WO2009051470A1

WO2009051470A1 - Flavour enhancers/food seasoning from seaweeds and a method for producing and uses thereof

Info

Publication number: WO2009051470A1
Application number: PCT/MY2008/000069
Authority: WO
Inventors: Suhaila Mohamed; Patricia Matanjun; Sharifah Kharidah Muhammad; Noordin Mohamad Mustapha; Farideh Namvar; Kheen Kuan Chan; Rosalina Roslan Tan; Samaneh Ghasemi
Original assignee: Universiti Putra Malaysia
Priority date: 2007-07-25
Filing date: 2008-07-15
Publication date: 2009-04-23
Also published as: MY150068A; WO2009051470A8

Abstract

A method for preparing a seaweeds based flavour enhancer/seasoning with cardiovascular health promoting, degenerative disease preventing and organ protective properties comprises the steps of pre-treating seaweeds; pulverization or extracting the pre-treated seaweeds using a solvent; with or without the addition of polyphenol rich plants/extracts and other food additives. Further, the present invention also relates to a method for preparing a non-toxic anti-cancer agent and healthcare, oral and topical nutraceutical ingredients for humans and animals. The steps of pre-treating alcoholic extracts derive from plants of the tropical seaweeds, Solanum and Piper spp.; extracting the pre-treated alcoholic extracts of by using a solvent; removing the pre-treated alcoholic extracts of from the used solvent; and concentrating the used solvent to acquire the nutraceutical ingredients. Also provided is a method for preparing a wound healing accelerator and general healthcare, oral and topical nutraceutical ingredients for diabetics and non-diabetics humans and animals. The steps of pre-treating vegetative parts derive from plants of the seaweeds and Arecaceae family; extracting the pre-treated vegetative parts by using a solvent; removing the pre-treated vegetative parts from the used solvent; and concentrating the used solvent to acquire the nutraceutical ingredients.

Description

FLAVOUR ENHANCERS/FOOD SEASONING FROM SEAWEEDS AND A METHOD FOR PRODUCING AND USES THEREOF

FIELD OF INVENTION

The present invention relates to flavour enhancers/food seasoning from seaweeds which are capable of preventing or assisting in the management of hypercholesterol, hyperlipidemia, atherosclerosis, obesity, oxidative stress, organ damage, cardiovascular disease and other ailments related to or caused by such conditions of an individual upon use of the seasonings or pertaining products.

Further, the present invention relates to nutraceuticals and products from extracts of tropical seaweeds, Solanum spp. fruits and Piper spp. leaves which is capable of inhibiting growth and killing cancer cells through apoptosis in human or animals by oral or topical application.

Further, the present invention relates to a nutraceuticals and products from palm vegetative parts and seaweeds, which is capable of accelerating or assisting wound healing for diabetic and non-diabetic human or animals by oral or topical application.

BACKGROUND OF THE INVENTION

Seasoning provides a purpose to enhance existing flavours of a food product. Two classes of well-known flavour enhancing compounds are monosodium glutamate and 5'- ribonucleotides. These flavour enhancing compounds are used as such, but are also, separately or in combination, part of flavour enhancing compositions. Seasonings are compounds that are added to a food in order to supplement or enhance its own natural flavour.

Degenerative diseases are of great apprehension in modern population. The main cause of degenerative diseases are cardiovascular disorders and oxidative stress partly due to imbalance diet, stress, inactive lifestyle, and over consumption of high cholesterol, fatty or high calorie food. Genetic disorder such as familial hypercholesterolemia, hypertension, obesity and diabetes also predisposes the inherited individuals to cardiovascular problems prior to the intervention of the external environmental factors. There are many therapies available for treatment and alleviation of the related cardiovascular problems, but it is good to have daily dietary seasoning, which can assist in either preventing the advancement of the cardiovascular diseases or improving the cardiovascular system of an individual upon regular use of such comestible products.

Metabolites from different flora are proven to help prevent cardiovascular diseases. Subsequently, different health-promoting products containing the plants metabolites as the active ingredients have been developed. For example, Villoo et al. have filed PCT patent application which was published as WO2006090198 which parts of the claims are directed to a comestible product incorporated with extracts from the plant of Terminalia arjuna. This application has claimed that the extract of the Terminalia arjuna is capable of treating cardiovascular disease upon consumption of the product.

According to a recent worldwide tendency to reevaluate seaweeds as food resources, the consumption of seaweeds in Korea shows a tendency to increase every year so that the seaweeds are highlighted as a material for healthy food. In Korea, one of the leading countries whose people use the seaweeds regularly, the seaweeds are so various in kind and rich in resources that they made up about 15% of the total production of marine products in the year 2000. The seaweeds are used in food, pastes, feedstuffs, fertilizers, and raw materials for the industry. Particularly, the seaweeds contain large amounts of minerals and vitamins, which are involved in the control of hormone metabolism in the human body. There are study results indicating that seaweed components, such as dietary fiber, alginate and fucoidan, have effects on intestinal cleaning, heavy metal release, fatness inhibition, anticancer, etc.

There are several prior art documents disclosing inventions relating to seaweed related products, method obtained from the seaweed products and the use of seaweed products.

For example, Korean patent application No. KR20020004717 discloses a seasoning containing medicinal materials such as oriental medicinal materials, seaweed and plants. The seasoning comprises a medicinal seasoning fruits consisting of apple, grape, pear, banana, apricot, melon and watermelon and oriental medicinal herbs. Japanese patent application no. JP20040343522 discloses a method for seasoning brown alga by which seaweed such as brown algae in particular maintains original green color (in color development) or restore the color to prevent deterioration when seasoned in an acidic environment, and having mild flavor suppressed in acidic taste.

In addition, another prior art US patent application which was published as US20060546677 discloses a method and composition for retardation of cardiovascular disorder in a mammal comprising the step of administering to a mammal an amount of seaweed extract in effective doses. This current invention also comprises a composition and method for preserving cardiovascular health in a mammal comprising the step of administering to a mammal an amount of seaweed extract in effective doses. Moreover, the seaweed is selected from the group consisting of brown algae, red algae and green algae, wherein the seaweed extract contains a polysaccharide.

Another prior art Korean patent application no. KR20000063563 describes a process of preparing a health food product by extracting seaweeds such as kelp, brown seaweed, Sargassum fulvellum or the like is provided. Whereby, the health food product has an excellent effect on prevention and treatment of hypertension, arteriosclerosis, cerebral apoplexy, obesity, diabetes, osteoporosis, kidney disease, liver disease, thrombosis, constipation, fecal impaction, hangover or the like.

Unrestrained cell proliferation is the characteristic of cancer, and tumor cells usually have damage to genes that directly control their cell cycles. Changes in cell survival contribute to a number of human diseases, including cancer, viral infections, autoimmune diseases, neurodegenerative disorders, and AIDS (Acquired Immuno-Deficiency Syndrome). Natural physiological cell death occurs primarily through apoptosis. Bioavailable nutraceuticals that cause apoptosis to cancer cells and inhibit cancer cell proliferation could retard the progression or even cause the regression of cancer. Apoptosis is the natural process of removing cells in normal or pathologic tissues. Rapid condensation and budding of the cell occur, with the formation of membrane-enclosed apoptotic bodies containing well-preserved organelles, which are phagocytosed and digested by adjacent local cells, without any inflammation. Characteristically, double- strand cleavages of nuclear DNA occurs at the linker regions between nucleosomes causing the production of oligonucleosomal fragments. Usually, apoptosis is suppressed by inhibitors of messenger RNA and protein synthesis. Apoptosis takes place spontaneously in malignant tumors, often significantly retarding their growth, and are increased in tumors responding to irradiation, cytotoxic chemotherapy, heating and hormone ablation. Apoptosis can be regulated by certain proto-oncogenes and the p53 tumor suppressor gene. C-myc expressions are involved in the initiation of apoptosis in some situations, and bcl-2 is a new type of proto-oncogene that inhibits apoptosis, rather than stimulating mitosis. Antibodies against a cell-surface protein designated APO-1 or Fas can enhance apoptosis in some human lymphoid cell lines which may have therapeutic uses.

Seaweeds, Solanum and Piper spp. are noted to contain not only labile antioxidants (i.e. ascorbate, glutathione) when fresh, but also more stable molecules such as carotenoids, mycosporine-like amino acids and a variety of phenolic compounds. Chemical composition of seaweeds, Solanum and Piper plants varies with individuals, species, habitats, maturity and environmental conditions. The potential antioxidant compounds identified in seaweeds include pigments (fucoxanthin, astaxanthin, carotenoid e.g.) and polyphenols (phenolic acid, flavonoid, tannins e.g.). The protective effects of dietary kelps and other red and green algae against mammary, skin and intestinal cancer were supported by epidemiological data and rodent model studies. An algal antioxidant- mediated mechanism was hypothesized as a contributing factor in the inhibition of mammary carcinogenesis by dietary kelp in the presence of enhanced antioxidant enzyme activity and reduced lipid peroxides in livers of treated rats.

Different metabolites from various organisms contain proven anti-cancer properties.

Subsequently, different anti-cancer products containing the organisms' metabolites as the active ingredients have been developed. For example, KIMURA TAKAYUKI; MIKI

YASUNARI; KAWAMOTO HITOSHI; MATSUDA HIDEYUKI; KAWAMUKI MAKOTO; TANAKA KATSUNORI; NAKAGAWA TSUYOSHI of KAISANBUTSUNO KIMURAYA KK have filed Japanese patent application no. JP2006306897 which is directed to provide a new anti-gastric cancer agent that suppresses gastric cancer cell proliferation which employs the fucoidan originating from Mozuku seaweed as the effective component. The fucoidan originating from hot water extract of Mozuku seaweeds, for example, lto mozudu (Nemacystus decipieus), Okinawa mozuku (Cladosiphon okamuranυs) or Futo mozuku (Tinocladia crassa) or the like or a purified product thereof obtained by treating the extract with a quaternary ammonium salt.

US patent application which was published as US20030157127A1 by Iwasaki, Teruaki claims a nutritious supplemental composition for suppression against onset of large intestinal cancer and manufacturing method thereof. The invention provide a composition containing well-balanced nutrition, having an effect to suppress the mutagenesis substances, having no sub-action even if the composition is continued to be taken as nutritious supplemental substance and capable of promoting healthy state. There are provided a nutritious supplemental composition for suppression against large intestinal cancer and its manufacturing method in which dietary fiber in a range of 15 wt % to 30 wt % in respect to a total amount of composition is contained in the dried koji fine powder including dead fungi of Aspergillus spp. while keeping a capability of catalysis of groups of enzyme produced by Aspergillus spp.

European patent application which was published as EP0919237 by SAKAI TAKESHI (JP); KITANO HIDEO (JP); YU FU-GONG (JP); NAKAYAMA SHINJI (JP); KOJIMA KAORU (JP); KIMURA HITOMI (JP); NAKANISHI YOSHIKUNI (JP); KATAYAMA KAORU (JP); TOMINAGA TAKANARI (JP); SHIMANAKA KAZUO (JP); IKAI KATSUSHIGE (JP); KATO IKUNOSHIN (JP) relate to anti-apoptosis inducers, anticancer drugs and carcinostatic drugs containing sulfated-fucose-containing polysaccharide(s) and/or degradation product(s) thereof, and a method for inducing apoptosis by using sulfated- fucose-containing polysaccharide(s) and/or degradation product(s) thereof as the active ingredient. A degrading enzyme which is useful in the production of the degradation products of sulfated-fucose-containing polysaccharides. US patent no. 4,708,962 by Higa; Tatsuo (Okinawa, JP), Snader; Kenneth M. (Vera Beach, FL) (November 24, 1987) that invents an Antiviral and antitumor cyclohexadienone compositions, and a method for inhibiting, remitting or controlling the growth of tumors or tumor cells utilizing antitumor compositions. More particularly, the antitumor compositions comprise, as active ingredient, an antitumor effective amount of halogenated chamigrenes extracted and derived from red alga and sea hares, which diet upon red alga.

Chronic wounds and their treatment are a big burden on the healthcare system, both in terms of cost, time and energy of care required. The lost in productivity and decreased quality of life is immeasurable. Thousands of people suffer from pressure ulcers and many more have chronic leg and foot wounds. This invention helps prevent chronic wound in diabetics from tropical herbal resources especially for those with a strong family history of diabetes.

