This invention relates to anti-diabetic potential in root & flower extracts of bhangira (perilla frutescens).
Background of the Invention
US20190090448 The present invention relates to a novel and distinct cultivar of Perilla frutescens designated as ‘CAP HEMA’ The new cultivar is rich in omega-3 and having high seed and fatty oil yield. A method for producing new cultivar of Perilla frutescens plant which comprises selection of half-sib family from wild type populations. The method further comprises evaluation for the yield attributing characters of selected strains in field conditions. The genotype produced is distinct, uniform and stably maintained by continuous rouging of off types in the population at early seedling stage from nursery itself and suitable for commercial. This is suited for high seed, fatty oil yield and rich in omega-3 fatty acid cultivation.
Research Gap: In this patent, only claimed for omega-3 in seeds used for heart problems. But not climed for antidiabetic property in root extract
None of the prior art indicate above either alone or in combination with one another disclose what the present invention has disclosed. Present invention is anti-diabetic potential in root & flower extracts of bhangira (perilla frutescens).
P2116138B1 It is intended to provide a syrup having a vivid red color which is extracted from Perilla frutescens var. crispa f. purpurea. The syrup is prepared by the following steps: (1) the step of blanching P. frutescens var. crispa f. purpurea and then draining; (2) the step of extracting the blanched P. frutescens var. crispa f. purpurea with water under heating; and (3) the step of adding an acid to the P. frutescens var. crispa f. purpurea extract
Research Gap: No work on root extract and not climed for antidiabetic property in root extract of Perilla frutescens
SUMMARY OF THE INVENTION
This summary is provided to introduce a selection of concepts, in a simplified format, that are further described in the detailed description of the invention.
This summary is neither intended to identify key or essential inventive concepts of the invention and nor is it intended for determining the scope of the invention.
To further clarify advantages and features of the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which is illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings.
Discloses herein an anti-diabetic Formulation of root & flower extracts of Bhangira (Perilla Frutescens) comprises Root extract of Perilla frutescens has a amylase inhibition from 70 to 80 % with concentration of 2 to 2.5 mg/ml of alcoholic or methanolic extract; Flower extract is Perilla frutescens (Bhangira ) having potential of 50 to 60% inhibition; IC 50 value of root extract about 1 to 1.1 mg/ml as compared to standard medicine/drug IC value 0.7 to 8 mg/ml; Wherein alpha amylase, which is indicator of anti-diabetic property shown by root extract (about 73% to 80 % inhibition at concentration of 1 to 25 mg/ml) is better than flower or stem other extract as compared to standard drug (88%) is significant and has potential of using as an anti-diabetic medicine. The roots, are separated and washed with distilled water and then sun dried in sunlight, flowers are dried in shade; after drying foreign matters and dust and dirt are removed. The roots and flowers were converted into powder with help of a grinder.
For Preparation of extracts for GC-MS analysis and antidiabetic activity: Powders (5 grams each) of root, flowers are extracted separately with methanol (each 50 ml) by using cold maceration technique. Later mixtures are filtered and evaporated the solvents till dryness at 800 C± 20 C on water bath; and the obtained semisolid liquid/extracts were kept in a refrigerator at 4°C for use.
An individual extract from Perilla frutescens (L) plant is injected into a Shimadzu GCMSQP2010Ultra system attached with a selective mass detector with an ion source of 230°C and 270°C surface temperature.
The operation of the instrument for analysis was as follows: Oven temperature 140°C for 6 min then 280°C at 10°C/min and maintained for 31 min; The sample injection is 1.0 microliter and the transporter gas was helium with velocity of 41.6 cm/s. used The evaluation of compounds/components was based on the retention time (RT) for GC and the elucidation of mass spectra was executed by relating the obtained spectral fragmentation, with the database provided by WILEY8 and NIST Library.
For Alpha amylase inhibitors assays Starch-Iodine colour assay: The starch- iodine assay is executed for screening Bhangira as alpha-amylase inhibitors, the overall quantity of combination includes 500 µl 0.02 M sodium phosphate buffer having pH and strength 6.9, 0.02M respectively and contains 6.0 mM sodium chloride, enzyme amylase (1000 µl, Diastage Loba Chemicals) and 200 µl extracts with concentrations ranging from 0.5 to 2.0 mg/ml (w/v); for 12 minutes, the combination was placed for incubation at 37 °C. Then, each mixture received 870 µl of newly ready starch (1%, w/v) and kept for 17min for incubation at 37 °C.
