Methods of isolating leaf proteins from moringa leaves

Abstract

Provided are methods of producing an isolate of soluble proteins from moringa leaves, and methods of preparing a powder of soluble proteins from moringa leaves. Also provided are compositions-of-matter comprising a chlorophyll-free preparation of soluble proteins of moringa leaves, wherein the soluble proteins comprise large and small Rubisco protein subunits, a preparation comprising the composition-of-matter, and edible, cosmetic and pharmaceutical compositions comprising same.

Description

[0001] METHODS OF ISOLATING LEAF PROTEINS

[0002] RELATED APPLICATION / S

[0003] This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 695,404, filed on September 17, 2024, the contents of which are incorporated herein by reference in their entirety.

[0004] SEQUENCE LISTING STATEMENT

[0005] The XML file, entitled 104969. xml, created on September 15, 2025, comprising 45,056 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

[0006] FIELD AND BACKGROUND OF THE INVENTION

[0007] The present invention, in some embodiments thereof, relates to methods of producing an isolate of soluble proteins from moringa leaves, and, more particularly, but not exclusively, to compositions comprising the soluble proteins from moringa leaves.

[0008] Various plants are fully edible with high protein contents include Moringa oleifera tree, Wolffia arrhiza and Wolffia globose. Leaf-based proteins, specifically those derived from the family of Moringaceae offer a non-GMO (genetically modified organism), non- allergenic source of nutrition with a superior profile of balanced essential amino acids. More than 70 % of the leaves proteins are in the chloroplast, where ribulose bisphosphate carboxylase (RuBisCO, herein “rubisco”), an enzyme involved in photosynthesis, is the dominating protein.

[0009] The family Moringaceae contains 13 species. The most widely known species is Moringa oleifera. Moringa is considered a superfood with several edible parts (e.g., leaves, roots, seeds, bark, fruit, flowers, and immature pods).

[0010] However, the bio-accessibility of nutrients, particularly proteins, in vegetable foods is impaired by non-starch polysaccharides (cellulose, hemicellulose), polyphenols, and anti- nutritional factors (inhibitors of proteases and amylases) that interfere with food proteins and digestive enzymes. The isolation of proteins from the vegetable matrices helps to improve the digestibility by removing fibers. However, food-grade extraction procedures, including, for example, the two-step alkaline extraction / isoelectric precipitation, used for preparing protein isolates from seeds, fails when applied to leaf powder because of the low solubility of membrane proteins compared to seed storage proteins.

[0011] Enzyme-assisted extraction of proteins from moringa leaf powder, using a cellulolytic enzyme mixture, which breaks down the matrix structure, made it possible to prepare a high protein concentrate (55.7%, w / w) that would not be achievable with alkaline extraction alone [Benhammouche, T.et al., 2021. “Nutritional quality of protein concentrates from moringa oleifera leaves and in vitro digestibility”. Food Chem. 348, 128858].

[0012] Additional prior art documents include, for example, US 116353386, US 2022 / 0163453, US 2021 / 0379139, WO 2020 / 127072, and US 10155019.

[0013] SUMMARY OF THE INVENTION

[0014] According to an aspect of some embodiments of the present invention there is provided a method of producing an isolate of soluble proteins from moringa leaves, comprising:

[0015] (a) grinding the moringa leaves in the presence of liquid, to thereby obtain a green juice of ground moringa leaves;

[0016] (b) disrupting organelles in the green juice so as to release soluble proteins from the organelles, and

[0017] (c) isolating the soluble proteins from the green juice, thereby producing the isolate of soluble proteins from the moringa leaves.

[0018] According to an aspect of some embodiments of the present invention there is provided a method of producing an isolate of soluble proteins from moringa leaves, comprising:

[0019] (a) grinding the moringa leaves in a buffer solution comprising an antioxidant, to thereby obtain a green juice (GJ) of ground moringa leaves;

[0020] (b) isolating a soluble protein phase from the green juice using a filter;

[0021] (c) removing chlorophyll from the soluble protein phase to obtain a chlorophyll-free preparation comprising the soluble proteins;

[0022] (d) removing solids from the chlorophyll-free preparation to obtain solid-free preparation comprising the soluble proteins; and

[0023] (e) removing phenolic compounds and salts from the solid-free preparation by ultrafiltration; thereby producing the isolate of soluble proteins from the moringa leaves.

[0024] According to an aspect of some embodiments of the present invention there is provided a method of preparing a powder of soluble proteins from moringa leaves,

[0025] (a) isolating soluble proteins from moringa leaves according to the method of some embodiments of the invention, to thereby obtain a solution of isolate soluble proteins, and

[0026] (b) spray drying the solution of the isolate soluble proteins, to thereby the powder of soluble proteins from the moringa leaves. According to an aspect of some embodiments of the present invention there is provided a composition-of-matter comprising a chlorophyll-free preparation of soluble proteins of moringa leaves, wherein the soluble proteins comprise large and small Rubisco protein subunits.

[0027] According to an aspect of some embodiments of the present invention there is provided a composition-of-matter comprising a chlorophyll-free preparation of soluble proteins of moringa leaves, wherein the soluble proteins comprise large and small Rubisco protein subunits, and wherein the large Rubisco protein comprises the amino acid sequence set forth by SEQ ID NO: 11.

[0028] According to an aspect of some embodiments of the present invention there is provided a composition-of-matter comprising a chlorophyll-free preparation of soluble proteins of moringa leaves which comprises Niazirin.

[0029] According to an aspect of some embodiments of the present invention there is provided a composition-of-matter produced according to the method of some embodiments of the invention, comprising a chlorophyll-free preparation of soluble proteins of moringa leaves, wherein the soluble proteins comprise large and small Rubisco protein subunits.

[0030] According to an aspect of some embodiments of the present invention there is provided a preparation comprising the composition-of-matter of some embodiments of the invention.

[0031] According to an aspect of some embodiments of the present invention there is provided an edible composition comprising the composition-of-matter of some embodiments of the invention, or the preparation of some embodiments of the invention.

[0032] According to an aspect of some embodiments of the present invention there is provided a cosmetic composition comprising the composition-matter of some embodiments of the invention or the preparation of some embodiments of the invention and a cosmetically acceptable carrier.

[0033] According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising the composition-matter of some embodiments of the invention or the preparation of some embodiments of the invention and a pharmaceutically acceptable carrier.

[0034] According to some embodiments of the invention, the liquid is a buffer solution.

[0035] According to some embodiments of the invention, the buffer solution comprises an antioxidant.

[0036] According to some embodiments of the invention, the leaves are attached to rachis and / or stem.

[0037] According to some embodiments of the invention, the leaves are isolated from rachis and / or stem. According to some embodiments of the invention, the grinding is performed by a shredder.

[0038] According to some embodiments of the invention, the grinding is performed by a crusher.

[0039] According to some embodiments of the invention, the method further comprising milling the ground moringa leaves prior to the isolating of the soluble proteins.

[0040] According to some embodiments of the invention, the method further comprising milling the ground moringa leaves prior to isolating of the soluble protein phase.

[0041] According to some embodiments of the invention, the liquid does not comprise alcohol or hydro-alcohol.

[0042] According to some embodiments of the invention, the buffer solution does not comprise alcohol or hydro-alcohol.

[0043] According to some embodiments of the invention, the filter is featured by a pore diameter of a size selected from a range of 50-400 microns.

[0044] According to some embodiments of the invention, the filter is featured by a pore diameter of a size selected from a range of 50-120 microns.

[0045] According to some embodiments of the invention, the filter is featured by a pore diameter of a size selected from a range of about 400 microns.

[0046] According to some embodiments of the invention, the disrupting the organelles is performed using a high-pressure homogenizer, a microfluidizer, and / or a colloid mill.

[0047] According to some embodiments of the invention, the organelles comprise chloroplasts.

[0048] According to some embodiments of the invention, method further comprising cooling the soluble proteins to a temperature of 4°C following the disruption of the organelles.

[0049] According to some embodiments of the invention, removing the chlorophyll is performed by heating the soluble protein phase for a pre-determined time period at a temperature in the range of 35-50 °C.

[0050] According to some embodiments of the invention, the method further comprising cooling the soluble protein phase to a temperature of 4 °C following the pre-determined time period.

[0051] According to some embodiments of the invention, following the pre-determined time period the soluble protein phase is characterized by an absorbance of about 0.5 or less at a wavelength of 595 nanometer (nm).

[0052] According to some embodiments of the invention, the pre-determined time period is in the range of 10-20 minutes.

[0053] According to some embodiments of the invention, the isolating the soluble proteins is effected by filtration. According to some embodiments of the invention, the removing the solids from the chlorophyll-free preparation is effected by filtration.

[0054] According to some embodiments of the invention, the filtration is performed by using a filter press, a filter drum, or a filter bag.

[0055] According to some embodiments of the invention, prior to the filtration the method comprises mixing the chlorophyll-free preparation with a filtration aid.

[0056] According to some embodiments of the invention, the filtration aid is selected from the group consisting of diatomaceous earth, perlite, cellulosic fibers, activated carbon, and synthetic porous particles.

[0057] According to some embodiments of the invention, isolating the soluble proteins is performed using a decanter centrifuge.

[0058] According to some embodiments of the invention, removing the solids from the chlorophyll-free preparation is effected by centrifugation at 8000-10000 g.

[0059] According to some embodiments of the invention, removing the solids from the chlorophyll-free preparation further comprises filtration of a non-precipitated fraction through a 2 micron coarse filter paper.

[0060] According to some embodiments of the invention, the soluble proteins are substantially solid-free.

[0061] According to some embodiments of the invention, the soluble protein phase is substantially solid-free.

[0062] According to some embodiments of the invention, the method further comprising removing phenolic compounds and salts from the soluble proteins.

[0063] According to some embodiments of the invention, the removing the phenolic compounds and salts is performed by ultrafiltration.

[0064] According to some embodiments of the invention, the ultrafiltration is performed using a cross flow filter cassette in a range of 10-100 KDa.

[0065] According to some embodiments of the invention, the ultrafiltration is performed using a cross flow filter cassette of about 10 KDa.

[0066] According to some embodiments of the invention, the ultrafiltration is performed using a cross flow filter cassette of about 50 KDa.

[0067] According to some embodiments of the invention, the method further comprising diafiltration following the ultrafiltration.

[0068] According to some embodiments of the invention, the method further comprising precipitating the soluble proteins. According to some embodiments of the invention, precipitating is performed by a chemical precipitation.

[0069] According to some embodiments of the invention, the moringa leaves are fresh leaves.

[0070] According to some embodiments of the invention, the moringa leaves and rachis are fresh.

[0071] According to some embodiments of the invention, the moringa leaves are dry leaves.

[0072] According to some embodiments of the invention, the moringa leaves are frozen leaves.

[0073] According to some embodiments of the invention, the antioxidant comprises sodiummetabisulfite (SMBS).

[0074] According to some embodiments of the invention, the antioxidant is provided at a concentration in a range of 0.2%-1.0% (gram / 100 ml).

[0075] According to some embodiments of the invention, the buffer solution comprises a citric acid buffer.

[0076] According to some embodiments of the invention, the pH of the buffer is in the range of 4- 5.

[0077] According to some embodiments of the invention, the pH of the buffer is further adjusted to the range of 7-9.

[0078] According to some embodiments of the invention, the pH of the buffer is adjusted to optimize protein solubility.

[0079] According to some embodiments of the invention, the buffer solution comprises a carbonate buffer.

[0080] According to some embodiments of the invention, the pH of the buffer is in the range of 6-9.

[0081] According to some embodiments of the invention, the pH of the buffer is about 9.

[0082] According to some embodiments of the invention, the pH of the buffer is about 6.7.

[0083] According to some embodiments of the invention, the isolate of soluble proteins from the moringa leaves comprises large and small Rubisco protein subunits in a level detectable by SDS- PAGE.

[0084] According to some embodiments of the invention, the soluble proteins from the moringa leaves comprise large and small Rubisco protein subunits in a level detectable by SDS-PAGE.

[0085] According to some embodiments of the invention, the soluble protein phase comprises large and small Rubisco protein subunits in a level detectable by SDS-PAGE.

[0086] According to some embodiments of the invention, the soluble proteins comprise the large Rubisco protein subunit which comprises the amino acid sequence set forth by SEQ ID NO: 11. According to some embodiments of the invention, the soluble proteins comprise the large Rubisco protein subunit which further comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 12-14, 15-16, 17-20, 22, 23, 25, 26, 28, 29-31 and 32.

[0087] According to some embodiments of the invention, the soluble proteins comprise the large Rubisco protein subunit which comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12-14, 15-16, 17-20, 22, 23, 25, 26, 28, 29-31 and 32.

[0088] According to some embodiments of the invention, the soluble protein phase comprises the large Rubisco protein subunit which comprises the amino acid sequence set forth by SEQ ID NO: 11.

[0089] According to some embodiments of the invention, the soluble protein phase comprises the large Rubisco protein subunit which further comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 12-14, 15-16, 17-20, 22, 23, 25, 26, 28, 29-31 and 32.

[0090] According to some embodiments of the invention, the soluble protein phase comprises the large Rubisco protein subunit which comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12-14, 15-16, 17-20, 22, 23, 25, 26, 28, 29-31 and 32.

[0091] According to some embodiments of the invention, the large Rubisco protein further comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 12-14, 15- 16, 17-20, 22, 23, 25, 26, 28, 29-31 and 32.

[0092] According to some embodiments of the invention, the method further comprising freezing the isolate of soluble proteins.

[0093] According to some embodiments of the invention, the method further comprising freezing the isolate of soluble proteins following the isolating the soluble proteins.

[0094] According to some embodiments of the invention, the method further comprising freezing the isolate of soluble proteins following the isolating the soluble protein phase.

[0095] According to some embodiments of the invention, the composition-of-matter of some embodiments of the invention being substantially free of phenolic compounds and salts.

[0096] According to some embodiments of the invention, the composition-of-matter being substantially free of phenolic compounds.

[0097] According to some embodiments of the invention, the composition-of-matter is devoid of phenolic compounds.

[0098] According to some embodiments of the invention, the preparation of some embodiments of the invention being a liquid.

[0099] According to some embodiments of the invention, the preparation of some embodiments of the invention comprising between 15-20% (gram / 100 ml liquid solution) of soluble proteins. According to some embodiments of the invention, the preparation of some embodiments of the invention being a powder.

[0100] According to some embodiments of the invention, the preparation of some embodiments of the invention comprising between 50-75% soluble proteins (gram proteins / 100 gram powder).

[0101] According to some embodiments of the invention, the composition-of-matter of some embodiments of the invention or the preparation of some embodiments of the invention, forming part of an edible preparation.

[0102] According to some embodiments of the invention, the composition-of-matter of some embodiments of the invention, or the preparation of some embodiments of the invention comprises at least 50% (gram / 100 gram composition or preparation) of the edible composition.

[0103] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0104] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0105] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0106] In the drawings:

[0107] Fig. 1 depicts a multi-step protein extraction process from Moringa leaves. A flowchart representing the multi-step protein extraction process from Moringa leaves, outlining each stage including the initial state of the raw material, grinding, milling, filtering, de-greening, solid separation, precipitation (an alternative route), washing / ultra-filtration, and drying (details under “General Materials and Experimental Methods”). The scheme also illustrates the side-streams of the different processing steps that are discarded. "Protein powder" versions 1 and 2 refer to protein products obtained without (version 1) or with (version 2) the inclusion of the precipitation step.

[0108] Fig. 2 is SDS-PAGE image demonstrating that the initial state of the raw material does not affect protein profile as visualized by SDS-PAGE. SDS-PAGE electrophoretic analysis of the proteins extracted from fresh (lane 1 and 2), dried (lane 3 and 4), or frozen (lane 5 and 6) Moringa leaves. The protein bands of the Rubisco proteins are marked as “LSU” (large Rubisco subunit) and “SSU” (small Rubisco subunit). The results demonstrate that the initial state of the raw material does not affect the protein profile. MW (molecular weight) ladder was determined based on protein standards (described in “General Materials and Experimental Methods”).

[0109] Figs. 3A-C are images depicting the result of the milling step of leaf material using a colloid mill. Following the initial grinding, the leaf material was milled. The gap between the rotor and stator was adjusted to 0 machine units (Figure 3A), 11 machine units (Figure 3B), or 20 machine units (Figure 3C) to achieve different tissue particle sizes.

