Title of Invention

A PROCESS FOR PREPARATION OF A HERBAL ANTICANCER AND RADIOPROTECTIVE EXTRACT

Abstract

The present invention particularly relates to preparation of a herbal extract from leaves/stem/bark/berries/seeds of Hippophae sps., which may be in particular H.rhamnoides and/or H. tibetine, wherein the herbal extract prepared thereby has particularly anticancer and radioprotective properties besides other possible applications. The process involves collection of plant material, air-drying, grinding, preparation of ethanolic extract, solvent partitioning, fractionation and vacuum concentration. The plant material is air-dried at ambient temperature of 30-38'C for 8-15 days, and grinded in a grinder at max. 45'C. An ethanolic extract is prepared, and then subsequently extracted with several organic solvents of increasing polarity. The extract thus obtained is then fractionated by passing through Silica column, eluted with different solvents, and the biologically active fractions are pooled, concentrated under vacuum in a rotavapor, dried and stored at room temperature.

Full Text

FIELD OF INVENTION This invention relates to a process for preparation of an extract from leaves/ stem / bark/ berries/ seeds of Sea Buckthorn, (Hippophae sps.), which is useful specifically, but without implying any limitation, for anticancer as well as radio protective purposes. PRIOR ART Development of non-toxic anticancer formulations is a necessity to bring about improvements in cancer therapy, while non-toxic formulations that provide radioprotection would be very useful in handling nuclear emergencies, which may arise in situations like reactor accidents. Individually, a large number of anticancer agents and radioprotective molecular drugs, both of synthetic and natural origin, have been tested in experimental models and human clinical trials for treatment of cancer and mitigation of radiation injury caused by whole-body exposure ranging from sub-lethal to supra-lethal doses. Some anticancer drugs known in the art, are etoposide (VP16-213) and teniposide (VM26), which are semi-synthetic derivatives of Podophyllum. A disadvantage with these drugs is that their use is associated with several side effects, which include toxicity, myelosuppression and alopecia. Some other anticancer agents, of plant origin, in the art are vinblastine (VLB) and vincristine (VCR) derived from Catharanthus roseus. A disadvantage with these compounds is that their use is associated with neurological side-effects and alopecia. Amongst radioprotective agents, aminoethyl isothiourea (AET) is one formulation that is known in the prior art. A major disadvantage of this radioprotector is that it is toxic at effective dosess. Some other formulations well known in the art include WR-151327, WR-3689, WR-638, WR-77913, and WR-44923, which are phosphorothioate compounds. Major disadvantages associated with these drugs are the side effects like nausea, vomiting, and hypotension. Among the various sulfhydryl radioprotectors, Amifostine or Ethiophos [S-2--(3-aminopropylamino) ethylphosphothioic acid] is used as an adjuvant in radiotherapy to selectively radioprotect normal cells. A major disadvantage of this compound, however, is the severe neurotoxicity, exhibited at clinically useful concentrations. Another disadvantage of this drug is that it provides effective radiation protection only when administered immediately prior to radiation exposure, since it has a plasma half-life of less than 10 min. Some other molecular radioprotectors known in the art include DNA-binding bisbenzimadoles like Hoechst 33342 and its close analogue Hoechst 33258. A significant disadvantage of these DNA ligands is that they are mutagenic. Yet another limitation of these DNA-binding radioprotectors is their dependence on P-glycoprotein pump activity for in vivo uptake, due to which their adequate concentrations in critical tissues is difficult to achieve. Other drugs known to provide radioprotection are spermine (SPM) and spermidine (SPD). These polyamines protect the cell's DNA against radiation damage via polyamine-induced compaction. A disadvantage with these compounds is that under in vivo conditions, they provide very little protection. However, a single compound, which acts both as a radioprotector and an anticancer agent, is not well known in the art. Drugs, which act concomitantly as an anticancer as well as a radioprotective agent can definitely be of much use in radiation therapy. A process for preparing a single herbal formulation, for