DESC:POLYSACCHARIDES ENRICHED DATE SEED EXTRACT AND EXTRACTION METHOD THEREOF

FIELD OF INVENTION
The present invention provides an extraction process to obtain polysaccharides enriched date seed extract with anti hyaluronidase activity.

BACKGROUND OF THE INVENTION
The following background discussion includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

Hyaluronic Acid or Hyaluronan is a glycosaminoglycan that is present in various connective and epithelial tissues. It increases viscosity of the fluid in synovial joints to reduce friction between bones. HA is a major component of the skin. When skin is exposed to UV, hyaluronin is degraded and skin cells reduce production of HA. Hyaluronic acid is degraded by hyaluronidase enzymes. Various biological sources are being explored to source hyaluronidase inhibitors. Polyphenols such as alkaloids, flavonoids and terpenes have demonstrated inhibition against HAases.

Date seeds are the by-product of date stoning, either for the production of pitted dates or for the manufacture of date paste (Ecocrop, 2011; Barreveld, 1993) and can act as a great source of Hyaluronic Acid. The date seed is a hard coated seed, usually oblong, ventrally grooved, with a small embryo. Date pits weigh 0.5 g to 4 g and represent 6 to 20% of the fruit weight depending on maturity, variety and grade (Ecocrop, 2011; Daghir, 2008; Zaid et al., 2002; Barreveld, 1993; Göhl, 1982).

Date seeds are traditionally used for animal feed. They can also be used as a source of oil (which has antioxidant properties valuable in cosmetics), as a coffee substitute, as a raw material for activated carbon or as an adsorbent for dye-containing waters (Lecheb, 2010; Banat et al., 2003; Barreveld, 1993).

The overall water footprint value for dry dates has been estimated to be as high as 8.6 m3/kg (Bazrafshan et al., 2020). As a result, the large volume of residues produced by the date processing industry is an indirect waste, if not efficiently valorized. The seeds of dry dates causes pollution when burned, in addition to being a shelter for rodents.

Furthermore, uncontrolled decomposition of date waste can potentially cause severe health and environmental problems (Alsafadi et al., 2020). To reduce the date waste's environmental effects, and to move towards a sustainable economy in the date processing industry, proper waste management and disposal strategies are needed. (Reference: Publication on Date fruit processing waste and approaches to its valorization).

Hence, there is needed an efficient and environment friendly process to manage date seed waste. Particularly, there is a need of a cost effective and efficient extraction process to obtain polysaccharides enriched date seed extract with anti hyaluronidase activity.

OBJECTIVE OF THE PRESENT INVENTION

An object of the present invention is to provide an efficient extraction process to obtain polysaccharides enriched date seed extract with anti hyaluronidase activity.
Another object of the present invention is to provide environmental friendly extraction process for waste valorization of date seeds.
Another object of the present invention is to provide a natural alternative to hyaluronidase inhibitors, and moisturizers.

Summary of the Invention:

In an aspect of the present invention, there is provided a polysaccharides enriched date seed extract wherein approx. 1.25 mg/ml of the date seed extract comprises 50% hyaluronidase inhibition activity.

In another aspect of the present invention, there is provided a method to prepare a polysaccharides enriched date seed extract comprising:

a) Grinding the Date seeds coarsely;
b) performing the extraction with reagents comprising water and methanol present in the ratio of 90:10 at 500celsius for approx.. 6 hrs cycle.

Detail Description of the Drawings:
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 in which:
Figure 1: illustrates percentage Hyaluronidase inhibition
Figure 2: illustrates inhibition at 0.5mg/ml

DESCRIPTION OF THE INVENTION:
In the present document, the word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any embodiment or implementation of the present subject matter described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.

While the disclosure is susceptible to various modifications and alternative forms, specific embodiment thereof has been shown by way of example in the drawings and will be described in detail below. It should be understood, however that it is not intended to limit the disclosure to the specific forms disclosed, but on the contrary, the disclosure is to cover all modifications, equivalents, and alternative falling within the spirit and the scope of the disclosure.

The present invention provides an extraction process to obtain polysaccharides enriched date seed extract with anti hyaluronidase activity. The Invention provides extraction process to develop polysaccharides enriched date seed extract proven for moisturization effects.

In an embodiment, the Date seeds are grind coarsely and the extraction is carried out at 90:10 water : methanol in the extraction ratio of 10:1 at 50 degree C for 6 hrs cycle.

In an embodiment, the extraction cycle were 6. This process gave the maximum quantity of polysaccharides.
In an embodiment, the green solvent extraction process provided by the present invention maintains the moisturization activity through enrichment of polysaccharides. The experiments demonstrates that 1.25 mg/ml of the date seed extract has 50% hyaluronidase inhibition activity which is an excellent moisturizing action.