[0110] Fig. 4 is a graph depicting de-greening process of the green juice. The gradual reduction in opacity of the liquid phase (GJ) with increasing de-greening duration is shown by absorbance at 595 nanometers (nm) after separation from the solid phase by centrifugation. The green juice (GJ) liquid was heated at 50°C for 0 minutes, 5 minutes, 10 minutes, and 20 minutes, followed by rapid cooling in cold water.

[0111] Fig. 5 is a histogram depicting the content of the phenolic compound before and after ultrafiltration. The brown juice (BJ) liquid was washed with double distilled water (DDW) using a cross-flow filter cassette (+UF). The phenolic compound content decreased significantly from 3.39 milligrams / milliliter (±0.14) in the BJ (before ultrafiltration) to 0.7 milligrams / milliliter (±0.54) after ultrafiltration (+UF).

[0112] Figs. 6A-B are images of protein powders produced by vacuum or spray drying. The protein concentrate was dried using two different methods: spray drying (Figure 6A) and vacuum drying (Figure 6B). The spray-dried protein powder was significantly whiter and had a fine, powdery texture, indicating a more refined product. In contrast, the vacuum-dried protein powder was brownish and exhibited a harder, crystalline structure.

[0113] Figs. 7A-C are images depicting the outcomes of functional tests for protein powder. Various functional tests were conducted to evaluate the properties of the protein powder. Figure 7A - the foaming test; Figure 7B - The emulsification test; and Figure 7C the fried test. The Foaming Test resulted in a stable and strong foam. The Emulsification Test produced a smooth and viscous texture, similar to hummus salad. When fried, the protein powder in an aqueous batter formed a texture resembling scrambled eggs.

[0114] Figs. 8A-D show a continuous sequence alignment of Rubisco large subunit from various plant species along with the mapping of detected peptides in samples of the soluble protein powder of some embodiments of the invention which was extracted from leaves, rachises and stems of Moringa oleifera using the “pilot process” described in the Examples section which follows. The sequences of the Rubisco large subunits from eight plant species were included in the alignments: Cuscuta sandwichiana (UNIPROT accession No. Q49CC1; SEQ ID NO: 3), Lemna minor (UNIPROT accession No. A9L9A4; SEQ ID NO: 4), Pisum sativum (UNIPROT accession No. P04717; SEQ ID NO: 5), Spinacia oleracea (UNIPROT accession No. P00875; SEQ ID NO: 6), Petunia hybrida (UNIPROT accession No. P04992; SEQ ID NO: 7), Malus domestica (UNIPROT accession No. A0A223A9S9; SEQ ID NO: 8), Musa acuminata (UNIPROT accession No. A9QBM3; SEQ ID NO: 9) and Moringa oleifera (UNIPROT accession No. A0A4Y5PRY2; SEQ ID NO: 10). It is noted that since each line in the sequence alignment is limited to 60 amino acids (e.g., from 1-60, from 61-120, from 121-180) the alignments span over 4 pages which are marked as FIG. 8A, FIG. 8B, FIG. 8C, and FIG. 8D. Amino acid sequences corresponding to the peptides identified by LC-MS / MS in the Moringa soluble protein powder are highlighted in bold and italics. Peptides identified as unique to Moringa oleifera are labeled by a continuous line and numbered Pl-12 and their sequences are depicted in Table 3 and in the sequence listing. Additional peptides (e.g., P13-P17) were identified by LC-MS / MS analysis in the Moringa soluble protein powder and are specified in Table 3 and in the sequence listing. It is noted that since P13-P17 correspond to highly conserved regions across the plant species that were included in the sequence alignments their peptide numbers are not indicated in the alignment shown in Figures 8A-D.

[0115] It is noted that peptide Pl is unique and matches only to the sequence of the Rubisco large subunit from the Moringa Oliefera and not from the other plant species that were used in the analysis.

[0116] Fig. 9 depicts extracted ion chromatograms (XICs) corresponding to representative Niazirin ions having the molecular formula of C14H17NO5 detected in the LC-QTOF-MS analysis (negative ESI mode) of Moringa leaf soluble protein powder which was further extracted with 80% methanol (upper panel) and Moringa leaves extracted with 80% methanol (lower panel). The ions shown (m / z 314.0794, 324.1083, and 341.0989) match literature-reported Niazirin fragments [Lin, H., et al., 2019. “Comparative analysis of chemical constituents of Moringa oleifera leaves from China and India by ultra-performance liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry”. Molecules, 24(5), 942; Wang, F., et al., 2022. “Pharmacokinetics of niazirin from Moringa oleifera Lam in rats by UPLC-MS / MS: Absolute bioavailability and dose proportionality”. eFood, 3(6), e39; Fantoukh, O. I., et al., 2021. “Profiling and quantification of the key phytochemicals from the drumstick tree (Moringa oleifera) and dietary supplements by UHPLC-PDA-MS”. Planta Medica, 87(05), 417-427; each of which is fully incorporated herein by reference]. Identification was made based on accurate mass and comparison with published spectra. It is noted that similar ions are present in both extracts from the Moringa soluble protein powder and the Moringa leaves.

[0117] Fig. 10 is SDS-PAGE image demonstrating the isolation of the Rubisco large and small subunits from fresh moringa leaves and rachis using the “pilot process” described in General Materials and Experimental Methods of the Examples section which follows. Lanes 1 and 2 represent two repeats which were done in 2 independent experiments in which the pilot process was used for isolation of the soluble protein powder from Moringa leaves, rachises and stems. The protein marker PM2700 ExcelBand™ (A2S) was used to determine protein molecular weight (MW). The protein bands of the Rubisco proteins are marked as “LSU” (large Rubisco subunit) and “SSU” (small Rubisco subunit).

[0118] Fig. 11 is a schematic illustration of a process of isolating soluble proteins from moringa leaves according to some embodiments of the invention. In step (I) the method comprises grinding of Moringa leaves (either attached to rachises and stems or isolated leaves). In step (II) the method comprises disrupting organelles such as chloroplasts. In step (III) the method comprises isolating soluble proteins following disruption of organelles. It is noted that step (III) can occur directly after disruption of organelles in step (II)) or following removal of phenolic compounds and salts (optional step (IV)(a)) or following precipitation of proteins (optional step (IV)(b).

[0119] Fig. 12 is a flow chart depicting a multi-step extraction process for soluble proteins from Moringa leaves according to some embodiments of the invention. The process begins with grinding of plant material (fresh or dry leaves) by shredding the plant material in liquid (I)(a) followed by crushing the shredded plant material (I)(b). Then the solids (e.g., pulps) are separated from the liquid (e.g., using a screw press or decanter centrifuge; (I)(c)) resulting in a “green juice”. Next, the process includes disrupting of organelles (e.g., chloroplasts; (II)) which are present in the green juice so as to facilitate the release of soluble proteins from the organelles. The method further comprises a step of isolating the soluble proteins (III) by separating the disrupted organelles (discarded solids or pellet) from the liquid (e.g., using either filtration or centrifugation). The resulting liquid (following disruption of organelles) has a brown color (“brown juice”). The method further comprises a step of removing phenolic compounds and salts from the soluble proteins and concentrating the proteins (IV)(a) by ultrafiltration and diafiltration. Additionally or alternatively, the method comprises a step of precipitating the soluble proteins by chemical precipitation and re-dissolution of the soluble proteins (IV)(b). In some embodiments step (IV)(a) is performed following step (IV)(b). In some embodiments step (IV)(b) is performed following step (IV)(a). The method further comprises a step of drying (V) the soluble proteins and obtaining a protein powder of soluble proteins from the moringa leaves. DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0120] The present invention, in some embodiments thereof, relates to methods of producing an isolate of soluble proteins from moringa leaves, and, more particularly, but not exclusively, to compositions comprising the soluble proteins from moringa leaves.

[0121] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details of construction and the arrangement of the components and / or methods set forth in the following description and / or illustrated in the drawings and / or the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0122] The interactions between proteins and polyphenols in Moringa leaf have a large impact on the protein functionality and polyphenols bioavailability. Proteins obtained from moringa leaf are endowed with "grassy flavor" and a greenish hue”, consequently, these attributes make moringa protein-based food products less attractive to the average consumer.

[0123] Therefore, a large-scale production process of a white proteinaceous preparation free of phenols should be developed in order to improve the functionality and overall organoleptic properties. However, such a process might affect the protein structure depending on the extraction conditions (e.g., solvents, temperature, pH, and / or salt).

[0124] The Examples section which follows exemplify an efficient method for extracting maximal quantities of soluble proteins from leaves and producing a high-quality protein powder. This powder is highly functional in a wide range of food applications, including foaming, emulsification, and frying, while maintaining an acceptable smell and no aftertaste. The production process has been refined to optimize each step, from establishing the most suitable initial state of the raw material, through grinding, milling, filtering, de-greening, solid separation, and precipitation (as an alternative route), to washing / ultra-filtration and drying. This ensures the production of a versatile and superior protein product suitable for various nutritional uses.

[0125] According to an aspect of some embodiments of the invention, there is provided a method of producing an isolate of soluble proteins from moringa leaves, comprising:

[0126] (a) grinding the moringa leaves in the presence of liquid, to thereby obtain a green juice of ground moringa leaves;

[0127] (b) disrupting organelles in said green juice so as to release soluble proteins from said organelles, and

[0128] (c) isolating said soluble proteins from said green juice, thereby producing the isolate of soluble proteins from the moringa leaves. According to an aspect of some embodiments of the invention, there is provided a method of producing an isolate of soluble proteins from moringa leaves, comprising:

[0129] (a) grinding the moringa leaves in a buffer solution comprising an antioxidant, to thereby obtain a green juice (GJ) of ground moringa leaves;

[0130] (b) isolating a soluble protein phase from the green juice using a filter;

[0131] (c) removing chlorophyll from the soluble protein phase to obtain a chlorophyll-free preparation comprising the soluble proteins;

[0132] (d) removing solids from the chlorophyll-free preparation to obtain solid-free preparation comprising the soluble proteins; and

[0133] (e) removing phenolic compounds and salts from the solid-free preparation by ultrafiltration; thereby producing the isolate of soluble proteins from the moringa leaves.

[0134] The phrase “an isolate of soluble proteins” refers to a composition which comprises proteins from the moringa leaves, but not all components present in a moringa leaf (e.g., not insoluble proteins). The soluble proteins from the moringa leaves are soluble in an aqueous solution.

[0135] A person of ordinary skill in the art can distinguish between soluble and insoluble proteins within an aqueous solution, e.g., by means of centrifugation, filtration and the like of a solution comprising soluble proteins.

[0136] In some embodiments the soluble proteins can be precipitated in an aqueous solution having an acid pH. In some embodiments, the acid pH is in the range of 3-6, e.g., 4-6, e.g., 4-5.

[0137] In some embodiments the soluble proteins are not precipitated in an aqueous solution having a neutral or basic pH. In some embodiments the neutral or basic pH is in the range of 6.1- 9, e.g., 6.1-8, e.g., 6.5-7.5.

[0138] The soluble proteins can be present in an extract or fraction of moringa leaves.

[0139] According to some embodiments of the invention, the moringa leaves are thrice-pinnate leaves which are separated from the main rachis.

[0140] According to some embodiments of the invention, the moringa leaves, e.g., leaflet, are attached to the rachises and stems.

[0141] According to some embodiments of the invention, the moringa leaves are isolated from the rachises and stems.

[0142] For the method of producing the isolate of soluble proteins the moringa leaves can be fresh moringa leaves, dried moringa leaves, or frozen moringa leaves. As used herein the term “dry” when describing plants tissues such as leaves, refers to a plant tissue that has undergone dehydration, either naturally (e.g., sun drying) or by using artificial methods, resulting in significantly reduced moisture content as compared to fresh leaves as defined below.

[0143] The moisture content can be described as the percentage of weight of water out of the total weight of tissue.

[0144] According to some embodiments of the invention, the dry plant tissue comprises less than 60 %, less than 50 %, less than 40 %, less than 30 %, less than 20 %, less than 15 %, less than 14 %, less than 13 %, less than 12 %, less than 11 %, less than 10 %, less than 9 %, less than 8 %, less than 7 %, less than 6 %, less than 5 %, less than 4 %, less than 3 %, less than 2 % moisture content (expressed as weight of water / total weight of the plant tissue).

[0145] As used herein the term “fresh” when describing plant tissues such as leaves, refers to a plant tissue (e.g., moringa leaves) which has been harvested not more than 4 days prior to processing, is being kept at a temperature range from 4 °C to 25 °C and is not being subjected to dehydration.

[0146] In some embodiments, the fresh leaves are harvested not more than 3 days, not more than 2 days, not more than 1 day, not more than 8 hours, not more than 4 hours, not more than 2 hours prior to processing, while being kept at a temperature range from 4 °C to 25 °C and is not being subjected to dehydration.

[0147] According to some embodiments, fresh leaves comprise 61-80 % moisture content, dry leaves comprise 8-15% moisture content, oven-dry leaves comprise 2-5% moisture content; and completely dry leaves have less than 2% moisture content.

[0148] According to some embodiments of the invention, the moringa leaves are fresh leaves. According to some embodiments of the invention, the moringa leaves are dry leaves. According to some embodiments of the invention, the moringa leaves are frozen leaves.

[0149] According to some embodiments of the invention, the moringa leaves are grounded in a liquid.

[0150] In some embodiments, the liquid is water per se, i.e., without any added reagents or salts.

[0151] In some embodiments, the liquid is a buffer solution.

[0152] According to some embodiments of the invention, the moringa leaves are grounded in a buffer solution comprising an antioxidant, to thereby obtain a green juice (GJ) of ground moringa leaves.

[0153] According to some embodiments of the invention, the buffer solution is an aqueous solution, i.e., a water-based solution, which includes specific compounds such as salts that control the pH of the solution and are capable of stabilizing the pH even when small amounts of acid or base are added.

[0154] The buffer solution of some embodiments of the invention maintains a pH of 6.7-7.5, 6.7- 7.4, 6.7-7.3, 6.7-7.2, 6.7-7.1, 6.7-7.0, 6.7-6.8 e.g., a pH of 6.7.

[0155] The buffer solution may include, for example, sodium bicarbonate and sodium carbonate (anhydrous). Non-limiting concentrations of the examples of buffer solutions may include sodium bicarbonate at a concentration range of 0.0125-0.0166 M (molar), e.g., about 0.0125 M, and sodium carbonate (anhydrous) at a concentration of 0.0875-0.120 M, e.g., about 0.0875 M.

[0156] In some embodiments the buffer solution comprises a citric acid buffer.

[0157] In some embodiments the buffer is at a pH in the range of 4-5, e.g., at a pH of about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, or about 5.

[0158] In some embodiments the citric acid buffer is used for grinding the moringa leaves, for obtaining the green juice.

[0159] In some embodiments the citric acid buffer is further adjusted to a pH in the range of 7-9 in order to optimize protein solubility.

[0160] In some embodiments the buffer solution comprises a carbonate buffer.

[0161] In some embodiments the buffer is at a pH in the range of 6.7-9.

[0162] In some embodiments the buffer is at a pH of about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, or about 8.9, about 9.0.

[0163] It should be noted that water per se is not a buffer solution since it cannot maintain a stable pH when adding acid or base thereto.

[0164] According to some embodiments of the invention, the buffer solution does not consist of water alone.

[0165] According to some embodiments of the invention, the buffer solution does not comprise alcohol or hydro-alcohol.

[0166] According to some embodiments of the invention, the method does not comprise contacting the moringa leaves with alcohol or hydro-alcohol. According to some embodiments of the invention, the method excludes addition of alcohol or hydro-alcohol to the moringa leaves.

[0167] According to some embodiments of the invention, the method of producing an isolate of soluble proteins from moringa leaves does not involve extraction of the moringa leaves with water (per se), methanol, ethanol, isopropanol, ethyl acetate, acetone or mixtures thereof. According to specific embodiments of the invention, the method of producing an isolate of soluble proteins from moringa leaves does not comprise extraction of niazimin A or niazirin.

[0168] According to specific embodiments of the invention, the method of producing an isolate of soluble proteins from moringa leaves does not involve using a chromophore.

