DESC:
Field of invention
The present invention relates to the field of dietary supplements, nutritional supplements. More specifically, the invention relates to nutraceutical compositions utilizing the leaves, roots, berries and stem. More particularly, it relates to the nutraceutical composition comprising of Capparis spinosa extract and a green chemistry process for preparation of the same.

Background of invention
Capparis spinosa L. (Caper) is a perennial spiny bush that bears rounded, fleshy leaves and big white to pinkish-white flowers. It is native to the Mediterranean region and growing wild on shells or in rocky coastal areas and a native variety of this grows in India. It contains a number of bioactive compounds, such as polyphenols and flavonoids. It has a history of use in traditional Indian system of medicines for various human diseases.
Fruits of C. spinosa, also known as caper berries, are ellipsoid, ovoid to pear-shaped. In Mediterranean countries, pickled bud is a commercial commodity. According to Sher and Alyemeni (2010). many parts of caper are being used as therapeutic drugs by traditional healers in Saudi Arabia. Sher and Alyemeni (2010) described that oral administration of dried fruits of C. spinosa with water treats hypertension and diabetic complications. Hot-water extract of C. spinosa prevents lead acetate-induced lipid oxidation in rats (Al-Soqeer, 2011).
CNI02091 104B discloses a method for obtaining refined extract from Capparis spinosa and application of extract. This patent essentially mentions a method for enrichment of a crude extract of Caper using ion exchange chromatography. Ethanol is used during the process. Quantitative information on the flavonoid content in the enriched extract is not available.

CN109303790A discloses the medical usage of Caper or Capparis spinosa extract. This patent reports a composition prepared from an extract of caper (berry/ stem/root leaf), Mandarin orange and tripterygium. The extraction solvents are water/ethanol/ isopropanol/butanol etc. The composition has been evaluated for a range of medical uses.

CNI07854656B discloses a lipid-lowering traditional Chinese medicine composition and preparation method and application thereof. Chinese traditional medicine composition in which caper fruits are a part. The process of making this composition involves pulverization of all the components/ water extraction/ ethanol extraction. Dextrin (a carbohydrate) has been used as a carrier/ filler. The disclosure informs of usage of spray drying for concentration in some of the steps.

US2019/0167569AI discloses enhanced herbal extraction and transdermal delivery. The patent describes a method where aqueous plant extract containing saponin is used for extraction of other plant parts using cavitation or ultrasound techniques. Caper is one of the many plants that were extracted using this method. Saponin might be having emulsifying activity that helps in transdermal delivery of the plant extracts.

DE102016107584Al discloses a composition and food or feed supplements for aiding cell protection in an organism. The patent reports a food supplement that contains 10% caper extract along with many other plant extracts. Which plant part of Caper not mentioned. Extraction process also not mentioned in this. This is an antioxidant supplement containing 5 to 10% Caper.

US10751282B2 discloses an edible product comprising reconstituted plant material. This patent specifically does not mention caper as an ingredient. It describes a process and reconstituted edible plant product. Here the plant part is extracted using a solvent and the extract is infused in a paper like sheet made of the remaining plant 20 fibre. The product is a tea.

IN0635/DEL/2009 discloses a formulation of a novel herbal antioxidant supplement for nutraceutical value. This patent is for an antioxidant supplement in which caper root is a component. The root is directly incorporated in this ayurvedic formulation. Do not involve any extraction process. The formulation contains many other plant materials other than caper root.

CN101406497A discloses Capparis spinosa extract as well as preparation method and application thereof. This patent reports a caper extract and extraction process. The extract is meant for anti-arthritic application. The extract is made using aqueous ethanol, which plant part of caper is used is not clear. The composition of the extract has been characterized qualitatively.

CNI02911216A discloses a method for separating and preparing cleome gynandra from capparis spinosa: Caper fruit / root are extracted using methanol. The crude methanol extract is purified using counter current chromatography using organic solvents as eluent, to isolate cleome glycoside.

CNI12251912 discloses Capparis spinosa drug-loaded nanofibre membrane as well as preparation method and application thereof: A medical dressing. Caper extract is prepared using ethyl acetate as solvent. The crude extract is loaded onto a polylactic acid membrane.

CNI01502544 discloses Capparis spinosa extract and capparis spinosa emulsifiable paste and method for producing the same: A caper cream containing ethanolic extract of caper. Probably caper root extract. The plant part is not mentioned clearly. The extract contains-2% alkaloid. The cream is used for treating psoriasis.