specific application both in cancer therapy and radioprotection is not known in the art, although a number of general methods for preparation of extracts from medicinal herbs are known. A disadvantage of the known processes employed for extraction of herbal compounds, is that they are complicated and cost-intensive. One such process known in the art is Ultrasound extraction, also called as 'sonication'. A disadvantage of this method is that it is not suitable for preparation of a herbal extract for use as a radioprotector, since it utilizes ultrasound energy, which results in loss of bioactive constituents responsible for providing radio-protection. Another general process known in the art is 'Supercritical Fluid Extraction technique. A significant disadvantage of this method is the cost involved. Another disadvantage of the above process is that extraction of polar solutes is difficult. OBJECTS OF THE PRESENT INVENTION The primary object of the present invention is to propose a process for preparation of a herbal extract from leaves/stem/bark/berries/seeds of Hippophae sps., which, besides other possible applications, is specifically useful as an anticancer agent and as a radioprotector. Another object of the present invention is to propose a process of preparation of a herbal extract fraction that exhibits a large therapeutic window, and thereby be utilized at concentrations far less than toxic doses. Still another object of the present invention is to propose a process for preparation of a herbal extract fraction from plant material, which has antioxidant, anticancer and radioprotective property. Yet another object of the present invention is to propose a process for preparation of a herbal extract fraction, which is sterile and can be administered intraperitoneally and orally. Further object of the present invention is to propose a process for preparation of a herbal extract fraction from plant material, which is simple and does not involve large captial investment costs. Still further object of the present invention is to propose a process for preparation of a herbal extract fraction from plant material, which is energy-efficient, as it requires very little heating, as compared to other known processes which require heating at higher temperatures. Yet further object of the present invention is to propose a process for preparation of a herbal extraction process which yields a fraction, in which anticancer and DNA condensation properties are not lost during the extraction process. Even further object of the present invention is to propose a process for preparation of a herbal extract which yields a fraction that induces apoptosis. Still further object of the present invention is to propose a process that yields a herbal extract (drug) that is fairly stable and can be stored at room temperature (30-40°C). Another object of this invention is to propose a herbal extract from Hippophae sps, which exhibits anticancer and radioprotective properties. According to this invention, there is provided a process for preparation of an anti-cancer and radio protective herbal extract comprising the steps of (a)air drying the leaves, stem, bark, berries and seeds of Hippophae sps. herb at ambient temperature of 30-38°C for 8-15 days, grinding the said dried porrions of the said herb, into a fine powder in a grinder at maximum temperature of 45°C and passing the finely ground material through nylon mesh of size 60, (b)soaking the said finely ground herb powder in ethanol in preferred ratio of 1:4 (w/v) for the preferred duration of 12-18 hours at preferred temperature of 35-45°C to obtain ethanol extract; repeating this step two times, (c)deeanting and filtering of the said ethanol extract through 3mm whatman filter and concentrating of the filtered extract at a preferred temperature of 45-50°C in a rotavapour to obtain concentrated extract, (d) solvent partitioning of the said concentrated extract at 25+2°C with the following solvents in w/v ratio of 1:50 to 1:250 comprising the steps of (i)mixing with petroleum ether for 12-24 hours, decanting and discarding the solvent fraction, (ii)mixing the residue material with benzene for 16-24 hours, decanting and discarding the solvent fraction, (iii)mixing the residue material with ethyl acetate for 16-18 hours, decanting and discarding the solvent fraction, (iv)mixing the residue material with acetone for 8-12 hours decanting and discarding the solvent fraction, (v)mixing the residue material with methanol for 30 minutes to 2 hours, concentrating the