DESC:-: Complete Specification :-

”Glycosidase inhibitor, process for its preparation, and applications thereof”

Cross references to related applications: This complete specification is filed further to application for patent No. 201921036657 filed on 12/09/2019 with provisional specification, the contents of which are incorporated herein in their entirety, by reference.

Field of the invention
This invention relates generally to compounds showing glycosidase inhibitory activity. More specifically, the present invention attempts to propound a selective glycosidase inhibitor derived from a plant source. Also disclosed herein are the inventive manner of elucidation and use of said selective glycosidase inhibitor.

Definitions and interpretations
Before undertaking the detailed description of the invention below, it may be advantageous to set forth definitions of certain words or phrases used throughout this patent document: the terms “include” and “comprise,” as well as derivatives thereof, mean inclusion without limitation; the term “or” is inclusive, meaning and/or; the phrases “associated with” and “associated therewith,” as well as derivatives thereof, may mean to include, be included within, interconnect, with, contain, be contained within, connect to or with, couple to or with, be communicable with, cooperate with, interleave, juxtapose, be proximate to, be bound to or with, have, have a property of, or the like; and the term “DDEB” refers Dehydrodieugenol B.

Background of the invention and description of related art
Glycoside hydrolases, also known as Glycosidases, are a type of carbohydrate-processing hydrolytic enzymes which catalyze breakdown of a wide range of substrates including di / oligo / poly - saccharides and glyconjugates.

In vivo, glycosidases are involved in myriad functions including glycosylation of proteins, plant cell wall biosynthesis, breakdown of ingested material in the gut and defense mechanisms against microbial infection. Glycosidases are harnessed, in-vitro, as proficient biocatalysts in processes involving degradation of biomass such as cellulose, hemicellulose, xylan, chitin, starch and so on.
To name a few, lactase, amylase, chitinase, sucrase, maltase, neuraminidase, invertase, hyaluronidase and lysozyme are popular glycosidases observed to be commercially exploited on industrial scale in bakery, detergent, textile, and fuel alcohol production industries. (Ajita Sundarram et al. a-Amylase Production and Applications: A Review. Journal of Applied & Environmental Microbiology, 2014, Vol. 2, No. 4, 166-175. doi:10.12691/jaem-2-4-10).

Furthermore, glycoside hydrolases are predicted to gain increasing roles as catalysts in biorefining applications (Linares-Pastén, J. A.; Andersson, M; Nordberg karlsson, E (2014). "Thermostable glycoside hydrolases in biorefinery technologies". Current Biotechnology. 3 (1): 26–44.).

In an isolated medical application, a combination of alpha-amylase and cellulase was shown to degrade polymicrobial bacterial biofilms and thus increase antibiotic effectiveness against bacterial infection (Fleming, Derek; Chahin, Laura; Rumbaugh, Kendra (February 2017). "Glycoside Hydrolases Degrade Polymicrobial Bacterial Biofilms in Wounds". Antimicrobial Agents and Chemotherapy. 61 (2): AAC.01998–16)

The reader shall appreciate therefore the manner and extent in which glycosidases are readily applicable all domains of life. In this parlance, it is extremely desirable to have some means for precise control and inhibition of their activity for desired / metered results and avoidance of adverse biochemical reactions and / or products.

State of art bears mention of many compounds which are known to inhibit the action of glycoside hydrolases. Notable to mention are naturally occurring heterocyclics such as deoxynojirimycin, swainsonine, australine and castanospermine as well as isofagomine and deoxygalactonojirimycin which are synthesized using these natural heterocyclic chemical structures. In clinical use, glycosidase inhibitors acarbose and miglitol are popularly applied as anti-diabetic medicaments, whereas oseltamivir and zanamivir find application as effective antivirals.