The prior art methods involve usage of high amount of organic solvents during extraction, which leave high environmental footprint due to effluents and solvent vapor and low quality of raw material

Accordingly, there exists a need for a nutraceutical composition based on Capparis spinosa extract prepared using green chemistry technology that uses no organic solvents and leaves minimum environmental footprint behind. At the same time the process does not compromise on the extraction efficiency.

Summary of invention
Accordingly, the present invention is in relation to a nutraceutical composition, comprising Capparis spinosa extract ranging from 30% - 50% w/w; and shell material 50%-70%w/w, wherein the composition is a microencapsulated powder.
A process for preparation of nutraceutical composition comprising Capparis spinosa extract ranging from 30% - 50% w/w and shell material 50%-70%, said method comprising steps of-
a) obtaining dried Capparis spinosa plant parts;
b) subjecting the dried plant parts to sequential enzyme assisted extraction with carbohydrase and protease;
c) subjecting the enzyme assisted extract to hydrothermal extraction and filtering; and
d) mixing the filtrate with shell material to obtain microencapsulated powder of nutraceutical composition.

Brief description of figures
The features of the present invention can be understood in detail with the aid of appended figures. It is to be noted however, that the appended figures illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope for the invention.
Figure 1: illustrates the flow chart for preparation of microencapsulated nutraceutical composition comprising Capparis spinosa leaves extract.
Figure 2: graphically represents Tocopherol estimation of caper leaves nutraceutical composition
Figure 3: graphically represents fatty acid composition estimation in the caper leaves nutraceutical composition.

Detailed description of invention

The foregoing description of the embodiments of the invention has been presented for the purpose of illustration. It is not intended to be exhaustive or to limit the invention to the precise form disclosed as many modifications and variations are possible in light of this disclosure for a person skilled in the art in view of the figures, description and claims. It may further be noted that as used herein and in the appended claims, the singular “a” “an” and “the” include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by person skilled in the art.

Source of materials: The Capparis spinosa used in the present invention is collected from fields owned by ISHKA farms situated in Niravi Pudhupatti village and Arungulam Villages, at Ettayapuram Taluk, Tuticorin District-628902, India, with Latitude 9.111716 and Longitude 78.053269. The enzymes are obtained from Novozyme India Pvt. Ltd. The other reagents used in the invention are obtained from commercial sources.

The present invention relates to a nutraceutical composition, comprising
Capparis spinosa extract - 30% - 50% w/w; and shell material 50%-70%w/w, wherein the composition is a microencapsulated powder obtained by freeze-dried/ spray dried extract of Capparis spinosa.

In another embodiment invention provides a nutraceutical composition, wherein the Capparis spinosa extract is obtained from leaves, stems, flowers, buds and roots.

In another embodiment of present invention, the Capparis spinosa extract is obtained from leaves.

In an embodiment of present invention the, Capparis spinosa ( Caper) grown in Ishka Farms by tissue culture contains considerable amounts of protein (15%), Polyunsaturated fatty acids (252.36 mg/100g), and essential amino acids. The leaves are also rich in flavonoids namely Rutin, Kaempferol, Kaempferol-3-rutinoside, and Quercetin when compared with literature published data.

In yet another embodiment of the present invention the shell material is selected from a group comprising maltodextrin, whey protein, pectin and combinations thereof.

In another embodiment of the invention wherein the shell material is a 1:1 w/w mix of maltodextrin and whey protein. The phytochemical extract gets trapped in this biopolymer matrix and the biopolymers form a protective coat.

In another embodiment of the invention wherein the protein-carbohydrate complex-shell material acts as an emulsifier and helps to trap the phytochemical extract in the shell material matrix. The maltodextrin-whey protein in 1:1 ratio is optimised and 10 % of the shell material is added to the extract (W/V). Following the high shear mixing the emulsion/dispersion is spray dried or freeze dried to obtain the final nutraceutical composition. The water activity of the composition thus obtained is less than 0.7.

In yet another embodiment wherein the nutraceutical composition i.e. a powder concentrate of the caper leaves is obtained using a sequential enzymatic process and microencapsulation through spray drying / freeze drying. The sequential enzymatic extraction process using enzymes, followed by microencapsulation through spray drying/ freeze drying process result in a "Capparis spinosa (caper) nutraceutical concentrate powder."

