conditions of 12 h light and dark cycle at 25 ± 2°C. Animals were supplemented with standard pelletized feed (Amrut Rat Feed, Pune, India) and water was made available ad libitum. The ethical clearance was approved by IAEC of R.C. Pate! Institute of Pharmaceutical Education and Research, Shirpur (M.S.) with resolution number RCCOP/IAEC/2007-08/42. Anti-inflammatory activity of the ethanolic extract of A. bettzichiana was evaluated using acute and chronic inflammatory models. Acute activity was evaluated by

Carrageenan induced rat paw edema method (Winter C.A., et al. 1962) and the chronic activity was evaluated by cotton pellet granuloma model (D, Arcy P.F., et al. 1960).
I) Carrageenan Induced Paw Edema Model:
Wistar albino rats were randomly divided in five groups (n=6) and 12 h prior to the experiments, the animals were deprived of food and water was made available during experiments. The first group (negative control) received normal saline (0.9% Nacl), second group (positive control) received 10 mg/kg p.o., diclofenac sodium and rest of the three groups received ethanolic extract of A. bettzichiana at the dose of 100 mg/kg, 200 mg/kg, and 400 mg/kg p.o., concentrations. In animals of all the groups acute inflammation was produced by sub plantar injection of 0.1 ml of 1% Carrageenan (A.4) in normal saline in right hind paw of rat. Animals were pre-medicated with drug/extracts 1 h before Carrageenan injection. Paw volumes (in ml) was measured by dislocation of the water Column in a plethysmometer (Ugo Basile 7140, Italy) immediately after Carrageenan application at 1, 2, and 3 h intervals after the stimulus (Winter C.A., et al. 1962).
The volume of paw recorded just before Carrageenan injection was recorded as initial volume (Vo) in each case. The mean percent rise in the paw volume was determined by the following formula (Chandragouda R. Patil, et al. 2009)

Where Vt = Volume at different intervals after Carrageenan injection
Different doses of ethanolic extract of A. bettzichiana showed dose dependant significant antiinflammatory activity. The dose concentration of 100 mg/kg did not exhibit significant activity in the early phase of I h (Table 10). But in the later phase (2 h and 3 h), the dose of 200 mg/kg and 400 mg/kg of ethanolic extract of A. bettzichiana significantly decreased rise in paw volume as compared to control (Table 11). It was observed that the dose 200 mg/kg and 400 mg/kg inhibited increase of paw volume (1.1 ± 0.096 and 1.1 ± 0.031 respectively) as compared with control (1.3 ± 0.021). The diclofenac sodium (Standard drug) also exhibited significant inhibition in rise of paw volume (Table 10). The test drug showed 52%, 39%, and 32% inhibition in rise of paw edema at the doses of 100, 200, and 400 mg/kg respectively at the end of 3 hrs. The percent rise of paw volume of diclofenac sodium, a reference standard drug, was found to be 23% (Table 11).

The Carrageenan inflammation is a useful model to detect oral action of anti-inflammatory agents (Di Rosa M., et al. 1971). The development of Carrageenan induced edema is biphasic, the early phase (1-2 h) is mainly mediated by histamine, serotonin and prostaglandins. After which increased vascular permeability is maintained by the release of kinins up to 2.30 hrs. The later phase (from 2.30 up to 6 hsr.) is sustained by prostaglandin release and mediated by bradykinin, leukotriens, polymorphonuclear cells and prostaglandins produced by tissue macrophages (Britto A.R.M.S., et al. 1998). The later phase is reported to be sensitive to most of the clinically effective antiinflammatory agents (Smucker E., et al. 1967). The ethanolic extract of A. bettzichiana revealed significant acute anti-inflammatory activity in early phase (Figure 7A and 7B) as well as later phase (Figure 7C).
Table 10: Effect of different doses of ethanolic extract and diclofenac sodium as reference standard on Carrageenan induced rat paw edema.

Group Dose (mg/kg) Mear i increase in paw volume

l hr. 2 hrs. 3 hrs.
Negative Control — 0.96 ±0.012 1.1 ±0.017 1.3 ±0.021
Diclofenac Sodium 10 0.92 ±0.013 1.0± 0.017** 1.0 ±0.046**
Ethanolic Extract 100 0.96 ±0.016 1.1 ±0.019 1.3 ±0.020
Ethanolic Extract 200 0.95 ±0.019 1.1 ±0.020 1.1 ±0.016*
Ethanolic Extract 400 0.96 ±0.020 1.1 ±0.022 1.1 ±0.031**
***P Table 11: Effect of different doses of ethanolic extract on % rise in paw volume of Carrageenan induced rat paw edema.