To break the enzymatic reaction, 60 µl of muriatic acid. (10.0M) is transferred, before transferring of 300 µl of iodine reagent (5 mM I2 1+ 5 mM KI); at 620nm wavelength absorbance was noted and a color change was observed.
Therapeutic use of extract of specific plant “Perilla frutescens “for proving Antidiabetic property through root extract. By using root extract of this plant diabetic tendency can be suppressd. Its root extract is having strong anti-diabetic potential thorugh amylase inhibition.
BRIEF DESCRIPTION OF THE DRAWINGS
The illustrated embodiments of the subject matter will be understood by reference to the drawings, wherein like parts are designated by like numerals throughout. The following description is intended only by way of example, and simply illustrates certain selected embodiments of devices, systems, and methods that are consistent with the subject matter as claimed herein, wherein:
Figure: - 1 GC-MS analysis of methanol root extract of Perilla frutescens (L.)
Figure: - 2 GC-MS analysis of methanol extract of flower of Perilla frutescens
The figures depict embodiments of the present subject matter for the purposes of illustration only. A person skilled in the art will easily recognize from the following description that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the disclosure described herein.
DETAILED DESCRIPTION OF THE INVENTION
The detailed description of various exemplary embodiments of the disclosure is described herein with reference to the accompanying drawings. It should be noted that the embodiments are described herein in such details as to clearly communicate the disclosure. However, the amount of details provided herein is not intended to limit the anticipated variations of embodiments; on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the scope of the present disclosure as defined by the appended claims.
It is also to be understood that various arrangements may be devised that, although not explicitly described or shown herein, embody the principles of the present disclosure. Moreover, all statements herein reciting principles, aspects, and embodiments of the present disclosure, as well as specific examples, are intended to encompass equivalents thereof.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments. As used herein, the singular forms “a",” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises,” “comprising,” “includes” and/or “including,” when used herein, specify the presence of stated features, integers, steps, operations, elements and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and/or groups thereof.
It should also be noted that in some alternative implementations, the functions/acts noted may occur out of the order noted in the figures. For example, two figures shown in succession may, in fact, be executed concurrently or may sometimes be executed in the reverse order, depending upon the functionality/acts involved.
In addition, the descriptions of "first", "second", “third”, and the like in the present invention are used for the purpose of description only, and are not to be construed as indicating or implying their relative importance or implicitly indicating the number of technical features indicated. Thus, features defining "first" and "second" may include at least one of the features, either explicitly or implicitly.
Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which example embodiments belong. It will be further understood that terms, e.g., those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.
These and other advantages of the present subject matter would be described in greater detail with reference to the following figures. It should be noted that the description merely illustrates the principles of the present subject matter. It will thus be appreciated that those skilled in the art will be able to devise various arrangements that, although not explicitly described herein, embody the principles of the present subject matter and are included within its scope.
Perilla frutescens(L.) which is commonly known as Bhangira is an herb which is found at hilly region of Uttarakhand or in Himalayan region of Nepal.
In this work, extracts of different parts of Perilla frutescens( PF.) were extracted in diferent solvents and then study of phytochemical constituents were done in different extracts Further anti-diabetic potential was determined by using alpha amylase inhibition ( in-vitro) method in root, flower and other extracts of this plant. . GC-MS analysis was done for identification of constituents. Root extract has shown significant antidiabetic potential by giving more than 85% a-amylase inhibition with IC50 value of 1.1 mg/ml . Concentration of 2 to 2.5 mg/ml of extract is sufficient to control inhibition more than 80% which is comparable to Standard compound used for anti-diabetic property which shows about 88% inhibition. Alcoholic extract of this plant shows more than 70% amylase inhibition at concentration of 2mg/ml. Flower extract is also having antidiabetic potential more than 60% amylase inhibition.
By this work, it is claimed that extract of Perilla frutescens(L.) has good poeteinal of anti-diabetic acitivity which has not been observed by any other so far.Alcoholic extract has shown maximum amylase inhibition. By .The GC MS analysis of PF roots extract revealed the presence of 14 major component and flower extract exhibited 12 major components. Many other small peaks represented other phytochemicals in trace amount in both methanolic extract of flower and roots of Perilla frutescens. Phytochemical constituents like 1,2-benzenedicarboxylicacid, hexadecanoicacid, gamma sitosterol, 8,11,14-Eicosatrienoic acid are supposed to support amylase inhibition in this encouraging study. By the study it can be concluded that methanolic or ethanolic extract of root extract of Perilla frutescens (L.) is comparable to standard drug and has good anti-diabetic property to be used in medicines as ayurvedic medicine in stead of allopathy medicine.