[0169] According to some embodiments of the invention, the ratio between the moringa leaves and the buffer solution can be from about 1: 1.5 (weight of plant material / volume of buffer; (w / v)) to about 1:25 (w / v), from about 1:2 (w / v) to about 1:20 (w / v), from about 1:2 (w / v) to about 1: 15 (w / v), e.g., about 1:3 (w / v), about 1:4 (w / v), about 1:5 (w / v), about 1:6 (w / v), about 1:7 (w / v), about 1:8 (w / v), about 1:9 (w / v), about 1: 10 (w / v), about 1: 11 (w / v), about 1: 12 (w / v), about 1: 13 (w / v), about 1: 14 (w / v), about 1: 15 (w / v), about 1: 16 (w / v), about 1: 17 (w / v), about 1: 18 (w / v), about 1: 19 (w / v) or about 1:20 (w / v).

[0170] Various antioxidants can be included in the buffer solution which is used for grinding the moringa leaves. Examples include, but are not limited to sodium ascorbate, acetic acid and sodiummetabisulfite.

[0171] According to some embodiments of the invention, the antioxidant is sodium-metabisulfite (SMBS).

[0172] According to some embodiments of the invention, the antioxidant is provided at a concentration in a range of 0.2%-1.0% (gram / 100 ml).

[0173] According to some embodiments of the invention, the concentration of the antioxidant in the buffer solution is in the range of 0.1% (gram / 100 ml) - 1.5% (gram / 100 ml), 0.15%-1.3% (gram / 100 ml), 0.2%-1.2% (gram / 100 ml), or 0.2%-1.0% (gram / 100 ml).

[0174] According to some embodiments of the invention, grinding can be performed with any suitable grinding machine. For example, the moringa leaves are shredder using a shredder following which the shredder plant material is further grounded using a crusher.

[0175] Following grinding, the green juice comprises liquid with “pulps” of the grounded moringa leaves.

[0176] According to some embodiments of the invention, grinding is performed using a blender.

[0177] Examples of a blender include, but are not limiting to, the hand blender MultiQuick 5 Vario MQ5235WH (Braun), the high power Fresh & Furious blender (Breville) or the CB353 power pitcher blender system (Ninja).

[0178] Grinding can be performed for 0.5-2 minutes, e.g., for about 1 minute, until a green juice is formed.

[0179] According to some embodiments of the invention, disrupting the organelles is performed using a high-pressure homogenizer, a microfluidizer, and / or a colloid mill. According to some embodiments of the invention, organelles comprise chloroplasts.

[0180] It should be noted that disrupting the organelles, such as chloroplasts, assists in releasing soluble proteins from the organelles.

[0181] According to some embodiments of the invention, the method further comprising cooling the soluble proteins to a temperature of 4 °C following said disruption of the organelles.

[0182] According to some embodiments of the invention, the method further comprising milling the ground moringa leaves prior to isolating of the soluble protein phase.

[0183] According to some embodiments of the invention, the method further comprising milling the ground moringa leaves prior to isolating of the soluble proteins.

[0184] Milling of the ground material in the antioxidant containing buffer can be performed using a milling machine, such as a colloid mill CM-65 (Beyond Machinery). The efficiency of the milling process can be controlled by parameters such as the gap between rotor and the stator of the milling machine. For example, a setting of “0” indicates a broad gap between the rotor and the stator, and a setting of “20” indicates a narrower gap between the rotor and the stator, and therefore results in smaller pieces.

[0185] According to some embodiments of the invention, milling is performed by applying between 0-20 machine units between the rotor and the stator, e.g., 0, 11, or 20 machine units between the rotor and stator.

[0186] According to some embodiments of the invention, following milling, the milled material is kept at 4°C prior to being filtered.

[0187] According to some embodiments of the invention, the grinding results in cell lysis (e.g., at least a partial cell lysis) which releases the intracellular proteins from the leaf tissue to the buffer solution.

[0188] According to some embodiments of the invention, the intracellular proteins which are comprised in the green juice are at least partially separated from the cell membrane and / or intracellular compartments and / or organelles of the leaves (e.g., at least partially separated from the chloroplast and nuclei).

[0189] According to some embodiments of the invention, the intracellular proteins which are comprised in the green juice are separated from the cell membrane and / or intracellular compartments and / or organelles of the leaves.

[0190] According to some embodiments of the invention, measures are taken not to degrade the intracellular protein fraction.

[0191] Once the moringa leaves are ground, and / or further milled, the method comprising isolating the soluble proteins or the soluble protein phase from the green juice using a filter. The filter is selected capable separating between a soluble phase (which comprises the soluble proteins from the moringa leaves) and a non-soluble phase.

[0192] According to some embodiments of the invention, the isolating of the soluble proteins is effected by filtration.

[0193] According to some embodiments of the invention, the filter is featured by a pore diameter of a size selected from a range of 50-400 microns, e.g., 50-380 microns, 50-350 microns, 50-300 microns, 50-250 microns, 50-200 microns, 50-180 microns, e.g., 50-150 microns.

[0194] According to some embodiments of the invention, the filter is featured by a pore diameter of a size selected from a range of 50-120 microns.

[0195] According to some embodiments of the invention, the filter is featured by a pore diameter having a size of about 400 microns (e.g., between 350-400 microns)

[0196] According to some embodiments of the invention, the filter is featured by a pore diameter of a size selected from a range of 50-140 microns, e.g., with a pore diameter of a size selected from the range of 50-120 microns, 50-110 microns, 50-100 microns, 50-90 microns, 50-80 microns, 50-70 microns, 50-60 microns, 80-130 microns, 80-125 microns, 80-120 microns, 90- 120 microns, 100-120 microns, or 100-110 microns.

[0197] Following the filtering, the filtered soluble phase (which comprises the soluble proteins) is characterized by a green color (filtered green juice) due to presence of chlorophyll.

[0198] The method of some embodiments of the invention, comprises the step of removing chlorophyll (also referred to as “de-greening”) from the soluble protein phase to obtain a preparation which is substantially chlorophyll-free, and comprising the soluble proteins.

[0199] As used herein the phrase “chlorophyll-free” refers to a preparation (e.g., powder) which comprises less than 0.1% w / w (weight of chlorophyll / weight of preparation).

[0200] According to some embodiments, the weight of the preparation is a dry weight.

[0201] According to some embodiments, the chlorophyll-free preparation comprises less than 0.09% (w / w), less than 0.08% (w / w), less than 0.07% (w / w), less than 0.06% (w / w), less than 0.05% (w / w), less than 0.04% (w / w), less than 0.03% (w / w), less than 0.02% (w / w), e.g., less than 0.01% (w / w).

[0202] For example, as shown in Example 5 of the Examples section which follows, the content of the chlorophyll in the powder that was prepared according to the pilot process described in the “General Materials and Experimental Methods” was 0.2 mg chlorophyll per gram of protein powder, which is equivalent to 0.02% (w / w).

[0203] According to some embodiments of the invention, the preparation which comprises the soluble proteins comprises less than 81%, less than 82%, less than 83%, less than 84%, less than 85%, less than 86%, less than 87%, less than 88%, less than 89%, less than 90%, less than 91%, less than 92%, less than 93%, less than 94%, less than 95%, less than 96%, less than 97%, less than 98%, less than 99%, less than 99.1%, less than 99.2%, less than 99.3%, less than 99.4%, less than 99.5%, less than 99.6%, less than 99.7%, less than 99.8%, less than 99.9% of the chlorophyll content present in the initial moringa leaves prior to being subjected to the grinding and purification process.

[0204] According to some embodiments of the invention, removing the chlorophyll is performed by heating the soluble protein phase for a pre-determined time period at a temperature in the range of 35-50 °C.

[0205] According to some embodiments of the invention, heating the soluble protein phase is effected at a temperature in the range of 45-50 °C, 46-50 °C, 47-50 °C, 48-50 °C, 49-50 °C, e.g., about 50 °C, e.g., at 50 °C.

[0206] It is noted that the pre-determined time period and the temperature used for heating the soluble phase are selected such that the proteins will not undergo denaturation in the de-greening process of removing the chlorophyll.

[0207] For example, when using high temperatures such as in the range of 45-50°C (e.g., at a temperature of about 50°C), the pre-determined time period can be in the range of 1-20 minutes. According to some embodiments of the invention, the pre-determined time period is in the range of 10-20 minutes.

[0208] According to some embodiments of the invention, when using lower temperatures such as in the range of 35-45 °C, the pre-determined time period can be longer, e.g., for 2-18 hours.

[0209] To preserve the integrity of the soluble proteins (e.g., to avoid degradation) following heating the soluble phase is subject to a rapid cooling.

[0210] According to some embodiments of the invention, the method further comprising cooling the soluble protein phase to a temperature of 4°C following the pre-determined time period. According to some embodiments of the invention, cooling is effected by immediately transferring the container (e.g., a tube) containing the soluble protein phase to a vessel containing ice.

[0211] According to some embodiments of the invention, following the pre-determined time period the soluble protein phase is characterized by absorbance of about 0.5 or less at a wavelength of 595 nanometer (nm).

[0212] For example, Figure 4 shows that following 5 minutes of heating the soluble phase (at 50 °C) the absorbance at a wavelength of 595 nm decreased from 2.53 to 0.86, and continued to decrease up to 0.3 units when the soluble phase was heated for 20 minutes. Following the de-greening process (removal of the chlorophyll from the soluble phase) the method comprises a step of removing solids from the chlorophyll-free preparation which comprises the soluble proteins.

[0213] According to some embodiments of the invention, removing the solids from the chlorophyll-free preparation is effected by filtration.

[0214] According to some embodiments of the invention, the filtration is performed by using a filter press, a filter drum, or a filter bag.

[0215] According to some embodiments of the invention, prior to the filtration the method comprises mixing said chlorophyll-free preparation with a filtration aid.

[0216] According to some embodiments of the invention, the filtration aid is selected from the group consisting of diatomaceous earth, perlite, cellulosic fibers, activated carbon, and synthetic porous particles.

[0217] According to some embodiments of the invention, isolating the soluble proteins is performed using a decanter centrifuge.

[0218] According to some embodiments of the invention, the solids are removed by centrifugation which results in a precipitated pellet and an upper liquid phase which comprises the nonprecipitated fraction.

[0219] A non-limiting example of a centrifuge which can be used to separate the solids from the chlorophyll-free preparation is the Sorvall RC 6 Plus Superspeed Centrifuge (e.g., available from Thermo Scientific).

[0220] According to some embodiments of the invention, removing the solids from the chlorophyll-free preparation is effected by centrifugation at 8000-10000 g.

[0221] According to some embodiments of the invention, removing the solids from the chlorophyll-free preparation is effected by centrifugation at 10000 g.

[0222] According to some embodiments of the invention, the centrifugation is performed for about 10-20 minutes, e.g., for 11-16 minutes, for 12-16 minutes, e.g., for 14-16 minutes, e.g., for about 15 minutes.

[0223] According to some embodiments of the invention, following the centrifugation, the upper liquid phase is subject to filtration, to remove solids that were included in the upper liquid phase following the centrifugation.

[0224] According to some embodiments of the invention, removing the solids from the chlorophyll-free preparation further comprises filtration of the non-precipitated fraction through a 2 micron coarse filter paper (Hobrafilt). According to some embodiments of the invention, the soluble proteins are substantially solid-free.

[0225] According to some embodiments of the invention, the soluble protein phase is substantially solid-free.

[0226] As used herein the phrase “solid-free” preparation refers to a liquid formulation or extract that has been processed to remove all particulate matter larger than 400 microns.

[0227] According to some embodiments of the invention, the method further comprising removing phenolic compounds and salts from said soluble proteins.

[0228] In some embodiments, removing the phenolic compounds and salts is performed by ultrafiltration.

[0229] Once the solids are removed from the chlorophyll-free preparation, the method comprises a step of removing phenolic compounds and buffer salts from the solid-free preparation by ultrafiltration.

[0230] The ultrafiltration is performed using a filter cassette which allows the buffer salts and phenolic compounds to cross the filtering membrane but retains the large protein molecules within the solid-free preparation. For example, if proteins of a size of at least 10 KDa are desired (are to be retained in the preparation), then the cross flow filter cassette would be of 10 KDa.

[0231] According to some embodiments of the invention, the ultrafiltration is tangential flow ultrafiltration (TFF).

[0232] According to some embodiments of the invention, the ultrafiltration is performed using a cross flow filter cassette in a range of 10-100 KDa.

[0233] According to some embodiments of the invention, the ultrafiltration is performed using a cross flow filter cassette of 10 about KDa.

[0234] According to some embodiments of the invention, the ultrafiltration is performed using a cross flow filter cassette of about 50 KDa.

[0235] According to some embodiments of the invention, during the ultrafiltration, Rubisco proteins and other large soluble proteins are retained by the membrane (also referred herein as “retentate”) and recirculate back into the feed reservoir, while buffer salts and phenolic compounds are washed through the filter (also referred to as “permeate”).

[0236] According to some embodiments of the invention, the ultrafiltration process results in a concentrated fraction of the soluble protein phase to about l / 10thof its initial volume.

[0237] According to some embodiments of the invention, the method further comprising diafiltration following the ultrafiltration. For diafiltration, an equal volume of double-distilled water (DDW) is added to the sample and passed through the filter cassette. According to some embodiments of the invention, the washing step is repeated for about eight times, resulting in a 99.9% removal of buffer salts and in about 90% removal of phenolic compounds from the solid-free preparation.

[0238] Additionally or alternatively, the method further comprises precipitating the soluble proteins by using chemical precipitation.

[0239] For example, proteins can be precipitated by adjusting the pH to about 4-5 by adding an acid (e.g., HC1 or citric acid).

[0240] Additionally or alternatively, the proteins can be precipitated by adding salts such as calcium chloride (CaCE), ammonium sulfate ((NHQiSOQ, or ammonium phosphate ((NHQsPOQ, with or without concurrent pH adjustment to acidic conditions.

[0241] In some embodiments, the resulting precipitate is separated using a decanter.

[0242] In some embodiments, the recovered precipitated proteins can be further neutralized to approximately pH 5.5-7, e.g., pH 6.5-7, e.g., pH 7.

[0243] In some embodiments, the soluble proteins comprise large and small Rubisco protein subunits in a level detectable by SDS-PAGE.

[0244] According to some embodiments of the invention, the isolate of soluble proteins from the moringa leaves comprises large and small Rubisco protein subunits in a level detectable by SDS- PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis).

[0245] According to some embodiments of the invention, the soluble proteins comprise the large Rubisco protein subunit which comprises the amino acid sequence set forth by SEQ ID NO: 11.

[0246] According to some embodiments of the invention, the soluble proteins comprise the large Rubisco protein subunit which comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 12-14, 15-16, 17-20, 22, 23, 25, 26, 28, 29-31 and 32.

[0247] According to some embodiments of the invention, the isolate of the soluble proteins can be used in a liquid form.

[0248] According to some embodiments of the invention, the method further comprising freezing the isolate of soluble proteins.

[0249] According to some embodiments of the invention, the method further comprising freezing the isolate of soluble proteins following isolating the soluble proteins or the soluble protein phase.

[0250] Thus, the isolate of the soluble proteins can be kept frozen until further use, or until it is subjected to a drying process.

[0251] In cases where the isolate of soluble proteins is kept frozen, prior to the drying procedure, the frozen isolate of soluble protein is gradually defrosted. Additionally or alternatively, the isolate of the soluble proteins can be subjected to a drying process without being frozen prior to drying.

[0252] According to an aspect to some embodiments of the invention, there is provided a method of preparing a powder of soluble proteins from moringa leaves, the method comprises:

[0253] (a) isolating soluble proteins from moringa leaves according to the method of some embodiments of the invention, to thereby obtain a solution of isolate soluble proteins, and

[0254] (b) spray drying the solution of the isolate soluble proteins, to thereby obtain the powder of soluble proteins from the moringa leaves.

[0255] According to some embodiments of the invention, the drying is performed by a freeze dryer.

[0256] According to some embodiments of the invention, the drying is performed by a low temp spray dryer (e.g., available from Buchi).

[0257] Reference is made to Figure 11.

[0258] Figure 11 is a schematic illustration of a process of isolating soluble proteins from moringa leaves according to some embodiments of the invention.

