FORM 2

THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
PROVISIONAL SPECIFICATION
[See section 10 and rule 13]
Title: Method of Extraction From Withania Somnifera and One or More Fractions Containing Pharmacologically Active Ingredients Obtained Therefrom


2. APPLICANT:
(a) NAME : BIO-VED Pharmaceuticals, Pvt. Ltd.
(b) NATIONALITY : Indian
(c) ADDRESS : 'BAIF' Bhavan Z Wing
Dr. Manibhai Desai Nagar Warje Malwadi, Pune 411 052 India.

The following specification describes the invention


METHOD OF EXTRACTION FROM WITHANIA SOMNIFERA AND ONE OR
MORE FRACTIONS CONTAINING PHARMACOLOGICALLY ACTIVE
INGREDIENTS OBTAINED THEREFROM
FIELD OF THE INVENTION
10001] The invention generally relates to the plant Withania somnifera (WS). More specifically, the invention relates to a method of extraction from WS and one or more fractions containing a combination of pharmacologically active ingredients extracted from WS by using the method of extraction
BACKGROUND
[0002] Several herbs have been known and used in India since thousands of years for treatment of various diseases. Withania somnifera (commonly known as Ashwagandha) is one of the several herbs known for its medicinal uses in India for more than three thousand years. Different pharmacologically active ingredients are present in WS that impart WS various medicinal qualities useful in the treatment of a number of human ailments and diseases. For example, the pharmacologically active ingredients of WS include alkaloids like withanine, withasomnin, etc. and steroidal glycosides like sitoindosides, withanolides, etc. These pharmacologically active ingredients present in WS have been used in the treatment of several diseases and disorders associated with the endocrine system, cardio vascular system, nervous system, reproductive system, and immune system. Research has also shown that some of the pharmacologically active ingredients present in WS have anti-cancer properties and thus WS is a promising alternative for cancer treatment and prevention.
[0003] Herbal medicines produced from herbs like WS cause minimal side effects due to the presence of naturally found pharmacological ingredients in them. Therefore, these medicines have proved advantageous over chemically synthesized pharmaceuticals. For example, the conventional treatment of various cancers usually includes chemotherapy

alone or along with radiotherapy. Chemotherapy using synthetic anti-cancer drugs alone or along with radiotherapy is known to cause several serious and unpleasant side effects like loss of hair, nausea, vomiting, fall in blood count, loss of immunity and weakness._-
[0004] Due to the various medicinal benefits associated with WS and the reduced side effects as mentioned above, there have been several efforts made to extract the different pharmacologically active ingredients from WS. For example, researchers have focused on extraction of withanolides and have developed methods for extraction of Withanolides. Withaferins are important withanolides present in WS. Studies have shown that Withaferin A in particular, is very useful in the treatment of cancer. However, pure Withaferin A has been associated with acute cytotoxicity. In addition, the existing methods for extracting Withaferins are limited. Further, the existing methods for extraction of Withaferin A rich fractions result in a very less final yield. Additionally, the existing methods of extraction from WS are not very suitable for industrial scaling up.
There is therefore a need for an improved method of extraction of pharmacologically active ingredients from WS that gives higher yields and has industrial scalability. There is also a need for improved extracts from that are less cytotoxic and more efficacious in the treatment of cancer and other ailments.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] FIG. 1 illustrates a chromatograph depicting the High Performance Liquid Chromatography (HPLC) profile of a fraction obtained by using the method of extraction in accordance with an embodiment of the invention.
[0006] FIG. 2 illustrates a table depicting the peak values of various pharmacologically active ingredients present in a fraction obtained by using the method of extraction in accordance with an embodiment of the invention.