Group Dose (mg/kg) Percent Rise in Paw Edema

1 hr. 2 hrs. 3 hrs.
Negative Control — 20 ±0.84 38 ±2.3 60 ± 1.2
Diclofenac Sodium 10 11 ± 0.35*.* 22 ±0.41** 23 ±3.8**
Ethanolic Extract 100 17 ±0.50* 30 ±0.53** 52 »1.6**
Ethanolic Extract 200 15 ±0.28** 27 ±0.23** 39 ±2.8**
■Ethanolic Extract 400 13 *0.30** 24 ±0.31** 32 ±4.6**
***P
II) Cotton Pellet Induced Granuloma Model:
Chronic or proliferative phase of inflammation was assessed by cotton pellet granuloma in rat (D' Arcy, et at I960). The model was based on the foreign body granuloma formed in rats by subcutaneous implantation of compressed cotton pellets. After several days, histologically giant cells and undifferentiated connective tissue can be measured by weighing the dried pellets after
removal.
The cotton pellets weighing exactly 15 ± 1 mg each, were made from cotton rolls. Then the pellets were sterilized in an autoclave for 30 min at 121°C and 15 lb pressure. Then the pellets were soaked in distilled water containing penicillin and streptomycin and implanted subcutaneously in to both sides of the groin region of each rat under light ether anesthesia, after shaving the fur of the animals. In this model, the animals were divided in to five groups as described in Carrageenan induced rat paw edema model. The drug and extracts were administered orally to the respective group of animals for seven consecutive days, from the day of cotton pellet implantation. On the 8lh day, the animals were anaesthetized again; the cotton pellets were removed surgically and made free from extragenous tissues. The pellets were incubated at 37°C for 24 h and dried at 60°C to constant weight. The weight of the cotton pellet before implantation was subtracted from the weight of the dried granuloma pellets. The increase in dry weight of the pellets was taken as a measure of granuloma formation.
The cotton pellet granuloma bioassay is considered a chronic inflammation study model for studies on chronic inflammation and considered as a typical feature of chronic inflammatory reaction (Spector W.G., 1969). During the study with reference to chronic model, pellet caused a significant increase in granuloma weight in control group. The study of ethanolic extract of A. beiizichiana on chronic and proliferative phase of inflammation indicated that ethanolic extract (200 mg/kg and 400 mg/kg, p.o.) slightly but significantly (P The cotton pellet method is widely used to evaluate the transudative and proliferative components of the chronic inflammation. In this model, inflammation and, granuloma develops during the period of several days (D. Sheshadrishekar, et al 2009). Inflammation involves proliferation of

macrophages, neutrophils and fibroblasts, which are basic sources of granuloma formation (Figure 8C). The wet weight of the cotton pellets correlates with the transuda; the dry weight of the pellets correlates with the amount of granulomatous tissue (Olajide O.A., et ah, 2000). Hence, the degree in the weight of granuloma indicates that the proliferative phase was effectively suppressed by the ethanolic extract of A. bettzichiana.
The present anti-inflammatory protocol showed that the ethanolic extract of A. bettzichiana elicited a significant anti-inflammatory activity in Carrageenan induced paw edema (acute) and cotton pellet granuloma (chronic) rat models. This effect may be due to presence of secondary metabolites, probable coumarins, steroids and alkaloids present in the plant. This study indicates that the ethanolic extract of A. bettzichiana possess significant anti-inflammatory activity.
Table 12: Effect of different doses of ethanolic extract of A, bettzichiana on % inhibition of Cotton Pellet Induced Granuloma.

Treatment Groups Dose (mg/kg) Mean increase % Inhibition
Control — 75 ± 2.5 —
Diclofenac Sodium 10 42±1 8** 45.70
Ethanolic Extract 100 71 ± 1.2 6.34
Ethanolic Extract 200 62 ± 2.7* 17.37
Ethanolic Extract 400 56 ±1.6** 26.35
P Example 3: Anti-tumour activity of EEWPM and EEMPM of A. bettzichiana'.
A) Animals used for Anti-tumour Studies:
The anti-tumour activity was performed with 6-7 week old Fj hybrid Swiss Albino mice. Animals with both sex and between weighing range of 27 ± 3 gm were utilized for the study. Animal care and research protocol were in accordance with the principles and guidelines of CPCSEA norms and approved by ethical committee (Registration No.500/la/CPCSEA72002) of Jawaharlal Nehru cancer Hospital and Research, Bhopai (MP.) with resolution number 500/1 a/CPCSEA/2002.
B) Division of Animals in Different Groups for in yivo Anti-tumour Studies:
The animals were divided in the following groups and in each animal the melanoma cell line (B16F10) was injected subcutaneously (S.C.)