[0259] In step (I) the method comprises grinding of Moringa leaves (either attached to rachises and stems or as isolated leaves).

[0260] In step (II) the method comprises disrupting organelles such as chloroplasts.

[0261] In step (III) the method comprises isolating soluble proteins following disruption of organelles. It is noted that step (III) can occur directly after disruption of organelles in step (II)) or following removal of phenolic compounds and salts (optional step (IV)(a)) and / or following precipitation of proteins (optional step (IV)(b)).

[0262] Reference is made to Figure 12.

[0263] Figure 12 is a flow chart depicting a multi-step extraction process for soluble proteins from Moringa leaves according to some embodiments of the invention. The process begins with grinding of plant material (fresh or dry leaves) by shredding the plant material in liquid (step (I)(a)) followed by crushing the shredded plant material (step (I)(b)). Then the solids (e.g., pulps) are separated from the liquid (e.g., using a screw press or decanter centrifuge; (step (I)(c)) resulting in a “green juice”. Next, the process includes disrupting of organelles (e.g., chloroplasts; (step (II)) which are present in the green juice so as to facilitate the release of soluble proteins from the organelles. The method further comprises a step of isolating the soluble proteins (step (III)) by separating the disrupted organelles (discarded solids or pellet) from the liquid (e.g., using either filtration or centrifugation). The resulting liquid (following disruption of organelles) has a brown color (“brown juice”). The method further comprises a step of removing phenolic compounds and salts from the soluble proteins and concentrating the proteins (step (IV)(a)) by ultrafiltration and diafiltration. Additionally or alternatively, the method comprises a step of precipitating the soluble proteins by chemical precipitation and re-dissolution of the soluble proteins (step (IV)(b)). In some embodiments step (IV)(a) is performed following step (IV)(b). In some embodiments step (IV)(b) is performed following step (IV)(a). The method further comprises a step of drying (step (V)) the soluble proteins and obtaining a protein powder of soluble proteins from the moringa leaves.

[0264] According to an aspect of some embodiments of the invention there is provided a composition-of-matter comprising a chlorophyll-free preparation of soluble proteins of moringa leaves, wherein the soluble proteins comprise large and small Rubisco protein subunits, produced according to the method of some embodiments of the invention.

[0265] According to an aspect of some embodiments of the invention there is provided a composition-of-matter comprising a chlorophyll-free preparation of soluble proteins of moringa leaves, wherein the soluble proteins comprise large and small Rubisco protein subunits.

[0266] The moringa Rubisco protein in the composition-of-matter or the preparation of some embodiments of the invention has a specific amino acid sequence which can be determined by sequence alignment to a known sequence of Rubisco from moringa.

[0267] According to an aspect of some embodiments of the invention there is provided a composition-of-matter comprising a chlorophyll-free preparation of soluble proteins of moringa leaves, wherein the soluble proteins comprise large and small Rubisco protein subunits, and wherein the large Rubisco protein comprises the amino acid sequence set forth by SEQ ID NO: 11.

[0268] In some embodiments, the large Rubisco protein further comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 12-14, 15-16, 17-20, 22, 23, 25, 26, 28, 29-31 and 32.

[0269] According to an aspect of some embodiments of the invention there is provided a composition-of-matter comprising a chlorophyll-free preparation of soluble proteins of moringa leaves, wherein the soluble proteins comprise large and small Rubisco protein subunits, and wherein the large Rubisco protein comprises the peptides depicted in Figures 8A-D and Table 3 below.

[0270] Representative sequences of the large subunit of the Rubisco protein from Moringa oleifera (rbcL ribulose- 1,5-bisphosphate carboxylase / oxygenase large subunit) are provided by GenBank Accession No. YP_009573401 (482 amino acids, SEQ ID NO: 1) and UNIPROT accession No. A0A4Y5PRY2 (475 amino acids; SEQ ID NO: 10). Methods of determining the presence and / or amount of the large and small Rubisco proteins in the composition-of-matter include, but are not limited to, SDS-PAGE followed by densitometric analysis against a protein marker, Western blotting or ELISA using specific anti- Rubisco large and small subunits antibodies, and mass spectrometry to identify and quantify the sub-units based on their mass-to-charge ratio.

[0271] According to an aspect of some embodiments of the invention there is provided a composition-of-matter comprising a chlorophyll-free preparation of soluble proteins of moringa leaves which comprises Niazirin.

[0272] The presence, the absence, and / or the amount of chlorophyll molecules of in the composition-of-matter of some embodiments of the invention can be determined by various methods, such as, high performance liquid chromatography (HPLC) or using a spectrophotometer, for example, by reading an absorbance at a wavelength of 595 nm.

[0273] According to some embodiments of the invention, the chlorophyll-free preparation has an absorbance which is equal or less than 0.3 units at a wavelength of 595 nm.

[0274] According to some embodiments of the invention, the chlorophyll-free preparation may comprise trace amounts of chlorophyll.

[0275] According to some embodiments of the invention, the chlorophyll-free preparation is devoid of chlorophyll.

[0276] According to some embodiments of the invention, the composition-of-matter being substantially free of phenolic compounds and salts.

[0277] According to some embodiments of the invention, the composition-of-matter may include trace amounts of phenolic compounds.

[0278] Methods of determining the presence and / or level of phenolic compounds are known in the art, e.g., as described in Ainsworth, E., Gillespie, K. 2007 (“Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin-Ciocalteu reagent”. Nat Protoc 2, 875- 877; which is fully incorporated herein by reference).

[0279] For example, the presence and / or level of phenolic compounds can be determined by incubating a sodium carbonate solution with a test sample, preferably in the dark, following which the absorbance at a wavelength of 760 nm is read.

[0280] Briefly, analyzed samples and Gallic Acid standards are mixed with Folin-Ciocalteu's phenol reagent (Merck) in microtiter multi-plate wells (DeepWell), followed by a 10-minute incubation. A 20% sodium carbonate solution is then added to each well and incubated for 2 hours at room temperature. All incubations are carried out in the dark. Following incubation, a sample from each reaction is transferred to an ELISA plate, and absorbance is read at a wavelength of 760 nm. Total phenolics (phenolic compounds) can be calculated as Gallic Acid equivalents using a standard curve derived from the blank-corrected A765 values of the gallic acid standards.

[0281] According to some embodiments of the invention, the composition-of-matter may include trace amounts of buffer salts.

[0282] According to an aspect of some embodiments of the invention, there is provided a preparation comprising the composition-of-matter of some embodiments of the invention.

[0283] According to some embodiments of the invention, the preparation being a liquid.

[0284] According to some embodiments of the invention, the preparation comprising about 15- 20% (gram / 100 ml solution), e.g., about 16.3% (gram / 100 ml solution) of soluble proteins.

[0285] According to some embodiments of the invention, the preparation being a powder.

[0286] According to some embodiments of the invention, the preparation comprising at least 50% (gram proteins / 100 gram powder), at least 55% (gram proteins / 100 gram powder), at least 60% (gram proteins / 100 gram powder), at least 65% (gram proteins / 100 gram powder), at least 70% (gram proteins / 100 gram powder), at least 75% (gram proteins / 100 gram powder) of soluble proteins of the moringa leaves.

[0287] According to some embodiments of the invention, the preparation comprising between 50- 75% (gram proteins / 100 gram powder) soluble proteins of the moringa leaves.

[0288] The protein content in the composition-of-matter or the preparation comprising same can be quantified using various known methods such as the Bicinchoninic acid protein assay (BCA) or the Bradford assay, against a standard curve using a protein with known quantities (e.g., bovine serum albumin (BSA), or the Kjeldahl method.

[0289] For example, for the BCA assay, triplicates from each extraction sample are placed on a microplate and incubated with the BCA working reagent (e.g., from WR, Pierce, USA) for 15 minutes at 37 °C. Absorbance of the samples is read in a microplate reader (e.g., the Infinite F200, Tecan) set to a wavelength of 550 nm.

[0290] For the Bradford assay, triplicates from each extraction sample are placed on a microplate and mixed with Bradford reagent (e.g., from Bio-Rad). Absorbance at wavelength of 590 nm is measured using a microplate reader (e.g., from Infinite F200, Tecan).

[0291] Kjeldahl quantifications can be done using the AO AC (Association of Official Agricultural Chemists) 981.10 protocol by the Microbiology Institute (Nesher, Israel; world wide web (dot) microb (dot) co (dot) il). Kjeldahl protocol is well established, and is used by the Microbiology Institute according to the “Teken” AOAC (Association of Official Agricultural Chemists) instructions. According to some embodiments of the invention, the composition-of-matter of some embodiments of the invention, or the preparation of some embodiments of the invention, forming part of an edible preparation.

[0292] As used herein the terms “edible” or “consumable”, which are interchangeably used herein, refer to a composition which is safe for human or animal eating. For example, an edible or a consumable composition includes, but is not limited to, a food product that is generally recognized as safe per a government or regulatory body (such as the United States Food and Drug Administration). In certain embodiments, the food product is considered safe to consume by a person of skill.

[0293] It should be noted that any edible food product suitable for a human consumption should also be suitable for consumption by another animal and such an embodiment is intended to be within the scope herein.

[0294] According to some embodiments of the invention, the materials used in the context of the invention are of “food grade” classification. Such substances are also referred to herein and in the art as “food contact substances” or “food contact materials.”

[0295] As used herein “food grade” refers to a substance which is either safe for human or animal consumption, or confirmed to come into direct contact with food products.

[0296] The phrase “food contact substance” or FCS, is used herein to describe substances that are generally safe for human consumption by virtue of being generally recognized as safe (GRAS) or by passing standard safety tests, and thus qualify for use as a component of materials used in manufacturing, packing, packaging, transporting, or holding food, in the same manner it is meant in the guideline and regulation of worldwide food administration authorities, such as, for example, the U.S. Food and Drug Administration (FDA), Center for Food Safety and Applied Nutrition (CFSAN), the Office of food Additive Safety.

[0297] The phrase “generally recognized as safe” or GRAS, as used herein, is meant in the same manner which is defined, for example, under sections 201(s) and 409 of the U.S. FD&C Act. The U.S. law states that any substance that intentionally contacts food or added to food is a food additive, that is subject to premarket review and approval by FDA, unless the substance is generally recognized, among qualified experts, as having been adequately shown to be safe under the conditions of its intended use, or unless the use of the substance is otherwise excluded from the definition of a food additive. GRAS substances are distinguished from food additives by the type of information that supports the GRAS determination, that it is publicly available and generally accepted by the scientific community, but should be the same quantity and quality of information that would support the safety of a food additive. Since the qualification to an FCS or GRAS category can be obtained through a process of applying, testing and qualifying to the requirements of the various official food and drug authorities, the present embodiments are meant to encompass all relevant substances and their derivatives which are to become FCSs and GRAS in the future, as well as those which already qualify as FCSs and GRAS.

[0298] According to a specific embodiment, the food or food ingredient is non-genetically modified (non-GMO).

[0299] According to an aspect of some embodiments of the invention, there is provided an edible composition comprising the composition-of-matter of some embodiments of the invention, or the preparation of some embodiments of the invention.

[0300] According to some embodiments of the invention, the composition-of-matter of some embodiments of the invention, or the preparation of some embodiments of the invention comprises at least 1% (gram / 100 gram composition) of the edible composition, at least 5%, at least 10%, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% (gram / 100 gram composition) of the edible composition.

[0301] According to some embodiments of the invention, the edible preparation is used for preparing a milk substitute; an egg substitute; meat, chicken, fish and seafood analogues; functional properties of different hydrocolloids in different applications, like methyl cellulose, egg yolk substitute for use as emulsifier for products like mayonnaise substitute, egg albumin substitute for whipping purpose in bakery products like English cake, whipping cream, and the like; casein protein emulsifying properties for real cheese, real meat products, protein enrichment for sport products, like shakes, protein and energy bars, snacks, non allergen; and / or non GMO texturizer and ingredient in the food industry.

[0302] Consumables include all food products, including but not limited to, cereal products, rice products, tapioca products, sago products, baker's products, biscuit products, pastry products, bread products, confectionery products, desert products, gums, chewing gums, chocolates, ices, honey products, treacle products, yeast products, baking-powder, salt and spice products, savory products, mustard products, vinegar products, sauces (condiments), tobacco products, cigars, cigarettes, processed foods, cooked fruits and vegetable products, meat and meat products, jellies, jams, fruit sauces, egg products, milk and dairy products, yoghurts, cheese products, butter and butter substitute products, milk substitute products, soy products, edible oils and fat products, medicaments, beverages, carbonated beverages, alcoholic drinks, beers, soft drinks, mineral and aerated waters and other non-alcoholic drinks, fruit drinks, fruit juices, coffee, artificial coffee, tea, cocoa, including forms requiring reconstitution, food extracts, plant extracts, meat extracts, condiments, sweeteners, nutraceuticals, gelatins, pharmaceutical and non-pharmaceutical gums, tablets, lozenges, drops, emulsions, elixirs, syrups and other preparations for making beverages, and combinations thereof.

[0303] The composition-of-matter of some embodiments of the invention, or the preparation of some embodiments of the invention can be used in various consumables including but not limited to water-based consumables, solid dry consumables and dairy products, dairy-derived products and dairy-alternative products. In some embodiments the composition is a foodstuff.

[0304] According to some embodiments of the invention the composition-of-matter or the preparation of some embodiments of the invention is a solid consumable.

[0305] According to some embodiments of the present invention the solid consumable is selected from the group consisting of cereals, baked food products, biscuits, bread, breakfast cereal, cereal bar, dairy product, energy bars / nutritional bars, granola, cakes, cookies, crackers, donuts, muffins, pastries, confectioneries, chewing gum, chocolate, fondant, hard candy, marshmallow, pressed tablets, snack foods, botanical materials (whole or ground), and instant powders for reconstitution.

[0306] Water-based consumables include but are not limited to beverage, water, aqueous drink, enhanced / slightly sweetened water drink, mineral water, carbonated beverage, non-carbonated beverage, carbonated water, still water, soft drink, non-alcoholic drink, alcoholic drink, beer, wine, liquor, fruit drink, juice, fruit juice, vegetable juice, broth drink, coffee, tea, black tea, green tea, oolong tea, herbal tea, cacao (water-based), tea-based drink, coffee-based drink, cacao-based drink, syrup, frozen fruit, frozen fruit juice, water-based ice, fruit ice, sorbet, dressing, salad dressing, sauce, soup, and beverage botanical materials (whole or ground), or instant powder for reconstitution (coffee beans, ground coffee, instant coffee, cacao beans, cacao powder, instant cacao, tea leaves, instant tea powder). In some embodiments, the composition can be a beverage such as Coca-Cola® and the like.

[0307] Solid dry consumables include but are not limited to cereals, baked food products, biscuits, bread, breakfast cereal, cereal bar, energy bars / nutritional bars, granola, cakes, cookies, crackers, donuts, muffins, pastries, confectioneries, chewing gum, chocolate, fondant, hard candy, marshmallow, pressed tablets, snack foods, and botanical materials (whole or ground), and instant powders for reconstitution as mentioned above.

[0308] For water-based or solid dry consumables a useful concentration may be from 0.2 ppm (e.g., 0.2-300) ppm or more.

[0309] In certain products a higher sweetener concentration is usually necessary to reach similar sweetness intensity, for example in dairy products, dairy-derived products and dairy-alternative products. Dairy-derived food products contain milk or milk protein. Dairy-alternative products contain (instead of dairy protein derived from the milk of mammals) protein from botanical sources (soy, rice, and other protein-rich plant materials). Dairy products, dairy-derived products and dairy-alternative products include but are not limited to milk, fluid milk, cultured milk product, cultured and noncultured dairy-based drinks, cultured milk product cultured with lactobacillus, yoghurt, yoghurt-based beverage, smoothy, lassi, milk shake, acidified milk, acidified milk beverage, butter milk, kefir, milk-based beverage, milk / juice blend, fermented milk beverage, icecream, dessert, sour cream, dip, salad dressings, cottage cheese, frozen yoghurt, soy milk, rice milk, soy drink, rice milk drink.

[0310] Milk includes, but is not limited to, whole milk, skim milk, condensed milk, evaporated milk, reduced fat milk, low fat milk, nonfat milk, and milk solids (which may be fat or nonfat).