DETAILED DESCRIPTION
[0007] Before describing in detail embodiments that are in accordance with the invention, it should be observed that the embodiments reside primarily in combinations of method steps related to extraction of a fraction containing a particular combination of pharmacologically active ingredients from WS. Accordingly, the method steps have been described to include only those specific details that are pertinent to understanding the embodiments of the invention so as not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having the benefit of the description herein.
[0008] In this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element preceeded by "comprises ...a" does not, without more constraints, preclude the existence of additional identical elements in the process, method, article, or apparatus that comprises the element.
[0009] Generally speaking, pursuant to various embodiments, the invention provides a method of extraction from WS and a fraction containing a combination of pharmacologically active ingredients extracted from WS using the method of extraction.
[0010] The plant WS belongs to the family Solanaceae or nightshade. According to the various embodiments of the invention, whole plant or parts including one or more of, but not limited to leaves, roots, bark, stems, flowers, and fruits may be used as a plant material for extraction. The plant material may be processed chemically or physically before extraction or optionally un-processed plant material may be used. For example, the plant material may be powdered roots of WS. Further, the powder may have particle size

ranging from ultra coarse to ultra fine. For example, coarse powder of the roots of WS may be used as the plant material for extraction.
[0011] Chemical reagents used in the method of extraction according to the various embodiments of the invention include, water, one or more alcohols, one or more organic solvents, one or more de-fatting agents and one or more de-pigmenting agents. Alcohols can be for example, but not limited to, methanol, ethanol, isopropyl alcohol and any other alcohol with comparable polarity and dipole moment. Organic solvents can be for example, but not limited to, chloroform, acetone, dichloromethane, tetrachloromethane and the like. De-fatting agents and de-pigmenting agents can be for example, but not limited to, hexane, heptanes, diethyl ether and petroleum ether.
[0012] In accordance with an embodiment of the invention, coarsely powdered roots of WS are used as the plant material. The plant material is taken in a suitable vessel and aqueous methanol and chloroform are added to the vessel to obtain a mixture in which the plant material is soaked. The concentration of aqueous methanol may range from 0% to 100 %, preferably, from 10 to 90% and more preferably form 20% to 80% and still more preferably from 30% to 70%. In an instance, the concentration of aqueous methanol used is 60%. It will be appreciated by a person skilled in the art that, the plant material, aqueous methanol and chloroform may be mixed in any suitable sequence and.proportion to get a mixture of the plant material, aqueous methanol and chloroform. In an instance, the coarsely powdered roots of WS are taken in a vessel of appropriate size. Aqueous Methanol is then added to the coarsely powdered roots of WS. Subsequently Chloroform is added to the vessel to get a mixture of the coarsely powdered roots of WS, aqueous methanol and chloroform.
[0013] In accordance with the above embodiment, the plant material is soaked in the mixture of aqueous methanol and chloroform for a pre-determined time period. The pre¬determined time period for soaking the plant material in the mixture of aqueous methanol and chloroform may range from 1 to 72 hours, more preferably from 1 to 24 hrs and still more preferably from 1 to 12 hrs. In an instance, the powdered roots of WS are soaked in the mixture of aqueous methanol and chloroform overnight (8 to 12 hours). Although, the

mixture may be kept at room temperature for 8 to 12 hours, it will be appreciated by a person skilled in the art that to reduce the extraction time, the mixture to be extracted may be heated up to an appropriate temperature ranging from 30°C to 70°C.
[0014] The mixture obtained in the above embodiment may be subjected to intermittent shaking during soaking of the plant material. The shaking of the mixture may be achieved by using any appropriate laboratory or industrial stirrer/shaker or optionally the mixture may be stirred manually by using an appropriate stirrer.
[0015] Soaking of the plant material in a solvent for a particular time constitutes maceration. Several solvents may be used for the process of maceration including, but not limited to, water and other organic solvents for example, acetone, methanol, dichloromethane and the like. Generally, the secondary metabolites that constitute the pharmacologically active ingredients in a plant material are located deep inside the plant tissues. Therefore, in order to extract the pharmacologically active ingredients effectively, the solvent should penetrate the tissues and cells of the plant material so that the secondary metabolites may get dissolved in the solvent and can be further extracted from that solvent.
[0016] In the above embodiment of the invention, water present in the aqueous methanol swells the tissues of the plant material and dissolves one or more pharmacologically active ingredients of WS. Further, methanol being a more potent extraction solvent with a wide range of solubility dissolves more pharmacologically active metabolites present in WS. The use of chloroform helps in extraction of other pharmacologically active ingredients of WS in addition to the pharmacologically active ingredients extracted by methanol and water. Thus, soaking of the plant material in the mixture of aqueous methanol and chloroform, results in dissolution of the pharmacologically active ingredients of WS in two fractions i.e. aqueous methanol fraction and chloroform fraction simultaneously. Further, using water, methanol and chloroform simultaneously leads to direct separation of Withaferin A into the chloroform fraction.

