This invention relates to a method of isolation of extract of Allium wallichii for herbal food preservative.
Background of the Invention
Today, food industry many food preservatives are being used, out of food preservative sodium benzoate or tartaric acid salt or common salt etc being used for preservation which are synthetic chemicals and after some duration they give adverse effect. Natural preservatives like mustard oil salts, sugar, vinegar is being used but as such plant extract is not being used as food preservative frequently. In this invention extract (aqueous and ethanolic) of allium wallichii have shown good sign as antibacterial and antioxidant nature due to combined effect it has potential to be used as natural food preservative in the form of extract.
CN104524139A Allium wallichii extract and application thereof provides an Allium wallichii extract. The combination of one or two or more of Allium wallichii roots, stems, leaves and flowers is used as a raw material, ethyl alcohol is added for extraction, an alcohol extract is combined, and the Allium wallichii extract is obtained. The invention further provides the new application of the Allium wallichii extract or other extracts. According to the research finding, Allium wallichii and the obtained Allium wallichii extract have the good antitumor activity, the antitumor activity of some parts is similar to a positive compound like cis-platinum or stronger than a positive compound like cyclophosphamide, and the Allium wallichii extract is good in safety and provides a new choice for clinical medication
Research Gap: In this , Allium walichii extracts have been shown for antitumor activity, But it is different from our use as food preservative due to high antimicrobial and antioxidant nature.
EP1721534A1 Use of extracts and compounds of allium-genus plants as preservatives in the food and agri-food industries discloses to the use of extracts and compounds of Allium-genus plants as preservatives in the food and agri-food industries, both in food products and on the surface thereof, as part of a liquid or solid coating or incorporated in any type of capsule, as well as on plants during harvest or post-harvest. The compounds used are thiosulfinates, such as ajoene and vinyldithiins and other products resulting from the decomposition of thiosulfinates.
Research Gap: In this invention, some other allium genus have been used for food preservation but in our invention specially allium wallichii has been used
None of the prior art indicate above either alone or in combination with one another disclose what the present invention has disclosed.
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.
Present invention involves application of extracts of Allium Wallichii as natural preservative for food due to presence of antiobacterial and antioxidant componants in extracts of this plant. Allium Wallichii is found at hill areas mainly at high altitutde region, mainly himalayn region of India and Nepal.
Allium Wallichii is a herb which is used as some local treatment of allergy or wound healing purposes.
Researcher has done work on extracts of Allium Allium Wallichii for antimicrobial, anti-cancer purposes but no work done reported on alcoholic extract extraction from leaves or flowers or roots and use as food preservative.
In this work Allium wallichii has been chosen for finding antimicrobial activity with using different gram negative and gram-positive bacterial strains. By showing significant and comparable free radical scavenging property, extract of the plant has shown its sign for use in pickles or other items as food preservative.
Leaves, stem and root samples were collected from Nepal region and then airdrying was done and extracts were taken by solvent extraction technique in different polar, non-polar and semi polar solvents (water/aqua, alcohol, n-hexane and other solvent).
Different Extracts of Allium Wallichii have shown good sign of antimicrobial property along with antioxidant nature. Alcoholic and aqueous extract have shown better antimicrobial and antioxidant properties as compared to standard reagent so when applied in ketchup or pickles it showed very good preservation property.
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.
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.
Material and Methods
Plant extracts
Allium Wallichii plants with leaves, flower, roots and stem were collected from Gorkha district of Nepal. The plant was taxonomically identified with flora of Nepal provided by the Department of Plant Resources, National Herbarium and Plant Laboratories, Govt. of Nepal. Aerial parts of the plants were taken and washed with distilled water. Materials were first chopped into small pieces and air-dried in shade for 15 days at room temperature (25oC to 30oC) The dried materials of Allium wallichii were ground to get a powder which was sieved with a mesh screen. After that, extraction of the powdered materials was carried out separately by the soxhlet method using ethanol, n-hexane, acetone and distilled water. Extracts were stored in the refrigerator at 4oC until further use.