In yet another embodiment of the invention dried and powdered caper plant parts are sequentially extracted with Viscozyme which is a cocktail of several carbohydrase; followed by extraction with Alkalase, and Flavourzyme . The caper plant parts/ leaf powder to extraction buffer weight/volume ratio is 1:20. The enzyme concentration in the extraction buffer is 1-2%. Prior to enzyme extraction, the biomass in the extraction buffer is sonicated for 30 min at an amplitude of 200 µm. The enzymes are finally deactivated in a hydrothermal autoclave at 100 bars, 150?, for 30 min. The extract is filtered, mixed with the shell material at 10% w/volume ratio of the extract with high shear homogenization, and finally freeze dried/spray dried. The composition of the shell material is maltodextrin and whey protein isolate at 1:1 weight by weight ratio.

The process for preparation of nutraceutical composition comprising Capparis spinosa extract ranging from 30% - 50% w/w and shell material 50%-70%w/w, said method comprising steps of-
a) obtaining dried Capparis spinosa plant parts;
b) subjecting the dried plant parts to sequential enzyme assisted extraction with carbohydrase and protease;
c) subjecting the enzyme assisted extract to hydrothermal extraction and filtering; and
d) mixing the filtrate with shell material to obtain microencapsulated powder of nutraceutical composition.
The method is exemplified as follows in flowchart A

Take 100g Caper leaf powder

2 L Sodium Acetate Buffer (pH – 4.5)

(Ultrasonication for 30 min)
Add 20 ml Vizcozyme

Incubate at 60°C for 12 hours

pH is Adjusted to 6 using Ammonium hydroxide Solution

Add 20g of Alkalse and 20 g of Flavourzyme

Incubate at 45°C for 12 hours

Hydrothermal extraction and deactivation of enzyme

Cool

Filter through Nutsche filter/Muslin clothe filter

Filtrate mixed with wall material for microencapsulation

Freeze drying or spray drying for microencapsulation

Microencapsulated powder nutraceutical composition
Flow chart -A
In another embodiment the extraction buffer solution is obtained by taking 510 mL of Solution A and 490 mL of Solution B, pH is adjusted to 4.5 and finally made up to 2 L where in Solution A is 0.2 M Acetic Acid (23.1 mL made up to 2 L and Solution B is 0.2 M Sodium Acetate (32.8 g made up to 2 L)
In another embodiment the invention provides a process for preparation of nutraceutical composition, wherein the cocktail of carbohydrase is selected from one of viscozyme, promozyme, liquozyme, celluclast and combinations thereof.

In yet another embodiment the invention provides a process for preparation of nutraceutical composition, wherein the cocktail of protease is selected from one of alcalase, flavourzyme, protamex, bromelain extract from Pineapple, papain and combinations thereof.

In another embodiment of the invention to provide a process for preparation of nutraceutical composition, wherein the enzyme assisted hydrothermal extraction(100 bars, 150?, for 30 min) comprises steps of subjecting the plant material to subcritical temperature water assisted with one of protease and carbohydrase.

In another embodiment the invention provides a process for preparation of nutraceutical composition, wherein the enzyme assisted hydrothermal extraction comprises steps of subjecting the plant material to subcritical temperature (100 bars, 150?, for 30 min) water.

In another embodiment the invention provides a process for preparation of nutraceutical composition, wherein the enzyme assisted ultrasonication extraction (30 min at an amplitude of 200 µm) is carried out using ultrasonic chamber with or without prior treatment with protease and carbohydrase.

In another embodiment the invention provides a process for preparation of nutraceutical composition, wherein the Capparis spinosa extract is obtained from leaves, stems, buds and roots.

In another embodiment the invention provides a process for preparation of nutraceutical composition, wherein the Capparis spinosa extract is obtained from leaves.

In another embodiment, the dried and powdered caper leaf powder in extraction buffer is sonicated for 30 minutes at an amplitude of 200 µm.

In another embodiment , the caper leaves are then sequentially extracted using Vizcozymes where in the vizcozyme is a cocktail of carbohydrases like cellulase, hemicellulase, arabanase, beta-glucanase and xylanase.

In another embodiment, the caper leaves are extracted using Alkalase and Flavourzyme.
In another embodiment the invention provides a nutraceutical composition, wherein the concentrate powder contains Protein (~15%), aminoacids (-9g amino acids/ 100g), polyunsaturated fatty acids (-252 mg/100g i.e 49% of FAME), Rutin (-892 ug/g), Kaempferol (2 ug/g), Kaempferol-3-rutinoside (-132 g/g). Quercetin (-15 Hg/g). and Tocopherols (-140 ug/g), Pyridoxine (~2.4 ug/g), Riboflavin (-7ug/g). Calcium (~19 mg/g), Iron (0.6 mg/g), Magnesium(-19 mg/g), Zinc (0.02 mg/g).