Table 13: Types of groups
Sr. No. Group Treatment No. of animals/ group
1. Control Group Tumour only 04
2. Test Group A1 Tumour + EEWPM (250 mg/kg) 04
Test Group A2 Tumour + EEWPM (500 mg/kg) 04

4. Test Group A3 Tumour + EEWPM (750 mg/kg) 04
5. Test Group B1 Tumour + EEMPM (250 mg/kg) 04
6. Test Group B2 Tumour + EEMPM (500 mg/kg) 04
7. Test Group B3 Tumour + EEMPM (750 mg/kg) 04
8. Radiation Group Tumour + Radiation (4 Gy) 04
Where £EWPM= ethanolic extract of wild plant material of A. bettzichiana and EEMPM= ethanolic extract of micro-propagated plant material of A. bettzichiana.
B16F10 melanoma cell line is a highly metastatic melanoma cell line (G.Kuttan, et ai 2005) which gives rise to a tumour nodule in most mouse lines. Ex-vivo melanoma tumour was -induced by B16F10 melanoma cell lines. Five lakh cells were injected subcutaneously and after 8-10 days tumour started growing- When the tumour was palpable, the tumour volume was measured at a given interval of days. When the tumour was observed as palpable, the tumour volume measurement was started from the 5th day and calculated the mean of every 5th day till 25th day. During first few days of drug treatment (5th-l0lh days) slow and steady tumour growth was observed in all groups (Figure 9 A). During 1 l'h-15th day tumour volume was increased rapidly in all the cases (Figure 9B). No tumour regression was noticed in test as well as radiation group. But from 15th-20th days and onward, a drastic regression in tumour size was noticed in test groups as compared to radiation group (Figure 9C). On the other hand, on the 25th day, the tumour volume in control group was doubled by 10 folds (Figure 9D). From the observed values it was cleared that, both the extract revealed statistically significant (P The most remarkable feature observed during in vivo antitumour activity was ethanolic extract of micropropagated plant material (EEMPM) of A. bettzichiana revealed significant dose dependant regression in tumour volume between 16th-25th day (Figure 9C and Figure 9D) of studies as compared to ethanolic extract of wild plant material (EEWPM). From the overall results, it was well deared that A bettzichiana has shown the tumour inhibition potential.

C] In vitro Anti-tumour Activity:
I) Cell Culture:
The B16F10 melanoma cell lines were provided by Department of Research, Jawaharlal Nehru Cancer Hospital and Research, Bhopal (M.P.). Cells were cultured in 5 ml Eagle's minimum essential media (EMBM) in Petri plates containing NaHCO3 (220 mg/100 ml). Media containing NaHCO3 was supplemented with 10% Fetal Calf Serum (FES) and streptomycin plus penicillin (100 μg/ml and 100 IU/ml, respectively). Cells were cultured in a 5% CO2 humidified atmosphere at 37°C until near confluence. A confluent monolayer was detached with 0.25% trypsin in PBS (pH 7.4, 0.01 M) and suspension was utilized for subculturing. Serially cultured cell lines at third stages were utilized for in vitro antitumour activity (Rodrigueg J., 2002). All the processes were carried out strictly under aseptic condition in a vertical laminar flow chamber.
The in vitro screening was performed by inoculating l x l05 cells/ml of suspension in culture media and incubated for 3 h at 37°C in 5% CO2. After 3 h, the cells were exposed to different concentrations of EEWPM and EEMPM of A. bettzichiana. Another set of parallel cultures was also run throughout the study. Duplicate plates of each culture were incubated for further 24 h. After incubation for 24 h at 37°C, the cells were trypsinized (0.25% in PBS) and then centrifuged at 1000 rpm for 5 min. Supernant was discarded and the cells were re-suspended in PBS to make protein free suspension. Then the cell viability was measured by trypan blue exclusion test and counted using neuber's WBC counting chamber.
II) Drug Treatment:
Ethanolic extract of wild plant material (EEWPM) and micropropagated plant material (EEMPM) of A. bettzichiana were prepared at a concentration of 250 mg/ml, 500 mg/ml, and 750 mg/ml in DDW. The drug extracts were treated with plates containing B16F10 melanoma cell lines. While a plate containing B16F10 melanoma cell lines only was maintained as control during the study.
III) Trypan Blue Exclusion Test:
The percentage of viable and non-viable cells was determined by using trypan blue exclusion test (James Kumi-Diaka, et al. 1999). It is based on the principle that live cells posses intact cell membrane that exclude certain dyes such as trypan blue, eosin or propidium. Cell growth and was measured by adding I part of 0.4% trypan blue with 1 part of cell suspension. The mixture was then allowed to incubate in room temperature for 3. min. and cells were immediately counted using hemocytometer. Percent cell viability was calculated by the formula: Number of viable cells (Unstained cells)
Cell Viability= X 100