In an embodiment, the water: methanol is present in the ratio of:
Solvent Ratio % polysaccharides
50:50 10%
70:30 14%
90:10 17%

The experiment shows that 17% polysaccharides gave the best anti hyaluronidase activity.

The Invention will be described with the help of non-limiting examples as mentioned below:
Example 1:
In this experiment, the inventors have evaluated the HAase inhibition activity of date seed extract (SesZenNHA™) against the well established plant based HA inhibitor which comes from Senna seeds. The polysaccharide content, which is believed to be the phytochemical responsible for HA inhibitory activity of Date seeds, was found to be 18.73% glucose equivalents as against Seena Polysaccharides of 7%. In hyaluronidase inhibition activity, IC20 of date-seed extract was found to be 0.5mg/ml whereas IC20 of senna seed extracts was 2 mg/ml. Hence the claimed extract derived from date seed holds 4 times better potential for HA inhibition, as per the below details:

Example 2:
For the experimental purposes, 500mg extracts of date seeds and senna seeds are sonicated for 5 minutes mixed with 100ml water. This solution is filtered and further diluted 1:50. This solution is henceforth referred to as seed extracts.
Estimation of polysaccharide content:
To estimate the polysaccharide content in the seed extracts, 1ml of 5% phenol is added to the 1ml of extract solution, followed by 5ml of conc. H2SO4. The absorbance was measured after 5 minutes at 488nm against blank. The experiment was carried out in triplicate. The glucose equivalent was calculated using:

Where V = Volume total DF = Dilution Factor(50) 106= 1000000(parts per million) Wt of sample in gram 100 = percentage

Example 3:
Estimation of Inhibition of Hyaluronidase enzymes:
To estimate the potential of extracts to inhibit the hyaluronidase enzymes the assay was performed as follows. 50µl of bovine hyaluronidase was mixed with 50µl of the extracts. Hyaluronidase was activated using 12.5mM calcium chloride and mixed with 250µl of sodium hyaluronate (1.2 mg/ml). The mixture was heated to 100°C. After it was subjected to cooling, 1.5ml of p-Dimethylamino benzaldehyde was added and incubated for 20 minutes. The absorbance was measure at 585nm. Oleanolic acid was used as a positive control. The inhibitory activity of the enzymes was calculated. The results are also shown in Figures 1 and 2.
Results:
Estimation of polysaccharide content: The polysaccharide content was evaluated for date-seed extract and found to be 18.73% (%w/w).
Hyaluronidase assay: The extracts were evaluated for the ability to inhibit hyaluronidase enzymes. The date seed extract was able to inhibit hyaluronidase enzymes better than senna seed extracts. IC20 of date-seed extract was estimated to be 0.5mg/ml, whereas IC20 of senna seed extracts was 2 mg/ml. Date seed extracts showed 50% hyaluronidase inhibitory activity at 1.25 mg/ml. Oleanolic acid had a mean anti-hyaluronidase activity at 92.53%

Example 4:
The experiments were performed to analyse physical and chemical characteristics, analytical qualities and microbial profile of the polysaccharides enriched date seed extract obtained by the method of the present invention:

,CLAIMS:We claim:
1. A polysaccharides enriched date seed extract wherein approx. 1.25 mg/ml of the date seed extract comprises 50% hyaluronidase inhibition activity.
2. The extract as claimed in claim 1, wherein IC20 of date-seed extract was estimated to be 0.5mg/ml.
3. A method to prepare a polysaccharides enriched date seed extract, as claimed in claim 1, comprising:
a) Grinding the Date seeds coarsely;
b) performing the extraction with reagents comprising water and methanol present in the ratio of 90:10 at 500celsius for approx. 6 hrs cycle.
4. The method as claimed in claim 2, wherein the extraction was performed in approx. 6 cycles.
5. The method as claimed in claim 2, wherein approx 500mg extracts of date seeds is sonicated for 5 minutes mixed with 100ml water, followed by filtering the solution and diluting it in the ratio of 1:50.
6. A method of inhibiting the activity of Hyaluronidase enzyme/s by the date seed extract as claimed in claim 1, comprising the steps of:
a) Mixing 50µl of bovine hyaluronidase mixed with 50µl of the extract of claim 1;
b) Adding12.5mM calcium chloride and 250µl of sodium hyaluronate (1.2 mg/ml);
c) Heating the mixture at 100°C followed by cooling ;
d) Adding 1.5ml of p-Dimethylamino benzaldehyde to the solution obtained in step (c) followed by incubation for 20 minutes.