Diabetes mellitus is recognized as a risk factor for compromised wound healing. The cellular and biochemical interplay that comprises the normal wound healing response is a complex process involving intricate interactions among a variety of different cell types, structural proteins, growth factors, and proteinases. The normal wound repair process consists of three phases — inflammation, proliferation, and remodeling — that occur in a predictable sequence and comprise a series of cellular and biochemical events. This study will examine leukocyte infiltration and the appearance of tumor necrosis factor-a (TNF) and IL-6 in wound in normal and streptozotocin-induced diabetic mice. Wound fluid aspirated from the wounds 1 , 3, and 7 days following treatment will be analyzed for the total number of leukocytes and TNF and IL-6 levels.

Acute wounds heal in an orderly progression, maturing through artificially defined phases of coagulation, inflammation, matrix synthesis and deposition, angiogenesis, fibroplasia, epithelialization, contraction, and remodeling. These processes were defined as three classic phases of wound healing — inflammation, fibroplasia, and maturation. The end result of uncomplicated healing is a fine scar with little fibrosis, minimal if any wound contraction, and a return to near normal tissue architecture and organ function. If a wound does not heal in an orderly or timely sequence, or if the healing process does not result in structural integrity, then the wound is considered chronic. Skin ulcers are probably the most common types of chronic wounds. These wounds can be created or perpetuated by many factors, including vascular insufficiency, either venous or arterial, prolonged inflammation, pressure necrosis, physical agents, infection, and cancer. Seventy percent of skin wounds, however, are due to pressure ulcers, diabetic foot ulcers, and venous ulcers. Chronic wound healing occurs by the same processes as acute wound healing, namely inflammation, fibroplasia, and epithelialization. Chronic wounds, however, differ from acute wounds in that healing occurs with the formation of abundant granulation tissue and often with excessive fibrosis leading to scar contraction and loss of function. Diabetes is defined as hyperglycemia associated with vascular, renal and neural changes. In children, there is usually a primary injury to the pancreas which causes insulin deficiency. In adults, diabetes is most often related to obesity and is manifested by a complex mechanism of insulin resistance and decreased insulin release from the pancreas. Diabetic patients still experience more wound failures, as shown by review of several studies of wounds of the lower extremities. Wound failure, defined as the need for major amputation, occurred in 2.5 to 12 per cent more of the diabetic patients studied than the non-diabetics. Experimentally, wound healing is defective in both insulin deficient and insulin resistant animals. Insulin improves healing in insulin deficient animals but not in animals with obesity and insulin resistance. There is clinical evidence that weight control with concurrent improvement in glucose tolerance is associated with better healing in insulin resistant obese patients. Diabetics have defects in leukocyte function. There is decreased phagocytosis which seems to be secondary to hyperglycemia. There is also decreased chemotaxis which may be an inherent defect in cells from diabetic patients. The defects in leukocyte function contribute to an increased rate of infection. For the inflammatory response, defects in leukocyte function may partially explain the defective healing seen in the absence of infection. The exact mechanism or mechanisms of the defect in healing in diabetes are still unclear. Each chronic wound is unique, with a unique set of physiologic and social circumstances preventing or retarding wound healing. Although chronic wound care will almost certainly remain highly individualized, ongoing research, especially in the area of growth factor manipulation, may allow these wounds to be treated more effectively than ever before. Researches have shown that metabolites from different organisms contain proven properties in assisting in wound healing. Subsequently, different wound healing products containing the organisms metabolites as the active ingredients have been developed. For example, Chien amd Bagchi have filed PCT application which was published as WO2007143631 which are directed to a nutraceutical treatments treatments for diabetic and non-diabetic wound healing formula comprising a combination of chromium.zinc berry, Polygonium cuspidatum, aloe vera, chlorophyll extracts and Z-arginine, which can reduce inflammation and assist wound healing.

European patent application which was published EP1871483 by Van den Elshout et al has been filed for food for wounded animals for increasing the speed of wound healing but the invention disclosed in this application does not contain neither palm vegetative parts or seaweeds.

UK patent no. GB2135426 by Richardson relate to wound healing system and dressing containing a dried form of liquid made from honey, essential oils, herbal extracts, alginate chitosan, carboxyl methyl cellulose or pectin.

PCT patent application which was published as WO2005115090 by Mehta et al claims a herbal composition having potent antimicrobial and wealing properties consisting of extracts of Ficus spp and Azadurachta as the major components to carry out the claimed effects.

SUMMARY OF THE INVENTION

The present invention aims to provide seaweeds based, herbal seasoning, hydrolysate or extract which is suitable to be used or incorporated into food as flavour enhancer/seasoning for improving cardiovascular health or reducing the risks of CVD upon use of the products.

The seaweeds are selected from brown, red or green seaweeds, whereby the seaweeds are preferably but not limited to Eucheuma spp, Sargassum spp and Caulerpa spp. The seaweed based seasoning composition further includes between about 1 % and 100 % by weight of red seaweed; between about 1 % and 100 % by weight of brown seaweed and between about 1% and 100% by weight of green seaweed. The seaweeds comestible product may be used as a mineral, antioxidant, dietary fiber supplement or enrichment ingredient and flavor enhancers in foodstuffs. The seaweeds comestible product may be mixed with foodstuffs; including cereals, bread, drinks, health bars, juices, concentrates, canned food, ice cream, water, staple goods such as wheat, corn, barley, and oat in any form, or taste maskers such as sugar to form new products. The herbs are selected from polyphenol rich flora. The composition is preferably in a form of dry or aqueous. The present invention further relates to the seaweeds that have the capability of seasoning means.

The present invention further relates to a method for preparing seasoning from seaweeds, wherein the method may comprise the steps of: hydrolyzing seaweeds in the presence or absence of catalysts at an optimum operating temperature and pH to form hydrolystate; maintaining the pH of the hydrolyzing process; deactivating the catalysts in the hydrolysate; adjusting pH of the hydrolysate to 3.5-4.5; and filtering the hydrolysate through layers of filter medium such as activated carbon and/or calcium carbonate to obtain the flavor enhancer. The method further relates to concentrating or drying the obtained flavor enhancer. In addition, the mentioned method includes adding any one or combinations of polyphenols rich plants or extracts, thickeners, preservatives, caramel, onion powder, ginger powder, garlic powder, cinnamon, spices, and black peppers to the obtained flavor enhancer. In this present method, seaweeds are cleaned, dried, ground with or without adding any one or combinations of polyphenols rich plants or extracts, thickeners, preservatives, caramel, onion powder, ginger powder, garlic powder, cinnamon, spices, and black peppers to the obtained seasoning, which can also be used as mineral, omega 3 and omega 6 fatty acids and dietary fiber supplements

In another embodiment of the present invention relates to the use of the seaweeds comestible product in a preparation to manufacture a functional ingredient for protecting or reducing hyperglycaenia, hypercholesterol, hyperlipidemia, atherosclerosis, diabetic, hypertension, cardiovascular ailments and/or oxidative stress in an individual. Further to that, the seaweeds comestible product may be used in a preparation to manufacture a functional ingredient for reducing cardiovascular related diseases, degenerative diseases and/or cancer related diseases in an individual.

The present invention also relates to seasoning containing seaweed flavour enhancer produced by the above-mentioned method and the seasoning includes providing heath benefits to a mammal.

The present invention aims to provide tropical seaweeds, Solanum and/or Piper spp. fruits and leaves extract which is suitable to be incorporated into daily diet and topically for improving the speed of anti-cancer upon use of the products. The alcoholic extract from species of tropical edible seaweeds including (but not limited to) red (Eucheuma cottonii)), green {Caυlerpa lentillifera) or brown seaweeds (Sargassum polycystυm) and Piper spp. leaves, are rich sources of natural antioxidants. The present invention claims the anti- cancer use of extracts from tropical seaweeds, on important cancer cell lines MCF-7 (human breast carcinoma cell line, oestrogen positive), MDA-MB- 231 , MDA 435 (human breast carcinoma cell line, oestrogen negative), HeLa, (human cervical adenocarcinoma cell line) HepG2, (human hepatocellular carcinoma cell line), DU145 (Human Prostate Cancer cell line) and HT-29 (human colon carcinoma). Further object of the present invention is to disclose a method to improve anti-cancer therapy or prevent chronic cancer diseases (with additional one or more effects such as anti-hypertensive, anti-atherogenic, anti-hypercholestrol, anti-obesity, anti-hyperlipidemic, anti-oxidative, antimicrobial, vasorelaxant, wound healing and the like) by administrating the aforesaid herbal extract or its derivatives products orally or topically to a subject.

Still another object of the present invention is to disclose an extraction method specifically useful in isolating the desired compounds from these marine and terrestrial plants of Piper spp. and Euchema spp., Appaphycυs spp., Caulerpa spp. and Sargassum spp. The disclosed method is able to isolate the active plant metabolites and provide efficient yield without degrading its therapeutic effect for anti-cancer and cardiovascular disease protection.

At least one of the preceding objects is met, in whole or in part, by the present invention, in which one of the embodiment of the present invention is a comestible or topical composition for promoting anti-cancer and overall healthcare of an individual comprises an extract derives from alcoholic extracts of a plant of Piper spp. and tropical seaweeds family using an extraction solvent.

Further embodiment of comestible composition is preferably having the extract prepared in a concentrated form thus can be conveniently incorporated to various products. The extract may be prepared in a concentrated form together with or without other known anti- cancer ingredients such as honey, curcumin, vanillin, vitamin E, thymoquinone, antioxidants, anti-angiogenic agents, sea cucumber extracts, etc.

In order to effectively isolate out the desired metabolites from the plants of the tropical seaweeds, Solarium and Piper leaves family, the preferred extraction solvent is selected from the group consisting of water, alcohol, acetone, chloroform and any combination thereof.

Further embodiment of the present invention is regarding a method for promoting anticancer and healthcare in a subject comprising the step of administrating orally the subject an effective amount of an extract derived from tropical seaweeds, Solarium and Piper spp. family in separation or together using an organic extraction solvent. The present invention also discloses a method for preparing an herbal extract comprising the steps of soaking dried alcoholic extracts derive from tropical seaweeds, Solanum and Piper spp. leaves family into a solvent; removing the soaked dried alcoholic extracts of from the solvent; and concentrating the solvent to acquire the extract.

Similarly, the solvent found to be efficient in the extraction is any one or combination of water, alcohol, acetone and chloroform.

Further embodiment to improve the extraction efficiency involve a pre-treatment step of fragmentizing the dried alcoholic extracts of into small fragment or pulverizing to paste or powdery form before soaking into the extraction solvent.

Still another embodiment of the present invention, the alcoholic extracts of the tropical seaweeds, Solanum and Piper leaves family is preferably derived from any one or combination of the species, but not limited to, Euchema cottonii, Appaphycus alvareziii, Caulerpa lentillifera and Sargassum polycystum, and Piper betel.

The present invention also aims to provide seaweeds or palm leaves extract, which is suitable to be incorporated into daily diet and topically for improving the speed of wound healing upon use of the products.

Further object of the present invention is to disclose a method to improve wound healing system or prevent chronic wound diseases (with additional one or more effects such as anti-hypertensive, anti-atherogenic, anti-hypercholestrol, anti-obesity, anti-hyperlipidemic, anti-oxidative, antimicrobial, vasorelaxant and the like) by administrating the aforesaid herbal extract or its derivatives products orally or topically to a subject.

Still another object of the present invention is to disclose an extraction method specifically useful in isolating the desired compounds from these marine and terrestrial plants of Arecaceae and Euchema cottonii, Appaphycus alvareziii, Caulerpa lentillifera and Sargassum polycystum family. The disclosed method is able to isolate the active plant metabolites and provide efficient yield without degrading its therapeutic effect for wound healing and cardiovascular disease protection. At least one of the preceding objects is met, in whole or in part, by the present invention, in which one of the embodiment of the present invention is a comestible or topical composition for promoting wound healing and overall healthcare of an individual comprises an extract derives from vegetative parts of a plant of Arecaceae and seaweeds family using an extraction solvent.

Further embodiment of comestible composition is preferably having the extract prepared in a concentrated form thus can be conveniently incorporated to various products. The extract may be prepared in a concentrated form together with or without other known wound healing ingredients such as honey, hyaluronic acid, Vaseline, menthol, thymol, penicillin, hydrogen peroxide, sea cucumber extracts, etc.

In order to effectively isolate out the desired metabolites from the plants of the Arecaceae and seaweeds family, the preferred extraction solvent is selected from the group consisting of water, alcohol, acetone, chloroform and any combination thereof.