Best Method of working:
This plant has been used by the local people for some therapeutically use for healing of some wounds or infections or local asthma or cough. But from the study there is no evidence of using root or flower extract of Perilla frutescens (L.) in anti-diabetic medicine or for anti-diabetic potential.
Perilla frutescens (L.) which is commonly known as Bhangira is an important herb of interior and remote villages of hilly region of Uttarakhand . In hilly regions people use herbal plant and their parts for therapeutic use. Peoples use seeds as a food which are also used as folklore medicine and remedy for various ailments cough, allergy, cough, intoxication, and some intestinal problems. In northern regions of India the stem part of the plant is used as pain reliever and anti-abortive agent the leaves are said to helpful for relief in asthma, colds and flu's. This plant is also found in hill areas of Nepal. Perilla frutescens(L.) i.e Bhangira is .having potential for medicinal use. earlier omega-3 content patent has been done but not claimed antidiabetic property in its root extract.
Perilla frutescens(L.) is listed in the Chinese Pharmacopoeia as a TCM and has been used for centuries as a medicinal plant for asthma, influenza , cough, and vomiting but its extract has not been used for anti-diabetic use.
Perilla frutescens(L.), belongs to the family Lamiaceae, generally called as “Bhanjira”.It is a less utilized product of Himalayan region in India. The plant is initially born to India and China and other countries (like Korea, , Japan, Thailand and other East Asian countries) are also produce this plant in large scale.
EXPERIMENTAL
Perilla frutescens L. was taken for this study in the months of October and November from the local village Sahiya of Chakrata, Dehradun, Uttarakhand, India. The plant was authenticated, and a voucher specimen was submitted in the Division of Pharmacognosy, The Himalaya Drug Company, Dehradun, India.
The roots, were separated and washed with distilled water and then sun dried in sunlight. However, flowers were dried in shade. After drying foreign matters and dust and dirt were removed. The roots and flowers were converted into powder with help of a grinder.
Preparation of extracts for GC-MS analysis and antidiabetic activity
Sample Preparation: Powders (5 grams each) of root, flowers were extracted separately with methanol (each 50 ml) by using cold maceration technique. Later mixtures were filtered and evaporated the solvents till dryness at 800 C± 20 C on water bath. The obtained semisolid liquid/extracts were kept in a refrigerator at 4°C for use.
Procedure for GCMS analysis of extracts
An individual extract from Perilla frutescens (L) plant was injected into a Shimadzu GCMSQP2010Ultra system attached with a selective mass detector with an ion source of 230°C and 270°C surface temperature. The operation of the instrument for analysis was as follows: Oven temperature 140°C for 6 min then 280°C at 10°C/min and maintained for 31 min. The sample injection was 1.0 microliter and the transporter gas was helium with velocity of 41.6 cm/s. used The evaluation of compounds/components was based on the retention time (RT) for GC and the elucidation of mass spectra was executed by relating the obtained spectral fragmentation, with the database provided by WILEY8 and NIST Library.
Alpha amylase inhibitors assays
Starch-Iodine colour assay: The starch- iodine assay was executed as defined by Xiao et al., [30] for screening Bhangira as alpha-amylase inhibitors, The overall quantity of combination includes 500 µl 0.02 M sodium phosphate buffer having pH and strength 6.9, 0.02M respectively and contains 6.0 mM sodium chloride, enzyme amylase (1000 µl, Diastage Loba Chemicals) and 200 µl extracts with concentrations ranging from 0.5 to 2.0 mg/ml (w/v). For 12 minutes, the combination was placed for incubation at 37 °C. Then, each mixture received 870 µl of newly ready starch (1%, w/v) and kept for 17min for incubation at 37 °C. To break the enzymatic reaction, 60 µl of muriatic acid. (10.0M) was transferred, before transferring of 300 µl of iodine reagent (5 mM I2 1+ 5 mM KI). At 620nm wavelength absorbance was noted and a color change was observed. There were no plant extracts added combination referred as control was used, which represent 100 percent enzyme activity. Appropriate control extracts without enzymes were also involved to abolish the absorbance produced by extracts. Starch is directed in dark blue; no starch is exposed in yellow; and partially decomposed starch is expressed as in brown in the solutions. Starch mixed to the enzyme assay mixture was not destroyed and produced a dark blue complex in the existence of an inhibitor of the extract, but no color complex was formed in the nonappearance of the inhibitor, that indicated the complete digested starch through a-amylase.