On above background, the identities, sources, elucidation protocols, and methodologies for use of glycosidase inhibitors has undeniably assumed great global importance today. Unfortunately, systematic study and development in this line of research has been majorly sidelined or otherwise eluded the scientific community till date. Therefore, major considerations that remain to be addressed, are-

1) Difficulties in identification of able glycosidase inhibitors;
2) Difficulties in identification of able natural sources of glycosidase inhibitors; and
3) Difficulties in extraction, purification and separation of active glycosidase inhibitors from natural sources,

The inventors named herein have contended with aforesaid issues, specifically concentrating on the difficulties in extraction, purification and separation of glycosidase inhibitors from natural sources, especially of plant origin.

Prior art, to the limited extent presently surveyed, does not list a single effective solution embracing all considerations mentioned hereinabove, thus preserving an acute necessity for new, efficient and safe glucosidase inhibitors. This, among other aims, have fueled research undertaken by the inventors named herein who consequently have come up with novel solutions for resolving all needs of the art once and for all.

Work of the presently named inventors, specifically directed against the technical problems recited hereinabove and currently part of the public domain including earlier filed patent applications and research publications, is neither expressly nor impliedly admitted as prior art against the present disclosures.

Attention of the reader is now requested to the detailed description to follow which narrates a preferred embodiment of the present invention and such other ways in which principles of the invention may be employed without parting from the essence of the invention claimed herein.

Objectives of the present invention
The present invention is identified in addressing at least all major deficiencies of art discussed in the foregoing section by effectively addressing the objectives stated under, of which:

It is a primary objective to provide a method capable of elucidating aselective glycosidase inhibitor from a plant source.
It is another objective further to the aforesaid objective(s) to establish able utility of the selective glycosidase inhibitor so elucidated for various applications.

The manner in which the above objectives are achieved, together with other objects and advantages which will become subsequently apparent, reside in the detailed description set forth below in reference to the accompanying drawings and furthermore specifically outlined in the independent claim 1. Other advantageous embodiments of the invention are specified in the dependent claims.

Detailed description
Principally, general purpose of the present invention is to assess disabilities and shortcomings inherent to known systems comprising state of the art and develop new systems incorporating all available advantages of known art and none of its disadvantages.

Accordingly, the disclosures herein are directed towards the establishment of a selective glycosidase inhibitor characterized in being derived from a plant source, therein specifically the inventive manner of its elucidation and furthermore its use for various applications.

Rationale of the present invention may be appreciated from that conventional methods for extraction of biologically-relevant molecules typically use harsh conditions such as organic solvents (such as toulene, acetone, chloroform, hexane) and high temperatures which generally degrades such molecules (which are otherwise active under ambient conditions) and thereby result in loss of activity. The approach propounded herein sequentially separates the bio-molecules based on their polarity in the extraction. Here, aqueous extraction separates water-soluble biomolecules from remaining molecules and in next step, use of less harsh solvents with increasing non-polarity helps in separation of bio-molecules. Thus, by using this approach, it is possible to achieve polarity-based selective screen for isolation of biomolecules from plants. This approach reduces the burden of additional purification steps and also leads to isolation of previously unknown bio-molecules and interfering substances from the plant material which would have been degraded and lost their biological activity by conventional methods.

According to one aspect of the present invention, the selective glycosidase inhibitor claimed herein was extracted from Ocimum tenuiflorum via a three stage elution process exemplified in performance of the following steps-
1) Leaves of Ocimum tenuiflorum were thoroughly cleaned, washed with tap water and crushed in presence of liquid nitrogen to obtain a powder.
2) 100 g of Powder obtained at step 1) was immediately added to distilled water in the ratio of 1:4. The admixture was agitated for 8 hours at room temperature.
3) The supernatant, that is Cold Water Extract (CWE), resulting from step 2) was separated from the solid powder by centrifugation at 8000 rpm, 10 minutes.
4) Powder remaining after step 3) was dried in shed at room temperature, which was then resuspended in water in 1:4 ratio and subjected first to agitation for 3 hours at 60oC and afterward to centrifugation at 8000 rpm for 10 minutes to yield supernatant, that is Hot Water Extract (HWE).
5) Residual powder obtained after centrifugation at step 4) was resuspended in methanol in ratio of 1:3 and agitated for 3 hours at 60°C. Centrifugation of this mixture yielded supernatant, that is Methanol extract (ME) after centrifugation.
6) Residual powder obtained after centrifugation at step 5) was resuspended in isopropanol in ratio of 1:3 and agitated for 3 hours at 60°C. Centrifugation of this mixture yielded supernatant, that is isopropanol extract (IPE) which was filtered and stored at -20°C. About 5.6 g of residual powder remained after above extraction process which was dried and stored in air tight container atcool dark place away from sunlight.