[0017] In another embodiment of the invention, methanol may be replaced by one or more of, but not limited to, ethanol, propanol or any other alcohol with a comparable polarity and dipole moment, to soak the plant material. In yet another embodiment of the invention, chloroform may be replaced by one or more of, but not limited to, acetone, dichloromethane, tetrachloromethane and the like, to soak the plant material. In an exemplary embodiment, coarsely powdered roots of WS are taken in a vessel. Aqueous propanol is then added to the vessel. Subsequently acetone is added to the vessel to get a mixture of the coarsely powdered roots of WS, aqueous propanol and acetone.
[0018] In the above embodiment, after, the plant material is soaked in the mixture of aqueous methanol and chloroform for the pre-determined time period, the mixture is filtered to obtain a first filtrate and a first residue. Filtration may be achieved by using appropriate laboratory or industrial filtration procedures. The first filtrate obtained after filtration may include two or more immiscible layers of solvents based on the solvents used. In this embodiment, the first filtrate includes a first layer and a second layer. The first layer and the second layer are immiscible. The first layer constitutes an aqueous methanol layer and the second layer constitutes a chloroform layer. The first layer and the second layer are then separated by using a separating funnel or any other appropriate laboratory or industrial procedures and/or apparatus. Subsequently, the first layer and the second layer are concentrated separately by subjecting the first layer and the second layer to an evaporation process. Appropriate industrial or laboratory evaporation apparatus and procedures may be used for the evaporation of the first layer and the second layer. Concentration of the first layer and the second layer may be done up to a duration so as to get a first dry extract and a second dry extract respectively. The first dry extract and the second dry extract are then weighed separately.
[0019] In accordance with the above embodiment, the first dry extract constitutes a first hydro-alcoholic fraction and the second dry extract constitutes a first chloroform fraction. The second dry extract is further dissoluted in hexane to obtain a first solution containing a first hexane soluble fraction and a second residue. The first solution is then filtered using any appropriate laboratory or industrial filtration procedures or apparatus.

Alternatively, two or more immiscible layers in the first solution may be separated by appropriate solvent partitioning techniques and instruments. The first hexane soluble fraction is discarded and the second residue is collected. In accordance with this embodiment of the invention, the second residue constitutes a first fraction containing predominantly Withaferin A in combination with other pharmacologically active ingredients of WS obtained in lesser quantities as compared to Withaferin A. Further, in accordance with this embodiment of the invention, the first hydro-alcoholic fraction contains additional pharmacologically active ingredients of WS.
[0020] In another embodiment of the invention, hexane may be replaced by one or more of, but not limited to, heptanes, diethyl ether, petroleum ether or any other suitable de-fatting agent and/or de-pigmenting agent. In an exemplary embodiment, petroleum ether is used to dissolve the second dry extract.
[0021] In accordance with the various embodiments of the invention, use of de-fatting agents and/or de-pigmenting agents as solvents in the method of extraction facilitates removal of lipids and pigments, which may impart toxicity to the one or more fractions extracted, if not isolated therefrom. Further, the first fraction obtained was found to have reduced cytotoxicity as the other pharmacologically active ingredients that are present in lesser quantities as compared to Withaferin A in the first fraction, possibly generate a more cyto-protective effect.
[0022] In accordance with another embodiment of the invention, the first residue may be subjected to re-extraction process according to the method as described in the above embodiment of the invention. For example, the first residue is soaked in a mixture of aqueous methanol and chloroform overnight (for 8 to 12 hours). Subsequently, the mixture is filtered to obtain a second filtrate and a third residue. The second filtrate includes two immiscible layers. The two immiscible layers include an aqueous methanol layer and a chloroform layer. The aqueous methanol layer and the chloroform layer are separated by using a separating funnel. Further, the separated aqueous methanol layer and chloroform layer are concentrated by subjecting to evaporation using a rotary evaporator to obtain a third dry extract and a fourth dry extract. In accordance with this embodiment