Qualitative Phytochemical analysis
Phytochemical testing is a qualitative analysis of primary and secondary metabolites present in the plants. Each 10% extract solution was prepared by using 0.1% dimethyl sulphoxide. The extracts were analyzed for the presence of bioactive phytoconstituent by using the following standard methods. It showed presence of some important phyto-chemicals which indicate towards antioxidant and antimicrobial property for preservative nature.
Test for alkaloid: 5ml of the extract was mixed with 2ml of 1% HCl and heated gently. Wagner’s reagents (iodine in potassium iodide) were then added to the mixture. The turbidity of reddish-brown resulting precipitation indicated the presence of alkaloids.
Test for Terpenoid: 5ml of the extract was dissolved in 2ml of chloroform and evaporated to dryness. To this, 2ml of concentrated H2SO4 was added and heated for about 2 minutes. A grayish/reddish colored precipitate was taken as evidence for the presence of terpenoid.
Tests for Flavanoid: 5mg dried extract was heated with 10ml of ethyl acetate over a steam bath for 3 minutes. The mixture was then filtered and 4ml of the filtrate was mixedwith 1ml of dilute ammonia solution. A yellow coloration indicated a positive test for flavonoids.
Tests for Steroid: Extract was mixed with 2ml of chloroform and concentrated H2SO4 was added sidewise. A red color produced in the lower chloroform layer indicated the presence of steroid
Tests for Phenol: Extract was mixed with 2ml of 2% solution of FeCl3. A blue-green or black coloration indicated the presence of phenol.
Tests for Coumarin: To 5 ml of extract, 3 ml of 10% aqueous solution of NaOH was added. The production of yellow color indicated the presence of coumarin.
Tests for Aminoacid: To 5ml of extract, few drops of ninhydrin reagent (10mg of ninhydrin in 200ml of acetone) were added. The mixture was gently heated for few minutes. The appearance of purple color indicated the presence of amino acid.
Tests for Saponin: 0.5 mg of the extract was vigorously shaken with a few ml of distilled water. The formation of frothing was positive for saponin.
Tests for Quinone: To 1ml of extract, 1ml of conc. H2SO4 was added. The appearance of red color indicated the presence of quinone.
Antioxidant Analysis
Total phenolic assay
Total phenolic content in plant extracts was determined by standard Folin-Ciocalteu reagent assay[12, 13]. 50mg of each extract were dissolved in 50ml DMSO (10% v/v) and centrifuged at 2000rpm for 5 minutes. Then, 0.5ml of supernatant of each extract was taken in separate, labeled test tubes and 0.5ml of 50% Folin-ciocalteu reagent was added in it. Then, the tubes were allowed to stand for 15 minutes at room temperature. After that, 2.5ml of 20% sodium carbonates were added and the tubes were incubated for 30 minutes in a dark place. Absorbance of all test solutions was recorded at 760nm against a reagent blank in a spectrophotometer (UV-1800 Shimadzu Spectrophotometer). All test solutions were analyzed in duplicate. Total phenolic content was expressed as mg gallic acid equivalent (mgGAE/100g) and data was obtained by using standard calibration curve of gallic acid with the equation y= 0.025x+0.006 (R2=0.983). The standard calibration curve of gallic acid was prepared with concentrations 2µg/ml, 4µg/ml, 6µg/ml, 8µg/ml, and 10µg/ml, and a graph was plotted between absorbance vs concentration (µg/ml) [14, 15].
DPPH scavenging assay
DPPH (2, 2-diphenyl -1-picrylhydrazyl radical) is a stable free radical that reacts with molecules that can donate a hydrogen atom. It has been widely used in the antioxidant assay of most compounds. This method is based on scavenging DPHH through the addition of a radical species or an antioxidant that decolorizes the purple-colored methanol solution of DPPH. Antioxidant activities of plant extracts can be assayed by the following standard protocol [9, 12]. DPPH reagent was prepared by dissolving 10mg DPPH (2, 2-diphenyl -1-picrylhydrazyl radical) in 100ml methanol i.e. 100 µg/ml. For DPHH control, 1ml DPPH reagent was added in 4ml methanol. Simultaneously, test samples were prepared as; 50mg of extracts were dissolved in 50ml methanol and centrifuged at 2000rpm for 5 minutes. After that, 2ml of supernatant of each extracts was taken in separate and labeled test tubes. Then, 1ml of DPHH reagent was added to each tubes and solutions were kept in dark and allowed to stand for exactly 30 minutes. The absorbance of all test solutions and DPPH control were recorded at 517nm against reagent blank with a spectrophotometer (UV-1800 Shimadzu Spectrophotometer). Finally, DPHH free radical scavenging assay was calculated as %RSA (Radical scavenging activity).
%RSA= (Control absorbance – Extracts absorbance)/ Control absorbance x 100
Table-1 Phytochemical constitutents in extract