Advancement of the present invention can be appreciated from that it carefully involves the extraction of relevant biomolecule from plant part (leaves) using a specific sequence of aqueous solvents followed by methanol and isopropanol. This invention avoids the use of harsh physico-chemical conditions like elevated temperatures and use of harsh organic non-polar solvents such as acetone, chloroform, hexane, and toluene. Thus, making the overall extraction process more environment-friendly, more safe for operation and helps in retaining the structural integrity and stability of bio-molecules from plant which his crucial for their biological activity.

Uniqueness of the sequential extraction process described hereinabove shall be appreciated from that conventional methods for extraction of small molecules from plant parts typically use organic solvents (ethanol, methanol, isopropanol, acetone, hexane, etc.) or their combinations. In contrast, the present invention propounds the sequential extraction using Ocimum tenuiflorum leaves, by using single solvent at a time with increasing the degree of non-polarity of the solvent. Consequently during this extraction process, which is in contrast to conventional extraction methods, different molecules get extracted in different solvents used based on their relative polarity with the solvent being used. This also results in removal of substances which would otherwise interfere in the extraction process of a biologically active molecule (that is DDEB in this case). Thus, based on the principle of like-dissolves-in-like, in the first two steps, all water-soluble molecules like sugars, proteins get extracted. To facilitate the extraction of water-soluble components, water was added in a higher (1: 4) sample: solvent ratio. Thus, the powder obtained after water extracts used for the extraction using methanol and isopropanol has no other interfering water-soluble components. For methanol and isopropanol extracts, the sample: solvent ratio used was 1:3, which allowed efficient extraction of molecules from the powder with minimal solvent. At the same time, the refluxing of the powder in solvents for 3 hours close to the boiling points results in increased extraction efficiency. Dehydrodieugenol B was extracted in isopropanol extract due to its more solubility in isopropanol than methanol.

For further purification of DDEB, silica gel column chromatography was used. After the step of silica gel column chromatography, different purification tools and techniques used conventionally for the purification of biomolecules from plant extracts, like solvent-solvent partitioning, preparative TLC, Sephadex LH-20, flash silica column chromatography lead to very low purification yield, unstable compound, cross-reactivity with the material leading to loss of biological activity, etc.

The reader shall note that the steps that are routinely followed for the isolation and purification of extracts to obtain natural products from plants are generally in reverse order from non-polar to polar resulting in differential extractions. But, this order is unable to extract and obtain DDEB in good yields and purity. A literature survey undertaken by the applicant named herein has confirmed that DDEB is not obtained from Ocimum using extraction in non-polar solvents, thereby qualifying novelty of this invention.

Silica gel column chromatography was followed by preparative HPLC, which was carried out at 25oC. The preparative HPLC resulted in the purification of a pure and stable (in terms of bio-activity) molecule without any cross-reactions and other issues discussed above. It is to be noted that the use of temperatures beyond 30 C resulted in the loss of biological activity. Further, to ascertain the structure, spectral and 1D, 2D NMR experiments were performed. On the comparison of data from these experiments with reported structure from plants other than the present plant (Ocimum tenuiflorum), DDEB structure was assigned. To be noted that, though various studies report many structures of small molecules from O. tenuiflorum, there is no report of isolation/purification of DDEB from Ocimum sp.