of the invention, the third dry extract constitutes a second hydro-alcoholic fraction and the fourth dry extract constitutes a second chloroform fraction. The fourth dry extract is then dissoluted in hexane to obtain a second solution containing a second hexane soluble fraction and a fourth residue. Thereafter, the second solution is filtered. The second hexane soluble fraction is discarded and the fourth residue is collected. In accordance with this embodiment of the invention, the fourth residue constitutes a second fraction containing predominantly Withaferin A in combination with other pharmacologically active ingredients of WS obtained in lesser quantities as compared to Withaferin A. Further, in accordance with this embodiment of the invention, the second hydro-alcoholic fraction contains additional pharmacologically active ingredients of WS.
[0023] In accordance with another embodiment of the invention, the total yield of a fraction containing predominantly Withaferin A along with other pharmacologically active ingredients in WS obtained in lesser quantities as compared to Withaferin A, is calculated as the sum of the first fraction and the second fraction. The total yield of a hydro-alcoholic fraction containing additional pharmacologically active ingredients is calculated as the sum of yield of the first hydro-alcoholic fraction and the second hydro-alcoholic fraction.
[0024] The fraction constituted by the second residue and the fourth residue was subjected to preliminary analysis by methods known in the art like, Thin Layer Chromatography (TLC) and High Performance Liquid Chromatography (HPLC). The fraction was found to contain predominantly Withaferin A along with other pharmacologically active ingredients of WS.
[0025] FIG. 1 illustrates a chromatograph depicting the HPLC profile of a fraction obtained by using the method of extraction in accordance with an embodiment of the invention. FIG. 1 was obtained as result of a preliminary analysis of the fraction obtained. Various peaks corresponding to different pharmacologically active ingredients present in the fraction obtained are shown in FIG. 1. Further, one peak corresponding to Withaferin

A that is predominant over other smaller peaks corresponding to the other pharmacologically active ingredients of WS is also shown in FIG. 1.
[0026] FIG. 2 illustrates a table depicting the peak values of various pharmacologically active ingredients present in a fraction obtained by using the method of extraction in accordance with an embodiment of the invention. As shown in FIG. 2, peak number 9 has the maximum area under it. FIG. 2 was obtained as a result of a preliminary analysis of the fraction and it was found that the fraction predominantly contained Withaferin A corresponding to peak number 9. In addition, other pharmacologically active ingredients corresponding to other peaks were also found to be present in lesser quantities. The other pharmacologically active ingredients present in the fraction are depicted by peaks 1 to 8 and peaks 10 to 11 in the FIG. 2.
[0027] The fraction as constituted by the second residue and the fourth residue, containing predominantly Withaferin A along with other pharmacologically active ingredients, as described in the above embodiments of the invention was studied for use in cancers during the preliminary evaluation of the fraction. One of the batch of the fraction was evaluated on the basis of parameters like 50% Inhibitory Concentration (IC50), Total Growth Inhibition (TGI) and 50% Lethal Concentration (LC50). Based on the evaluation of 1C50, TGI and LC50, it was concluded that the fraction inhibited the proliferation of human cancer cells and thus was found to be effective and efficacious in treatment of cancers. In another preliminary study the fraction was also found to reduce the side effects associated with chemotherapy and radiotherapy when the fraction was used in combination with chemotherapy and/or radiotherapy. The fraction when used in combination with chemotherapy and/or radiotherapy was found to exhibit synergistic effects and enhanced efficacy.
[0028] During the preliminary studies, the hydro-alcoholic fraction containing additional pharmacologically active ingredients of WS, obtained by the method of extraction as described in the above embodiments of the invention, was found to inhibit pro¬inflammatory cytokines including, Tumor Necrosis Factors (TNF), Interleukins (IL), Cyclo-oxygenases (COX) and some other target receptors. Hence, the hydro-alcoholic