Further embodiment of the present invention is regarding a method for promoting wound healing and healthcare in a subject comprising the step of administrating orally the subject an effective amount of an extract of vegetative parts derived from plant of Arecaceae and seaweeds family in separation or together using an organic extraction solvent.

The present invention also discloses a method for preparing an herbal extract comprising the steps of soaking dried vegetative parts derive from a plant of Arecaceae and seaweeds family into a solvent; removing the soaked dried vegetative parts from the solvent; and concentrating the solvent to acquire the extract.

Further embodiment to improve the extraction efficiency involve a pre-treatment step of fragmentizing the dried vegetative parts into small fragment or pulverizing to paste or powdery form before soaking into the extraction solvent. Still another embodiment of the present invention, the vegetative parts of the plant of Arecaceae and seaweeds family is preferably derived from any one or combination of the species, but not limited to, Euchema cottonii, Appaphycυs alvareziii, Caulerpa lentillifera and Sargassυm polycystum, Elaeis guineensis, Elaeis oleifera, Phoenix dactylifera and Cocos nucifera.

The present invention relates to provide seaweeds based seasoning, which is suitable to be incorporated into daily diet for improving cardiovascular health upon use of the seasonings. Further object of the present invention is to disclose a method to improve cardiovascular health or prevent cardiovascular related diseases (one or more effects such as anti- hypercholesterol, anti-atherogenic, anti-hyperglycaemic, anti-obesity, anti-hyperlipidemic, anti-oxidative, vasorelaxant and the like) by administrating the aforesaid seaweeds based seasoning or its derivatised seasonings orally to a subject.

At least one of the preceding objects is met, in whole or in part, by the present invention, in which one of the embodiment of the present invention is a comestible product for promoting cardiovascular health of an individual comprises seasoning derived from seaweeds with or without the addition of polyphenol rich herbs such as, but not limited to, spices, ginger, green tea, mint, pepper, capsicum spp, onions, garlic, citrus spp. and the like.

Further embodiment of the comestible product will include the seasoning prepared in a concentrated form thus can be conveniently incorporated into food. The present invention is regarding a method for promoting cardiovascular health in a subject comprising the step of administrating orally the subject an effective amount of seasoning of seaweeds after removal of excess salt through water or polar solvent extraction.

Still another embodiment of the present invention, the seaweeds of the plant of Seaweeds family is preferably derived from any one or combination of the species, but not limited to, Eucheumia spp., Caulerpa spp., Sargassum spp. and other seaweeds. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the percentages of apoptotic breast cancer MCF-7 cells in the presence of Sargassum polycystum alcoholic extract SPME (0-200 μg/ml) compared to normal cells.

Fig. 2 shows the Reduction of viability of five human cancer cells after 48 h incubation, measured by MTT test (mean ±S.D. ; n=3) by each seaweeds alcoholic dry extracts (concentration 12.5 to 200 μg/ml)).

Fig. 3 are morphological changes of breast cancer MCF-7 cells after treatment by S. polycystum extract followed by Hoechst 33342 staining, (a) Fluorescence microscope photographs of control cells treated with 0.1% DMSO, (b) cells treated with 25 μg/ml S. polycystum extract after 24 h incubation.

Fig. 4 (a)-(b) shows the viability of MDA 435 and MCF 7 breast cancer cells after incubating in various concentrations of extracts of Piper spp., Turmeric (Kunyit), and Terung Susu Kambing (TSK) for 48 hrs and 72 hours respectively (MTT Assay).

Fig. 5 (a)-(c) shows the viability (MTT assay) and Cell Death (LDH assay) of DU 145 Human Prostate cancer cells after incubating in various concentrations of extracts of Piper spp., for 24-72 hrs respectively (MTT Assay).

Fig.6 shows the wound healing of the tested normal Sprague Drawley rats at different time interval of the experiments; and

Fig.7 shows the wound healing effect of the product given to the tested diabetic animals.

Fig. 8 shows the effects on body weight indicating safety and non-toxicity of the comestible product given to the tested animals DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those inherent therein. The embodiment describes herein is not intended as limitations on the scope of the invention. As used herein, the "present invention" refers to any one of the embodiments of the invention described herein, and any equivalents. Furthermore, reference to various feature(s) of the "present invention" throughout this document does not mean that all claimed embodiments or methods must include the referenced feature(s).

The "comestible product" of the present invention refer to a composition of ingredients that may be provided in liquid or dry forms. For example, the liquid form may be an aqueous mixture that may be concentrated or ready-to-ingest. The dry form may include loose powder, or compressed forms that may be added to and partially or completely dissolved and/or held in suspension.

The term "promoting cardiovascular health" used herein throughout the specification refers to the one or more effects such as anti-atherogenic, anti-hypercholestrol, anti- hyperglycaemic, anti-obesity, anti-hyperlipidemic, anti-hypertensive, anti-oxidative, vasorelaxant and the like. The aforementioned effects can be initiated variedly upon different ingested amount or dose, form of supply of the seasoning, coupled with other components, age and sex of the administrated subject and so on. With respect to the preferred embodiment of the present invention, a method for preparing seasoning preferably herbal seasoning from plants of Seaweeds family is disclosed. The method comprises the steps of pre-treating seaweeds. The disclosed method is found to be effective in isolating the favored phenolic compounds/saccarides, minerals, peptides, omega 3 and omega 6 fatty acids, or active metabolites which provides a therapeutic effect or health promoting effect towards the cardiovascular system upon ingestion orally of a subject. According to another preferred embodiment of the disclosed method, the seaweeds are pre-treated with desalting. The pre-treatment process may involve a washing step to clean any dirt or physical pollutants captured at the surface of the seaweeds. Other pretreatment step is presoaking, followed by preferably reducing the moisture content of the seaweeds by any known means or approaches in the art to improve it. In the preferred embodiment, the seaweeds are subjected to drying in an oven at 40-120⁰C. The drying temperature shall not be set too high as such practice may degrade the active metabolites and phenolic compounds/saccarides contained within the seaweeds. The preferred temperature shall range from 40⁰C to 60⁰C. Other pretreatment steps that can be employed are preparing the dried seaweeds into small fragments, or pulverization to paste or powdery form. Possibly, the pre-treated seaweeds may be subjected to a plurality occasion of seasoning using different types of polyphenol rich herbs such as, but not limited to, spices, ginger, green tea, mint, pepper, capsicum spp, onions, garlic, citrus spp. and the like to obtain the optimal product and efficacy.

It is important to note that the seaweeds used in the disclosed method in the preferred embodiment derives from the plant species of, but not limited to, Eucheumia spp., Caulerpa spp., Sargassum spp. and other seaweeds. The seasonings obtained from the abovementioned plant species are found pleasant in both taste and fragrance thus render them suitable to be incorporated into edible seasonings especially comestibles. Attention is now drawn to another embodiment of the present invention which involves a comestible product for promoting cardiovascular health of an individual comprises seasonings derive from seaweeds with or without combination with polyphenol rich herbs or extracts such as, but not limited to, spices, ginger, green tea, mint, pepper, capsicum spp, onions, garlic, citrus spp. and the like. The comestibles mentioned herein can be any common daily consumed processed food such as bread, noodles, confections, chocolates, beverages, and the like. One skilled in the art shall appreciate the fact that the aforesaid seasoning can be incorporated into the processed comestibles during the course of food processing. Therefore, any modification thereon shall not depart from the scope of the present invention. As setting forth in the above description, the seaweeds used for preparing the seasoning are derived from the plants of Seaweeds family. More preferably, the plant is any one or combination of the plant species of , but not limited to, Eucheυmia spp., Caulerpa spp., Sargassum spp. and other seaweeds. The inventors of the present invention discovered that the seasoning derives from the aforementioned species possesses both pleasant taste and fragrance that confers the derived seasoning to be comfortably incorporated with the comestibles seasoning with minimal additional refining process.

According to the preferred embodiment, the seasoning to be incorporated into the comestibles can be acquired from any known method not limited only to the foregoing disclosed method. Following to another embodiment, the seasoning is prepared in a concentrated form, preferably paste or powdery form which enables the seasoning to be prepared in various formulations of the comestibles. In line with the preferred embodiment, the phenolic compounds, minerals, saccarides, minerals, small peptides, omega 3 and omega 6 fatty acids, with the cardiovascular health-promoting properties are those metabolites in the seaweeds. Therefore, the seasoning from the seaweeds may be combined with polyphenol rich herbs such as, but not limited to, spices, ginger, green tea, mint, pepper, capsicum spp, onions, garlic, citrus spp. and the like. In view of the prominent property of promoting cardiovascular health of the seasoning, further embodiment of the present invention includes a method for promoting cardiovascular health in a subject comprising the step of administrating orally the subject an effective amount of seasoning of seaweeds with or without combination to polyphenol rich herbs such as, but not limited to, spices, ginger, green tea, mint, pepper, capsicum spp, onions, garlic, citrus spp. and the like. From the experiments conducted, it is clear that the seasoning employed in this embodiment shows anti-hypercholesterol, anti-oxidative, antiatherogenic, anti-hyperglycaemic, anti-hyperlipidemic, anti-obesity, vasorelaxant and similar effects. Moreover, the plant of Seaweeds family is any one or combination of , but not limited to, Eucheumia spp., Caulerpa spp., Sargassum spp. and other seaweeds. Upon intake of the seasoning, the ingested composition not only acts like prophylaxis to prevent development or advancement of the cardiovascular diseases, but also function as therapeutic composition to relieve or even help heal the diseased state.

The following examples are intended to further illustrate the invention, without any intent for the invention to be limited to the specific embodiments described therein.

EXAMPLE 1

Samples of seaweeds were obtained locally, whereby the red seaweeds {Eucheuma cottonii) was harvested from Universiti Malaysia Sabah cultivation farm from Kudat waters (north coast of Borneo), green seaweeds (Caulerpa lentillifera) was collected from Semporna waters (North-East coast of Borneo) and the brown seaweeds (Sargasυm polycystum) was collected from Kota Kinabalu waters (Noth-west coast of Borneo). Fresh seaweeds samples were thoroughly washed with distilled water and their holdfasts and epiphytes were removed, air dried at 15⁰C for 4 days, and then pulverized using a Cyclotech mill to pass through a 0.5 mm screen.

Optimum hydrolysis conditions were conducted by filling up reaction vessel with a known amount of deionised water at the optimum operating temperature and seaweeds (e.g. Eucheuma spp., Caulerpa spp. and Sargassum spp.) substrate concentration for the protease (e.g. Flavourzyme, mixtures of exo and endopeptidases) used. The pH of the slurry was adjusted using alkali or acid, to the optimum pH of the protease. A known amount of protease was then added to the reactor. As the hydrolysis proceeds, protons released were neutralised by addition of 0.1 M alkali into the reactor. The mixture was constantly stirred when the alkali was added to ensure uniform distribution. After 5 hours, the samples were collected, boiled for 10 minutes, adjusted to pH 3.5-4.5, using citric acid or alkali, and vacuum filtered through a layer of activated carbon and CaCO₃.

The hydrolysates may be added with polyphenols rich extracts which have cardiovascular protective properties, thickeners, preservatives, caramel, onion powder, ginger powder, garlic powder, cinnamon, spices and black pepper to taste and boiled at 100⁰C to concentrate it to form vegemite alternative which have cardiovascular protective properties.

EXAMPLE 2

Anti-obesity, anti-atherogenic, antioxidative and organ protective properties evaluation were conducted on the different products from the different source of seaweeds. The anti- obesity, anti-atherogenic and antioxidative seasoning/flavour enhancers from three species of edible seaweed seasoning/flavour enhancers (Eucheuma spp., Caulerpa spp. and Sargassum polystum) are described. Animals on high-cholesterol food supplemented with seaweed seasoning/flavour enhancers for 16 weeks, showed lower body weight gain, lower total plasma cholesterol, lower plasma triglycerides (TG) and lower plasma malondialdehyde (MDA), lower tissues GSH-Px, SOD and CAT activities which were elevated by high cholesterol diets. High cholesterol diet animals supplemented with seaweed seasoning/flavour enhancers particularly Eucheuma spp. and Caulerpa spp. showed a trend of increased plasma high density lipoprotein cholesterol (HDL-C), and lower Al (atherogenic index) compared to animals on high cholesterol diet alone. These edible seaweed seasoning/flavour enhancers showed significant anti-obesity, antioxidative, lower Atherogenic index, hypocholesterol effect and organ protective effects for use as functional ingredient seasoning/flavour enhancers for reducing degenerative diseases risks.