Thus, the above observations prove that the unique process detailed in this invention has to be followed to obtain DDEB, a bioactive molecule that is unable to be obtained using conventional methods.

Industrial Applicability
The edible oil-based formulations of crude IPE and DDEB were developed and assessed by the applicant named herein for bio-activities (inhibition of glycosidases) and stability (shelf-life) at room temperature for 15 days.

Keeping in mind the oral route of administration, commercially available edible sunflower oil fortified with vitamin A, D, and E was used as a vehicle for formulation. Both crude IPE and DDEB retained PPA and HPA inhibition for 15 days in oil at room temperature (24-27oC).

Also, for other industrial applications, inhibition of various glycosidases was assayed with formulations of crude IPE and DDEB. Amylases from human, porcine, bacterial, and fungal sources were well-inhibited by both DDEB and crude IPE at 0th and 15th-day indicating applications in food and feed processing industries.

Strong xylanase inhibition was observed for crude IPE and DDEB for the 0th and 15th days. This indicates a possible role of crude IPE and DDEB in xylan processing industries. Similarly, strong Epoxide hydrolase activity inhibition is also noted suggesting its potential in the pharmaceutical industry.

For crude IPE and DDEB, very less/minimal (<10%) inhibition of cellulase was observed on the 0th and 15th day.

To study the stability the formulations of crude IPE and DDEB were checked for microbial contamination against bacterial and fungal contamination. No microbial contamination was observed at the end of the 20-day shelf-storage of formulations of crude IPE and DDEB at room temperature. This confirms the storage-stability of the formulations as well as the anti-microbial efficacy of crude IPE and DDEB.

As will be further readily appreciated, the inventive procedure of this invention has resulted into the isolation of a bio-molecule, viz. DDEB, with potential industrial and research applications, including-
1) Pancreatic alpha amylase inhibitor for reducing hyperglycemia
2) Epoxide hydrolase inhibitor for reducing inflammation and related metabolic disorders
3) Beta amylase / glucosidase inhibitor to prevent early ripening of agricultural produce (fruits, vegetables etc)
4) Increasing the shelf life of food (ready to eat foods) products and non alcoholic beverages by preventing enzymatic degradation
5) Preventing adulteration of milk, by acting as lactase inhibitor
6) Controlling the rate of industrially important processes like fuel alcohol production, in paper and textile industry by removal of xylans and starch sizing agent from woven fabric,
7) Controlling the rate of cellulose digestion in textile, paper and wood pulp industries
8) Controlling the rate of starch of digestion in dough to control production of smaller fermentable sugar
9) Quenching / stopping / controlling the reaction as a part of reagent based assay and diagnostic kits
10) As an antimicrobial

The overall glycosidase inhibition and stability study highlight the importance of the crucial role of the invention, i.e., the sequential solvent extraction process using which crude IPE was obtained leading to the isolation of DDEB, a molecule previously not reported from Ocimum tenuiflorum. Inhibition of glycosidases such as amylase, epoxide hydrolase, and xylanase by formulations of crude IPE and DDEB open the door for other similar industrial applications, highlighting the patentability of the invention

As will be realized further, the present invention is capable of various other embodiments and that its several components and related details are capable of various alterations, all without departing from the basic concept of the present invention. Modifications and variations of the system and apparatus described herein will be obvious to those skilled in the art. Such modifications and variations are intended to come within ambit of the present invention, which is limited only by the appended claims. ,CLAIMS:1] A method for elucidation of a glycosidase inhibitor from plant material, comprising-

a) Conditioning the plant material, leaves of Ocimum tenuiflorum in particular, to result in a powder of said plant material;

b) Subjecting the conditioned plant material, leaves of Ocimum tenuiflorum in particular, to a three-step extraction process using a specific sequence of solvents, being distilled water, methanol and isopropanol in corresponding elution steps, to progressively allow differential elution of molecules present in the plant material for elucidation of a solution containing the glycosidase inhibitor, Dehydrodieugenol B in particular;

c) Subjecting the solution reached at step b) to a downstream protocol arranged to result in a dried preparation of the glycosidase inhibitor, Dehydrodieugenol B in particular.