Total number of cells (Stained and unstained cells)
The in vitro cytotoxic effect of different concentrations of ethanolic extract of wild plant material (EEWPM) and ethanolic extract of micropropagated plant material (EEMPM) of A bettzichiana species on cell viability was determined by using B16FI0 melanoma cell lines. Treatment of extracts against B16FI0 melanoma cell lines, at all concentrations showed decrease in percent cell viability as compared to that control when examined by trypan blue exclusion test. In overall variations of test samples, 750 mg/ml concentration of both types of extracts (EEWPM and EEMPM) showed its best activity (Figure 10A).
Both the extracts revealed significant dose dependant activity with decrease in the total number of viable cells with increasing number non-viable cells (Table 15). Below 45% of ceil viability at 750 mg/ml concentration as compared with control as 100% was observed in case of both the extracts (Figure 10B).
Table 14: Effect of different doses of ethanolic extract (Wild and Micropropagated plant) on tumour volume for anti-tumour studies

Groups Tumour vo j————™
lume(cm )

5th-l0th,Day 11th-15th Day 16,th-20thDay 2th-25th Day
Control 0.38 ±0.01 0.87 ±0.018 2 ± 0.0085 3.7 ±0.029
Radiation 0.23 ±0.01 0.79 ± 0.01 0.9 ± 0.036 • 1.2 ±0.029
EEWPM 250 mg/kg 0.015 ±0.00041 0.074 ± 0.00041 0.29 ± 0.00063 0.36 ± 0.00075
EEWPM 500 mg/kg 0.01 ±0.00065 0.053 ± 0.00065 0.091 ±0.00063 0.83 ± 0.0063
EEWPIM 750 mg/kg 0.015 ±0.00065 0.072 ± 0.00065 0.033 ± 0.00065 0.025 ± 0.00063
EEMPM 250 mg/kg 0.016 ±0.00085 0.11 ±0.00085 0.3 ± 0.00087 0.55 ±0.0022
EEMPM 500 mg/kg 0.014 ±0.00063 0.12 ±0.00063 0.23.± 0.00065 0.085 ±0.0019
EEMPM 750 mg/kg 0.014±0.014 0.074 ±0.0013 0.062 ±0.001 0.014 ±0.00048
Where EEWPM= ethanolic extract of wild plant material of A. bettzichiana and EEMPM= ethanolic extract of micropropagated plant material of A. bettzichiana
Values are expressed as Mean ± SEM. Statistical analysis was performed by one way ANOVA followed by Bonferroni's multiple comparison test.
P
Table 15: In vitro cytotoxic effect of different concentration of A. bettzichiana extracts on B16F10 cell lines.

Different Cone, of Extract Total No. of viable cells Total No. of non-viable cells
Control 125000 —
EEWPM 250 mg 108750 16250
EEWPM 500 mg 80500 46250
EEWPM 750 mg 53000 71250
EEWPM 250 mg 112500 18000
EEWPM 500 mg 80250 43000
EEWPM 750 mg 54250 70750
TECHNICAL ADVANCEMENT AND ECONOMIC SIGNIFICANCE:
o The present invention provides physicochemical, phyto-chemical and pharmacological
evaluation of the plant Alternanthera betlzichiana. o Further, the present invention provides novel Alternanthera betlzichiana extracts
characterized by the presence of steroids; glycosides; proteins; carbohydrate; and alkaloids. o Still further, the present invention provides simple, high yielding and economic processes
for preparing the Alternanthera bettzichiana extract. o Furthermore, the present invention provides Alternanthera bettzichiana extracts which
exhibit significant anti-bacterial, anti-inflammatory and anti-cancer activity.
While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the invention. These and other changes in the preferred embodiment of the invention will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the invention and not as a limitation.