Phytochemicals A. wallichii
ethanol extract n-hexane extract aqueous extract
Alkaloid + + +
Terpenoid + + +
Flavanoid + - +
Steroid + + ++
Phenol + - ++
Coumarin - - -
Tannin + - +
Glycoside + + +
Carbohydrate + + +
Aminoacid + + +
Quinone - - -

Table 2; Result of total phenolics content and DPPH free radical scavenging assay in selected plants
( also compared with other species of allium species

Plants Total phenolic content (mgGAE/100g) DPPH free radical scavenging assay (% RSA)
ethanol extract n-hexane extract aqueous
extract ethanol extract n-hexane extract aqueous
extract
A. Wallichi 152 42 90 65.44 41.29 58.60
Other Allium species 132 40 82 49.53 30.44 44.20
*Ascorbic acid was used as a reference compound which % RSA was found to be 68.11
Determination of antibacterial/anticandidal activity
The agar well diffusion method (Perez et al; 1990) was modified. Soyabean casein digest agar (SCDA) was used for bacterial cultures. The culture medium is inoculated with the microorganisms suspended in soyabean casein digest broth. A total of 8mm diameter wells were punched into agar and filled with plant extracts and solvent blank s(distilled water, hexane and methanol as the case may be).Standard antibiotic was simultaneously used as positive control. The plates were then incubated at 37°C for 18 h. The antibacterial/anticandidal activity was evaluated by measuring the inhibition zone diameter observed. Wells were filled with 0.1 ml of 20 mg/ml concentration of each sample (2 mg/well). Bioactivity was determined by measuring Diameter of Inhibition Zones (DIZ) in mm.
Table: 3. Antimicrobial activity of leaves and root extract of A.Wallichii
S.N. Test microorganisms Diameter of zone of inhibition(mm)
Hexane extract (leaves) Eethanol extract(leaves) Aqueous extract(leaves) Hexane (root) Ethanolic
Root Aq (root)
1.0. Staphylococcus aureus MTCC 737
22
18
10
18
20
12
2.0. E.coli MTCC 1687
18
14
14
16
17
14
3.0. Pseudomonas aeruginosa MTCC 1688
15
12
-
14
15
12
4.0. Salmonella enteric
MTCC 3858
15
13
11
16
17
11
5.0. Candida albicans
MTCC 3017
17 16 10 20 20 13

Results- From the table-1 it is concluded that alcoholic extract consists of more than 10 active phytochemicals like alkaloid, terpenoid, flavonoid, phenol tannin etc, which are contributing for active antioxidant nature. From table it was observed that Ethanolic extract of A. Wallichi has shown DPPH activity almost comparable to standard anti-oxidant (ascorbic acid). From table-3 it can be observed that plant extract has shown significant antimicrobial activity against selected strains Due to combined effect of antimicrobial and antioxidant effect, it can be used as food preservative.

We Claims:

1. A method of isolation of extract of ALLIUM WALLICHII for herbal food preservative comprising the steps of: collecting Allium Wallichii plants with leaves, flower, roots and stem;
Taking Aerial parts of the plants and washed with distilled water;
Chopping the Materials first into small pieces and air-dried in shade for 15 days at room temperature (25oC to 30oC);
Grounding the dried materials of Allium wallichii to get a powder which is sieved with a mesh screen; After that, extraction of the powdered materials is carried out separately by the soxhlet method using ethanol, n-hexane, acetone and distilled water; and storing in the refrigerator at 4oC so as to obtained extract.
2. The method as claimed in claim 1, wherein the agar well diffusion method is modified and Soyabean casein digest agar (SCDA) is used for bacterial cultures.
3. The method as claimed in claim 1, wherein the culture medium is inoculated with the microorganisms suspended in soyabean casein digest broth; wherein a total of 8mm diameter wells are punched into agar and filled with plant extracts and solvent.
4. The method as claimed in claim 1, wherein Standard antibiotic is simultaneously used as positive control; and the plates are then incubated at 37°C for 18 h. The antibacterial/anticandidal activity is evaluated by measuring the inhibition zone diameter observed.
5. The method as claimed in claim 1, wherein wells are filled with 0.1 ml of 20 mg/ml concentration of each sample (2 mg/well); and Bioactivity is determined by measuring Diameter of Inhibition Zones (DIZ) in mm.