fraction obtained by the method of extraction as described in the above embodiments of the invention may be useful in the treatment of one or more inflammatory diseases. The one or more inflammatory diseases may include, but not limited to, Ankylosing Spondylitis, Psoriasis, Rheumatoid Arthritis, Osteoarthritis, Multiple sclerosis, Atherosclerosis, Alzheimer's disease, and other types of Arthritis. During the preliminary studies, the hydro-alcoholic fraction was also found to inhibit phosphodiesterase (PDE) and hence may be useful in the treatment of Asthma and other PDE mediated diseases. The hydro-alcoholic fraction may also be useful in the treatment of Diabetes.
[0029] The one or more fractions obtained by the method of extraction as described in the above embodiments of the invention were also tested for cytotoxicity. LD50 for a fraction containing predominantly Withaferin A along with other pharmacologically active ingredients was found to be very high as compared with the LD50 of pure Withaferin A. Hence, it was concluded that the fraction containing predominantly Withaferin A along with other pharmacologically active ingredients is significantly less cytotoxic as compared to pure Withaferin A.
[0030] The overall yield of the one or more fractions was also found to be significantly higher when the one or more fractions were prepared according to the method of extraction as described in the above embodiments the invention. On the other hand, when extraction was carried out using the methods known in the art. the yield of the pharmacologically active ingredients of WS like Withaferin A and other pharmacologically active ingredients was found to be very low as compared to the yield obtained by the method of extraction as described in the invention.
[0031] Various embodiments of the invention provide a method for extraction from WS and one or more fractions containing pharmacologically active ingredients extracted from WS by using the method of extraction. The invention provides a fraction that predominantly contains Withaferin A along with other pharmacologically active ingredients in lesser quantities as compared to Withaferin A. Further, the invention provides one or more fractions that have significantly reduced cytotoxicity. The invention also provides a fraction that mhibits the proliferation of cancer cells and is thus found to

be effective and efficacious in the treatment of cancers. The invention further provides a method for extraction of a fraction that gives higher yields of the fraction and particularly higher proportion of Withaferin A in the fraction. The invention also provides a fraction that causes synergistic effects and enhanced efficacy when used in combination with chemotherapy and/or radiotherapy. The invention further provides a fraction that reduces side effects when used in combination with chemotherapy and/or radiotherapy. The invention also provides another fraction containing additional pharmacologically active ingredients of WS. The invention further provides another fraction containing additional pharmacologically active ingredients that exhibits anti-inflammatory activity and may be used for the treatment of inflammatory diseases.
[0032] Those skilled in the art will realize that the above recognized advantages and other advantages described herein are merely exemplary and are not meant to be a complete rendering of all of the advantages of the various embodiments of the invention.
[0033] In the foregoing specification, specific embodiments of the invention have been described. However, one of ordinary skill in the art appreciates that various modifications and changes can be made to the invention without departing from the scope of the invention. Accordingly, the specification is to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of the invention.
Signature:
Gowree Gokhale Constituted Patent Agent for the Applicant