[0311] For dairy products, dairy-derived products and dairy-alternative products, a useful concentration will be from about 0.3 to 500 ppm or higher, and may be up to 550 ppm, 600 ppm, 650 ppm, 700 ppm, or 750 ppm.

[0312] The composition of the invention can also include one or more additional flavor ingredients, such as additional sweeteners. A non-limiting list of suitable flavor ingredients useful with the composition of the invention includes sucrose, fructose, glucose, high fructose corn syrup, xylose, arabinose, rhamnose, erythritol, xylitol, mannitol, sorbitol, inositol, AceK, aspartame, neotame, sucralose, saccharine, naringin dihydrochalcone (NarDHC), neohesperidin dihydrochalcone (NDHC), rubusoside, rebaudioside A, stevioside, stevia and trilobtain.

[0313] Sweeteners commonly used in consumables include, but are not limited to, Acesulfame K

[0314] - Artificial Sweetener (E950); Agave Syrup - Modified Sugar; Alitame - Artificial Sweetener (E956); Aspartame - Artificial Sweetener (E951); Aspartame- Acesulfame Salt - Artificial Sweetener (E962); Barley Malt Syrup - Modified Sugar; Birch Syrup - Sugar Extract; Blackstrap Molasses - Sugar Extract; Brazzein - Natural Sweetener; Brown Rice Syrup - Modified Sugar; Cane Juice - Sugar Extract; Caramel - Modified sugar; Coconut Palm Sugar - Sugar Extract; Com Sugar (HFCS) - Modified sugar; Com Sweetener (HFCS) - Modified sugar; Corn Syrup (HFCS)

[0315] - Modified sugar; Curculin - Natural Sweetener; Cyclamate - Artificial Sweetener (E952); Dextrose - Sugar; Erythritol - Sugar Alcohol (E968); Fmctose Glucose Syrup (HFCS) - Modified sugar; Fructose - Sugar; Galactose - Sugar; Glucitol (Sorbitol) - Sugar Alcohol (E420); Glucose

[0316] - Sugar; Glucose Fmctose Symp (HFCS) - Modified sugar; Glycerol (Glycerin) - Sugar Alcohol (E422); Glycyrrhizin - Natural Sweetener (E958); Golden Symp - Modified sugar; High Fmctose Com Symp (HFCS) - Modified Sugar; HFCS-42 - Modified Sugar; HFCS-55 - Modified Sugar; HFCS-90 - Modified Sugar; Honey - Natural Sugar; HSH - Sugar Alcohol; Hydrogenated Starch Hydrolysate (HSH)- Sugar Alcohol; Isoglucose (HFCS) - Modified sugar; Inulin - Sugar Fiber; Inverted Sugar - Modified sugar; Isomalt - Sugar Alcohol (E953); Lactitol - Sugar Alcohol (E966); Lactose - Sugar; Levulose (Fructose) - Sugar; Luo Han Guo - Natural Sweetener; Maltitol - Sugar Alcohol (E965); Maltodextrin - Sugar; Maltose - Sugar; Mannitol - Sugar Alcohol (E421); Maple Syrup - Sugar Extract; Miraculin - Natural Sweetener; Molasses - Sugar Extract; Monellin - Natural Sweetener; Monk Fruit (Luo Han Guo) - Natural Sweetener; Neohesperidin DC - Artificial Sweetener (E959); Neotame - Artificial Sweetener (E961); Oligofructose - Sugar Fiber; Palm Sugar - Sugar Extract; Pentadin - Natural Sweetener; Rapadura - Sugar Extract; Refiners Syrup - Modified Sugar; Saccharin, - Artificial Sweetener (E954); Saccharose (Sucrose) - Sugar; Sorbitol - Sugar Alcohol (E420); Sorghum Syrup - Sugar Extract; Stevia - Natural Sweetener; Stevioside - Natural Sweetener (E960); Sucralose - Artificial Sweetener (E955); Sucrose - Sugar; Tagatose - Modified Sugar; Thaumatin - Natural Sweetener (E957); Trehalose - Sugar; Xylitol - Sugar Alcohol (E967); Yacon Syrup - Natural Sweeten;

[0317] The composition-of-matter or the preparation of some embodiments of the invention can be used in the cosmetic industry, for example for protein enrichment, emulsifying properties, and / or non-hazardous texturizer.

[0318] According to an aspect of some embodiments of the invention, there is provided a cosmetic composition comprising the composition-matter of some embodiments of the invention or the preparation of some embodiments of the invention and a cosmetically acceptable carrier.

[0319] As used herein a "cosmetic composition" refers to a preparation which includes the active ingredients described hereinabove (e.g., the composition-matter or the preparation of some embodiments of the invention) and additional chemical components such as physiologically suitable carriers and excipients. The purpose of a cosmetic composition is to facilitate administration of the active ingredient to an organism.

[0320] The composition-of-matter or the preparation of some embodiments of the invention can be used for treating a subject in need of a high-protein content food in his / her diet.

[0321] The subject can be for example, an elderly person which consumes high-protein content shakes in order to supply the need of sufficient proteins in his / her diet. Additionally or alternatively, the subject can be for example a subject in need of high protein content food due to enhanced physical activity, metabolism or diet restrictions.

[0322] According to some embodiments of the invention, the subject suffers from a pathology, disease or disorder related to protein deficiency, or deficiencies of several macronutrients which include proteins. Examples include, but are not limited to, diseases such as kwashiorkor (a disease characterized by protein deficiency), marasmus (a disease characterized by deficiency of protein, carbohydrates and fats). For example, the composition-of-matter or the preparation of some embodiments of the invention can be a protein enhancer, or can form part of a protein enhancer formulation for increasing the uptake of proteins in a subject in need thereof.

[0323] According to an aspect of some embodiments of the invention, there is provided a pharmaceutical composition comprising the composition-matter of some embodiments of the invention or the preparation of some embodiments of the invention and a pharmaceutically acceptable carrier.

[0324] As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism (e.g., a subject).

[0325] As used herein, the term “subject” includes mammals, preferably human beings at any age which suffer from pathology.

[0326] Herein the term "active ingredient" refers to the composition-matter or the preparation of some embodiments of the invention accountable for the biological effect.

[0327] Hereinafter, the phrases "physiologically acceptable carrier", "pharmaceutically acceptable carrier", or "suitable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound, i.e., the composition-matter or the preparation of some embodiments of the invention. An adjuvant is included under these phrases.

[0328] Herein the term "excipient" refers to an inert substance added to a cosmetic or pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0329] Cosmetic or pharmaceutical compositions of the present invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0330] Cosmetic or pharmaceutical compositions for use in accordance with the present invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations. Proper formulation is dependent upon the administration approach chosen.

[0331] The cosmetic or pharmaceutical composition may be applied in a local manner, for example, via administration of the cosmetic composition directly into a tissue region of a patient. Suitable routes of administration may, for example, include topical, subcutaneous and intradermal injections.

[0332] For injection, the active ingredients of the cosmetic or pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer.

[0333] Alternatively, the active ingredient may be in a powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

[0334] Determination of a therapeutically or cosmetically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0335] For any preparation used in the methods of the invention, the therapeutically or cosmetically effective amount or dose can be estimated initially from in vitro assays. Depending on the need dosing can be of a single or a plurality of administrations.

[0336] The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0337] The composition is preferably of high purity and substantially free of potentially harmful contaminants, e.g., at least National Food (NF) grade, generally at least analytical grade, and preferably at least pharmaceutical grade. To the extent that a given compound must be synthesized prior to use, such synthesis or subsequent purification shall preferably result in a product that is substantially free of any potentially contaminating toxic agents that may have been used during the synthesis or purification procedures.

[0338] Following is a non-limiting list of epidermal penetrants which can be used with the cosmetic composition of some embodiments of the invention.

[0339] In order to enhance the percutaneous absorption of the active ingredients, one or more of a number of agents can be added to the cosmetic composition including, but not limited to, dimethylsulfoxide, dimethylacetamide, dimethylformamide, surfactants, azone, alcohol, acetone, propylene glycol and polyethylene glycol.

[0340] The cosmetic composition of some embodiments of the invention also includes a dermatologically acceptable carrier.

[0341] The phrase "dermatologically acceptable carrier", refers to a carrier which is suitable for topical application onto the skin, i.e., keratinous tissue, has good aesthetic properties, is compatible with the active agents of the present invention and any other components, and is safe and nontoxic for use in mammals. An effective amount of carrier is selected from a range of about 50 % to about 99.99 %, preferably from about 80 % to about 99.9 %, more preferably from about 90 % to about 98 %, and most preferably from about 90 % to about 95 %, by weight, of the composition.

[0342] The carrier utilized in the compositions of the invention can be in a wide variety of forms. These include emulsion carriers, including, but not limited to, oil-in-water, water-in-oil, water-in- oil-in-water, and oil-in-water-in-silicone emulsions, a cream, an ointment, an aqueous solution, a lotion or an aerosol. As will be understood by the skilled artisan, a given component will distribute primarily into either the water or oil / silicone phase, depending on the water solubility / dispersibility of the component in the composition.

[0343] Emulsions according to the present invention generally contain a pharmaceutically or cosmetically effective amount of an agent disclosed herein and a lipid or oil. Lipids and oils may be derived from animals, plants, or petroleum and may be natural or synthetic (i.e., man-made). Preferred emulsions also contain a humectant, such as glycerin. Emulsions will preferably further contain from about 1 % to about 10 %, more preferably from about 2 % to about 5 %, of an emulsifier, based on the weight of the carrier. Emulsifiers may be nonionic, anionic or cationic. Suitable emulsifiers are described in, for example, U.S. Pat. No. 3,755,560, issued to Dickert, et al. Aug. 28, 1973; U.S. Pat. No. 4,421,769, issued to Dixon, et al., Dec. 20, 1983; and McCutcheon's Detergents and Emulsifiers, North American Edition, pages 317-324, 1986.

[0344] The emulsion may also contain an anti-foaming agent to minimize foaming upon application to the keratinous tissue. Anti-foaming agents include high molecular weight silicones and other materials well known in the art for such use.

[0345] Suitable emulsions may have a wide range of viscosities, depending on the desired product form. Exemplary low viscosity emulsions, which are preferred, have a viscosity of about 50 centistokes or less, more preferably about 10 centistokes or less, most preferably about 5 centistokes or less. The emulsion may also contain an anti-foaming agent to minimize foaming upon application to the keratinous tissue. Anti-foaming agents include high molecular weight silicones and other materials well known in the art for such use.

[0346] One type of emulsion is a water-in- silicone emulsion. Water-in-silicone emulsions contain a continuous silicone phase and a dispersed aqueous phase. Preferred water-in-silicone emulsions of the present invention comprise from about 1 % to about 60 %, preferably from about 5% to about 40%, more preferably from about 10 % to about 20 %, by weight of a continuous silicone phase. The continuous silicone phase exists as an external phase that contains or surrounds the discontinuous aqueous phase described hereinafter.

[0347] The continuous silicone phase may contain a polyorganosiloxane oil. A preferred water- in-silicone emulsion system is formulated to provide an oxidatively stable vehicle for delivery of a pharmaceutically or cosmetically effective amount of an agent disclosed herein. The continuous silicone phase of these preferred emulsions comprises between about 50 % and about 99.9 % by weight of organopoly siloxane oil and less than about 50 % by weight of a non-silicone oil. In an especially preferred embodiment, the continuous silicone phase comprises at least about 50 %, preferably from about 60 % to about 99.9 %, more preferably from about 70 % to about 99.9 %, and even more preferably from about 80 % to about 99.9 %, polyorganosiloxane oil by weight of the continuous silicone phase, and up to about 50 % non-silicone oils, preferably less about 40 %, more preferably less than about 30 %, even more preferably less than about 10 %, and most preferably less than about 2 %, by weight of the continuous silicone phase. These useful emulsion systems may provide more oxidative stability over extended periods of time than comparable water-in-oil emulsions containing lower concentrations of the polyorganosiloxane oil. Concentrations of non-silicone oils in the continuous silicone phase are minimized or avoided altogether so as to possibly further enhance oxidative stability of the active compound of the invention in the compositions. Water-in- silicone emulsions of this type are described in U.S. Pat. No. 5,691,380 to Mason et al., issued Nov. 25, 1997.

[0348] The organopolysiloxane oil for use in the composition may be volatile, non-volatile, or a mixture of volatile and non-volatile silicones. The term "nonvolatile" as used in this context refers to those silicones that are liquid under ambient conditions and have a flash point (under one atmospheric of pressure) of or greater than about 100 degrees Celsius. The term "volatile" as used in this context refers to all other silicone oils. Suitable organopolysiloxanes can be selected from a wide variety of silicones spanning a broad range of volatilities and viscosities. Examples of suitable organopolysiloxane oils include poly alkylsiloxanes, cyclic polyalkylsiloxanes, and poly alkylarylsiloxanes, which are known to those skilled in the art and commercially available.

[0349] The continuous silicone phase may contain one or more non-silicone oils. Concentrations of non-silicone oils in the continuous silicone phase are preferably minimized or avoided altogether so as to further enhance oxidative stability of the pharmaceutically effective agent in the compositions. Suitable non-silicone oils have a melting point of about 25 °C or less under about one atmosphere of pressure. Examples of non-silicone oils suitable for use in the continuous silicone phase are those well known in the chemical arts in topical personal care products in the form of water-in-oil emulsions, e.g., mineral oil, vegetable oils, synthetic oils, semisynthetic oils, etc.

[0350] Useful topical compositions of the present invention comprise from about 30 % to about 90 %, more preferably from about 50 % to about 85 %, and most preferably from about 70 % to about 80 % of a dispersed aqueous phase. The term "dispersed phase" is well-known to one skilled in the art it implies that the phase exists as small particles or droplets that are suspended in and surrounded by a continuous phase. The dispersed phase is also known as the internal or discontinuous phase. The dispersed aqueous phase is a dispersion of small aqueous particles or droplets suspended in and surrounded by the continuous silicone phase described hereinbefore. The aqueous phase can be water, or a combination of water and one or more water soluble or dispersible ingredients. Non-limiting examples of such optional ingredients include thickeners, acids, bases, salts, chelants, gums, water-soluble or dispersible alcohols and polyols, buffers, preservatives, sunscreening agents, colorings, and the like.

[0351] The topical compositions of the present invention typically comprise from about 25 % to about 90 %, preferably from about 40 % to about 80 %, more preferably from about 60 % to about 80 %, water in the dispersed aqueous phase by weight of the composition.

[0352] The water-in- silicone emulsions of the present invention preferably comprise an emulsifier. In a preferred embodiment, the composition contains from about 0.1 % to about 10 % emulsifier, more preferably from about 0.5 % to about 7.5 %, most preferably from about 1 % to about 5 %, emulsifier by weight of the composition. The emulsifier helps disperse and suspend the aqueous phase within the continuous silicone phase.

[0353] A wide variety of emulsifying agents can be employed herein to form the preferred water- in-silicone emulsion. Known or conventional emulsifying agents can be used in the composition, provided that the selected emulsifying agent is chemically and physically compatible with essential components of the composition, and provides the desired dispersion characteristics. Suitable emulsifiers include silicone emulsifiers, e.g., organically modified organopolysiloxanes, also known to those skilled in the art as silicone surfactants, non-silicon-containing emulsifiers, and mixtures thereof, known by those skilled in the art for use in topical personal care products.

[0354] Suitable emulsifiers are described, for example, in McCutcheon's, Detergents and Emulsifiers, North American Edition (1986), published by Allured Publishing Corporation; U.S. Pat. No. 5,011,681 to Ciotti et al., issued Apr. 30, 1991; U.S. Pat. No. 4,421,769 to Dixon et al., issued Dec. 20, 1983; and U.S. Pat. No. 3,755,560 to Dickert et al., issued Aug. 28, 1973.