The chromogenic DNSA technique was also used to perform the inhibitory assay Miiller., 1959-[31] &Hara and Honda., 1990[32]. The whole assay tubes were comprised of 500 µl of sodium phosphate buffer of 0.02M strength and pH 6.9, this buffer solution contained 6 mM sodium chloride, 1 ml alpha amylase enzyme (DiastageLoba Chemicals), and 400 µl extracts at concentrations ranging from 0.5-2.0 mgml-1(w/v). The assay vials were placed at 37°C for 11 minutes in an incubator. After pre-incubation, vials was filled with 1 percent (v/v) 580 µl of starch (1%) moved to incubator for 17 minutes at 37°C.After completion of 17 minutes removed all tubes and stopped the reaction by dropping 1.0 ml DNSA solution .All tubes were dipped into boiling water for 3-5 minutes. Now all tubes were left on working condition for cooling. Absorbances of all mixture were taken at 540 nm using. No plant extract was poured in the control tube, which represented 100 percent enzyme activity. To exclude the absorbance caused by plant extract, appropriate extract controls were introduced in combination without the enzyme. The IC50 values were calculated as per the concentration of extract containing the a -amylase
Below given formula was applied to calculate Inhibition percentage. Inhibition (%) = Abs at 540 (control) – Abs 540 (extract) ´X 100/ Abs 540(control)
RESULTS AND DISCUSSION
Table:-1 Major Components of methanolic extract of Perilla frutescens root as identified by GCMS
Peak# R.Time Area% Name
1 19 17.442 19.22 1,2-benzenedicarboxylicacid,bis (2-methylp Antimicrobial and
alpha glucosidase inhibition and the in vivo hypoglycemic efficacy [33]
2 21 17.951 1.17 hexadecanoicacid, methylester Antioxidant,
hypocholesterolemic
nematicide, pesticide,
antiandrogenic flavor,
hemolytic, 5-Alpha reductase inhibitor
Antibacterial and antifungal [34]
3 23 18.454 9.87 n-Hexadecanoic acid Anti-inflammatory,
Antioxidant, Pesticide, Nematicide, Inhibitor [35-37]

4 25 19.590 1.68 9,12-Octadecadienoic acid (Z,Z)-,methyl ester Anti-cancer [40].
5 26 19.650 1.63 8,11,14-Eicosatrienoic acid,methylester Not reported
6 29 20.164 9.58% (R)- (-)-14-Methyl-8-hexadecyn-1-ol Not reported
8 31 20.468 1.29 6-Methyl-4,5-tetramethylene-6-ethyl-2-phenyl-5,6-dihydro-4 Not reported
9 33 21.365 1.48 1-Phenanthrene methanol,1,2,3,4,4a,9,10,10a-octahydro-6-m Not reported
10 41 23.419 1.12 Ethyl (9Z,12Z)-9,12-octadecadienoate# Not reported
11 43 23.967 4.18 Hexadecanoic acid,2-hydroxy-1- (hydroxymethyl) ethyl ester Antimicrobial [33]
12 45 27.087 24.95 ETHYL (9Z,12Z)-9,12-OCTADECADIENOATE# Not reported
13 56 38.648 1.01 4,22-Stigma stadiene-3-one Not reported
14 58 40.602 4.13 .gamma.-Sitostenone Not reported

Table:-2 Major Components of methanolic extract of Perilla frutescens flower as identified by GCMS
Peak# R.Time Area% Name
1 1 9.857 5.34 1- (Furan-2-yl)-4-methylpentan-1-one Not Reported
2 3 10.593 2.02 4- (2-Methylcyclohex-1-enyl)-but-2-enal Not Reported
3 5 12.635 1.15 (2,6,6-trimethyl-cyclohex-1-enylmethanesu Not Reported
4 12 16.324 1.30 Tetradecanoic acid
5 15 17.096 1.55 2-Pentadecanone,6,10,14-trimethyl- Not Reported
6 17 17.413 24.41 1,2-benzenedicarboxylicacid,bis (2-methylp Antimicrobial and
alpha glucosidase inhibition and the in vivo hypoglycemic efficacy-[33]

7 22 18.428 13.16 n-Hexadecanoic acid Anti-inflammatory, Antioxidant-[35], hypocholesterolemic nematicide, pesticide, anti androgenic flavor, hemolytic, 5-Alpha reductase inhibitor-[36], , potent mosquito larvicide-[37]