The seaweed seasoning/flavour enhancers supplemented animals on high-cholesterol food (group CE, CC and CS) showed significant lower body weight gain as compared to animals fed on high-cholesterol food (C) alone (Table 1). The seaweed seasoning/flavour enhancers supplemented animals on normal diet showed no significant differences in body weight gain as compared to the normal diet animals suggesting that the seaweed seasoning/flavour enhancers do not significantly increase or decrease body weight of normal healthy animals. The animals fed on high-cholesterol food (C), had their plasma

TC increased by 57% in 4 weeks and 107% in 16 weeks which were significantly higher than normal control animals (N). The animals fed on high-cholesterol food supplemented with seaweed seasoning/flavour enhancers showed lower plasma TC value (lower by - 11.8% in CE, -18.5% in CC and -11.4% in CS) as compared to animals fed on high- cholesterol food alone (C). The plasma TC value in normal animals supplemented with seaweed seasoning/flavour enhancers (NE, NC and NS) showed no significant difference with animals fed on control diet (N). There was an increase of plasma TG level of animals fed with high-cholesterol food alone (C) from the beginning till the end of the experiment (16 weeks). After 16 weeks, animals on high cholesterol diet supplemented with Eucheuma spp. and Caulerpa spp. Seasoning/flavour enhancers showed a significant lower plasma TG level of -36.1% and -33.7% respectively as compared to animals without supplementation (C).

Table 2 shows, plasma HDL-C of high-cholesterol fed animals supplemented with Eucheuma spp. (CE) and Caulerpa spp. (CC). Seasoning/flavour enhancers were significantly higher than high-cholesterol food alone (C). The plasma HDL-C in high- cholesterol fed animals supplemented with Eucheuma spp. and Caulerpa spp. Seasoning/flavour enhancers showed a significant increase of 55% and 48.8% respectively compared to high-cholesterol food (C) alone. The high-cholesterol group (C) showed significantly higher LDL-C level as compared to the control group (N). The plasma LDL-C in high-cholesterol fed animals supplemented with Eucheuma spp., Caulerpa spp. and Sargassum spp. Seasoning/flavour enhancers showed a significant decrease of - 49.3%, -34.7% and -22% respectively relative to animals fed on high-cholesterol food (C) alone.

The data demonstrated that animals fed on high cholesterol diet supplemented with seaweed seasoning/flavour enhancers (particularly Eucheuma spp. and Caulerpa spp.) reduced the levels of plasma TC and LDL-C. In addition, the results showed that animals fed on high cholesterol diet supplemented with seaweed seasoning/flavour enhancers (particularly Eucheuma spp. and Caulerpa spp.) increased the concentration of HDL-C when compared with high cholesterol diet (C) alone. High-cholesterol food supplemented with Eucheuma spp. and Caulerpa spp. showed a trend of increased plasma HDL-C, reduced TG, reduced LDL-C levels and lower atherogenic index (Al) as compared to high- cholesterol group without seaweed seasoning/flavour enhancers supplementation. Atherogenic index (Table 2) expressed by the ratio of LDL-C/HDL-C, significantly decreased in high-cholesterol food supplemented with seaweed seasoning/flavour enhancers as compared to high-cholesterol food alone. These effects is beneficial as the observed decreased in these indices indicate that seaweed seasoning/flavour enhancers play an important protective role in reducing risk against atherosclerosis and CVD.

Table 2 shows the animals fed on high-cholesterol food (C) showed higher MDA level compared to control animals (N), especially the MDA elevation at week 12. Animals fed on high-cholesterol food supplemented with Eucheuma spp. and Cauleφa spp. Seasoning/flavour enhancers showed significantly lower MDA level (12.8% and 10.9% lower respectively) as compared to animals without supplementation (C) after 12 weeks, indicating that these seaweed seasoning/flavour enhancers retarded lipid peroxidation. High lipid peroxidation as indicated by the terminal product MDA, is associated with high oxidative injury by free radicals and oxidative status.

At weeks 8 and 12, the erythrocytes SOD activities of high-cholesterol food animals (C) was significantly increased by 75.2% and 93.1% respectively compared to control animals (N) (Table 3). The high-cholesterol food animals with Eucheuma spp. and Caulerpa spp. Seasoning/flavour enhancers, (CE and CC) also showed increased erythrocytes SOD activities, but was insignificantly different from the control animals (N). SOD is the first line of antioxidative enzyme defense against oxygen stress converting reactive oxygen to hydrogen peroxide.

Table 3 shows at week 12, the GSH-Px activity in high-cholesterol animals erythrocytes (C) increased significantly by 61.5% compared to control animals (N). The high- cholesterol food with seaweed seasoning/flavour enhancers, CE, CC and CS groups had increased erythrocytes GSH-Px activities (30.2%, 15.3% and 7.2% respectively), but was insignificantly different (p>0.05) from the control animals (N). The erythrocytes CAT activity in high-cholesterol animals (C) were insignificantly different from control animals (N) at 4, 8, 12 and 16 weeks. The CAT activities in all groups remain unchanged. At week 16, all 3 antioxidant enzymes (SOD, GSH-Px and CAT) activities remained unaffected in animals erythrocytes suggesting no accumulation of superoxide anion radical, and may explain the unaltered MDA concentration in all the animals plasma. The antioxidant enzyme activities of erythrocytes in normal animals with seaweed seasoning/flavour enhancers (NE, NC and NS) were also insignificantly different to the control (N).

Table 1: Body weight, Plasma Total Cholesterol (TC), and Plasma triglycerides (TG) of animals fed with seaweed seasoning/flavour enhancers on normal and high cholesterol diets (mean ± SEM).

Group Body weight (g) WeekO Week 16 Body weight gain(g/animal/week)

N 276±11^a 397±23^{a ■} 7.59±1.42^a

NE 275 ±9^a 423±14^a 9.48±1.31^a

NC 289 ±8^a 407 ± 11 ^a 7.40 ± 0.51 ^a

NS 270 ±7^a 381 ± 11^a 7.14±0.74^a

C 269 ±6^a 499±22^b 14.26±1.40^b

CE 273 ±7^a 436±19^a 10.05±1.06^a

CC 284 ± 8^a 423 ± 25^a 8.31 ± 0.99^a

CS 289±9^a 421 ±14^a 8.18 ± 1.16^a

Plasma TC in animals TC (mmol/l)

N 1.39±0.07^a 1.48±0.03^a

NE 1.38 ± 0.11^a 1.46 ± 0.12^a

NC 1.47 ± 0.07^a 1.57±0.09^a

NS 1.38 ± 0.12^a 1.38 ± 0.10^a

C 1.31 ±0.06^a 2.71 ± 0.07^c

CE 1.30 ± 0.04^a 2.39 ± 0.07"°

CC 1.36±0.03^a 2.21 ±0.16^b

CS 1.45 ± 0.10^a 2.40 ± 0.15"°

Plasma TG (mmol/l)

N 0.76 ± 0.03^a 0.86 ± 0.10^a

NE 0.86 ± 0.12^a 0.89 ± 0.09^a

NC 0.85 ± 0.08^a 0.92 ± 0.04^a

NS 0.87 ± 0.08^a 0.94 ± 0.13^a

C 0.72 ± 0.05^a 1.69 ± 0.15^b

CE 0.88 ± 0.21 ^a 1.08 ± 0.25^a

CC 0.80 ± 0.18^a 1.12±0.13^a

CS 0.88 ± 0.04^a 1.56 ± 0.17^b

Values are means ± SEM of ten animals per group. Values in the same column with different superscript letters are significantly different

(P<0.05).

Table 2: Plasma HDL-C, LDL-C, Atherogenic index (Al) and Lipid peroxidation levels in animals fed on normal and high cholesterol diets supplemented with seaweed seasoning/flavour enhancers (mean ± SEM).

Plasma HDL-C (mmol/l)

Group Week O Week 16

N 0.84 ± 0.03^a 1.081 0.1O³"

NE 0.87 ± 0.03^a 1.041 0.09³^

NC 0.87 ± 0.04^a 1.22 ± 0.04^a

NS 0.90 ± 0.05^a 1.101 0.08^

C 0.81 ± 0.05^a 0.80 ± 0.04°

CE 0.81 ± 0.05^a 1.24 ± 0.08^a

CC 0.88 ± 0.04^a 1.19 ± 0.07^a

CS 0.85 ± 0.08^a 0.93 ± 0.10"°

LDL-C (mmol/l ) AI(LDL-C/HDL-C)

N 0.32 ± 0.05^a 0.24 ± 0.02^a 0.23 ± 0.03^a

NE 0.34 ± 0.03^a 0.27 ± 0.05^a 0.26 ± 0.03^a

NC 0.39 ± 0.07^a 0.32 ± 0.03^a 0.26 ± 0.02^a

NS 0.35 ± 0.04^a 0.29 ± 0.03^a 0.26 ± 0.01 ^a

C 0.29 ± 0.05^a 1.50 ± 0.09^b 1.88 ± 0.14^b

CE 0.34 ± 0.04^a 0.76 ± 0.08^c 0.55 ± 0.05^c

CC 0.32 ± 0.09^a 0.98 ± 0.12°" 0.74 ± 0.08°

CS 0.40 ± 0.04^a 1.17 ± 0.12^d 1.08 ± 0.08^d

Lipid peroxidation MDA levels in animals plasma (nmol/ml)

N 7.24 ± 0.41 ^a 10.69 ± 0.73^a

NE 7.59 ± 0.23^a 10.76 ± 0.27^a

NC 7.45 ± 0.35^a 11.19 ± 0.32^a

NS 7.78 ± 0.42^a 10.87 ± 0.26^a

C 7.55 ± 0.51 ^a 11.50 ± 0.25^a

CE 7.70 ± 0.40^a 11.03 ± 0.29^a

CC 7.15 ± 0.36^a 11.29 ± 0.37^a

CS 8.23 ± 0.33^a 11.74 ± 0.24^a

Values are means ± SEM of ten animals per group.

Values in the same column with different superscript letters are significantly different (P<0.05). Table 3: Antioxidant enzymes (SOD, GSH-Px, and CAT) activities in erythrocytes of animals fed on normal and high cholesterol diets supplemented with seaweed seasoning/flavour enhancers (mean ± SEM).

SOD l lU/min)

Group > Week 0 Week 16

N 2.86 ± 0.44^a 5.22 1 1.Ie³"

NE 2.72 ± 0.42^a 6.60 ± 1.14*

NC 2.25 ± 0.30^a 3.16 ± 0.69^a

NS 2.43 ± 0.23^a 7.20 ± 1.33^b

C 2.62 ± 0.32^a 7.02 ± 0.69^b

CE 2.28 ± 0.24^a 6.36 ± 1.02^

CC 2.59 ± 0.33^a 7.06 ± 1.64"

CS 2.74 ± 0.40^a 8.09 ±1.48^b

GSH-Px (U/ml)

N 5.85 ± 0.72^a 11.25 ±

2.05^ab

NE 4.48 ± 0.47^a 12.63 ±

2.36^ab

NC 6.05 ± 0.62^a 9.37 ± 1.79^

NS 6.54 ± 0.80^a 8.52 ± 1.70^b

C 5.29 ± 0.51 ^a 15.08 ± 0.89^a

CE 4.72 ± 0.86^a 13.88 ±

O.δδ³"

CC 5.60 ± 0.39^a 12.40 ±

1.54*

CS 5.72 ± 0.65^a 9.45 ± 2.93^ab

CAT (U/sec)

N 0.28 ± 0.01^a 0.26 1 0.01^

NE 0.25 ± 0.02^a 0.25 ± 0.01 ^

NC 0.30 ± 0.02^a 0.27 ± 0.02*"

NS 0.27 ± 0.02^a 0.26 ± 0.03^{a b}

C 0.28 ± 0.01 ^a 0.31 ± 0.01^a

CE 0.25 ± 0.01 ^a 0.23 ± 0.01 ^b

CC 0.26 ± 0.02^a 0.30 ± 0.02^

CS 0.25 ± 0.02^a 0.26 ± 0.03^ab

Values are means ± SEM of ten animals per group.Values in the same column with different superscript letters are significantly different (P<0.05). EXAMPLE 3

The nutrient composition, vitamin C, α-tocopherol, dietary fibres, minerals, fatty acid and amino acid profiles of three tropical edible seaweed seasoning/flavour enhancers, Eυcheuma spp. (red), Caulerpa spp. (green) and Sargassum spp. (brown) are shown in Table 4. The seaweed seasoning/flavour enhancers were high in mineral (37.15-46.19%) and dietary fibres (25.05-39.67%) and low in lipids content (0.29-1.11%) on dry weight (DW) basis. These seaweed seasoning/flavour enhancers contained 12.0-15.53% macrominerals (Na, K, Ca and Mg) and 7.53-71.53 mg/100g trace minerals (Fe₁ Zn, Cu and Se). The crude protein content of Eucheuma spp. (9.76% DW) and Caulerpa spp. (10.41% DW) were higher than that of Sargassum spp. (5.4% DW), and the seaweed chemical scores are between 20-67% which is common for seaweeds. Sargassum spp. had the highest chemical score of 67.4% that was slightly less than for beef (69%), but was better than casein (58%). The PUFA content in the fat of Eucheuma spp. were 51.55%; Caulerpa spp., 16.76% and Sargassum spp., 20.34%. Eicosapentanoic acid (EPA), accounted for 24.98% of all fatty acids in Eυcheuma spp.. The seaweed seasoning/flavour enhancers have significant vitamin C (~35mg/100g) and α-tocopherol (5-12mg/100g) contents.