The figure 2 and 3 depicts the and tocopherol composition in the different parts of caper plant and Fatty acid composition estimation in caper leaves nutraceutical composition. respectively. The table 1 tabulates the Antioxidant and total phenolic content in finished extracts of different parts of caper plant. The Table 2 tabulates the flavonoid composition in finished extract from different parts of caper plant.
Table 1: Antioxidant and total phenolic content in finished extracts of different parts of caper plant

Table 2: Flavonoid composition in finished extracts from different parts of Caper plant

Experimental:
General procedure:
Dried and powdered caper plant leaves are sequentially extracted with Viscozyme, which is a cocktail of carbohydrases; followed by extraction with Alkalse, and Flavourzyme. The caper leaf powder to extraction buffer weight/volume ratio is maintained at the ratio 1:20. The enzyme concentration in the extraction buffer is 1-2%. Prior to enzyme extraction, the biomass in the extraction buffer was sonicated for 30 min at an amplitude of 200 µm. The enzymes are finally deactivated in a hydrothermal autoclave at 100 bars, 150?, for 30 min. The step also helps in further extraction of phytochemicals. The extract is filtered, mixed with the shell material at 10% w/volume ratio of the extract with high shear homogenization, and finally freeze dried/spray dried. The composition of the shell material is maltodextrin and whey protein isolate at 1:1 weight by weight ratio.

Example 1
100 g of Dried and powdered caper plant leaves are sequentially extracted with 20 g of Viscozyme, which is a cocktail of carbohydrases; followed by extraction with 20g of Alkalse, and 20g of Flavourzyme. The caper leaf powder to extraction buffer weight/volume ratio is maintained at the ratio 1:20. The enzyme concentration in 2L extraction buffer is 1-2%. Prior to enzyme extraction, the biomass in the extraction buffer was sonicated for 30 min at an amplitude of 200 µm. The enzymes are finally deactivated in a hydrothermal autoclave at 100 bars, 150?, for 30 min. The step also helps in further extraction of phytochemicals. The extract is filtered, mixed with the shell material at 10% w/volume ratio of the extract with high shear homogenization, and finally freeze dried/spray dried. The composition of the shell material is maltodextrin and whey protein isolate at 1:1 weight by weight ratio.
Example 2
100g of Dried and powdered caper plant leaves are sequentially extracted with 10 g of Viscozyme, which is a cocktail of carbohydrases; followed by extraction with 10 g of Alkalase, and 10g of Flavourzyme. The caper leaf powder to extraction buffer weight/volume ratio is maintained at the ratio 1:50. The enzyme concentration in 5 L extraction buffer is 0.2-0.5%. Prior to enzyme extraction, the biomass in the extraction buffer was sonicated for 60 min at an amplitude of 200 µm. The enzymes are finally deactivated in a hydrothermal autoclave at 100 bars, 150?, for 30 min. The step also helps in further extraction of phytochemicals. The extract is filtered, mixed with the shell material at 10% w/volume ratio of the extract with high shear homogenization, and finally freeze dried/spray dried. The composition of the shell material is maltodextrin and whey protein isolate at 2:1 weight by weight ratio

Example 3
100g of Dried and powdered caper plant leaves are sequentially extracted with 30 g of Viscozyme, which is a cocktail of carbohydrases; followed by extraction with 30 g of Alkalase, and 30g of Flavourzyme. The caper leaf powder to extraction buffer weight/volume ratio is maintained at the ratio 1:10. The enzyme concentration in 1 L extraction buffer is 2-3%. Prior to enzyme extraction, the biomass in the extraction buffer was sonicated for 15 min at an amplitude of 200 µm. The enzymes are finally deactivated in a hydrothermal autoclave at 100 bars, 150?, for 30 min. The step also helps in further extraction of phytochemicals. The extract is filtered, mixed with the shell material at 10% w/volume ratio of the extract with high shear homogenization, and finally freeze dried/spray dried. The composition of the shell material is maltodextrin and whey protein isolate at 5:1 weight by weight ratio.