[0355] Other preferred topical carriers include oil-in-water emulsions, having a continuous aqueous phase and a hydrophobic, water-insoluble phase ("oil phase") dispersed therein. Examples of suitable carriers comprising oil-in-water emulsions are described in U.S. Pat. No. 5,073,371 to Turner, D. J. et al., issued Dec. 17, 1991, and U.S. Pat. No. 5,073,372, to Turner, D. J. et al., issued Dec. 17, 1991. An especially preferred oil-in-water emulsion, containing a structuring agent, hydrophilic surfactant and water, is described in detail hereinafter. A preferred oil-in-water emulsion comprises a structuring agent to assist in the formation of a liquid crystalline gel network structure. The structuring agent may also function as an emulsifier or surfactant. Preferred compositions of this invention comprise from about 0.5 % to about 20 %, more preferably from about 1 % to about 10 %, most preferably from about 1 % to about 5 %, by weight of the composition, of a structuring agent. The preferred structuring agents of the present invention are selected from the group consisting of stearic acid, palmitic acid, stearyl alcohol, cetyl alcohol, behenyl alcohol, the polyethylene glycol ether of stearyl alcohol having an average of about 1 to about 21 ethylene oxide units, the polyethylene glycol ether of cetyl alcohol having an average of about 1 to about 5 ethylene oxide units, and mixtures thereof.

[0356] A wide variety of anionic surfactants are also useful herein. See, e.g., U.S. Pat. No. 3,929,678, to Laughlin et al., issued Dec. 30, 1975.

[0357] The preferred oil-in-water emulsions comprise from about 0.05 % to about 10 %, preferably from about 1 % to about 6 %, and more preferably from about 1 % to about 3 % of at least one hydrophilic surfactant which can disperse the hydrophobic materials in the water phase (percentages by weight of the topical carrier). The surfactant, at a minimum, must be hydrophilic enough to disperse in water. Suitable surfactants include any of a wide variety of known cationic, anionic, zwitterionic, and amphoteric surfactants. See, McCutcheon's. Detergents and Emulsifiers, North American Edition (1986), published by Allured Publishing Corporation; U.S. Pat. No. 5,011,681 to Ciotti et al., issued Apr. 30, 1991; U.S. Pat. No. 4,421,769 to Dixon et al. issued to Dec. 20, 1983; and U.S. Pat. No. 3,755,560. The exact surfactant chosen depends upon the pH of the composition and the other components present. Preferred are cationic surfactants, especially dialkyl quaternary ammonium compounds, examples of which are described in U.S. Pat. No. 5,151,209 to McCall et al. issued to Sep. 29, 1992; U.S. Pat. No. 5,151,210 to Steuri et al., issued to Sep. 29, 1992; U.S. Pat. Nos. 5,120,532; U.S. Pat. No. 4,387,090; U.S. Pat. No. 3,155,591; U.S. Pat. No. 3,929,678; U.S. Pat. No. 3,959,461; McCutcheon's, Detergents & Emulsifiers (North American edition 1979) M.C. Publishing Co.; and Schwartz, et al., Surface Active Agents, Their chemistry and Technology, New York: Interscience Publishers, 1949.

[0358] Alternatively, other useful cationic emulsifiers include amino-amides. Non-limiting examples of these cationic emulsifiers include stearamidopropyl PG-dimonium chloride phosphate, behenamidopropyl PG dimonium chloride, stearamidopropyl ethyldimonium ethosulfate, stearamidopropyl dimethyl (myristyl acetate) ammonium chloride, stearamidopropyl dimethyl cetearyl ammonium tosylate, stearamidopropyl dimethyl ammonium chloride, stearamidopropyl dimethyl ammonium lactate, and mixtures thereof. The preferred oil-in-water emulsion comprises from about 25 % to about 98 %, preferably from about 65 % to about 95 %, more preferably from about 70 % to about 90 % water by weight of the topical carrier.

[0359] The cosmetic composition can be formulated in any of a variety of forms utilized by the cosmetic industry for skin application including solutions, lotions, sprays, creams, ointments, salves, gels, etc., as described herein.

[0360] Preferably, the cosmetic composition is formulated viscous enough to remain on the treated skin area, does not readily evaporate, and / or is not easily removed by rinsing with water, but rather is removable with the aid of soaps, cleansers and / or shampoos.

[0361] Methods for preparing compositions having such properties are well known to those skilled in the art, and are described in detail in Remington's Pharmaceutical Sciences, 1990 (supra); and Pharmaceutical Dosage Forms and Drug Delivery Systems, 6th ed., Williams & Wilkins 1995.

[0362] The topical compositions of some embodiments of the invention, include but are not limited to lotions and creams, may comprise a dermatologically acceptable emollient. Such compositions preferably contain from about 2% to about 50% of the emollient. As used herein, "emollient" refers to a material useful for the prevention or relief of dryness, as well as for the protection of the skin. A wide variety of suitable emollients are known and may be used herein. See, e.g., Sagarin, Cosmetics, Science and Technology, 2nd Edition, Vol. 1, pp. 3243 (1972), which contains numerous examples of materials suitable as an emollient. A preferred emollient is glycerin. Glycerin is preferably used in an amount of from or about 0.001 to or about 20 %, more preferably from or about 0.01 to or about 10 %, most preferably from or about 0.1 to or about 5 %, e.g., 3%.

[0363] Lotions and creams according to some embodiments of the invention generally comprise a solution carrier system and one or more emollients. Lotions typically comprise from about 1 % to about 20 %, preferably from about 5 % to about 10 % of emollient; from about 50 % to about 90 %, preferably from about 60 % to about 80 % water; and a pharmaceutically effective amount of an agent described herein. A cream typically comprises from about 5 % to about 50 %, preferably from about 10 % to about 20 % of emollient; from about 45 % to about 85 %, preferably from about 50 % to about 75 % water; and a pharmaceutically effective amount of an agent described herein.

[0364] The topically applied cosmetic composition of the present invention may also include additional components which are added, for example, in order to enrich the cosmetic compositions with fragrance and skin nutrition factors. Such components are selected suitable for use on human keratinous tissue without inducing toxicity, incompatibility, instability, allergic response, and the like within the scope of sound medical judgment. In addition, such optional components are useful provided that they do not unacceptably alter the benefits of the active compounds of the invention.

[0365] The CTFA Cosmetic Ingredient Handbook, Second Edition (1992) describes a wide variety of non-limiting cosmetic ingredients commonly used in the skin care industry, which are suitable for use in the compositions of the present invention. Examples of these ingredient classes include: abrasives, absorbents, aesthetic components such as fragrances, pigents, coloring s / colorants, essential oils, skin sensates, astringents, etc. (e.g., clove oil, menthol, camphor, eucalyptus oil, eugenol, menthyl lactate, witch hazel distillate), anti-acne agents, anti-caking agents, antifoaming agents, antimicrobial agents (e.g., iodopropyl butylcarbamate), antioxidants, binders, biological additives, buffering agents, bulking agents, chelating agents, chemical additives, colorants, cosmetic astringents, cosmetic biocides, denaturants, drug astringents, external analgesics, film formers or materials, e.g., polymers, for aiding the film-forming properties and substantivity of the composition (e.g., copolymer of eicosene and vinyl pyrrolidone), opacifying agents, pH adjusters, propellants, reducing agents, sequestrants, skin-conditioning agents (e.g., humectants, including miscellaneous and occlusive), skin soothing and / or healing agents (e.g., panthenol and derivatives (e.g., ethyl panthenol), aloe vera, pantothenic acid and its derivatives, allantoin, bisabolol, and dipotassium glycyffhizinate), skin treating agents, thickeners, and vitamins and derivatives thereof.

[0366] The cosmetic composition can be applied directly to the skin. Alternatively, it can be delivered via normal skin application by various transdermal drug delivery systems which are known in the art, such as transdermal patches that release the composition into the skin in a time released manner. Other drug delivery systems known in the arts include pressurized aerosol bottle, iontophoresis or sonophoresis. Iontophoresis is employed to increase skin permeability and facilitate transdermal delivery. U.S. Pat. Nos. 5,667,487 and 5,658,247 discloses an ionosonic apparatus suitable for the ultrasonic-iontophoretically mediated transport of therapeutic agents across the skin. Alternatively, or in addition, liposomes or micelles may also be employed as a delivery vehicle.

[0367] The emollients include, but are not limited to, hydrocarbon oils and waxes, such as mineral oil, petrolatum, and the like, vegetable and animal oils and fats, such as olive oil, palm oil, castor oil, corn oil, soybean oil, and the like, and lanolin and its derivatives, such as lanolin, lanolin oil, lanolin wax, lanolin alcohols, and the like. Other emollients include esters of fatty acids having 10 to 20 carbon atoms, such as including myristic, stearic, isostearic, palmitic, and the like, such as methyl myristate, propyl myristate, butyl myristate, propyl stearate, propyl isostearate, propyl palmitate, and the like. Other emollients include fatty acids having 10 to 20 carbon atoms, including stearic, myristic, lauric, isostearic, palmitic, and the like. Emollients also include fatty alcohols having ten to twenty carbon atoms, such as cetyl, myristyl, lauryl, isostearyl, stearyl and the like.

[0368] Although some are water soluble, polyhydric alcohols and polyether derivatives are included as emollients, including glycols, glycerol, sorbitol, polyalkylene glycols and the like, such as propylene glycol, dipropylene glycol, polyethylene glycol 200-500, and the like. The water soluble examples are preferred.

[0369] Examples of surfactants include, but are not limited to, spolyoxyalkylene oxide condensation products of hydrophobic alkyl, alkene, or alkyl aromatic functional groups having a free reactive hydrogen available for condensation with hydrophilic alkylene oxide, polyethylene oxide, propylene oxide, butylene oxide, polyethylene oxide or polyethylene glycol Particularly effective are the condensation products of octylphenol with about 7 to about 13 moles of ethylene oxide, sold by the Rohm & Haas Company under their trademark TRITON 100® series products.

[0370] Other ingredients such as, fragrances, stabilizing agents, dyes, antimicrobial agents, antibacterial agents, anti agglomerates, ultraviolet radiation absorbers, and the like are also included in the composition comprising the composition-of-matter or the preparation of some embodiments of the invention.

[0371] A conditioner agent stable to acid hydrolysis, such as a silicone compound having at least one quaternary ammonium moiety along with an ethoxylated monoquat is preferably also utilized in order to stabilize and optionally thicken the composition with the composition-of-matter or the preparation of some embodiments of the invention.

[0372] An optional thickener also can be included to improve composition esthetics and facilitate application of the composition to the hair. Nonionic thickeners in an amount of 0% to about 3% by weight are preferred. Exemplary thickeners are methylcellulose, hydroxybutyl methylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, hydroxyethyl ethylcellulose and hydroxyethylcellulose, di (hydrogenated tallow) phthalic acid amide, crosslinked maleic anhydride-methyl vinyl ether copolymer, guar gum, xanthan gum and gum arabic.

[0373] The carrier of the conditioning composition is predominantly water, but organic solvents also can be included in order to facilitate manufacturing of the composition or to provide esthetic properties, such as viscosity control. Suitable solvents include the lower alcohols like ethyl alcohol and isopropyl alcohol; glycol ethers, like 2-butoxyethanol, ethylene glycol monoethyl ether, propylene glycol and diethylene glycol monoethyl ether or monomethyl ether; and mixtures thereof. Non-aqueous solvents can be present in the conditioning composition of some embodiments of the invention in an amount of about 1 % to about 50 %, and in particular about 5% to about 25%, by weight of the total weight of the carrier in the composition.

[0374] Non-limiting conditioning agents which may be used in opaque conditioners include: stearyltrimethylammonium chloride; behenetrimethylammonium chloride; cetrimonium bromide; soytrimonium chloride; tallowtrimonium chloride; dihyrogenatedtallowdimethylammonium chloride; behentrimethylammonium methosulfate; Peg-2 Oleammonium chloride; dihyrogenatedtallowdimethylammonium bromide; dihyrogenatedtallowdimethylammonium methosulfate; palmityltrimethylammonium chloride; hydrogenated tallowtrimethylammonium chloride; hydrogenated tallowtrimethylammonium bromide; dicetyidimethylammonium chloride; distearyldimethylammonium chloride; dipalmityidimethylammonium chloride; hydrogenated tallowtrimethylammonium methosulfate; cetrimonium tosylate: eicosyltrimethylammonium chloride, and ditallowdimethylammonium chloride.

[0375] Materials that can be used to opacify compositions of the invention include fatty esters, opacifying polymers, such as styrene polymers, like OPACIFIER 653 from Morton, International, Inc.; and fatty alcohols. The following is a non-limiting list of fatty alcohols: cetyl alcohol; stearyl alcohol; cetearyl alcohol; behenyl alcohol; and arachidyl alcohol. Conditioning compositions of the invention which are not clear also can include Lexamine S-13, dicetylammonium chloride, and ceteareth-20.

[0376] Cosmetic carriers are well known in the art, e.g., reviewed by Tanya M. Barnes et al., 2021. Pharmaceutics 2021, 13(12), 2012; “Vehicles for Drug Delivery and Cosmetic Moisturizers: Review and Comparison”, which is fully incorporated herein by reference.

[0377] Techniques for formulation and administration of drugs for pharmaceutical compositions may be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

[0378] Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, intraocular injections, intracerebral injection or intracerebroventricular infusion.

[0379] Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0380] For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0381] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0382] Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

[0383] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0384] For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0385] The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0386] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

[0387] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

[0388] The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

[0389] Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose.

[0390] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0391] For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans. Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 P-l).

[0392] Dosage amount and interval may be adjusted individually to provide levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0393] Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

[0394] The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0395] Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

[0396] The composition-of-matter or the preparation of some embodiments of the invention, which comprises the chlorophyll-free preparation of soluble proteins of moringa leaves can be used in various food, cosmetics, and pharmaceutical applications, performing at least at a comparable level to other protein preparations.

[0397] As used herein the term “about” refers to ± 10 %.

[0398] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0399] The term “consisting of’ means “including and limited to”.

[0400] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0401] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0402] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0403] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0404] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0405] As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0406] When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

[0407] It is understood that any Sequence Identification Number (SEQ ID NO) disclosed in the instant application can refer to either a DNA sequence or a RNA sequence, depending on the context where that SEQ ID NO is mentioned, even if that SEQ ID NO is expressed only in a DNA sequence format or a RNA sequence format. Similarly, though some sequences are expressed in a RNA sequence format (e.g., reciting U for uracil), depending on the actual type of molecule being described, it can refer to either the sequence of a RNA molecule comprising a dsRNA, or the sequence of a DNA molecule that corresponds to the RNA sequence shown. In any event, both DNA and RNA molecules having the sequences disclosed with any substitutes are envisioned.

[0408] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0409] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0410] EXAMPLES

[0411] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion. GENERAL MATERIALS AND EXPERIMENTAL METHODS

[0412] Plant Material

[0413] Moringa oleifera (e.g., types Karmei-Yosef, Brown, 42, 5, 2-21, pink, LHV, 74, and 36) was cultivated under diverse growth conditions across various geographical regions Sub-tropical climate conditions). The thrice-pinnate leaves of Moringa were harvested, and the leaflets were separated from the main rachis. The Moringa leaves were used as fresh, dried or frozen samples. For example, fresh leaflets were dried and stored at room temperature. Alternatively, fresh leaflets were frozen and stored at -20°C before proceeding with protein extraction.

[0414] Additionally or alternatively, both the leaves and main rachis (named “green biomass” hereinafter) were used. The biomass was used as fresh, dried or frozen samples. For example, fresh leaflets and stems were dried and stored at room temperature. Alternatively, fresh leaflets and stems were frozen and stored at -20°C before proceeding with protein extraction.

[0415] Protein Extraction Procedure

[0416] Proteins of the Moringa leaves and / or green biomass were extracted in a multi-step process, as follows.

[0417] Lab Process

[0418] Grinding: Leaves were ground using the hand blender MultiQuick 5 Vario MQ5235WH (Braun), the high power Fresh & Furious blender (Breville) or the CB353 power pitcher blender system (Ninja) in SMBS buffer at pH 6.7 at a ratio of 1:2 (w / v) plant material / volume of SMBS buffer. It is noted that this ratio can be extended up to 1:20 (w / v) plant material / volume of SMBS buffer. The SMBS buffer contains Sodium bicarbonate 0.0125 M, Sodium carbonate (anhydrous) 0.0875 M, and 0.2% (gram / 100 ml) or 1% (gram / 100 ml) sodium-metabisulfite (SMBS) as an antioxidant.

[0419] Milling: In most instances, the ground material in buffer was further milled using a colloid mill CM-65 (Beyond Machinery), by applying either 0, 11, or 20 machine units between the rotor and stator. A setting of 0 indicates a broad gap, while a setting of 20 results in smaller pieces due to a narrower gap. The milled material was kept at 4°C prior to filtering.