8 30 20.156 29.19 (Z,Z)-6,9-CIS-3,4-epoxy-nonadecadiene Increase zinc bioability [33]
9 31 20.318 4.53 Octadecanoic acid Antimicrobial activity
10 35 22.136 1.54 Eicosanoic acid
Anti-inflammatory effects [38]

11 43 36.874 1.51 .gamma.-Sitosterol
antihyperlipidemic activity, antidiabetic [39]
12 46 40.552 1.44 .gamma.-Sitostenone Not Reported

Gas chromatogram and mass spectra of methanolic extract of flower and roots of Perilla frutescens are presented in fig. 1, and 2 respectively. The GC MS analysis of PF roots extract revealed the presence of 14 major component (table-1) and flower exhibited 12 major components (table-2). Many other small peaks represent other phytochemicals in trace amount in both methanolic extract of flower and roots Perilla frutescens.
1,2-benzenedicarboxylicacid,bis (2-methylp, and n-Hexadecanoic acid are common compound found both in extract of flower and roots of this plant.
Table 3: a Amylase Inhibition( %) of Extracts Vs Standard Drug
Conc. ( mg/ ml Std Flower extract. Root Extract.
0.00 0.0 0.0 0.0
0.50 43 32.0 38.63
1.00 78 45.23 49.59
1.50 82.55 50.96 60.08
2.00- 85.0
to 88.1 56.12
to 60.01 73.12 to
80.2
2.50

Table 3: a Amylase Inhibition( %) of Extracts Vs Standard Drug
Conc. ( mg/ ml Std Flower extract. Root Extract.
0.00 0.0 0.0 0.0
0.50 43 32.0 38.63
1.00 78 45.23 49.59
1.50 82.55 50.96 60.08
2.00- 85.0
to 88.1 56.12
to 60.01 73.12 to
80.2
2.50

Alpha amylase inhibition of methanolic extract of roots and flower of Perilla frutescens was performed. The results are displayed in table-3. The standard (at various concentration range 0.5to-2.5 mg/ ml) revealed greatest a-amylase inhibitory action from 43 to 88% as depicted in table -3 . From table-4, IC50 value for standard observed is 0.75 mg/ml while 1.1 mg/ml value for root extract and 1.40 mg/ml IC50 value for flower extract. At a similar focus methanol concentrates of root showed 38 % inhibition at 0.5 mg/ml concentration and reached up to 73% amylase inhibition at concentration of 2mg/ml with an IC50 worth of 1.10 milligram/ ml. flower extract of Perilla frutescens showed the % inhibitory action from 32.18±0.11 to56.12±0.20 with an IC50 worth of 1.40 milligram/ ml. The a amylase inhibitors known as starch blockers
CONCLUSION: It can be concluded that both roots and flower of Perilla frutescens are a rich source of structurally novel and biologically active metabolites. Secondary or primary metabolites present in both part may be potential bioactive compounds of interest in the pharmaceutical industry and a novel medicinal compounds. The present study suggests that extracts possess the Secondary metabolites have different activities like antimicrobial, antioxidant, anticancer antidiabetic activities, that supports their folkloric use of these part of Perilla frutescens,
We concluded that the biological values of Perilla frutescens contain pharmacologically active compounds that may enhance its use as a traditional drug.
The in vitro anti-diabetic activity was assessed by a-amylase inhibitory assay from the inhibitory assay, it was estimated that the methanol extract of root as well as flower of Perilla frutescens exhibited significant inhibition of a-amylase enzyme The results of a- amylase inhibitory assay proved the antidiabetic potentiality of root and flower Perilla frutescens.