methanol extract in rotavapour at 50-55°C under reduced pressure and dissolving the dried methanol extract in a small amount of 0.5 to 1ml of methanol. (e)Loading the said methanol extract on top of a silica column of size 2.5 x 20cm, mesh size 60-120 and subjecting it to fractionalisation comprising the steps of (i) passing hexane and chloroform sequentially through the said column for 6-8 hours at a temperature of 25+ 2°C and discarding the fraction so obtained, (ii)successively eluting the said column with a mixture comprising of chloroform and methanol in following ratios and in the following sequence chloroform:methanol=(95:5);chloroform:methanol (92.5:7.5); chloroform :methanol=(90:10); chloroform :methanol (80:20); chloroform:methanol= (70:30); chloroform:methanol(60:40); and discarding the fractions, (ii)successively eluting the said column with a mixture comprising of chloroform and methanol in the following ratios and in the following sequence chlorofornr.methanol=(50.50); chloroform:methanol=(40:60); chloroform:methanol=(30:70), pooling the fractions and collecting the pooled fraction, (f) concentrating the pooled fractions in a rotavapour at a temperature of 50-55°C, and drying it to obtain the said anti-cancer and radio protective herbal extract in the form of yellowish-white flakes. Description of Figures Radioprotective property, chromatin condensing property and anticancer property of the herbal extract (drug) is illustrated by the accompanying figures, wherein: Fig. -1 depicts DNA protective action and chromatic condensing property of the herabal extract. Fig. a) shows no evidence of DNA damage in untreated mice thymocytes subjected to alkaline single cell gel electrophoresis. Fig. b) shows severe DNA damage in thymocytes subjected to gamma radiation (20 Gy) (Fig. lc-e) shows varying degrees of chromatin condensation in mice thymocytes when subjected to different concentrations (10, 20 and 40 µg/ml) of the herbal extract. (Fig. lf-h) shows radioprotective effect of the herbal extract (40µg/ml) evaluated in mice thymocytes by exposure to 40, 80 and 200 Gy gamma radiation which shows that there is no DNA damage even at 200 Gy, in the presence of the drug. (Figure-2) depicts the effect of herbal extract on induction of apoptosis in mice thymocytes, as determined by flow cytometry. Figure-3 shows the percent induction of apoptosis by the drug of the present invention which shows that the drug at all concentrations tested apoptosis, as early as 6 hr after treatment. Description of the Invention According to this invention, the exact is prepared by a process comprising of the following steps: 1. Drying and Grinding: Plant material comprising leaves, steam bark, berries and seeds of 'Hippophae sps'. is collected during the months of April-Nov. from the Himalayas at a height of over 3000 m. Care is taken to ensure that there is no contamination with any other herbal material. The plant material is then air-dried at 30-3 8°C for 8-15 days, and the powder prepared in a grinder at maximum 45°C. The powdered material is than passsed through a nylon mesh (mesh size: 60). 2. Preparation of Ethanolic Extract: (a) The powdered plant material is soaked in Ethanol for 12-18 hr. Preferably 12-16 hr at 35-45°C. The plant material and solvent are taken in a weight to volume ratio of 1:4. The process is repeated two times. (b) The solvent is then decanted, centrifuged and/or filtered through whatman filter 3MM and pooled. (c) The extract thus obtained is then concentrated in a rotavapor at 50°C, to complete dryness. 3. Solvent Partitioning: Solvent partitioning is carried out in the following five stages: (a) Dried ethanolic extract is taken and treated with Petroleum ether, taken in w/v ratio from 1:50 to 1:250, for 12-24 hr, preferably for 16-24 hr. The preferred temperature is 25+2°C. This solvent fraction is then discarded. (b] The residual material is then extracted with Benzene, taken in quantity from 1:50 to 1: 250 (w/v) for 12-24 hr, preferably for 16-24 hr. The preferred range of temperature is 25 ± 2°C This solvent fraction is also discarded. (c) The residua! material is then left for 16-18 hr in Ethyl acetate, taken in a w/v ratio from 1:50 to 1: 250. The preferred temperature range is 25 ±2°C The ethyl acetate fraction is decanted and discarded. (d) The remaining material is then extracted for 8-12 hr, preferably for 8-10 hr in Acetone, taken in a w/v ratio from 1:50 to 1:250, preferably at 25 ±2°C. The acetone fraction is also decanted and discarded. (e) The left material is then kept in Methanol, in a w/v ratio from 1:50 to 1: 250 for 30 min-2 hr, preferably for 30 min-lhr. The extraction is preferably performed at 25 ±2°C.The methanol fraction is then concentrated in a rotavapor at 50-55°C under reduced pressure. The thoroughly dried extract is then dissolved in minimum amount (0.5-1 ml) of methanol. lit. Fractipnation The methanol extract thus obtained, after extraction with different organic solvents, is then loaded on top of a Silica Column (Size: 2.5x20 cm; Mesh Size: 60-120) and subjected to fractionations as under: (a) Hexane and Chloroform are sequentially passed through the column for 6-8 hrs, preferably at 25 ±2°C, and discarded since the biological activity of interest is not observed in these fractions. (b) The column is then eluted with a mixture comprising of chloroform and methanol, in the following sequence: CHCb: MeOH (95:5), followed by CHCb: MeOH (92.5:7.5), CHCb: MeOH (90:10), CHCb: MeOH (80:20), CHCb: MeOH (70:30). CHCb: MeOH (60:40), CHCb: MeOH (50:50), CHCb: MeOH (40:60), and CHCb: MeOH (30:70). The fractions are separately collected. (c) The last three fractions viz., CHCb: MeOH (50:50), CHCb: MeOH (40:60), and CHCb: MeOH (30:70) are pooled together, while the rest are discarded. IV. Vacuum Concentration: (a) The pooled fractions are then concentrated in a rotavapor at 50-55°C, and the final extract thus obtained dried completely to yield yellowish-white flakes. (b) These yellowish-white flakes are then stored at 30-40°C. Prior to use, the fraction is dissolved in demineralized water and filtered through 0.22 µm filter to ensure sterility. The present invention will be more fully understood from the discussion of the following representative example studied during the evaluation of the present invention, which is intended to be an illustrative example and is not intended to be taken in any way to imply any limitation on the scope of the present invention. WORKING EXAMPLE: The plant material (500 g) is soaked in ethanol for 12 hr in a ratio of 1: 4 at 45°C. The solvent is decanted, filtered through Whatman 3MM, concentrated in a rotavapor at 50 °C, and completely dried. 200 mg of this ethanolic extract (dried) is taken and extracted with Petroleum ether (50 ml) at 25 ±2°C for 24 hr. This solvent fraction is then discarded. The residual material is then extracted with 50 ml of Benzene at 25 ±2°C for 24 hr, This solvent fraction is also discarded. The residual material is then left for 18 hr at 25 ±2°C in Ethyl acetate (50 ml). The ethyl acetate fraction is also decanted and discarded. The remaining material is then extracted for 12 hr in Acetone (40 ml) at 25 ±2°C. The acetone fraction is similarly decanted and discarded. The left material is then kept in Methanol (40 ml) for 30 min at 25 ±2°C. The methanol fraction is then concentrated in a rotavapor at 50°C under reduced pressure. The thoroughly dried extract is then dissolved in 1 ml of methanol. The methanol extract thus obtained, after extraction with different organic solvents, is then loaded on top of a Silica Column (Size: 2.5x20 cm; Mesh Size: 60). Hexane (500 ml) and Chloroform (500 ml) are then sequentially passed through the column, and discarded since the biological activity of interest is not observed in these fractions. The column is then eluted with 250 ml of a mixture of chloroform and methanol in the following sequence: CHCb: MeOH (95:5), followed by CHCI3- MeOH (92.5:7.5), CHCb: MeOH (90:10), CHCb: MeOH (80:20), CHCb: MeOH (70:30), CHCb: MeOH (60:40), CHCb: MeOH (50:50), CHCb: MeOH (40:60), and CHCb: MeOH (30:70). The fractions are separately collected. The last three fractions viz., CHCb: MeOH (50:50), CHCb: MeOH (40:60), and CHCb: MeOH (30:70) are pooled together, while the rest are discarded. The pooled fractions are then concentrated in a rotavapor at 50°C, and the final extract thus obtained dried completely to yield yellowish-white flakes. Just prior to use, the fraction is dissolved in demineralized water and filtered through 0.22 µm filter to ensure sterility. Evaluation as a Radioprotector and an Anticancer Agent The following studies clearly indicate the radioprotective and anticancer properties of the drug of the present invention: 1. Toxicity Evaluation: Table-1 illustrates the absence of toxicity of the drug of the present invention injected by intravenous route in Strain' A' mice. Fractionated extract (drug of the present invention) administered . route. (Table Removed) It can, therefore, be concluded that the drug of the present invention may be administered either by i.v. route or orally and is well tolerated at doses ranging from 2-30 mg/kg b.w. 2. Comet Assay: The effect of the drug on radiation-induced DNA strand breaks has been depicted in Fig-1. The fractionated extract (drug of the present invention) in a dose-dependent manner inhibited the DNA strand breaks, as is revealed by a gradual reduction in the comet tail formation. Exposure of cells to 20Gy of gamma radiation resulted in a significant amount of DNA strand breaks. Pre-treatment of thymocytes with the drug of the present invention, resulted in gradual reduction of DNA strand breaks, and at a concentration of 20 µg /ml the drug completely blocked the tail formation (Fig-Id). Beyond these concentrations the extract induced a strong condensation of chromatin, as can be seen from the complete absence of tail formation and appearance of intensely stained circular bodies (Fig-1e). The condensation of chromatin induced by the drug of the present.invention seems to be very strong as exposure of such cells even to 200 Gy did not yield any tail formation (Fig-1 h). 3. Flow Cytometric Analysis: Flow cytometry data reveals that mice thymocytes, under cultured conditions, undergo time-dependent apoptosis. Flow cytometric analysis of thymocytes treated with different concentrations of the drug has been shown in Fig-2. The drug of the present invention inhibited the progression of the cell cycle in a dose- dependent manner, as revealed by a decrease in the G2 phase and a small increase in the S-phase. The drug in a dose and time-dependent manner induced apoptosis, as revealed by the appearance of hypodiploid peak (pre-Gl population). Figs-2 and Fig-3 show that in control there was no apoptosis upto 6hr of incubation as can be seen from complete absence of pre-Gl population (hypodiploid). However, in later time periods the percentage of apoptosis (cell death) increased with the passage of time. The drug induced apoptosis at all concentrations tested (5-80 µg/ml) as early as 6hr after treatment, which increased further with the passage of time. Maximum apoptosis is evident at a concentration of 80 µg/ml, where 37%, 40% and 46% of cells underwent apoptosis in 6, 12 and 24 hr respectively. This shows that the drug induces a fairly high degree of apoptosis. Similar results have been obtained with squamous cell carcinoma (4451), when tested in vitro. 4. Detection of DNA Ladder Formation Induced bv the Herbal Extract: Induction of apoptosis by the drug of the present invention was confirmed by electrophoresis, where intemucleosomal degradation of DNA into multiple fragments of 180- 200 bp in length was clearly observed, conforming that the drug induces apoptosis. 5. Thermal Denaturation Studies: The interaction of the drug with DNA (Calf Thymus) under in vitro conditions was studied using DNA melting profile. Thermal denaturation profile of Calf Thymus DNA (Table-2) revealed that in control (no drug treatment), DNA melts at 74 ± 1.2 °C under buffer conditions (14mM NaCI). Addition of increasing amount of the drug before heating resulted in shift of Tm (Table-2). Table-2 Effect of varied concentrations of the extract on Tm (melting temperature) of calf thymus DNA. (Table Removed) At lower DNA: Drug concentration of 1:0.5 and 1:1, the drug induced lowering of Tm, and shifted the Tm towards left by 2°C and 5°C. Where as higher DNA: Drug ratio induced a strong shift of Tm towards right by 11 °C and 21 °C. The initial shift of Tm towards left by lower concentration and shift towards right by higher concentrations indicate interaction of the drug with DNA. This might account for the radioprotection exhibited by the drug. 5. Survival Studies: Survival studies in mice exposed to lethal (10 Gy) radiations have revealed over 60% radioprotection under following conditions (Dose: lOmg/kg b.w.; single injection given intraperitoneaily 30 min prior to irradiation). With use of more appropriate doses of the drug, it should be possible to enhance post-irradiation survival. The foregoing studies clearly reveal that the drug of the present invention, in a dose-dependent manner, exhibits both anticancer and radioprotective properties. At higher concentrations, the drug acts as an anticancer agent, inducing cell cycle inhibition and apoptosis. On the other hand, the drug at lower concentrations provides radioprotection by bringing about chromatin condensation, which is reversible in nature. At the molecular level, the drug binds to DNA causing altered gene expression because of reduced access