Characterized in that
? the sequential extraction process is undertaken using a single solvent at a time with increasing the degree of non-polarity of the solvent used, to thereby result in different molecules present in the plant material being progressively eluted, on basis of their relative polarity, with the sequence of solvents used, therein ensuring that each successive solvent has no interference from molecules soluble in the preceding solvent; and

? the preparation of the glycosidase inhibitor, Dehydrodieugenol B in particular reached in step c), is pure, biologically active, and self-stable.

2] The method for elucidation of a glycosidase inhibitor from plant material as claimed in claim 1, wherein the step of conditioning the plant material, leaves of Ocimum tenuiflorum in particular, prior to being subjected to a sequential extraction process consists of-

a) Cleaning and washing the plant material, leaves of Ocimum tenuiflorum in particular,with tap water for removal of soil, dust, and other debris;

b) Crushing the cleaned and washed plant material, leaves of Ocimum tenuiflorum in particular, in presence of liquid nitrogen to obtain a powder of said plant material.

3] The method for elucidation of a glycosidase inhibitor from plant material as claimed in claims 1 and 2, wherein the first elution step in the extraction process using the specific sequence of solvents consists of-

(i) admixing 100g of the plant material, which is powdered in presence of liquid nitrogen, with distilled water in a relative proportion of 1:4 to result in an admixture;

(ii) subjecting the admixture to agitation for 8 hours at room temperature to result in a cold water extract;

(iii) Subjecting the cold water extract to centrifugation at 8000 RPM for 10 minutes to result in a powder of said plant material;

(iv) Drying the powder obtained at step (iii) in shed at room temperature; and

(v) Suspending the dried powder in water at a ratio of 1:4, and subjecting suspension to agitation for 3 hours at 60oC followed by centrifugation at 8000 rpm for 10 minutes to yield a supernatant, being a hot water extract containing the glycosidase inhibitor, Dehydrodieugenol B in particular.

4] The method for elucidation of a glycosidase inhibitor from plant material as claimed in claim 1, wherein the second elution step in the extraction process using the specific sequence of solvents consists of–

(i) Suspending the dried powder obtained from the first elution step in methanol at a ratio of 1:3 respectively to result in an admixture;

(ii) subjecting the admixture to agitation for 3 hours at 60°C and centrifugation to result in a methanol extract containing the glycosidase inhibitor, Dehydrodieugenol B in particular.

5] The method for elucidation of a glycosidase inhibitor from plant material as claimed in claim 1, wherein the third elution step in the extraction process using the specific sequence of solvents consists of -

(i) Suspending the dried powder obtained from the second elution step in isopropanol in ratio of 1:3 respectively to result in an admixture;

(ii) subjecting the admixture to agitation for 3 hours at 60°C and centrifugation to result in an isopropanol extract containing the glycosidase inhibitor, Dehydrodieugenol B in particular.

6] The method for elucidation of a glycosidase inhibitor from plant material as claimed in claim 1, wherein the downstream protocol arranged to result in a dried preparation of the glycosidase inhibitor consists of-

a) Filtration of the dried powder obtained from the third elution step to result in a dried pure preparation of the glycosidase inhibitor, Dehydrodieugenol B in particular; and

b) Storing the dried pure preparation of the glycosidase inhibitor, Dehydrodieugenol B in particularat -20°C.

7] The dried pure preparation of the glycosidase inhibitor, Dehydrodieugenol B in particular obtained by the method of claim 1, characterised in having-
a) A yield of 5.6 g per 100 g of the conditioned plant material;

b) Biological activity demonstrable by marked inhibition of glycosidases, xylanases, amylases and cellulases in particular; and

c) self-stability and anti-microbial propensity as marked by no microbial contamination up to 20 days of storage at room temperature.

8] The method for elucidation of a glycosidase inhibitor from plant material as substantially described in the accompanying description.