I CLAIM:
1. A process for the preparation of Alternanthera bettzichiana extract; said process comprising
the following steps:
a. selecting the Alternanthera bettzichiana plant material followed by washing and
cleaning;
b. drying the cleaned Alternanthera bettzichiana plant material;
c. grinding the dried material to obtain powdered material followed by passing the
powdered material through a sieve to obtain the Alternanthera bettzichiana
powdered material ready for extraction; and
d. subjecting the material to solvent extraction at a temperature of about 20 to 60 °C to
obtain the Alternanthera bettzichiana extract.
wherein said solvent extraction comprises extraction with at least one solvent or successive extraction or cold maceration.
2. The process as claimed in claim 1, wherein the successive solvent extraction comprises extraction of the plant material using at least one solvent having low polarity followed by extraction with at least one solvent having high polarity.
3. The process as claimed in claim 1, wherein the solvent used for extraction is at least one selected from the group consisting of water, petroleum ether, chloroform, methanol and ethanol.
4. The process as claimed in claim 1, wherein the solvent extraction further comprises i) filtering the extract in hot condition; it) distilling on water bath; and iii) drying at low temperature under reduced pressure in a evaporator.
5. The process as claimed in claim 1, wherein the cold maceration comprises i) adding a solvent to the flask containing plant material under vigorous shaking; ii) plugging the flask with cotton plug and aluminium foil followed by keeping the flask aside for 12 hours; iii) shaking the flask vigorously and allowing to stand the flask for 7 days with frequent shaking; and iv) separating the fluid extract by decantation and filtration.
6. Alternanthera bettzichiana extract; said extract comprising:
i. steroids; ii. glycosides; iii. carbohydrate; and

iv. alkaloids. wherein said extract is alcoholic extract with extractive value of about 32% and is useful in the treatment of at least one disease selected from the group consisting of cancer, inflammatory diseases and bacterial infections.
7. Alternanthera bettzichiana extract; said extract comprising:
i. proteins;
ii. glycosides;
iii. carbohydrate; and
iv. alkaloids. wherein said extract is aqueous extract with extractive value of about 19% and is useful in the treatment of at least one disease .selected from the group consisting of cancer, inflammatory diseases and bacterial infections.
8. Alternanthera bettzichiana extract; said extract comprising:
i. steroids; and ii. glycosides. wherein said extract is chloroform extract with extractive value of about 25% and is useful in the treatment of at least one disease selected from the group consisting of cancer, inflammatory diseases and bacterial infections.
9. The Alternanthera bettzichiana extract as claimed in claim 6,7 and 8 wherein the glycosides comprises cardiac glycoside, anthraquinone glycoside, saponin glycoside and coumarin glycoside.
10. A medicament for the treatment of at least one disease selected from the group consisting of cancer, inflammatory diseases and bacterial infections comprising administering in said subject an effective amount of Alternanthera bettzichiana extract.

Documents:

http://ipindiaonline.gov.in/patentsearch/GrantedSearch/viewdoc.aspx?id=IDcosyP2hmH6fCyfZEqhog==&loc=vsnutRQWHdTHa1EUofPtPQ==

« Previous Patent

Next Patent »

Patent Number

270502

Indian Patent Application Number

3259/MUM/2010

PG Journal Number

01/2016

Publication Date

01-Jan-2016

Grant Date

28-Dec-2015

Date of Filing

29-Nov-2010

Name of Patentee

HUNDIWALE JOGENDRA CHANDRAKANT

Applicant Address

HOUSE NO.1167, HUNDIWALE LANE, CHOPDA, DISTRICT : JALGAON 425 107, MAHARASHTRA, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	HUNDIWALE JOGENDRA CHANDRAKANT	HOUSE NO.1167, HUNDIWALE LANE, CHOPDA, DISTRICT : JALGAON 425 107, MAHARASHTRA, INDIA.
2	N GANESH	JAWAHARLAL NEHRU CANCER HOSPITAL, IDGAH HILLS, BHOPAL, (M.P.), INDIA.
3	KULKARNI MOHAN VINAYAK	DIVISION OF BIOCHEMISTRY, DEPARTMENT OF CHEMISTRY, UNIVERSITY OF PUNE, PUNE, MAHARASHTRA, INDIA.
4	SALUNKHE KISHOR SAHEBRAO	PLOT NO. 86, DANDEKAR NAGAR, PIMPRALA SIVAR, DIST JALGAON 425 001, MAHARASHTRA, INDIA.
5	GAGRANI MANISH BANSILAL	20, SAWALI,VASANT NAGAR, DONDAICHA ROAD, SHAHADA 425 409, MAHARASHTRA, INDIA.

PCT International Classification Number

A61K36/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA