The present invention relates to a method for the preparation of a phyto-product using plant material. More specifically the present invention relates to a method for the preparation of phyto product extract which is used in cancer treatment.
BACKGROUND OF INVENTION
Plants that are used as foodstuffs or medicinal herbs contain a wide variety of chemicals that are assimilated into the body following ingestion. Some of these chemicals are important nutrients for men and animals (e.g. fats, carbohydrates, proteins, vitamins, minerals) while others have none, or little or no known nutritional value. Over the past century, the phytochemicals in plants have been a pivotal pipeline for Pharmaceutical discovery.
Annonaceousacetogenins (AGEs) constitute a series of polyketides found almost exclusively from plants in the family Annonaceae, with some of their species of origin being important economic crops in Asia and North and South America. Numerous AGEs have been isolated and identified from various parts of annonaceous plants. Annonaceous AGEs are a unique compound class of C35 or C37 secondary metabolites, derived from the polyketide pathway, which include structurally a ?-lactone ring along with several oxygenated functionalities. For example, hydroxy group, ketone, epoxide, tetrahydrofuran (THF), and tetahydropyran (THP) moieties, and even double and triple bonds are structural features encountered among the AGEs. Annonaceousacetogenins have been found to exhibit a broad range of biological properties, such as antineoplastic, antiparasitic, cytotoxic, immunosuppressive, neurotoxic, and pesticide effects. Among the broad array of biological properties documented in the biomedical literature for the AGEs, their cytotoxic and antitumor effects and the underlying mechanisms for such effects have received the most attention.
A. muricata L., commonly known as soursop, graviola, guanabana, paw-paw, and sirsak, is a member of the Annonaceae family comprising approximately 130 genera and 2300 species. A. muricata is native to the warmest tropical areas in South and North America and is now widely distributed throughout tropical and subtropical parts of the world, including India, Malaysia, and Nigeria. A. muricata is an evergreen, terrestrial, erect tree reaching 5–8 m in height and features an open, roundish canopy with large, glossy, dark green leaves. The edible fruits of the tree are large, heart-shaped, and green in color, and the diameter varies between 15 and 20 cm.
Earlier studies have reported that the extracts of A.muricata are selectively toxic to a certain type of tumor cells but none of the studies has shown an effective result with high yield and sustain biological activity. None of the prior art inventions has been shown to provide an effective solution to the desired problems.
RU2259841C1 discloses the suggested remedy with a wide spectrum of pharmacological action for maintenance therapy in oncological patients is a dry extract of birch fungus obtained due to processing reduced raw material with water at the ratio of 1:4-6 for 6-12 h at periodic mixing. Then one should separate supernatant and repeatedly treat the residue with water at the ratio of 1:3-4 for 4-6 h, then extracts should be combined and dried, moreover, the target product contains flavonoids of not less than 15% on conversion to quercetin. The remedy could be designed as powder, granules, capsules, or tablets. Due to the complex of substances in the extract its application causes no side reactions being nontoxic and highly efficient in decreasing unfavorable medicinal loadings upon the patient's body.
BR102016029177A2 discloses soursop seed oil extraction process (annona muricata l.) and the product obtained by the present invention (pi) relates to an extraction process for obtaining crude oil using soursop seed oil (annona muricata) l.) The potential for pharmacological and biotechnological use and application. More specifically the process involves the fractionation and obtaining of the liquid fraction of the oil, where the said oil presents reduced amount/concentration of acetogenins, not showing, thus, toxic action, being able to be used in models of biological properties evaluation, with immunomodulatory effects and further, as an active agent for the treatment of type 1 diabetes mellitus
The existing inventions are not able to overcome the problem associated with high yield and biological activity. The existing inventions are complex and are not cost-effective. Thus there is a need for the present invention to overcome the above-discussed problems.
OBJECT OF THE INVENTION
The main objective of the present invention is to provide an improved A. muricate phytoproducts.
Yet another objective of the present invention is to provide a significant improvement in the processing of fruit of A. muricate providing a stable and optimum composition, thereby improving the yield of secondary metabolites or other therapeutic molecules.
Yet another objective of the present invention is to provide a stable process that improves the yield of secondary metabolites or other therapeutic molecules.
Yet another objective of the present invention is to provide an effective bioactivity potential of the processed A. muricate products.
Further objectives, advantages, and features of the present invention will become apparent from the detailed description provided hereinbelow, in which various embodiments of the disclosed invention are illustrated by way of example.

SUMMARY OF THE INVENTION
The present invention relates to a method for the preparation of a phyto-product using plant material. The method includes a plant material. The leaves of the plant material are washed and dried in shade. The dried leaves of the plant material are powdered and sieved in a 40 µm mesh. The powdered leaves are extracted with a low polar solvent at 50oC temperature for 3hrs resulting in the removal of lipids, fats, oils, and colored pigments. The processed leaf extracts are combined with ethanol and water forming different types of solution. The solutions were extracted by the decoction method with reflux attachment at a temperature below 50oC for 6 hr. The excess solvents are removed by distillation and drying process from the extract at 50oC temperature for 12 hrs. The dried extracts are mixed in the same ratio to get the desired product. A method for the preparation of phyto extract from the ripened fruit of plant material. The method includes a ripen fruit collected from the plant material and is washed. The mesocarp region of the fruit is collected by hand. The pulpy material is mixed with natural antioxidants at a 0.1% level resulting in processed pulpy material. The processed pulpy material is dried using continuous cycles of heating and cooling (60oC to - 60oC with Cryoliquid) for 12hr to achieve the drying process under vacuum condition, and the dried material was grind under DHR condition to get dry fruit powder with original flavor and taste of the fruit with all the active material intact. Herein, the plant material is selected from but not limited to Annona muricata. Herein, the leaves of the plant material should have one year and above age. Herein, the polar solvent is selected from but not limited to water, acetic acid, ethanol, and propanol.
The main advantage of the present invention is that the present invention provides an improved A. muricate phytoproducts.
Yet another advantage of the present invention is that the present invention provides a significant improvement in the processing of fruit of A. muricate that provides a stable and optimum for thereby improving the yield of secondary metabolites or other therapeutic molecules.
Yet another advantage of the present invention is that the present invention provides a stable process that improves the yield of secondary metabolites or other therapeutic molecules.
Yet another advantage of the present invention is that the present invention provides an effective bioactivity potential of processed A. muricate products.
Further objectives, advantages, and features of the present invention will become apparent from the detailed description provided herein below, in which various embodiments of the disclosed invention are illustrated by way of example.
DETAILED DESCRIPTION OF THE INVENTION
While this invention is susceptible to embodiment in many different forms, there is shown in the drawings and will herein be described in detail specific embodiments, with the understanding that the present disclosure of such embodiments is to be considered as an example of the principles and not intended to limit the invention to the specific embodiments shown and described. In the description below, reference numerals are used to describe the same, similar or corresponding parts in the several views of the drawings. This detailed description defines the meaning of the terms used herein and specifically describes embodiments in order for those skilled in the art to practice the invention.
Definition
The terms “a” or “an”, as used herein, are defined as one or as more than one. The term “plurality”, as used herein, is defined as two or as more than two. The term “another”, as used herein, is defined as at least a second or more. The terms “including” and/or “having”, as used herein, are defined as comprising (i.e., open language). The term “coupled”, as used herein, is defined as connected, although not necessarily directly, and not necessarily mechanically.
The term “comprising” is not intended to limit inventions to only claiming the present invention with such comprising language. Any invention using the term comprising could be separated into one or more claims using “consisting” or “consisting of” claim language and is so intended. The term “comprising” is used interchangeably used by the terms “having” or “containing”.
Reference throughout this document to “one embodiment”, “certain embodiments”, “an embodiment”, “another embodiment”, and “yet another embodiment” or similar terms means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of such phrases or in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics are combined in any suitable manner in one or more embodiments without limitation.
The term “or” as used herein is to be interpreted as an inclusive or meaning any one or any combination. Therefore, “A, B or C” means any of the following: “A; B; C; A and B; A and C; B and C; A, B and C”. An exception to this definition will occur only when a combination of elements, functions, steps, or acts are in some way inherently mutually exclusive.
The drawings featured in the figures are to illustrate certain convenient embodiments of the present invention and are not to be considered as a limitation to that. The term "means" preceding a present participle of operation indicates the desired function for which there is one or more embodiments, i.e., one or more methods, devices, or apparatuses for achieving the desired function and that one skilled in the art could select from these or their equivalent is given in the disclosure herein and use of the term "means" is not intended to be limiting.
The present invention relates to a method for the preparation of a phyto-product using plant material. A method for the preparation of phyto extracts from the leaf of plant material. The method includes a plant material. The leaves of the plant material are washed and dried in shade. The dried leaves of the plant material are powdered and sieved in a 40 µm mesh. The powdered leaves are extracted with a low polar solvent at 50oC temperature for 3hrs resulting in the removal of lipids, fats, oils, and colored pigments. The processed leaf extracts are combined with ethanol and water forming different types of solution. The solutions were extracted by the decoction method with reflux attachment at a temperature below 50oC for 6 hr. The excess solvents are removed by distillation and drying process from the extract at 50oC temperature for 12 hrs. The dried extracts are mixed in the same ratio to get the desired product.
A method for the preparation of the phyto extract from the ripened fruit of plant material, the method includes:
a ripen fruit is collected from the plant material and is washed,
the mesocarp region of the fruit is collected by hand,
the pulpy material is mixed with natural antioxidant at a 0.1% level resulting in a processed pulpy material,
the processed pulpy material is dried using continuous cycles of heating and cooling (60oC to - 60oC with Cryoliquid) for 12hr to achieve the drying process under vacuum condition, and
the dried material was grinded under DHR condition to get dry fruit powder with original flavor and taste of the fruit with all the active material intact.
Herein, the plant material is selected from but not limited to Annona muricata. Herein, the leaves of the plant material should have one year and above age. Herein, the polar solvent is selected from but not limited to water, acetic acid, ethanol, and propanol.
In another embodiment, the Phytoproduct from different plant parts (leaf & fruit) of Annona muricata shows anticancer effect. The processing of fruit is carried out to get the powder from the pulpy & fibrous mesocarp regions. The drying process was carried out by using a continuous hot & cold cycle with intermediate freezing by cyro liquid in order to get fine powder of the pulp in quick time with retention of original colour, taste, flavor, and bioactivity. The fruit powder is prepared without using any drying agent which is generally added during drying of fruits by other techniques like spray drying, oven drying, or by a freeze-drying process. The leaf of Annona muricata is processed with different permissible organic solvents mixture from d value ranging from 0 to 100 with 5 d increments. Further pooling the fractions in equal ratio to get the full range of Phyto constitutes from the leaf of Annona muricata with high anticancer activity.
In another embodiment, the bioactivity and toxicity of Annona muricata leaf extracts are disclosed.
Hydroalcoholic (aqueous ethanolic) extracts mixture gave the highest yields which gave it an edge in being more commercially valuable than the rest. The previous report of increased viability of non-cancerous cells and induced healing only added to its advantage, thus leaf extracts (ethanol and water extract with an increment of five d value of 0 to 100 mixed in the same ratio) qualified for further anticancer evaluation by MTT assay.
The cancer cell lines chosen for our study was keeping in mind the increasing incidences of breast and colon cancer worldwide (WHO). The % cytotoxicity and IC50 values of the aqueous ethanolic extract were contemplated to be better than the other extracts obtained from solvents of different polarity. The results of the MTT assay were like those obtained in previous work on cell lines. However, this is the first report of the anticancer efficacy of aqueous ethanolic extract on breast and colon cancer cell lines. Thus, this extract was further evaluated for toxicokinetic and pharmacokinetic parameters.
28-day repeated dose toxicity on Wistar rats was performed according to the OECD guidelines.
In this toxicity study, no changes in the behavior of rats were observed except for 10% of the rats in Group II and Group III which exhibited hypokinetic symptoms like a loss in grip strength, irregular gait and posture, less than normal activity, and slow movements suggestive of low degree symptoms of Parkinsonism.
The body weight changes serve as a sensitive indicator of the general health status of animals. A varied response of male and female rats to AMHE (Annona muricata leaf hydroalcoholic extract mixture) at high dose levels was observed. 1000 and 2500 mg/kg/day resulted in a decline in the bodyweight of female rats throughout the duration of the experiment. This finding was exactly similar as reported in the previous study (Arthur et al., 2011). However, weight gains were observed in all animals administered 100 mg/kg/day. It can be stated that at 100 mg, AMHE did not interfere with the normal metabolism of animals as corroborated with non-significant differences from animals in the vehicle control group. However, at a higher dose (1000 and 2500 mg/kg/day), the crude extract may have been metabolized to a toxic product that could interfere with gastric function and decreased food conversion efficiency. In addition, the diets were well-accepted by animals treated with 100 mg suggesting AMHE did not possibly cause any alterations in carbohydrate, protein, or fat metabolism in these experimental animals. It also shows that AMHE at 100 mg did not adversely interfere with the nutritional benefits such as weight gain and stability of appetite expected of animals that are continually supplied with food and water ad libitum. However, the same could not be said for animals administered 1000 and 2500 mg AMHE. Thus, the overdose of this extract could result in loss of appetite and a decrease in body weight in females. There were some significant changes in blood indices in the toxic group. However, no significant changes were detected in the hematological profile of the therapeutic group indicating its safety.
The liver releases alanine aminotransferase (ALT) and an elevation in its plasma concentration is an indicator of liver damage. Any necrosis of liver cells leads to a significant increase in SGOT, SGPT, and other enzymes. The results of our study showed significant changes in SGOT, SGPT, and ALP levels at high doses suggesting minimal toxicity, this was also supported by the histopathological findings of the liver of both males and females at medium and high toxic doses which showed first-degree pathological changes. This was like the results of a previous study conducted to evaluate the toxicity of A. muricata extract on the liver and kidney. These findings however are contradictory with the effects of Annona muricata aqueous extract (AMAE) which reported non-significant changes in ALT and AST in both male and female rats at all doses. Another interesting observation from the biochemical profile analysis of the rat’s blood was that there was a decrease in blood urea and serum creatinine levels in males thus suggesting a kidney protective function. But in females, there was a significant increase in blood urea level only suggesting possible kidney damage, especially by renal infiltration mechanism.
Acetogenins are potent mitochondrial poisons, like other parkinsonism-inducing compounds, and can cross the blood-brain. These studies reinforce the concept that the consumption of Annonaceae may contribute to the pathogenesis of atypical Parkinsonism. Our study thus gave proof of this mechanism. The histopathology of the brain showed very few pyknotic changes in the hippocampal region at the highest doses suggesting neurodegeneration, which was in accordance with the clinical observances like, mild hypokinesia, loss in grip strength, irregular gait, and posture.
Thus, our findings suggest that prepared A. muricate leaf extract is completely nontoxic at therapeutic doses and is minimally toxic to the liver at high doses. However, it strengthens the existing proofs of neurotoxicity which was observed only at very high doses.
In another embodiment, the bioactivity of Annona muricata fruit powder is disclosed.
Cytotoxic effects of Annonamuricata fruit powder in prostate cancer (PC3) cell line
Step 1: Preparation of the fruit extract
15g of prepared powder was extracted with 100% ethanol for 20 min.
The extracted product was filtered and dried using the Rota vacuum evaporator.
The extracted material was weighed, and extractive yield was calculated.

Step 2: Preparation of the PC3 cell culture
The extract was then analyzed for their cytotoxicity by MTT assay in PC3 cell line
DMEM media was selected for the culturing of cells. 50 ml of liquid DMEM media was mixed with 10% FBS (50ml) and 1X penicillin-streptomycin was added to the media.
Thawing and recovery of frozen cells • Cells were stored in liquid nitrogen (N2) in cryo vials. • During the beginning of the experimentation, frozen vials with cells were placed directly on ice from the liquid N2 tank. • The vials were then immersed in the water bath at 37 o C for 10 minutes and were again placed on ice. • When fully thawed, the contents of the vial were transferred into a T-25 flask containing cold media (with added DMSO). • After 12 hours the DMSO enriched media was discarded and a small volume of complete media was added to re-suspend the cells. • The cell viability and morphology were then determined at regular intervals by using light microscopy. • The cells were now ready for experimental usage. Complete Growth Media -The base medium for the cell lines was RPMI-1640 Medium. To make the complete growth medium, the following components were added to the base medium: foetal bovine serum to a final concentration of 10%. Sub-culturing -The following volumes were given for a 75 cm2 flask. Corning T- 75 flasks were used for sub-culturing the cells. a) The culture medium was removed and discarded. b) The cell layer was rinsed with 0.25% (w/v) Trypsin - 0.53 mM EDTA solution, in order to remove all traces of serum that contains trypsin inhibitor. 2.0 to 3.0 mL of Trypsin-EDTA solution was added to the flask and the cells were observed under an inverted microscope until cell layer is dispersed (usually within 5 to 15 minutes).

Step 3: Cytotoxic assay Fruit extract on the PC3 cell culture
Cytotoxic assay was done by MTT ((3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay method
The MTT assay is a colorimetric assay for assessing cell metabolic activity. NAD(P) H-dependent cellular oxidoreductase enzymes may, under defined conditions, reflect the number of viable cells present. These enzymes can reduce the tetrazolium dye MTT 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan, which has a purple color. Viable cells contain NAD(P)H-dependent oxidoreductase enzymes which reduce the MTT reagent to formazan, an insoluble crystalline product with a deep purple color. Formazan crystals are then dissolved using a solubilizing solution and absorbance is measured at 500-600 nm using a plate reader. The darker the solution, the greater is the number of viable and metabolically active cells. These assays are also used for measuring the cytotoxicity i.e. loss of viable cells of potential medical agents and toxic materials. MTT assays are usually done in the dark since the MTT reagent is sensitive to light.

Preparation of MTT reagent: MTT is soluble in water (10 mg/mL), ethanol (20 mg/mL), and buffered salt solutions and culture media (5 mg/mL). A 5 mg/mL solution in PBS was used. This was mixed by vertexing or sonication.
The solution was sterilized after adding MTT using a 0.22µ syringe filter.
MTT solution was stored at -20°C (stable for at least 6 months).

Assay procedure: After 24 and 48 hours of treatment, the cells were gently washed using IX phosphate buffer saline (PBS; 10mM, 2.7mM potassium chloride, 137mM sodium chloride, and 1.76mM potassium phosphate, pH 7.4 ±0.2), and 50µL of 1 mg/mL of methylthiazole tetrazolium (MTT; Sigma-Aldrich, CAS number: CAS Number 298-93-1) solution was added such in each well. After 2-4 hours of incubation, the formazan crystals were solubilized using 100 µL of DMSO and incubated in shaking conditions for 5 min to dissolve the formazan crystals. Subsequently, the absorbance was measured at 570 nm with 630 nm as a reference filter. Absorbance measured in un-treated cells was taken as 100% survival.
A. Average of duplicate reading was calculated for each sample.
B. The culture medium background was subtracted from assay readings.
C. IC50 values are calculated using Graphpad.

IC 50 values of Annona fruit from MTT assay
Sample/Standards IC50 value
24 Hr 48Hr
Paclitaxel 13.77 6.06
Annona Fruit Powder 425.27 202.51

Hence, from this study, we can efficiently conclude that Annona muricata leaf extract and fruit powder can be used as a commercially valuable oncology herbal /nutraceutical product and can also be used as a combination/ co-therapy with synthetic remedies to escalate their cytotoxic actions with fewer side effects. This will also have beneficial effects on the survival, immune modulation, and QOL (Quality of Life) of cancer patients when used in combination with conventional therapeutics.
It will thus be seen that the objects set forth above, among those made apparent from the preceding description, are efficiently attained and, since certain changes may be made in carrying out the above method and steps set forth without departing from the spirit and scope of the invention is intended that all matter contained in the above description and not in a limiting sense.
Although this invention has been described by examples and with reference to possible embodiments thereof, it is to be understood that modifications or improvements may be made thereto without departing from the scope of the invention.

Further objectives, advantages, and features of the present invention will become apparent from the detailed description provided hereinbelow, in which various embodiments of the disclosed present invention are illustrated by way of example and appropriate reference to accompanying drawings. Those skilled in the art to which the present invention pertains may make modifications resulting in other embodiments employing principles of the present invention without departing from its spirit or characteristics, particularly upon considering the foregoing teachings. Accordingly, the described embodiments are to be considered in all respects only as illustrative, and not restrictive, and the scope of the present invention is, therefore, indicated by the appended claims rather than by the foregoing description or drawings. Consequently, while the present invention has been described with reference to particular embodiments, modifications of structure, sequence, materials and the like apparent to those skilled in the art still fall within the scope of the invention as claimed by the applicant.

I/ WE CLAIMS
1. The method for the preparation of a phyto-product using a plant material, the method comprising:
a method for the preparation of phyto extracts from the leaf of plant material, the method includes:
a plant material, leaves of the plant material are washed and dried in shade,
the dried leaves of the plant material are powdered and sieved in a 40 µm mesh,
the powdered leaves are extracted with a low polar solvent at 50oC temperature for 3hrs resulting in the removal of lipids, fats, oils, and colored pigments,
the processed leaf extract are combined with ethanol and water forming different types of solution,
the solution was extracted by the decoction method with reflux attachment at a temperature below 50oC for 6 hr,
the excess solvents are removed by distillation and drying process from the extract at 50oC temperature for 12 hrs, and
the dried extracts are mixed in the same ratio to get the desired product;
a method for the preparation of phyto extract from the ripened fruit of plant material, the method includes:
a ripen fruit is collected from the plant material and is washed,
the mesocarp region of the fruit is collected by hand,
the pulpy material is mixed with natural antioxidant at 0.1% level resulting in a processed pulpy material,
the processed pulpy material is dried using continuous cycles of heating and cooling (60oC to - 60oC with Cryoliquid) for 12hr to achieve the drying process under vacuum condition, and
the dried material was grinded under DHR condition to get dry fruit powder with original flavor and taste of the fruit with all the active material intact.
2. The method as claimed in claim 1, wherein, the plant material is selected from but not limited to Annona muricata.
3. The method as claimed in claim 1, wherein, the leaves of the plant material should have one year and above age.
4. The method as claimed in claim 1, wherein the process to form the extract from the fruit of the plant material is carried out in inert atmosphere under reduced oxygen level in order to reduce the oxidation process,
5. The method as claimed in claim 1, wherein, the polar solvent is selected from but not limited to water, acetic acid, ethanol, and propanol.