To conclude, this is the first report on phytochemical compounds of methanolic extract of root as well as flower of Perilla frutescens This study confirmed the presence of various new compounds in the Perilla frutescens and also justified the use of roots and flowers of Perilla frutescens as a remedy to diabetes in traditional medicine. From these results, it could be concluded that Perilla frutescens contained various bioactive metabolites. Additionally, However, the isolation of individual secondary metabolites and investigating the biological activity possessed by these would give impetus for further research on the antidiabetic potential of this plant.
ADVANTAGES OF THE INVENTION:
By this invention, Perilla frutescens which is commonly known as Bhanjira can be utilized as anti-diabetic agent. Its root and flower extract can be used for controlling type-2 diabetes
1. Root extract of Perilla frutescens has a amylase inhibition from 70 to 80 % with concentration of 2 to 2.5 mg/ml of alcoholic or methanolic extract
2. Flower extract is Perilla frutescens( Bhangira ) having potential of 50 to 60% inhibition
3. IC 50 value of root extract about 1 to 1.1 mg/ml as comared to standard medicine/drug IC value 0.7 to 8 mg/ml.
4. Overall alpha amylase , which is good indicator of anti-diabetic property shown by root extract ( about 73% to 80 % inhibition at concentration of 1 to 25 mg/ml ) is better than flower or stem other extract as compared to standard drug( 88%) is significant and has potential of using as an anti-diabetic medicine.

We Claim:

1. An anti-diabetic Formulation of root & flower extracts of Bhangira (Perilla Frutescens) comprises Root extract of Perilla frutescens has a amylase inhibition from 70 to 80 % with concentration of 2 to 2.5 mg/ml of alcoholic or methanolic extract; Flower extract is Perilla frutescens (Bhangira ) having potential of 50 to 60% inhibition; IC 50 value of root extract about 1 to 1.1 mg/ml as compared to standard medicine/drug IC value 0.7 to 8 mg/ml;
Wherein alpha amylase, which is indicator of anti-diabetic property shown by root extract (about 73% to 80 % inhibition at concentration of 1 to 25 mg/ml) is better than flower or stem other extract as compared to standard drug (88%) is significant and has potential of using as an anti-diabetic medicine.
2. A method of preparation of anti-diabetic Formulation as claimed in claim 1, wherein the roots, are separated and washed with distilled water and then sun dried in sunlight, flowers are dried in shade; after drying foreign matters and dust and dirt are removed. The roots and flowers were converted into powder with help of a grinder.
3. The method as claimed in claim 1, wherein for Preparation of extracts for GC-MS analysis and antidiabetic activity: Powders (5 grams each) of root, flowers are extracted separately with methanol (each 50 ml) by using cold maceration technique. Later mixtures are filtered and evaporated the solvents till dryness at 800 C± 20 C on water bath; and the obtained semisolid liquid/extracts were kept in a refrigerator at 4°C for use.
4. The method as claimed in claim 1, wherein an individual extract from Perilla frutescens (L) plant is injected into a Shimadzu GCMSQP2010Ultra system attached with a selective mass detector with an ion source of 230°C and 270°C surface temperature.
5. The method as claimed in claim 1, wherein the operation of the instrument for analysis was as follows: Oven temperature 140°C for 6 min then 280°C at 10°C/min and maintained for 31 min; The sample injection is 1.0 microliter and the transporter gas was helium with velocity of 41.6 cm/s. used The evaluation of compounds/components was based on the retention time (RT) for GC and the elucidation of mass spectra was executed by relating the obtained spectral fragmentation, with the database provided by WILEY8 and NIST Library.
6. The method as claimed in claim 1, wherein for Alpha amylase inhibitors assays
Starch-Iodine colour assay: The starch- iodine assay is executed for screening Bhangira as alpha-amylase inhibitors, the overall quantity of combination includes 500 µl 0.02 M sodium phosphate buffer having pH and strength 6.9, 0.02M respectively and contains 6.0 mM sodium chloride, enzyme amylase (1000 µl, Diastage Loba Chemicals) and 200 µl extracts with concentrations ranging from 0.5 to 2.0 mg/ml (w/v); for 12 minutes, the combination was placed for incubation at 37 °C. Then, each mixture received 870 µl of newly ready starch (1%, w/v) and kept for 17min for incubation at 37 °C.
7. The method as claimed in claim 1, wherein to break the enzymatic reaction, 60 µl of muriatic acid. (10.0M) is transferred, before transferring of 300 µl of iodine reagent (5 mM I2 1+ 5 mM KI); at 620nm wavelength absorbance was noted and a color change was observed.