to the replicating and transcribing machinery. The ability of the drug at higher concentrations in bringing about irreversible condensation instantaneously, may be utilized effectively in cancer therapy, since prolonged condensation of chromatin leads to alterations in DNA synthesis, transcription and induction of apoptosis (cell death). In addition, the DNA compaction property of the drug could be exploited for radiosensitizing tumors that are intrinsically radioresistant. Lower concentrations on the other hand bring about reversible modulation of DNA, and can be exploited for radioprotection purposes. The drug may thus have possible applications in the treatment of cancer, particularly solid tumors that characteristically exhibit slow progression of apoptosis, and radioprotection. It is to be understood that the process of the present invention is susceptible to modifications, adaptations, changes by those skilled in the art. Such changes, adaptations, modifications are intended to be within the scope of the present invention which is set-forth under the following claims: WE CLAIM 1. A process for preparation of an anti-cancer and radio protective herbal extract comprising the steps of: a) air drying the leaves, stem, bark, berries and seeds of Hippophae sps. herb at ambient temperature of 30-38°C for 8-15 days, grinding the said dried portions of the said herb into a fine powder in a grinder at maximum temperature of 45°C and passing the finely ground material through nylon mesh of size 60, b) soaking the said finely ground herb powder in ethanol in preferred ratio of 1:4 (w/v) for the preferred duration of 12-18 hours at preferred temperature of 35-45°C to obtain ethanol extract; repeating this step two times, c) decanting and filtering of the said ethanol extract through 3mm whatman filter and concentrating of the filtered extract at a preferred temperature of 45-50°C in a rotavapour to obtain concentrated extract, d) solvent partitioning of the said concentrated extract at 25+2°C with the following solvents in w/v ratio of 1:50 to 1:250 comprising the steps of: i) mixing with petroleum ether for 12-24 hours, decanting and discarding the solvent fraction, ii) mixing the residue material with benzene for 16-24 hours, decanting and discarding the solvent fraction, iii) mixing the residue material with ethyl acetate for 16-18 hours, decanting and discarding the solvent fraction, iv) mixing the residue material with acetone for 8-12 hours decanting and discarding the solvent fraction, v) mixing the residue material with methanol for 30 minutes to 2 hours, concentrating the methanol extract in rotavapour at 50-55°C under reduced pressure and dissolving the dried methanol extract in a small amount of 0.5 to 1ml of methanol. e) Loading the said methanol extract on top of a silica column of size 2.5 x 20cm, mesh size 60-120 and subjecting it to fractionalisation comprising the steps of: (i) passing hexane and chloroform sequentially through the said column for 6-8 hours at a temperature of 25+ 2°C and discarding the fraction so obtained, (ii) successively eluting the said column with a mixture comprising of chloroform and methanol in following ratios and in the following sequence: chloroform:methanol=(95:5); chloroform:methanol (92.5:7.5); chloroform -.methanol=(90:10); chloroform methanol (80:20); chloroform:methanol=(70:30); chloroform:methanol(60:40); and discarding the fractions, (iii) successively eluting the said column with a mixture comprising of chloroform and methanol in the following ratios and in the following sequence: chloroform: methanol= (50:50); chloroform: methanol=(40:60); chloroform:methanol=(30:70), pooling the fractions and collecting the pooled fraction. (f) concentrating the pooled fractions in a rotavapour at a temperature of 50-55°C, and drying it to obtain the said anti-cancer and radio protective herbal extract in the form of yellowish-white flakes. 2. A process for preparation of an anti-cancer and radio protective herbal extract substantially as herein described and illustrated.

Documents:

946-del-2001-abstract.pdf

946-del-2001-claims.pdf

946-del-2001-complete specification (granted).pdf

946-del-2001-Correspondence-Others-(04-11-2010).pdf

946-del-2001-correspondence-others.pdf

946-del-2001-correspondence-po.pdf

946-del-2001-description (complete).pdf

946-del-2001-drawings.pdf

946-del-2001-form-1.pdf

946-DEL-2001-Form-15-(15-09-2010).pdf

946-del-2001-form-2.pdf

946-del-2001-form-4.pdf

946-del-2001-GPA-(04-11-2010).pdf

946-del-2001-gpa.pdf