The highest mineral content was in Eucheuma spp. (46.19%), followed by Sargassum spp. (42.40%) and Caulerpa spp. (37.15%). These seaweed seasoning/flavour enhancers contained high amounts of the macrominerals (12.01 - 15.53 g/100g) and trace elements (7.53 to 71.53 mg/100g) needed in human nutrition. The Na/K ratios were very low for Eucheuma spp. and Sargassum spp. (0.14 to 0.16). The total dietary fibre in Sargassum spp. was the highest (39.67%) followed by C.lentillifera (32.99) and E.cottonii (25.05%). However, soluble fibre in E.cottonii (18.25%) and Caulerpa spp. (17.21%) were significantly higher than Sargassum spp. (5.57%). The vitamin C content in all three seaweed seasoning/flavour enhancers studied showed no significant difference among them. Sargassum spp. (11.29 mg/100g) contain higher α-tocopherol content than C.lentillifera (8.41 mg/10Og) and E.cottonii (5.85 mg/10Og). Fatty acids profile

The total fat content in E.cottonii, Caulerpa spp. and Sargassum spp. were 1.10%, 1.11% and 0.29% DW respectively. Thirty-three fatty acids were present and the fatty acid composition of the different seaweed seasoning/flavour enhancers may vary considerably (Table 5).

The seaweed seasoning contained the essential fatty acids linoleic acid (C18:2ω6) and linolenic acid (C18:3ω3), and the eicosanoid precursors arachidonic acid (C20:4ω6) (except Caulerpa spp.) and EPA (C20:5ω3). The most abundant fatty acids in Eucheuma spp. was the omega-3 fatty acid, EPA which accounted for 24.98% of all fatty acid. Palmitic (C16:0) and oleic acids (C18:1ω9) were the most abundant fatty acid in Caulerpa spp. and Sargassum spp.. Sargassum spp. contained a small quantity of docosahexaenoic acid. Eucheuma spp. has the highest amount of omega-3 fatty acids (45.73%) as compared to Sargassum spp. (9.63%) and Caulerpa spp. (7.55%).

Amino acid profile

The amino acid analysis on Eucheuma spp., Caulerpa spp. and Sargassum spp. (Table 6) Seasoning/flavour enhancers shows sixteen amino acids. For assessing dietary protein quality, the chemical score is shown. Sargassum spp. which had the lowest seaweed content (5.4% DW) had the highest chemical score of 67.4% which were slightly less than for beef (69%), but were better than casein (58%), oats (57%), rice (56%), soybeans

(47%), wheat (43%) or peanuts (55%) (Brody, 1999). The most limiting amino acid was lysine followed by methionine.

Table 4: Nutrient composition of seaweed seasoning/flavour enhancers Eucheuma spp., Caulerpa spp. and Sargassum spp. (mean ± SD, % dry weight of sample)

Nutrient Eucheuma spp. Caulerpa spp. Sargassum spp.

Seaweed (%) 9.76±1.33^a 10.41 ±0.26^a 5.40 ± 0.07^b

Lipid (%) 1.10±0.05^a 1.11 ±0.05^a 0.29 ± 0.01 ^b

Mineral (%) 46.19 ±0.42^a 37.15 ±0.64^c 42.40 ± 0.41 ^b

Crude fibre (%) 5.91 ±1.21^b 1.91 ±0^c 8.47 ±1.21 ^a

Carbohydrate (%) 26.49 ± 3.01 ^c 38.66 ± 0.96^a 33.49 ±1.70^b

Moisture content (%) 10.55 ±1.60^a 10.76±0.80^a 9.95 ± 0.55 ^a

Soluble fibre (%) 18.25±0.93^a 17.21 ±0.87^a 5.57 ± 0.28^b

Insoluble fibre (%) 6.80 ± 0.06° 15.78 ±1.20^b 34.10 ±0.28^a

Total dietary fibre (%) 25.05 ± 0.99 32.99 ± 2.07 39.67 ± 0.56

Vitamin C (mg/100g 35.3 ± 0.01 ^a 34.7 ± 0.02^a 34.5 ± 0.01 ^a

WW) α-tocopherol 5.85 ± 0.27° 8.41 ±0.12^b 11.29±0.61^a

(mg/100g DW)

Na(mg/100gDW) 1771.84 ± 0.01 ^b 8917.46 ±0.00^a 1362.13 ±0.00°

K(mg/100g DW) 13,155.19±1.14^a 1142.6810.00° 8371.23 ± 0.01 ^b

Ca(mg/100g DW) 329.69 ± 0.33^b 1874.74 ±0.20^a 1792.06 ± 0.51 ^a

Mg(mg/100g DW) 271.33 ± 0.20° 1028.62 ±0.58^a 487.81 ± 0.24^b

Fe(mg/100gDW) 2.61 ± 0.00° 21.37±0.00^b 68.21 ± 0.03^a

Zn(mg/100gDW) 4.30 ± 0.02^a 3.51 ± 0.00^b 2.15 ±0.00°

Cu(mg/100gDW) 0.03 ± 0.00^b 0.11 ±0.00^a 0.03 ± 0.00^b

Se(mg/100g DW) 0.59 ± 0.00° 1.07±0.00^b 1.14±0.03^a

Na/K ratio 0.14 7.8 0.16

Total cations (%) 15.535 12.990 12.085

Values in the same row with different superscripts letters are significantly different (P<0.05).

Table 5: Fatty Acid Content (% of total fatty acid content) of Eucheuma spp., C. lentillefera and Sargassum spp.

Fatty Acid Seaweed seasoning/flavour enhancers (% of total fatty acid content)

Name Carbon Eucheuma Caulerpa Sargassum spp.

No spp. spp.

Caprylic C8:0 - - 0.36

Capric C10:0 0.17 0.16 0.36

Undecanoic C11 :0 3.67 0.85 -

Laurie C12:0 0.88 0.13 3.62

Tridecanoic C13:0 - 0.12 -

Myristic C14:0 1.65 1.65 3.26

Myristoleic C14:1 - 0.33 0.05

Pentadecanoic C15:0 - 0.11 0.18

Palmitic C16:0 15.10 33.78 37.97

Palmitoleic C16:1 11.10 1.31 3.81

Heptadecanoic C17:0 - 0.16 0.28

Cis-10-Heptadecanoic C17:1 8.16 1.55 0.06

Stearic C18:0 2.11 7.83 4.20

Elaidic C18:1ω9 0.11 0.22 0.04

Oleic C18:1ω9 3.44 32.49 24.21

Linolelaidic C18:2ω6 1.44 0.09 0.05

Linoleic C18:2ω6 1.15 7.64 8.44 γ-Linolenic C18:3ω6 0.80 0.31 0.27 α-Linolenic C18:3ω3 3.88 5.54 1.41

Arachidic C20:0 0.21 0.47 0.40

Cis-11 -Eicosenoic C20:1ω9 0.29 0.17 0.12

Cis-11 ,14-Eicosadienoic C20:2 - 0.07 0.22

Cis-11 ,14,17- C20:3ω3 16.87 1.15 6.38

Eicosatrienoic

Arachidonic C20:4ω6 1.29 - 0.63

Cis-5,8,11 ,14,17- C20:5ω3 24.98 0.86 1.71

Eicosapentaenoic

Henocasanoic C21 :0 - - 0.09

Behenic C22:0 - 0.31 0.15

Erucic C22:1ω9 0.20 0.27 0.08

Cis13, 16-Docisadienoic C22:2 0.86 0.95 1.10

Cis-4,7,10,13,16,19- C22:6ω3 - - 0.13

Docosahexaenoic

Tricosanoic C23:0 0.92 0.14 0.28

Lignoceric C24:0 0.47 0.70 0.17

Nervonic C24:1ω9 0.27 0.66 -

Saturated FAs 25.17 ± 0.38° 46.41 ± 0.56" 51.30 ± 0.51 ^a

MUFAs 23.28 ± 0.47° 36.83 ± 0.55^a 28.36 ± 0.48^b

PUFAs 51.55 ± 0.57^a 16.76 ± 0.27° 20.34 ± 0.43^b

PUFAs ω6 4.68 ± 0.05 8.04 ± 0.12 9.40 ± 0.17 PUFAs ω3 45.72 ± 0.59 7.55 ± 0.09 9.63 ±0.15 Ratio ω6/ω3 0.10 1.07 0.98

FA=fatty acid; MUFAs=monounsaturated fatty acid;PUFA=polyunsaturated fatty acid Values in the same row with different superscripts letters are significantly different (P<0.05).

Table 6: Amino Acid Content (mg/g dry weight of sample) of Eucheuma spp., C. lentillefera and Sargassum spp.

Amino Acid # (mg/g Seaweed seasoning/flavour enhancers (mg/g dry reference sample) protein)

Eucheuma spp. Caulerpa spp. Sargassum spp.

Aspartic Acid (Asp) 2.65 ±0.15 8.33 ± 0.11 4.47 ± 0.87

Glutamic Acid (GIu) 5.17 ±0.13 13.47 ±0.23 8.08 ±1.08

Serine (Ser) 1.92 ± 0.04 5.49 ± 0.20 2.58 ±0.16

Glycine (GIy) 2.27 ± 0.32 5.14 ±0.03 3.19 ±0.35

Histidine (His) 0.25 ±0.10 1.44 ±0.13 0.26 ±0.11

Arginine (Arg) 2.60 ±0.14 5.71 ± 0.22 2.88 ±0.17

Threonine (Tnr) 34 @2.09 ± 0.01 5.84 ± 0.22 2.60 ±0.16

Alanine (Ala) 3.14 ±0.11 6.88 ±0.19 4.25 ±0.15

Proline (Pro) 2.02 ± 0.09 4.29 ± 0.11 2.55 ±0.14

Thyrosine (Tyr) 1.01 ±0.12 3.33 ± 0.08 1.26 ± 0.06

Valine (VaI) 35 @2.61 ± 0.07 6.18 ±0.02 3.13±0.14

Methionine (Met) 25 @0.83±0.17 @1.58 ±0.08 @1.25±0.04 lsoleucine (lie) 28 @2.41 ± 0.04 5.06 ±0.12 2.94 ±0.16

Leucine (Leu) 66 @3.37 ± 0.06 7.79 ±0.19 4.67 ± 0.25

Phenylalanine (Phe) 63 19.07 ±2.48 19.95 ±1.41 30.42 ± 4.43

Lysine (Lys) 58 @1.45±0.48 @1.22 ±0.05 @2.11 ±0.77

Chemical score (%) 25.6 20.2 67.4

Most limiting amino acid lysine lysine lysine

Total Amount 52.86 ± 3.37° 101.63 ± 76.38 ±2.31"

2.92^a

Essential Amino Acid 32.07 ± 3.13^b 48.98 ± 2.19^a 47.13 ± 4.12^a

(EAA)

EAA (%) 60.59 ±1.36^a 48.19 ±0.77^b 61.66 ± 4.73^a

Seaweed (%) 9.76±1.33^a 10.41 ±0.26^a 5.40 ± 0.07^b Values in the same row with different superscripts letters are significantly different (P<0.05). Chemical score (%) = mα limiting amino acid per α of test seaweed x 100 mg limiting amino acid per g of reference seaweed

(# based on FAO/WHO/UNU amino acid requirement pattern) @ limiting amino acids

EXAMPLE 5

The organ protective properties of the seaweed seasoning/flavour enhancers on liver, heart, brain and kidneys are shown in the Tables below.