[0420] Filtering: The liquid phase (“green juice”, also termed “GJ” herein), containing the soluble proteins, was separated from the solid phase (pulp), by filtering through a 73 or 120 micron filterbag (e.g., using Cannabeg net mesh), or using an industrial filter-press (e.g., A.D. Sinun Tech LTD; worldwideweb(dot)sinun(dot)co(dot)il ) through a 50, 80 or 120 micron filter clothes.

[0421] De-greening: The green coloring and non-soluble protein sedimentation caused by chlorophylls that were present in the GJ were eliminated by heating the liquid GJ in containers in a water bath at 50°C for 5 to 20 minutes, followed by rapid cooling in cold water. Solid Separation: Residual chloroplast material and insoluble proteins were removed by centrifugation at 8000g or 10000g (Sorvall RC 6 Plus Superspeed Centrifuge) for 15 minutes, followed by optional filtration through a 2-micron depth filter sheet (Hobrafilt). In most case the centrifugation was at a speed of 10000 g. The upper liquid phase, containing the total soluble proteins, was collected (termed “brown juice”, or “BJ” herein) and separated from the pellet.

[0422] Washing and Concentration Using Tangential Flow Ultrafiltration (TFF): To remove phenolic compounds and buffer salts, concentrate the soluble protein phase, and prepare it for drying, the BJ liquid was processed through a Tangential Flow Ultrafiltration (TFF) system using a 10 or 100 kDa cross-flow filter cassette (Sartorius, Vivaflow 200). In most cases, the 10 kDa filter cassette was used. During filtration, Rubisco and other large soluble proteins were retained by the membrane (retentate) and recirculated back into the feed reservoir, while buffer salts and phenolic compounds were washed through the filter (permeate). This process concentrated the soluble protein phase to l / 10th of its initial buffer volume.

[0423] Subsequently, the protein samples were subjected to diafiltration for further purification. An equal volume of double-distilled water (DDW) was added to the sample and passed through the filter cassette. This washing step was repeated eight times, resulting in a 99.9% removal of buffer salts from the sample.

[0424] The soluble proteins concentrate was kept frozen until the drying step. Prior to the drying procedure, the protein concentrate samples were gradually defrosted and transferred to freeze dryer (MCGS) or a low temp spray dryer (Buchi) to obtain a dry protein powder. In most cases the preferred drying method was the low temp spray dryer (Buchi).

[0425] Pilot Process

[0426] The protein extraction process at pilot scale was performed in two main stages:

[0427] (I). Upstream Processing - Production of green juice (GJ);

[0428] (II). Downstream Processing - Purification and concentration of soluble proteins

[0429] (I). Upstream Process

[0430] Grinding:

[0431] Green biomass was shredded and crushed using a mechanical shredder and crusher.

[0432] Two options were applied: either a carbonate buffer with sodium metabisulfite (SMBS) adjusted to pH ~9, or a two-stage acidic-neutral process in which a citric acid buffer with SMBS adjusted to pH 4-5 was first applied, and the resulting green juice was subsequently adjusted to pH 7-9 to optimize protein solubility.

[0433] This step facilitated protein solubilization while minimizing oxidative degradation. Filtering:

[0434] The liquid phase (green juice, GJ), containing soluble proteins, was separated from the solid pulp using a screw press equipped with a 400-micron mesh filter. In some cases, a decanter centrifuge was used as an alternative method for separation.

[0435] Storage:

[0436] Immediately after extraction, the green juice was either cooled or frozen to preserve protein integrity and prevent oxidation prior to further processing.

[0437] (II). Downstream Process

[0438] Chloroplast Disruption:

[0439] After thawing under controlled conditions, the green juice was processed using either a high- pressure homogenizer, microfluidizer, or colloid mill to disrupt chloroplast structures and release the soluble protein content. The process was conducted under cold conditions to avoid thermal degradation and oxidation.

[0440] Chlorophyll and Chloroplast Removal:

[0441] Two alternative methods were used to separate chlorophyll-containing particles and residual insoluble matter:

[0442] (i) Filtration Approach:

[0443] The green juice was combined with a filtration aid to facilitate removal of insoluble particles and pigment-containing plant material, such as chlorophyll- or chloroplast-derived components. Suitable filtration aids include, but are not limited to, diatomaceous earth, perlite, cellulosic fibers, activated carbon, or synthetic porous particles.

[0444] In a specific example, the green juice was mixed with diatomaceous earth according to the operational specifications of the filtration apparatus employed and further incubated with for a period of time sufficient to promote adsorption and particle aggregation (e.g., ~30 minutes), and then filtered using a filter press, filter drum, or filter bags. This process yielded a clarified liquid phase, referred to as “brown juice” (BJ)."

[0445] (ii) Centrifugation Approach:

[0446] Alternatively, the green juice was processed through a decanter centrifuge, which used centrifugal force to separate insoluble particles and yielded the brown juice fraction.

[0447] Protein Concentration and Purification:

[0448] Two alternative approaches were evaluated:

[0449] (i) Tangential Flow Ultrafiltration (TFF) and Diafiltration:

[0450] The brown juice was concentrated using cross-flow ultrafiltration with 10 or 50 kDa filter cassettes. Rubisco and other large soluble proteins were retained in the retentate and recirculated, while buffer salts and phenolic compounds passed through as permeate. The volume of the soluble protein solution was reduced to -1 / 10 of the original.

[0451] Following concentration, diafiltration was performed by adding an equal volume of double-distilled water (DDW) to the sample and filtering it. This washing step was repeated eight times to achieve -99.9% removal of salts.

[0452] (ii). Chemical Precipitation and Re-dissolution:

[0453] The pH of the brown juice was adjusted to 4-5, corresponding to the approximate isoelectric point of the proteins, and the mixture was stirred for 0-30 minutes to induce protein precipitation. Alternatively, proteins were precipitated by the addition of salts — such as calcium chloride (CaCh), ammonium sulfate ((NH^SCb), or ammonium phosphate ((NH^sPCb) — with or without concurrent pH adjustment to acidic conditions.

[0454] The resulting precipitate was separated using a decanter. The recovered solid protein phase was then neutralized to approximately pH 7, yielding a concentrated protein slurry containing -15% dry matter. Additional washing steps may be employed to reduce residual salt content and further enhance protein purity.

[0455] Drying:

[0456] The concentrated protein solution or slurry was dried using either a freeze dryer (MCGS) or a low-temperature spray dryer (Buchi). In most cases, the preferred method was spray drying due to its efficiency and preservation of protein functionality.

[0457] Protein quantification

[0458] Protein content was quantified using the Bicinchoninic acid protein assay (BCA) or the Bradford assay, against a bovine serum albumin (BSA) standard curve, or the Kjeldahl method. It is noted that in cases of discrepancies between the results obtained from different measuring methods, the Kjeldahl method was used as the definitive standard for protein quantification.

[0459] For the BCA assay, triplicates from each extraction sample were placed on a microplate and incubated with the BCA working reagent (WR, Pierce, USA) for 15 minutes at 37 °C. Absorbance of the samples was read in a microplate reader (Infinite F200, Tecan) set to a wavelength of 550 nm.

[0460] For the Bradford assay, triplicates from each extraction sample were placed on a microplate and mixed with Bradford reagent (Bio-Rad). Absorbance at wavelength of 590 nm was measured using a microplate reader (Infinite F200, Tecan).

[0461] Kjeldahl quantifications were done using the AOAC (Association of Official Agricultural Chemists) 981.10 protocol by the Microbiology Institute (Nesher, Israel; world- wide- web (dot) microb (dot) co (dot) il). Kjeldahl protocol is well established, and is used by the Microbiology Institute according to the “Teken” AOAC (Association of Official Agricultural Chemists) instructions.

[0462] SDS-PAGE protein electrophoresis

[0463] Protein preparations were separated and analyzed by SDS-PAGE (Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis). Protein samples were boiled in reducing SDS- sample buffer (Laemmli buffer; 62.5 mM Tris-HCl (pH 6.8), 2% (w / v) SDS, 10% (v / v) glycerol, 0.01% (w / v) bromophenol blue, 5% (v / v) 2-mercaptoethanol)) and Dithiothreitol (DTT) (1%) and loaded on a 12% acrylamide SDS-PAGE gel. Following the electrophoresis, gels were stained with Coomassie Brilliant Blue (APExBIO) for 1 hour, destained with water and photographed. Precision Plus Protein™ (Biorad) was used to determine protein molecular weight (MW).

[0464] Estimation of total phenolic content

[0465] The procedure was done based on the method described in Ainsworth, E., Gillespie, K. 2007 (“Estimation of total phenolic content and other oxidation substrates in plant tissues using Folin-Ciocalteu reagent”. Nat Protoc 2, 875-877; which is fully incorporated herein by reference) with minor adjustments. Analyzed samples and Gallic Acid standards were mixed with Folin- Ciocalteu's phenol reagent (Merck) in microtiter multi-plate wells (DeepWell), followed by a 10- minute incubation. A 20% sodium carbonate solution was then added to each well and incubated for 2 hours at room temperature. All incubations were carried out in the dark. Following incubation, a sample from each reaction was transferred to an ELISA plate, and absorbance was read at a wavelength of 760 nm. Total phenolics were calculated as Gallic Acid equivalents using a standard curve derived from the blank-corrected A765 values of the gallic acid standards.

[0466] Functional Tests of Protein Powder

[0467] Foaming Test: 96.5 grams of water were first added to a Milk Frother (Nespresso). Subsequently, 3.5 grams of protein powder were introduced. The Frother was then operated for one minute.

[0468] Emulsification Test: A mixture of 135 grams of water and 15 grams of protein powder was blended (Fresh & Furious blender, Breville) for two minutes until all the powder was dissolved. Following this, oil (Canola oil; Etz Hazait) was gradually added to the mixture while continuing to blend, forming an emulsion.

[0469] Frying Test: 90 grams of water and 10 grams of protein powder were added to a blender and mixed for two minutes until the powder was completely dissolved. An electric induction stove was preheated for several minutes to a temperature of 160°C-180°C. The liquid mixture containing the dissolved protein powder (referred to as the "batter") was then poured into a frying pan placed on the electric induction stove. The batter was stirred continuously until it achieved the consistency of an omelette.

[0470] Taste and Odor Tests: Five panelists assessed the samples for both flavor and aroma, providing qualitative feedback on their sensory perceptions.

[0471] Proteomic Analysis

[0472] A soluble protein powder extracted from Moringa oleifera leaves (with the rachis and stems) was subjected to proteomic analysis. The sample was digested with trypsin and analyzed by LC-MS / MS on a Thermo Exploris 480 mass spectrometer. Peptide identification was performed using Thermo Scientific Proteome Discoverer 2.4 software with the Sequest HT search engine, against a custom database composed of the Moringa oleifera proteome (Taxon ID 3735) and the full UniProtKB database. Protein identifications were filtered at a 1% false discovery rate (FDR), and semi-quantification was based on the average peak area of the three most intense peptides per protein.

[0473] Protein Grouping and Quantification

[0474] Identified proteins were grouped by function and family (e.g., Rubisco large subunit, chlorophyll a-b binding proteins) for clarity. Their relative abundance was calculated as a percentage of the total detected protein signal.

[0475] Peptide Mapping and Sequence Alignment

[0476] To assess taxonomic specificity, the detected peptides of the Rubisco large subunit were aligned to the Moringa oleifera reference sequence and compared with homologous sequences from seven other plant species: Cuscuta reflexa (Pl 1894), Lemna minor (A0A1S3E4X7), Pisum sativum (P00870), Spinacia oleracea (P00875), Petunia hybrida (Q6ZNJ1), Malus domestica (Q84JH2), and Musa acuminata (Q8GTB6). Multiple sequence alignments were performed using Clustal Omega. Peptides identified in the Moringa powder were mapped onto the aligned sequences to evaluate their specificity.

[0477] Niazirin presence in protein powder

[0478] To assess the presence of Niazirin in the Moringa leaf + rachis protein powder, approximately 200 mg of the powder was extracted with 3 mF of 80% methanol. The extract was analyzed by liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) using negative electrospray ionization (ESI) mode. Identification of Niazirin was based on accurate mass detection of characteristic ions and comparison with data from reference literature, including published spectra and retention time profiles. Chlorophyll Quantification

[0479] Fresh leaf discs were homogenized in organic solvent (analytical-grade acetone or methanol) at a defined ratio of solvent to tissue weight. The homogenate was centrifuged, and the clarified supernatant containing extracted pigments was recovered. Absorbance of the extract was measured spectrophotometrically at 663 nm and 645 nm using the respective solvent as a blank. Concentrations of chlorophyll a, chlorophyll b, and total chlorophyll were calculated according to the equations described by Arnon [Arnon, D. I. (1949). “Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris”. Plant physiology, 24(1), 1], applying solvent-specific extinction coefficients. Results were normalized to the fresh weight of the plant tissue.

[0480] EXAMPLE 1

[0481] EXTRACTION OF SOLUBLE PROTEINS FROM MORINGA LEAVES

[0482] Experimental Results

[0483] A multi-step process for extraction of soluble proteins from Moringa leaves - The extraction of soluble proteins from Moringa leaves and their conversion into a high-quality, functional protein powder for a wide range of food applications is a multi-step process. Figure 1 depicts a process of extraction of soluble proteins from Moringa leaves according to some embodiments of the invention. The process begins with a raw material (can be done using a raw material at various initial states such as fresh, dried, or frozen), grinding, milling, filtering, degreening, solid separation, precipitation (an alternative route), washing / ultra-filtration, and drying. For each step, multiple parameters were tested to determine the most suitable, industrially scalable, and techno-economically feasible procedure.

[0484] Moringa leaves can be used in afresh, dry or frozen state - Moringa leaves used as raw plant materials were harvested from various geographical locations and processed as fresh, dried, or frozen material. The raw material was grinded in a buffer containing an antioxidant such as sodium-metabisulfite. The initial state of the source material did not result in significant differences in the final protein product. This is evidenced by the similar protein profiles from each raw material type, as observed on SDS-PAGE (Figure 2).

[0485] Milling using different gaps between the rotor and stator of a colloid mill affect tissue particle sizes - Following the initial grinding, the leaf material was further processed using a colloid mill. The gap between the rotor and stator was adjusted to 0, 11, or 20 machine units to achieve optimal cell rupture for soluble protein extraction, resulting in distinctly different tissue particle sizes (Figures 3A-C). It is noted that despite the distinct differences in particle sizes obtained with the various colloid gap units, the percentage of extracted soluble protein remained similar (data not shown). In addition, since milling with the 0 gap unit was faster in most experiments the working gap was 0.

[0486] De-greening of the green juice by heating for a limited time following by rapid cooling - The liquid phase (GJ), containing the soluble proteins, was separated from the solid phase (pulp) by filtering through a filter bag or using an industrial filter press. The green coloration, caused by chlorophylls present in the GJ, was eliminated by heating the liquid in tubes at 50°C for 0, 5, 10, 15 and 20 minutes (De-greening; Figure 4), followed by rapid cooling by immediately transferring the sample tubes to ice. The residual chloroplast material and insoluble proteins were removed by centrifugation, followed by optional filtration through a 2-micron depth filter sheet. The upper liquid phase, containing the soluble proteins, was collected (termed “brown juice” or “BJ”) and separated from the pellet.

[0487] Removal of phenolic compounds and concentration of the soluble protein phase - To remove phenolic compounds, concentrate the soluble protein phase, and prepare it for drying, the B J liquid was repeatedly washed with DDW using a 100 kDa cross-flow filter cassette (designated +UF). As shown in Figure 5, this washing and filtration stage effectively removed most of the phenolic compounds, reducing their concentration from 21.5 grams (±10) in the BJ (before ultrafiltration) to 0.7 grams (±0.5) after the ultrafiltration (designated as “+UF” in Figure 5). The liquid was then concentrated to one-tenth of its initial volume, and the resulting soluble protein concentrate was kept frozen until the drying step.