Example 4
100 g of Dried and powdered caper plant stem are sequentially extracted with 20 g of Viscozyme, which is a cocktail of carbohydrases; followed by extraction with 20g of Alkalse, and 20g of Flavourzyme. The caper stem powder to extraction buffer weight/volume ratio is maintained at the ratio 1:20. The enzyme concentration in 2L extraction buffer is 1-2%. Prior to enzyme extraction, the biomass in the extraction buffer was sonicated for 30 min at an amplitude of 200 µm. The enzymes are finally deactivated in a hydrothermal autoclave at 100 bars, 150?, for 30 min. The step also helps in further extraction of phytochemicals. The extract is filtered, mixed with the shell material at 10% w/volume ratio of the extract with high shear homogenization, and finally freeze dried/spray dried. The composition of the shell material is maltodextrin and whey protein isolate at 1:1 weight by weight ratio.
Example 5
100 g of Dried and powdered caper plant roots are sequentially extracted with 20 g of Viscozyme, which is a cocktail of carbohydrases; followed by extraction with 20g of Alkalse, and 20g of Flavourzyme. The caper root powder to extraction buffer weight/volume ratio is maintained at the ratio 1:20. The enzyme concentration in 2L extraction buffer is 1-2%. Prior to enzyme extraction, the biomass in the extraction buffer was sonicated for 30 min at an amplitude of 200 µm. The enzymes are finally deactivated in a hydrothermal autoclave at 100 bars, 150?, for 30 min. The step also helps in further extraction of phytochemicals. The extract is filtered, mixed with the shell material at 10% w/volume ratio of the extract with high shear homogenization, and finally freeze dried/spray dried. The composition of the shell material is maltodextrin and whey protein isolate at 1:1 weight by weight ratio.

Example 6
100 g of Dried and powdered caper buds are sequentially extracted with 20 g of Viscozyme, which is a cocktail of carbohydrases; followed by extraction with 20g of Alkalse, and 20g of Flavourzyme. The caper buds to extraction buffer weight/volume ratio is maintained at the ratio 1:20. The enzyme concentration in 2L extraction buffer is 1-2%. Prior to enzyme extraction, the biomass in the extraction buffer was sonicated for 30 min at an amplitude of 200 µm. The enzymes are finally deactivated in a hydrothermal autoclave at 100 bars, 150?, for 30 min. The step also helps in further extraction of phytochemicals. The extract is filtered, mixed with the shell material at 10% w/volume ratio of the extract with high shear homogenization, and finally freeze dried/spray dried. The composition of the shell material is maltodextrin and whey protein isolate at 1:1 weight by weight ratio.
The composition can be used as nutraceutical dietary supplement providing significant amounts of antioxidant flavonoids with known health benefits. The composition also has significant amounts of proteins, amino acids, polyunsaturated acids, Tocopherols, Calcium, iron, Magnesium, Zinc and the like. The process is a green chemistry method, hence free from harmful organic solvent residues. Environmental footprint of the production process is also significantly low.
,CLAIMS:WE CLAIM
1. A nutraceutical composition comprising Capparis spinosa extract ranging from 30% - 50% w/w; and shell material 50%-70%w/w, wherein the composition is a microencapsulated powder.
2. The nutraceutical composition as claimed in claim 1, wherein the extract is from part of Capparis spinosa plant selected from a group comprising leaf, root, stem, bud and combination thereof.
3. The nutraceutical composition as claimed in claim 1, wherein the shell material is selected from a group comprising maltodextrin, whey protein, pectin and combination thereof.
4. The nutraceutical composition as claimed in claim 1, wherein the extract comprises Protein 15%, aminoacids , Polyunsaturated fatty acids, Rutin, Kaempferol, Kaempferol-3-rutinoside, Quercetin, Tocopherols, Pyridoxine, Riboflavin, Calcium, Iron, Magnesium and Zinc.
5. The nutraceutical composition as claimed in claim 1, wherein the Capparis spinosa is harvested by tissue culture.
6. A process for preparation of nutraceutical composition comprising Capparis spinosa extract ranging from 30% - 50% w/w and shell material 50%-70% w/w, said method comprising steps of-
a) obtaining dried Capparis spinosa plant parts;
b) subjecting the dried plant parts to sequential enzyme assisted extraction with carbohydrase and protease;
c) subjecting the enzyme assisted extract to hydrothermal extraction and filtering; and
d) mixing the filtrate with shell material to obtain microencapsulated powder of nutraceutical composition.
7. The process as claimed in claim 6, wherein the shell material comprises maltodextrin and whey protein in 1:1w/w ratio.
8. The process as claimed in claim 6, wherein the enzyme extraction is at a temperature ranging from 45 ? to 60? for 12 hours.