Table 7: Effects of seaweed seasoning/flavour enhancers supplementation on MDA (malondialdehyde - a lipid peroxidation product formed that can damage DNA, cell membrane, proteins) concentration, in various organs of normal and hypercholesterol animals after 16 weeks.

Group MDA (nmol/ml) liver N 8.62 ± 0.25^b

NE 8.49 ± 0.35^B

NC 8.21 ± 0.21^B

NS 8.13 ± 0.12^B

C 14.22 ± 3.01 ^A

CE 9.22 ± 0.42^B

CC 7.99 ± 0.32^B

CS 8.52 ± 0.31 ^B heart N 9.13 ± 0.30^BO

NE 8.67 ± 0.26^BC

NC 9.76 ± 0.60^BA

NS 9.04 ± 0.30^BC

C 10.84 ± 0.54^A

CE 8.80 ± 0.63^BC

CC 7.98 ± 0.49^c

CS 8.46 ± 0.80^BC kidney N 8.99 ± 0.49^B

NE 9.40 ± 0.60^B

NC 9.21 ± 0.46^B

NS 8.28 ± 0.45^B

C 11.36 ± 0.83^A

CE 7.87 ± 0.24^B CC 7.87 ± 0.52^b

CS 7.80 ± 0.13^B brain N 7.96 ± 0.51 ^AB

NE 6.97 ± 0.10^B

NC 8.38 ± 0.37^AB

NS 7.76 ± 0.46^AB

C 9.13 ± 1.00^A

CE 8.02 ± 0.63^AB

CC 8.00 ± 0.29^AB

CS 8.07 ± 0.35^AB spleen N 8.70 ± 0.23^A

NE 7.50 ± 0.05^B

NC 7.96 ± 0.10^B

NS 8.42 ± 0.35^AB

C 8.45 ± 0.17^AB

CE 8.17 ± 0.25^AB

CC 7.90 + 0.17^B

CS 8.38 ± 0.47^AB

Lungs N 7.72 ± 0.20^a

NE 7.97 ± 0.45^a

NC 7.34 ± 0.27^a

NS 7.25 ± 0.09^a

C 7.34 ± 0.15^a

CE 7.29 ± 0.13^a

CC 7.29 ± 0.16^a

CS 7.30 ± 0.28^a

Values are means ± SEM of ten animals per group. Different superscript letters are significantly different (P<0.05) within the same columns for each organ.

The high-cholesterol food consumption for 16 weeks significantly increased MDA levels in the tissues of most organs namely, liver, heart, kidneys, brain and spleen (Table 4). Seaweed seasoning/flavour enhancers to animals on high cholesterol diets (CE, CC and CS groups) had significantly reduced MDA concentrations in the livers, hearts and kidneys compared to C group (untreated high-cholesterol animals):

(i) In the livers by -35.2%, -43.8% and -40.1% respectively. (ii) in the hearts by -18.8%, -26.4% and -22% respectively (iii) in the kidneys by -30.7%, -30.7% and -31.3% respectively. The MDA levels in the brain, lungs, erythrocytes and spleen were also reduced but insignificantly. Animals fed on basal diet supplemented with seaweed seasoning/flavour enhancers (NE, NC and NS groups) showed no significant difference in MDA concentrations in the livers, hearts, kidneys, brains and lungs except for NC group which showed a significantly lower MDA concentration in the spleens as compared to the control group (N).

Antioxidant enzymes activities

Hypercholesterol diet (C) significantly reduced SOD activity by -51% in the livers, and - 33.4% in the hearts as compared to the normal control animals (N). Hypercholesterol also reduced the SOD activities insignificantly in some other organs (-26.6% in the kidneys; - 47.1% in the spleens) and insignificantly increased the SOD activities in others (31% in the brains and 34% in the erythrocytes).

Seaweed seasoning/flavour enhancers to animals on high cholesterol diets significantly showed the following changes:

(i) For CE, CC and CS animals, there were increased SOD activities in livers by +97.5%, +111.3% and +71.5% respectively, compared to untreated

Hypercholesterol (C) animals, similar to the level of normal control animals (N). (ii) For the CE and CC animals, there were increased SOD activities in the hearts by +34.5% and +29% respectively, compared to untreated Hypercholesterol (C) animals. (iii) For the CS animals, there were reduced SOD activities in the hearts by -58.1% as compared to the Hypercholesterol (C) animals (iv) For the CS animals, there were reduced SOD activities in the kidneys by -52.4% as compared to normal control animals (N).

(v) All groups showed no significant differences in SOD activities in the brain, lungs, erythrocytes and spleen as compared to normal control animals (N). High cholesterol food (C) significantly lowered the GSH-Px activity in the liver (-43%), heart (-26%), and kidneys (-63%) compared to normal control animals (N). High cholesterol food supplemented with seaweed seasoning/flavour enhancers, significantly showed:

(i) For CE, CC and CS animals, increased GSH-Px activity in livers by +54%, +39% and

+71% respectively compared with the hypercholesterol animals (C), similar to the level of the normal control animals (N).

(ii) For CS animals, lower GSH-Px activity in the hearts, compared to the C group (-46%) or N group (-60%)

(iii) For CE and CC animals, increased GSH-Px activities in the kidneys by +104.4% and

80.3% respectively compared to C animals.

The GSH-Px activities in brain, lungs, erythrocytes and spleen in high-cholesterol foods (with or without seaweed seasoning/flavour enhancers) showed no significant differences with the control animals (N). The CAT activities in liver, heart, kidney, brain, lungs and spleen in high-cholesterol food showed no significant differences with the control diet (N).

Generally, the antioxidant enzyme activities in tissues of animals fed with basal diet supplemented with seaweed seasoning/flavour enhancers (NE, NC and NS) showed no significant difference to the control animals (N).

High-cholesterol food induced hypercholesterol which was accompanied by increased plasma LDL-C, increased atherogenic index (Al = LDL-C/HDL-C), elevated levels of MDA concentrations, and reduced SOD and GSH-Px activities in tissues (livers, hearts and kidneys) of animals. Malondialdehyde (MDA) is generated as a relatively stable end product from the oxidative degradation of polyunsaturated fatty acids (PUFA). This free radical-driven lipid peroxidation has been causatively implicated in the aging process, atherosclerosis, Alzheimer's disease, and cancer. Increased superoxide anion production in hypercholesterol conditions, contribute to the atherosclerotic process and increased tissue MDA levels.

The addition of seaweed seasoning in the high-cholesterol food resulted in a decrease in MDA concentrations in liver, heart and kidney. No significant changes in MDA levels were found in the brain, lungs, erythrocytes and spleen as compared to the control diet (N). These data suggest that animals fed with seaweed seasoning/flavour enhancers are less susceptible to peroxidative damage.

Animals fed on high-cholesterol food (C) expressed significantly lower SOD activities in the livers and hearts compared to control diet (N). However, animals on high-cholesterol food supplemented with seaweed seasoning/flavour enhancers expressed significantly higher SOD activity in the liver, compared to those fed on high-cholesterol food alone. Free radicals are the source of lipid peroxidation derived from oxygen, and SOD is the first line of defense against them. Hence, the increased in SOD activity in liver would reduce the accumulation of superoxide anion radical, and are partially responsible for the decreased MDA concentration observed in these tissues. A good correlation exist between the MDA concentration and the SOD activity (r=-0.78) in the liver tissues of the animals, but not in the tissues of the other organs.

The high-cholesterol food (C) reduced the GSH-Px activity in liver, heart and kidney of animals. The increased levels of superoxide anion could inactivate GSH-Px, which lead to an enhancement of H₂O₂ level which, in turn, would inactivate SOD₁ which would explain the high MDA and low antioxidant enzymes activities found in these tissues. Higher GSH- Px activities were observed in the livers and kidneys of animals on high-cholesterol food supplemented with seaweed seasoning/flavour enhancers (CE, CC and CS) compared to untreated hypercholesterol animals (C). A weak correlation exist between the MDA concentration and the GSH-Px activity (r=-0.55) in the liver tissues of the animals, but not in the other organs. GSH-Px main function are to neutralize oxidative attack, are competent peroxidases, but they may also exercise cell- and tissue-specific tasks in metabolic control.

The MDA concentrations, SOD, GSH-Px and CAT activities were unaffected by the different diets in the other tissues, namely the brains, lungs, erythrocytes and spleens of animals, suggesting no increased accumulation of superoxide anion radical in these tissues. The catalase within normal tissues handles approximately 5 - 50% of the generated H₂O₂, and glutathione peroxidase/reductase handles the rest. It appears that catalase and the glutathione peroxidase/reductase system function intracellular^ to dispose H₂O₂ in a coordinated way, sequentially, in which the glutathione peroxidase/reductase system is the rate-limiting step. Catalase was unaffected by the various diets in most of the tissues studied, and was not correlated to MDA levels or any other enzyme activities analyzed.

The term "promoting anti-cancer and healthcare of cardiovascular system" used herein throughout the specification refers to the one or more effects such as healing to cancer, injury, anti-diabetic, anti-hypertensive, anti-atherogenic, anti-hypercholestrol, anti-obesity, anti-hyperlipidemic, anti-oxidative, vasorelaxant and the like. The aforementioned effects can be initiated variedly upon different ingested amount or dose, form of supply of the extract, coupled with other components, age and sex of the administrated subject and so on.

The term "pharmaceutically effective amount" used herein throughout the specification refers to the amount of the active ingredient, the extract, to be administered orally to the subject to trigger the desired effect without or causing minimal toxic adverse effect against the subject. One skilled in the art should know that the effective amount can vary from one individual to another due to the external factors such as age, sex, diseased state, races, body weight, formulation of the extract, availability of other active ingredients in the formulation and so on.

With respect to the preferred embodiment of the present invention, a method for preparing extracts from plants of the tropical seaweeds, Solanum spp. fruits and Piper spp. leaves and/or Piper family is disclosed. The method basically comprises the steps of pre-treating parts from Tropical seaweeds, Solanum spp. fruits and Piper spp. leaves and/or Piper family; extracting the pre-treated parts using a solvent; removing the pre-treated parts from the used solvent; and concentrating the used solvent to acquire the herbal extract. The disclosed method is found to be effective in isolating the favored compounds, peptides or active metabolites which provides a anti-cancer enhancement effects or health promoting effect towards the injured system upon ingestion orally or topically of a subject. According to another preferred embodiment of the disclosed method, the alcoholic extracts of are pre-treated before subjecting to extraction. The pre-treatment process may involve a washing step to clean any dirt or physical pollutants captured at the surface of the alcoholic extracts of. Other pretreatment step is preferably reducing the moisture content of the alcoholic extracts of by any known means or approaches in the art to improve the extraction rate and yield. In the preferred embodiment, the alcoholic extracts of are subjected to drying in an oven at 40-120°C. The drying temperature shall not be set too high as such practice may degrade the active metabolites and compounds contained within the alcoholic extracts of. The preferred temperature shall range from 40⁰C to 70⁰C. Other pretreatment steps that can be employed are preparing the dried alcoholic extracts of into small fragments, or pulverization to paste or powdery form prior to the soaking step. The fragmentized portion or pulverized alcoholic extracts of can greatly increase the available contact surface of the dried alcoholic extracts of that are exposed to extraction solvent thus enhance the rate as well as the yield of the extraction method. Possibly, the pre-treated alcoholic extracts of may be subjected to a plurality occasion of extraction using different types of extraction solvent to obtain the optimal yield.

Though mere soaking the pre-treated alcoholic extracts into the extraction solvent shall enable the extraction of the active compounds due to the polarity attraction of the extraction solvent, the process may be accelerated by stirring the extraction mixture, or the use of heat and pressure, both of the extraction solvent and the pre-treated alcoholic extracts of, during the time the extraction is conducted. It is important to note that the extracted compounds from the alcoholic extracts of are mainly constituted of proteins, biophenols, pigments, minerals, polysaccharides, lipids, small peptides and other bioactive compounds. Therefore, the efficiency rate of the extraction is sensitive towards pH changes in the extraction solvent. In the most preferred embodiment of the disclosed method, the pH of the extraction content shall fall within the interval of pH 1 to 10 for achieving optimal yield and extraction rate. Preferably, the pH of the extraction solvent is monitored closely during the extraction to ensure that the pH of the extraction solvent favors the reaction. Any shift of the pH can be adjusted back to the preferred range by using pH adjusters such as hydrochloric acid, sodium hydroxide, and the like. Moreover, applying appropriate amount of heat energy to the extraction system is another feasible approach to enhance the extraction rate and yield. Relying upon the solvent utilized, the heating is most preferred within the range of 40⁰C to 120⁰C. Precaution should be taken into consideration that denaturalization possibly occurs at high temperature of heating.