[0488] EXAMPLE 2 IDENTIFICATION OF OPTIMAL DRYING METHODS OF THE SOLUBLE PROTEIN EXTRA CT FROM MORINGA LEA VES

[0489] Experimental Results

[0490] Identification of optimal drying method for protein concentrate - To identify the optimal drying method for protein concentrate, the characteristics of protein powders obtained via vacuum drying and spray drying were compared (Figures 6A-B). The protein powder produced through spray drying was found to be much whiter in color and had a refined, powdery consistency (Figure 6A). In contrast, the vacuum drying method resulted in a protein powder that was brownish and had a harder, crystalline structure (Figure 6B). These findings indicate that spray drying produces a superior quality protein powder in terms of color and texture. EXAMPLE 3

[0491] FUNCTIONAL AND SENSORY PROPERTIES OF THE MORINGA PROTEIN POWDER

[0492] Experimental Results

[0493] Functional properties of the protein powder - The functional and sensory properties of the protein powder were evaluated through various assays including a foaming test, an emulsification test, and a frying test. The results are shown in Figures 7A-C.

[0494] In the foaming test, the protein powder demonstrated excellent foaming capacity, producing a stable and strong foam (Figure 7A).

[0495] The emulsification test resulted in an emulsion with a smooth and viscous texture (Figure 7B), similar to that of hummus salad.

[0496] When subjected to the frying test, the protein powder batter achieved a texture resembling scrambled eggs (Figure 7C).

[0497] Sensory analysis of the protein powder - Sensory analysis conducted by five panelists revealed that the protein powder possessed a "milky" aroma and a "nutty" flavor.

[0498] EXAMPLE 4

[0499] THE EXTRACTION PROCESS FROM MORINGA LEAVES YIELDS HIGH QUANTITIES OF SOLUBLE PROTEINS

[0500] Experimental Results

[0501] Table 1 summarizes the percentage of protein per sample in each step of the extraction of the soluble proteins from the moringa leaves.

[0502] Table 1

[0503] Table 1: Analysis for samples taken from the stages of production of an isolate of soluble proteins from moringa leaves using the Kjeldahl determination method. Percentage of protein balance reflects the total protein at a stage / total protein in the starting material. Percentage of mass balance reflects the total mass at a stage / total mass in staring material.

[0504] The results presented in Table 1 above demonstrate that following the washes and ultrafiltration of the brown juice the retentate comprises 5.6% protein (gram / 100 gram solution

[0505] Table 2 below summarizes the yield of the process in several representative experiments. The brown juice was weighted (soluble protein phase) and kept frozen until further use. The soluble protein phase was then subjected to ultrafiltration followed by diafiltration and then to spray drying until a dry powder was obtained. It is noted that in case the brown juice was frozen prior to spray drying, the present inventors have first defrosted the brown juice and then subjected it to spray drying. The protein content of the dry powder was determined using the Kjeldahl method and the results are summarized in Table 2 below.

[0506] Table 2

[0507] Table 2. Provided are the weight of the wet soluble protein (brown juice), the weight of the dry soluble proteins (powder), and the protein content in the dry powder presented in percentages of gram of protein / 100 gram powder weight. The results presented in Table 2 show that the yield of the process was very high, ranging from 57.12-72.88 % (gram protein / 100 gram powder), with an average of 67.7% (gram protein / 100 gram powder).

[0508] EXAMPLE 5

[0509] Chlorophyll content in the protein powder

[0510] The protein powder produced from Moringa oleifera leaves according to the pilot process contained approximately 0.2 mg total chlorophyll per gram of powder. This value is substantially lower than the levels typically found in fresh Moringa leaves, which have been reported to contain ~2 mg / g fresh weight total chlorophyll [Gonzalez-Romero, J., et al., 2020. Bioactive Compounds and Antioxidant Capacity of Moringa Leaves Grown in Spain Versus 28 Leaves Commonly Consumed in Pre-Packaged Salads. Processes, <S(10), 1297; Artadana, I. B. M., & Pandji, E. (2023). Moringa oleifera L. Microgreens and their Antioxidant Activity. In E3S Web of Conferences (Vol. 374, p. 00018). EDP Sciences].

[0511] EXAMPLE 6

[0512] PROTEIN COMPOSITION OF MORINGA LEAVES POWDER

[0513] Experimental Results

[0514] Protein Composition of Moringa Leaves (with rachis and stems) Powder - Rubisco (ribulose- 1,5-bisphosphate carboxylase / oxygenase) was the most abundant protein in the Moringa oleifera soluble protein powder accounting for approximately 66% of the protein detected. Other proteins were also consistent with a leaf-derived extract (e.g. chlorophyll a-b binding proteins, oxygen-evolving enhancer proteins, and fructose-bisphosphate aldolase.

[0515] Rubisco Sequence Specificity

[0516] Peptide mapping onto the Rubisco large subunit sequence revealed 11 distinct peptides (Table 3 and Figure 8). Among these, one peptide (Pl) was found exclusively in Moringa oleifera, while six additional peptides (P2-P7) appeared in one or two of the other species included in the alignment. The pattern and distribution of these peptides along the Rubisco sequence support the conclusion that the protein detected in the powder is consistent with Rubisco derived from Moringa oleifera. These findings support the potential use of the peptide profile for establishing species- specific identification of the protein source. Table 3 below provides the sequences of the peptides identified by LC-MS / MS in the powder prepared from the Moringa leaves and rachis according to some embodiments of the invention. Table 3 shows that the identified peptides have specificity to the Rubisco Large Subunit (SEQ ID NO: 10) and that they share conserved domains across various plant species. The peptides were matched against a multiple sequence alignments constructed from eight plant species: Cuscuta sandwichiana (Q49CC1; SEQ ID NO: 3), Lemna minor (A9L9A4; SEQ ID NO: 4), Pisum sativum (Garden Pea) (P04717; SEQ ID NO: 5), Spinacia oleracea (Spinch) (P00875; SEQ ID NO: 6), Petunia hybrida (P04992; SEQ ID NO: 7), Malus domestica (Apple) (A0A223A9S9; SEQ ID NO: 8), Musa acuminata (Banana) (A9QBM3; SEQ ID NO: 9), and Moringa oleifera (A0A4Y5PRY2; SEQ ID NO: 10). The Table indicates which peptides were detected in the Moringa sample alone (e.g., SEQ ID NO: 11, peptide Pl) and whether each sequence is conserved across the listed species. The peptide numbers and sequence accession codes (UniProt) correspond to those shown in Figure 8. Table 3

[0517] Peptides identified in the powder prepared from moringa leaves and rachis Table 3: (*) - Peptide “P4B” (SEQ ID NO: 32) comprises the amino acid sequence of peptide “P4” (LTYYTPDYETK (SEQ ID NO: 14), which is unique to Moringa and apple and an additional sequence (DTDILAAFR, SEQ ID NO: x33) which is common in all 7 species;

[0518] (**) - the sequence of peptide “P5B” (SEQ ID NO: 16) is comprised in the sequence of peptide “P5” (SEQ ID NO: 15), yet, both of these peptides are unique to Moringa and Apple;

[0519] (***) - the sequence of peptide “PIO” (SEQ ID NO:21) was identified by the LC-MS / MS analysis as DNGILLHIHR (SEQ ID NO: 21), however, in all of the sequences available in GenBank and Uniprot this sequence appears as DNGLLLHIHR (SEQ ID NO:22);

[0520] (****) the peptide sequence of peptide “P12” (SEQ ID NO: 24) was identified by the LC-MS / MS analysis as DTDLLAAFR (SEQ ID NO:24), however, in all of the sequences available in GenBank and Uniprot this sequence appears as DTDILAAFR (SEQ ID NO:25);

[0521] (*****) the sequence of peptide “P14” (SEQ ID NO: 27) was identified by the LC-MS / MS analysis as YGRPILGCTIKPK (SEQ ID NO: 27) however, in all of the sequences available in GenBank and Uniprot the conserved sequence is YGRPLLGCTIKPK (SEQ ID NO:28).

[0522] Table 4

[0523] Table 4 summarizes the percentage of protein per sample in each step of the extraction of the soluble proteins from moringa leaves (with the rachises and stems).

[0524] Table 4: Analysis for samples taken from different stages of isolating soluble proteins from moringa leaves and rachises using the Kjeldahl determination method. Percentage (%) of protein balance reflects the total protein at a stage / total protein in the starting material. Percentage of mass balance reflects the total mass at a stage / total mass in staring material, “g” = gram;

[0525] From Table 4 it is noted that the % of protein in stage in the dry powder using the pilot process for isolating soluble proteins from the moringa leaves (e.g., 76% gram / 100 gram material) is at least comparable to the % of protein in stage when using the lab process for isolating the soluble proteins from the moringa leaves (e.g., 72.8% gram / 100 gram material, Table 2 above).

[0526] Figure 10 depicts the SDS-PAGE analysis showing the presence of the large and small rubisco subunits isolated from fresh moringa leaves and rachis using the pilot process described in the “General Material and Experimental Methods” above. It is noted that both the lab process and the pilot process result in clear presence of the large and small rubisco subunits from Moringa.

[0527] EXAMPLE 7

[0528] NIAZIRIN PRESENCE IN THE PROTEIN POWDER

[0529] Experimental Results:

[0530] LC-QTOF-MS analysis of the Moringa leaf protein powder extract revealed chromatographic peaks corresponding to the accurate masses of representative ions of Niazirin. These ions included m / z values 314.0794, 324.1083, and 341.0989, which align with expected fragments of Niazirin under negative ESI mode. The extracted ion chromatograms (XICs) for these ions showed consistent peaks, suggesting the presence of Niazirin in the extract of the Moringa protein powder (upper panel in Fig. 9) which corresponds to the niazirin in the fresh Moringa leaves (lower panel in Fig. 9).

[0531] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0532] It is the intent of the Applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

Claims

WHAT IS CLAIMED IS:

1. A method of producing an isolate of soluble proteins from moringa leaves, comprising:(a) grinding the moringa leaves in the presence of liquid, to thereby obtain a green juice of ground moringa leaves;(b) disrupting organelles in said green juice so as to release soluble proteins from said organelles, and(c) isolating said soluble proteins from said green juice, thereby producing the isolate of soluble proteins from the moringa leaves2. A method of producing an isolate of soluble proteins from moringa leaves, comprising:(a) grinding the moringa leaves in a buffer solution comprising an antioxidant, to thereby obtain a green juice of ground moringa leaves;(b) isolating a soluble protein phase from said green juice using a filter;(c) removing chlorophyll from said soluble protein phase to obtain a chlorophyll-free preparation comprising said soluble proteins;(d) removing solids from said chlorophyll-free preparation to obtain solid-free preparation comprising said soluble proteins; and(e) removing phenolic compounds and salts from said solid-free preparation by ultrafiltration; thereby producing the isolate of soluble proteins from the moringa leaves.

3. The method of claim 1, wherein said liquid is a buffer solution.

4. The method of claim 3, wherein said buffer solution comprises an antioxidant.

5. The method of any one of claims 1-4, wherein said leaves are attached to rachis and / or stem.

6. The method of any one of claims 1-4, wherein said leaves are isolated from rachis and / or stem.

7. The method of any one of claims 1-6, wherein said grinding is performed by a shredder.

8. The method of any one of claims 1-6, wherein said grinding is performed by a crusher.

9. The method of any one of claims 1 and 3-8, further comprising milling the ground moringa leaves prior to said isolating of said soluble protein.

10. The method of any one of claims 1 and 3-9, wherein said liquid does not comprise alcohol or hydro-alcohol.

11. The method of any one of claims 1 and 3-10, wherein said filter is featured by a pore diameter of a size selected from a range of 50-400 microns.

12. The method of any one of claims 1 and 3-11, wherein said disrupting said organelles is performed using a high-pressure homogenizer, a microfluidizer, and / or a colloid mill.

13. The method of any one of claims 1 and 3-12, wherein said organelles comprise chloroplasts.

14. The method of any one of claims 1 and 3-13, further comprising cooling said soluble proteins to a temperature of 4°C following said disruption of said organelles.

15. The method of any one of claims 1 and 3-14, wherein said isolating said soluble proteins is effected by filtration.

16. The method of claim 15, wherein said filtration is performed by using a filter press, a filter drum, or a filter bag.

17. The method of any one of claims 15-16, wherein prior to said filtration the method comprises mixing said chlorophyll-free preparation with a filtration aid.

18. The method of any one of claims 1, 3-17, wherein said isolating said soluble proteins is performed using a decanter centrifuge.

19. The method of any one of claims 1 and 3-18, wherein said soluble proteins are substantially solid-free.

20. The method of any one of claims 1 and 3-19, further comprising removing phenolic compounds and salts from said soluble proteins.

21. The method of claim 20, wherein said removing said phenolic compounds and salts is performed by ultrafiltration.

22. The method of any one of claims 2-21, wherein said ultrafiltration is performed using a cross flow filter cassette in a range of 10-100 KDa.

23. The method of any one of claims 2-22, further comprising diafiltration following said ultrafiltration.

24. The method of any one of claims 1 and 3-19, further comprising precipitating the soluble proteins.

25. The method of any one of claims 1-24, wherein said moringa leaves are fresh leaves.

26. The method of any one of claims 1-24, wherein said moringa leaves are dry leaves.

27. The method of any one of claims 1-24, wherein said moringa leaves are frozen leaves.

28. The method of any one of claims 2 and 4-27, wherein said antioxidant comprises sodium-metabisulfite (SMBS).

29. The method of claim 28, wherein said antioxidant is provided at a concentration in a range of 0.2%-1.0% (gram / 100 ml).

30. The method of any one of claims 2-29, wherein said buffer solution comprises a citric acid buffer.

31. The method of any one of claims 2-29, wherein said buffer solution comprises a carbonate buffer.

32. The method of any one of claims 1 and 3-31 when depending on claim 1, wherein the soluble proteins comprise large and small Rubisco protein subunits in a level detectable by SDS-PAGE.

33. The method of any one of claims 1 and 3-31 when depending on claim 1, wherein the soluble proteins comprise the large Rubisco protein subunit which comprises the amino acid sequence set forth by SEQ ID NO: 11.

34. The method of claim 33, wherein the soluble proteins comprise the large Rubisco protein subunit further comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 12-14, 15-16, 17-20, 22, 23, 25, 26, 28, 29-31 and 32.

35. The method of any one of claims 1 and 3-34, further comprising freezing the isolate of soluble proteins following isolating said soluble proteins.

36. A method of preparing a powder of soluble proteins from moringa leaves,(a) isolating soluble proteins from moringa leaves according to the method of any one of claims 1-34, to thereby obtain a solution of isolated soluble proteins, and(b) spray drying said solution of said isolate soluble proteins, to thereby the powder of soluble proteins from the moringa leaves.

37. A composition-of-matter comprising a chlorophyll-free preparation of soluble proteins of moringa leaves, wherein said soluble proteins comprise large and small Rubisco protein subunits, and wherein said large Rubisco protein comprises the amino acid sequence set forth by SEQ ID NO: 11.

38. A composition-of-matter comprising a chlorophyll-free preparation of soluble proteins of moringa leaves which comprises Niazirin.

39. A composition-of-matter comprising a chlorophyll-free preparation of soluble proteins of moringa leaves, wherein said soluble proteins comprise large and small Rubisco protein subunits produced according to the method of any one of claims 1-35.

40. The composition-of-matter of any one of claim 37-39, being substantially free of phenolic compounds and salts.

41. A preparation comprising the composition-of-matter of any one of claims 37-40.

42. The preparation of claim 41, being a liquid.

43. The preparation of claim 42, comprising between 15-20% (gram / 100 ml solution) of soluble proteins.

44. The preparation of claim 41, being a powder.

45. The preparation of claim 44, comprising between 50-75% soluble proteins (gram proteins / 100 gram powder).

46. The composition-of-matter of any one of claims 37-40 or the preparation of any one of claims 41-45, forming part of an edible preparation.

47. An edible composition comprising the composition-of-matter of any one of claims 37-40, or the preparation of any one of claims 41-45.

48. The edible composition of claim 46, wherein said composition-of-matter of any one of claims 37-40, or the preparation of any one of claims 41-45, comprises at least 50% (gram / 100 gram composition) of the edible composition.

49. A cosmetic composition comprising the composition-matter of any one of claims 37-40 or the preparation of any one of claims 41-45 and a cosmetically acceptable carrier.

50. A pharmaceutical composition comprising the composition-matter of any one of claims 37-40 or the preparation of any one of claims 41-45 and a pharmaceutically acceptable carrier.

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