It is important to note that the alcoholic extracts of used in the disclosed method in the preferred embodiment derives from the plant species of, but not limited to, Euchema cottonii, Appaphycus alvareziii, Caυlerpa lentillifera and Sargassum polycystum, Piper betel and Piper spp. The extracts obtained from the abovementioned plant species are suitable to be incorporated into edible products or as capsules and tablets.

Pursuant to further embodiment of the disclosed method, the extraction solvent preferably employed is, but not limited to, any one or combination of water, alcohol, acetone and chloroform. The desired compounds to be extracted from the alcoholic extracts of are mainly constituted of, but not limited to, proteins, biophenols, lipids, saccharides, minerals and small peptides. Due to polarity of these compounds, the solvent is found to be effective in extracting these desired compounds from the plant matrix. To acquire optimal yield by using effective amount of extraction solvent, the ratio the pre-treated alcoholic extracts of to the extraction solvent is preferably 1-3: 6-10 (w/v).

Preferably, the pre-treated alcoholic extracts of after subjecting to extraction can be separated by any known means and approaches in the art for disposal. Vacuum filtration is most preferred as such approach is commonly available. Similarly, vaporization of the used extraction solvent to concentrate compounds can be performed by different approaches. For, example drying the used extraction solvent using heat energy or vacuum drying. Concentrating the compounds by dissipating the used extraction solvent shall finally reach to the stage where the compounds are concentrated to a paste or powdery form. This paste or powdery form of compounds extract can then be utilized for various applications.

Attention is now drawn to another embodiment of the present invention which involves a comestible and topical composition for promoting anti-cancer and healthcare of the cardiovascular system of an individual comprises extracts derive from alcoholic extracts of a plant of tropical seaweeds, Solanum spp. fruits and Piper spp. leaves and Piper family using an extraction solvent. The comestibles mentioned herein can be any common daily consumed processed food such as bread, noodles, confections, chocolates, beverages, and the like. One skilled in the art shall appreciate the fact that the aforesaid extract can be incorporated into the processed comestibles, capsules, tablets or topical medicine during the course of processing. Therefore, any modification thereon shall not depart from the scope of the present invention.

As setting forth in the above description, the alcoholic extracts of used for preparing the extract are derived from the plants of tropical seaweeds, Solanum and Piper spp. More preferably, the plant is any one or combination of the plant species of, but not limited to,

Euchema cottonii, Appaphycus alvareziii, Caυlerpa lentillifera and Sargassum polycystum,

Piper betel and Piper spp.. The inventors of the present invention discovered that the extract derives from the aforementioned species possesses both acceptable taste that confers the derived extract to be comfortably incorporated with the comestibles product, capsules, tablets or topical medicine with minimal additional refining process.

According to the preferred embodiment, the extract to be incorporated into the comestibles and medicine can be acquired from any known method not limited only to the foregoing disclosed method. Following to another embodiment, the extract is prepared in a concentrated form, preferably paste or powdery form which enables the extract to be prepared in various formulations of the comestibles, capsules, tablets or topical applications.

In line with the preferred embodiment, the extract shall be the plant metabolites which are susceptible to an extraction solvent. The compounds and small peptides with the anticancer and cardiovascular system health-promoting properties are those metabolites with polarity in the alcoholic extracts of. Therefore, the extract from the alcoholic extracts of plants of tropical seaweeds, Solanum spp. fruits and Piper spp. leaves and Piper family is preferably derives from the extraction solvent of water, alcohol, acetone, chloroform and any combination thereof. In view of the prominent property of promoting anti-cancer and general healthcare of the cardiovascular system by the extracts in a subject, further embodiment of the present invention includes a method comprising the step of administrating orally or topically to the subject an effective amount of an extract of alcoholic extracts derive from a plant of tropical seaweeds, Solanum spp. fruits and Piper spp. leaves and/or Piper family using an extraction solvent. From the experiments conducted, it is clear that the extract employed in this embodiment shows anti-cancer as well as previously demonstrated anti-diabetic, antihypertensive, anti-oxidative, anti-atherogenic, anti-hypercholestrol, anti-hyperlipidemic, anti-obesity, vasorelaxant and similar effects. Moreover, the plant of tropical seaweeds, Solanum spp. fruits and Piper spp. leaves and Piper family is any one or combination of, but not limited to, Euchema cottonni, Kappaphycus alvareziii, Caulerpa lentillifera and Sargassum polycystum, Piper betel and Piper spp..

EXAMPLE 6

The extraction conditions were conducted as follows. Vegetative parts of the plants were collected, cleaned, washed and cut into small pieces and oven dried at 4O⁰C overnight. The dried material was ground using a blender and extracted three times with hot and cold alcohol (1 :10 v/v) and three times with hot and cold water or with mixtures of chloroform and alcohol. Other solvent such as acetone may be used as a medium for the extraction. This is a process designed to separate soluble compound by diffusion from a solid matrix (plant tissue) using a liquid matrix (solvent). Alcohol, water, chloroform and acetone has produced good yield in extracting the active components. The extraction was done a few times. The pooled extracts were vacuum-dried at 40⁰C and stored until used.

EXAMPLE 7

The alcoholic extract of all these three seaweed species tested showed no toxicity to normal Vera cell line (Table 1 ). The alcoholic seaweeds extracts exhibited dose and time depended inhibition against the proliferation of the five tested cancer cell lines. The IC50 values (Table 1 ) for MDA-MB 231 cell line, were approximately 50 μg/ml for S. Polycystum, 60 μg/ml for C.lentillifera, and 100 μg/ml for E. Cottonii. The IC50 values for HeLa cell line, were approximately 30 μg/ml for S. Polycystum, 55 μg/ml for C.lentillifera, and 70 μg/ml for E. Cottonii. Amongst the seaweeds extracts S. Polycystum extract (SPME) showed the strongest anti-proliferation activity against all the cancer cell lines tested with IC50 values of 25, 50, 30, 100, 150 μg/ml after 24 h treatment on MCF-7, MDA-MB- 231 , HeLa, HepG2, and HT-29 cells, respectively. A very good correlationship were found between the cancer cells anti-proliferation activity with the extracts total polyphenols content, and electron donor activities but not with their proton donor activities.

Table 8: The IC50 values of seaweeds alcoholic dry extracts on various human cancer cell lines expressed as μg/ml, and Cytotoxicity on Vero cells

Vero cells 1000 1250 1000

Values are expressed as Mean ± Standard deviation, n=3. EXAMPLE 8

Induction of apoptosis by SPME on MCF-7 cells

Percentages of apoptotic MCF-7 cells in the presence of SPME (0-200 μg/ml) compared to normal cells are depicted in Fig.1. Apoptosis was evaluated using fluorescence microscopy by calculating the percentage of cells showing nuclear morphology of apoptosis after staining with Hoechst 33342. Percentages of apoptotic cells increased from 20% to 68% by increasing the concentration of the seaweeds extract from 12.5 to 200 μg/ml. This increasing apoptosis did not occur in normal cell lines. The MTT assay showed them to be cytotoxic against all the cell lines in a dose dependent manner, with brown seaweeds (S. polycystum) having the greatest inhibition. Cytotoxicity was not observed in normal Vero cell line. The estrogen positive breast cancer MCF-7 and human cervical adenocarcinoma HeLa cell lines were most sensitive to S. Polycystum crude methanol extract (SPME), with IC50 of 20 μg/ml, after 72 hours incubation. The IC₅₀ value of SPME on MCF-7, MDA-MB- 231 , HeLa, HepG2, and HT-29 cells was approximately 25, 50, 30, 100, 150 μg/ml after treatment for 24 h. Treatment of MCF-7 cells with various concentrations of SPME resulted in growth inhibition and induction of apoptosis in a time- and dose-dependent manner. The cancer cells anti-proliferative activity in the extracts are due to the phenolic compounds and electron donor free radical scavenging activities.

The term "promoting wound healing and healthcare of cardiovascular system" used herein throughout the specification refers to the one or more effects such as healing to injury, anti-diabetic, anti-hypertensive, anti-atherogenic, anti-hypercholestrol, anti-obesity, anti- hyperlipidemic, anti-oxidative, vasorelaxant and the like. The aforementioned effects can be initiated variedly upon different ingested amount or dose, form of supply of the extract, coupled with other components, age and sex of the administrated subject and so on.

The term "pharmaceutically effective amount" used herein through out the specification refers to the amount of the active ingredient, the extract, to be administered orally to the subject to trigger the desired effect without or causing minimal toxic adverse effect against the subject. One skilled in the art should know that the effective amount can vary from one individual to another due to the external factors such as age, sex, diseased state, races, body weight, formulation of the extract, availability of other active ingredients in the formulation and so on.

With respect to the preferred embodiment of the present invention, a method for preparing extracts from plants of the seaweeds and/or Arecaceae family is disclosed. The method basically comprises the steps of pre-treating parts from plants of seaweeds and/or Arecaceae family; extracting the pre-treated parts using a solvent; removing the pre- treated parts from the used solvent; and concentrating the used solvent to acquire the herbal extract. The disclosed method is found to be effective in isolating the favored compounds, peptides or active metabolites which provides a wound healing enhancement effects or health promoting effect towards the injured system upon ingestion orally or topically of a subject.

According to another preferred embodiment of the disclosed method, the vegetative parts are pre-treated before subjecting to extraction. The pre-treatment process may involve a washing step to clean any dirt or physical pollutants captured at the surface of the vegetative parts. Other pretreatment step is preferably reducing the moisture content of the vegetative parts by any known means or approaches in the art to improve the extraction rate and yield. In the preferred embodiment, the vegetative parts are subjected to drying in an oven at 40-120 °C. The drying temperature shall not be set too high as such practice may degrade the active metabolites and compounds contained within the vegetative parts. The preferred temperature shall range from 40⁰C to 70⁰C. Other pretreatment steps that can be employed are preparing the dried vegetative parts into small fragments, or pulverization to paste or powdery form prior to the soaking step. The fragmentized portion or pulverized vegetative parts can greatly increase the available contact surface of the dried vegetative parts that are exposed to extraction solvent thus enhance the rate as well as the yield of the extraction method. Possibly, the pre-treated vegetative parts may be subjected to a plurality occasion of extraction using different types of extraction solvent to obtain the optimal yield. Though mere soaking the pre-treated vegetative parts into the extraction solvent shall enable the extraction of the active compounds due to the polarity attraction of the extraction solvent, the process may be accelerated by stirring the extraction mixture, or the use of heat and pressure, both of the extraction solvent and the pre-treated vegetative parts, during the time the extraction is conducted. It is important to note that the extracted compounds from the vegetative parts are mainly constituted of proteins, biophenols, pigments, minerals, polysaccharides, lipids, small peptides and other bioactive compounds. Therefore, the efficiency rate of the extraction is sensitive towards pH changes in the extraction solvent. In the most preferred embodiment of the disclosed method, the pH of the extraction content shall fall within the interval of pH 1 to 10 for achieving optimal yield and extraction rate. Preferably, the pH of the extraction solvent is monitored closely during the extraction to ensure that the pH of the extraction solvent favors the reaction. Any shift of the pH can be adjusted back to the preferred range by using pH adjusters such as hydrochloric acid, sodium hydroxide, and the like. Moreover, applying appropriate amount of heat energy to the extraction system is another feasible approach to enhance the extraction rate and yield. Relying upon the solvent utilized, the heating is most preferred within the range of 40⁰C to 120⁰C. Precaution should be taken into consideration that denaturalization possibly occurs at high temperature of heating.

It is important to note that the vegetative parts used in the disclosed method in the preferred embodiment derives from the plant species of, but not limited to, Euchema cottonii, Appaphycus alvareziii, Caulerpa lentillifera and Sargassum polycystυm, Elaeis guineensis, Elaeis oleifera, Phoenix dactylifera and Cocos nucifera. The extracts obtained from the abovementioned plant species are found acceptable in both taste and fragrance thus render them suitable to be incorporated into edible products or as capsules and tablets.

Pursuant to further embodiment of the disclosed method, the extraction solvent preferably employed is, but not limited to, any one or combination of water, alcohol, acetone and chloroform. The desired compounds to be extracted from the vegetative parts are mainly constituted of, but not limited to, proteins, biophenols, lipids, saccharides, minerals and small peptides. Due to polarity of these compounds, the solvent is found to be effective in extracting these desired compounds from the plant matrix. To acquire optimal yield by using effective amount of extraction solvent, the ratio the pre-treated vegetative parts to the extraction solvent is preferably 1-3: 6-10 (w/v).

Preferably, the pre-treated vegetative parts after subjecting to extraction can be separated by any known means and approaches in the art for disposal. Vacuum filtration is most preferred as such approach is commonly available. Similarly, vaporization of the used extraction solvent to concentrate compounds can be performed by different approaches. For, example drying the used extraction solvent using heat energy or vacuum drying. Concentrating the compounds by dissipating the used extraction solvent shall finally reach to the stage where the compounds are concentrated to a paste or powdery form. This paste or powdery form of compounds extract can then be utilized for various applications.

Attention is now drawn to another embodiment of the present invention which involves a comestible and topical composition for promoting wound healing and healthcare of the cardiovascular system of an individual comprises extracts derive from vegetative parts of a plant of seaweeds and Arecaceae family using an extraction solvent. The comestibles mentioned herein can be any common daily consumed processed food such as bread, noodles, confections, chocolates, beverages, and the like. One skilled in the art shall appreciate the fact that the aforesaid extract can be incorporated into the processed comestibles, capsules, tablets or topical medicine during the course of processing. Therefore, any modification thereon shall not depart from the scope of the present invention.

As setting forth in the above description, the vegetative parts used for preparing the extract are derived from the plants of seaweeds and Arecaceae family. More preferably, the plant is any one or combination of the plant species of, but not limited to, Eυchema cottonii, Appaphycυs alvareziii, Caulerpa lentillifera and Sargassum polycystum, Elaeis guineensis, Elaeis oleifera, Phoenix dactylifera and Cocos nυcifera. The inventors of the present invention discovered that the extract derives from the aforementioned species possesses both acceptable taste that confers the derived extract to be comfortably incorporated with the comestibles product, capsules, tablets or topical medicine with minimal additional refining process.

In line with the preferred embodiment, the extract shall be the plant metabolites which are susceptible to an extraction solvent. The compounds and small peptides with the wound healing and cardiovascular system health-promoting properties are those metabolites with polarity in the vegetative parts. Therefore, the extract from the vegetative parts of plants of seaweeds and Arecaceae family is preferably derives from the extraction solvent of water, alcohol, acetone, chloroform and any combination thereof.

In view of the prominent property of promoting wound healing and general healthcare of the cardiovascular system by the extracts in a subject, further embodiment of the present invention includes a method comprising the step of administrating orally or topically to the subject an effective amount of an extract of vegetative parts derive from a plant of seaweeds and/or Arecaceae family using an extraction solvent. From the experiments conducted, it is clear that the extract employed in this embodiment shows wound healing as well as previously demonstrated anti-diabetic, antihypertensive, anti-oxidative, anti- atherogenic, anti-hypercholestrol, anti-hyperlipidemic, anti-obesity, vasorelaxant and similar effects. Moreover, the plant of seaweeds and Arecaceae family is any one or combination of, but not limited to, Euchema cottonni, Kappaphycus alvareziii, Caulerpa lentillifera and Sargassum polycystum, Elaeis gυineensis, Elaeis oleifera, Phoenix dactylifera and Cocos nucifera. EXAMPLE 9

The extraction conditions were conducted as follows. Oil palm vegetative parts were collected, cleaned, washed and cut into small pieces and oven dried at 4O⁰C overnight. The dried material was ground using a blender and extracted three times with hot and cold alcohol (1 :10 v/v) and three times with hot and cold water or with mixtures of chloroform and alcohol. Other solvent such as acetone may be used as a medium for the extraction. This is a process designed to separate soluble compound by diffusion from a solid matrix (plant tissue) using a liquid matrix (solvent). Alcohol, water, chloroform and acetone has produced good yield in extracting the active components. The extraction was done a few times. The pooled extracts were vacuum-dried at 40⁰C and stored until used.

EXAMPLE 10

Enhanced wound healing, anti-diabetic, anti-oxidative, antimicrobial and organ protective properties evaluation had been conducted on the different products from the different source of palm vegetative parts. The wound healing properties of palm vegetative parts extract and palm vegetative parts functional ingredients are shown below.

± 0.0

value are mean ± SE of six animals in each group EO1 : Cold alcoholic extract; E02: Hot alcoholic extract; EW1 : Cold water extract: EW2: Hot water extract

Wound contraction

Claims

1. A comestible product consisting of a seaweed origin.

2. The comestible product as claimed in claim 1 , further consist of herbal extracts/ powder and/or hydrolysate.

3. The comestible product as claimed in claim 1 , wherein the seaweed is selected from brown, red, and/or green seaweeds.

4. The comestible product as claimed in claim 1 , wherein the seaweed composition includes: a) between about 1 % and 100 % by weight of red seaweed; b) between about 1% and 100% by weight of brown seaweed; and c) between about 1% and 100% by weight of green seaweed.

5. The comestible product as claimed in claim 2, wherein the herbsis from polyphenol rich plants.

6. The comestible product as claimed in claims 1 and 2, wherein the composition is provided in a dry or aqueous form.

7. The comestible product as claimed in claims 1 and 2, wherein the seaweed having the capability of seasoning/flavour enhancer means.

8. The comestible product as claimed in claim 4, wherein the red seaweed is Eucheuma spp, the brown seaweed is Sargassum spp and the green seaweed is Caulerpa spp.

9. The comestible product as claimed in claim 4, wherein the seaweed composition may be used as a mineral, antioxidant, dietary fiber supplement or enrichment ingredient and flavor enhancers in foodstuffs.

10. The comestible product as claimed in claim 4, wherein the seaweed composition may be mixed with foodstuffs; including cereals, bread, drinks, health bars, juices, concentrates, canned food, ice cream, water, staple goods such as wheat, corn, barley, and oat in any form, or taste maskers such as sugar to form new products.

11. Use of the seaweed composition in a preparation to manufacture a functional ingredient for protecting or reducing cardiovascular disease risk, hyperglycaenia, hypercholesterol, hyperlipidemia, atherosclerosis, diabetes, hypertension and/or oxidative stress in an individual.

12. Use of the seaweed composition in a preparation to manufacture a functional ingredient for reducing cardiovascular related diseases, degenerative diseases and/or cancer related diseases in an individual.

13. Seasoning containing seaweed produced according to any of claims 1 to 12.

14. The seasoning as claimed in claim 13, wherein the seasoning includes providing health benefits to a mammal.

15. A method for preparing seasoning from seaweed, wherein the method comprising the steps of:

(a) 25 optimum operating temperature and pH to form hydrolystate;

(b) maintaining the pH of the hydrolyzing process;

(c) deactivating the catalysts in the hydrolysate;

(d) adjusting pH of the hydrolysate to 3.5-4.5; and (e) filtering the hydrolysate through layers of filter medium such as activated carbon and/or calcium carbonate to obtain the flavor enhancer.

16. The method as claimed in claim 15, further comprising the step of concentrating or drying the obtained flavor enhancer.

17. The method as in claimed in claims 15 and 16, further comprising the step of adding any one or combinations of polyphenols rich extracts and or herbs, thickeners, preservatives, caramel, onion powder, ginger powder, garlic powder, cinnamon, spices, and black peppers to the obtained flavor enhancer.

18. A method as in claims 15, wherein seaweeds are cleaned, dried, ground with or without adding any one or combinations of polyphenols rich extracts or herbs, thickeners, preservatives, caramel, onion powder, ginger powder, garlic powder, cinnamon, spices, and black peppers to the obtained seasoning, which can also be used as mineral, omega 3, omega 6 fatty acids and dietary fiber supplements.

19. A composition for promoting anti-cancer and healthcare of cardiovascular system of an individual comprises an extract derives from alcoholic extracts of a plant of tropical seaweeds, Solanum spp. fruits and Piper spp. leaves and Piper family using an extraction solvent.

20. The composition according to claim 19, wherein the extract is prepared in a concentrated form together with or without other known anti-cancer ingredients such as honey, curcumin, vanillin, vitamin E, thymoquinone, antioxidants, anti-angiogenic agents, sea cucumber extracts, etc.

21. The composition according to claims 19 and 20, wherein the plant of Tropical seaweeds, Solanum spp. fruits and Piper spp. leaves and Piper family is any one or combination of Euchema cottonii, Appaphycus alvareziii, Caulerpa lentillifera and Sargassum polycystum, Piper betel and Piper spp.

22. The composition according to claims 19 and 20, wherein the extraction solvent is selected from the group consisting of water, alcohol, acetone, chloroform and any combination thereof.

23. A use of an effective amount of extracts of alcoholic extracts derived from a plant of the tropical seaweeds, Solanum spp. fruits and Piper spp. leaves and Piper family using an extraction solvent for the manufacture of a medicament for promoting anti-cancer and healthcare of cardiovascular system in a subject.

24. The use according to claim 23, wherein the plant of Tropical seaweeds, Solanum spp. fruits, Piper spp. leaves and Piper family is any one or combination of Euchema cottonii, Appaphycus alvareziii, Caulerpa lentillifera and Sargassum polycystum Piper betel and Piper spp.

25. A method for preparing an herbal extract comprises the steps of:

(a) pre-treating alcoholic extracts derive from plants of the tropical seaweeds, Solanum spp. fruits and Piper spp. leaves and Piper family;

(b) extracting the pre-treated alcoholic extracts of by using a solvent; (c) removing the pre-treated alcoholic extracts of from the used solvent; and

(d) concentrating the used solvent to acquire the herbal extract.

26. A method according to claim 25, wherein the plant of tropical seaweeds, Solanum spp. fruits and Piper spp. leaves and Piper family is selected from the group consisting of, Euchema cottonii, Appaphycus alvareziii, Caulerpa lentillifera and Sargassum polycystum, Piper betel and Piper spp.

27. A method according to claim 25, wherein the solvent is any one or combination of water, alcohol, acetone and chloroform.

28. A method according to claim 25, wherein the pre-treatment is any one or combination of cleaning the alcoholic extracts of and drying the alcoholic extracts.

29. A method according to claim 25, wherein ratio of the dried alcoholic extracts of to the extraction solvent is 1 -3: 6-10 (w/v).

30. A composition for promoting wound healing and healthcare of cardiovascular system of an individual comprises an extract derives from vegetative parts of a plant of seaweeds and Arecaceae family using an extraction solvent.

31. The composition according to claim 30, wherein the extract is prepared in a concentrated form together with or without other known wound healing ingredients such as honey, hyaluronic acid, Vaseline, menthol, thymol, penicillin, hydrogen peroxide, sea cucumber extracts, etc.

32. The composition according to claims 30 and 31 , wherein the plant of seaweeds and Arecaceae family is any one or combination of Euchema cottonii, Appaphycus alvareziii, Caulerpa lentillifera and Sargassum polycystum, Elaeis guineensis, Elaeis oleifera, Phoenix dactylifera and Cocos nucifera.

33. The composition according to claims 30 and 31 , wherein the extraction solvent is selected from the group consisting of water, alcohol, acetone, chloroform and any combination thereof.

34. A use of an effective amount of extracts of vegetative parts derived from a plant of the seaweeds and Arecaceae family using an extraction solvent for the manufacture of a medicament for promoting wound healing and healthcare of cardiovascular system in a subject.

35. A method according to claim 34, wherein the plant of seaweeds and Arecaceae family is any one or combination of Euchema cottonii, Appaphycus alvareziii, Caulerpa lentillifera and Sargassum polycystum Elaeis guineensis, Elaeis oleifera, Phoenix dactylifera and Cocos nucifera.

36. A method for preparing an herbal extract comprises the steps of: (e) pre-treating vegetative parts derive from plants of the seaweeds and Arecaceae family; (f) extracting the pre-treated vegetative parts by using a solvent; (g) removing the pre-treated vegetative parts from the used solvent; and (h) concentrating the used solvent to acquire the herbal extract.

37. A method according to claim 36, wherein the plant of seaweeds and Arecaceae family is selected from the group consisting of Euchema cottonii, Appaphycus alvareziii,

Caulerpa lentillifera and Sargassum polycystum, Elaeis guineensis, Elaeis oleifera, Phoenix dactylifera and Cocos nucifera.

38. A method according to claim 36, wherein the solvent is any one or combination of water, alcohol, acetone and chloroform.

39. A method according to claim 36, wherein the pre-treatment is any one or combination of cleaning the vegetative parts and drying the vegetative parts.

40. A method according to claim 36, wherein ratio of the dried vegetative parts to the extraction solvent is 1-3: 6-10 (w/v).