Patent Application
20130310332
(a) Field
The subject matter disclosed generally relates to products derived from harwood trees, especially any variety of maple tree, such as sugar maple (Acer Saccharum) or red maple (Acer Rubrum L.), and more specifically to neutraceutical compositions comprising maple tree extract, essential oil compositions comprising oil extracted from sugar maple or other maple, sweetening compositions containing sugar extracted from sugar maple, infusion compositions prepared from maple tree leaves, food ingredients comprising maple tree extract, cosmetic composition comprising maple tree extracts as well as compounds isolated from sugar maple biomass and the methods of extracting the same.
(b) Related Prior Art
Products derived from the sap of the sugar maple tree Acer Saccharum or other maple trees are well known in the art. For many years, it has been known that the sap of the hard or sugar maple trees, known as winter sap, could be concentrated very substantially from its original, very watery and only slightly sweet condition to well known maple syrup which, when further boiled, finally reaches crystalline stage wherein the product is maple sugar. For example, U.S. Pat. No. 3,397,062 to Nessly describes carbonated beverages derived from concentrated maple sap to which flavouring may be added. Also, U.S. Pat. No. 5,424,089 to Munch et al. describes carbonated beverages prepared from non-concentrated maple sap to which flavouring ingredients may have been added.
Other products concern principally maple sugar tree products such as maple syrup. For example U.S. Pat. No. 6,485,763 to Jampen describes a method for producing a shelf stable, spreadable high viscosity maple syrup by adding a sucrose-cleaving enzyme.
Using maple based products appears to present several advantages for a healthy diet, as maple-sugar products or products derived from any other variety of maple tree which contain molecules such as polyphenols as well as other nutrients such as vitamins and oligoelements that can contribute to good health. However, very little is known concerning the potential health benefits of other types of maple-based products such as products derived from the sap, the samara (including the fruits, the seeds as well as the stem), leaves (including the stem), twigs, roots, heartwood and sap wood, whole branches, wood from branches, bark from branches and bark of any Acer tree. Also, maple-derived sugar remains a relatively little known nutrient from which only recently has it been found potential properties as an anti-oxidant, anticancer, antibacterial, anti-diabetic agent, anti-inflammatory, anti-arthritic, anti-hyperglycemic, as well as beneficial effects on cardiovascular health, neurodegenerative diseases, Alzheimer's diseases, liver disorders (such as metabolic syndrome, damaged hepatic function, hepatic and liver dyslipidemia, hepatitis, liver cancer), atherosclerosis, hypertension, and skin diseases (such as eczema, psoriasis and the likes).
Therefore, there is a need for maple tree-derived products to improve or enhance health and well-being.
Therefore, there is a need for maple tree-derived sugar products to improve or enhance health and well-being
According to an embodiment, there is provided a composition for the prophylaxis of an ailment comprising a therapeutically effective amount of an extract of an Acer tree in association with a pharmaceutically acceptable carrier.
The extract of Acer tree may be an extract from a non-concentrated or concentrated sap, a samara fruit, a samara seed, a stems of leaf, a stem of a samara, a twig, a root, a leaf, a bark, a heartwood, a sapwood, a whole branch, a bark of a branch, a wood of a branch, a sugar, a syrup, a syrup extract, a syrup-derived product, a rejection of syrup or syrup-derived product production, a residue of syrup or syrup-derived product production, or combinations thereof.
The Acer tree may be chosen from Acer nigrum, Acer lanum, Acer acuminatum, Acer albopurpurascens, Acer argutum, Acer barbinerve, Acer buergerianum, Acer caesium, Acer campbeffii, Acer campestre, Acer capillipes, Acer cappadocicum, Acer carpinifolium, Acer caudatifolium, Acer caudatum, Acer cinnamomifolium, Acer circinatum, Acer cissifolium, Acer crassum, Acer crataegifolium, Acer davidii, Acer decandrum, Acer diabolicum, Acer distylum, Acer divergens, Acer erianthum, Acer etythranthum, Acer fabri, Acer garrettii, Acer glabrum, Acer grandidentatum, Acer griseum, Acer heldreichii, Acer hentyi, Acer hyrcanum, Acer ibericum, Acer japonicum, Acer kungshanense, Acer kweilinense, Acer laevigatum, Acer laurinum, Acer lobelii, Acer lucidum, Acer macrophyllum, Acer mandshuricum, Acer maximowiczianum, Acer miaoshanicum, Acer micranthum, Acer miyabei, Acer mono, Acer mono×Acer truncatum, Acer monspessulanum, Acer negundo, Acer ningpoense, Acer nipponicum, Acer oblongum, Acer obtusifolium, Acer oliverianum, Acer opalus, Acer palmatum, Acer paxii, Acer pectinatum, Acer pensylvanicum, Acer pentaphyllum, Acer pentapomicum, Acer pictum, Acer pilosum, Acer platanoides, Acer poliophyllum, Acer pseudoplatanus, Acer pseudosieboldianum, Acer pubinerve, Acer pycnanthum, Acer rubrum, Acer rufinerve, Acer saccharinum, Acer saccharum, Acer sempervirens, Acer shirasawanum, Acer sieboldianum, Acer sinopurpurescens, Acer spicatum, Acer stachyophyllum, Acer sterculiaceum, Acer takesimense, Acer tataricum, Acer tegmentosum, Acer tenuifolium, Acer tetramerum, Acer trautvetteri, Acer triflorum, Acer truncatum, Acer tschonoskii, Acer turcomanicum, Acer ukurunduense, Acer velutinum, Acer wardii, Acer x peronai, and Acer x pseudoheldreichii.
The Acer tree may be chosen from Acer Saccharum and Acer Rubrum L.
The syrup-derived product may comprise butter, granulated sugar, hardened sugar, soft sugar, taffy, flakes an extract from lyophilisation of a sap, a maple concentrate or a maple syrup, an extract from drying of a sap, a maple concentrate or a maple syrup, an extract from crystallization of a sap, a maple concentrate or a maple syrup, an extract from pulverization of a sap, a maple concentrate or a maple syrup, an extract from atomization of a sap, a maple concentrate or a maple syrup, an extract from centrifugation of a sap, a maple concentrate or a maple syrup, or combinations thereof.
The syrup extract may be chosen from a methanol extract, a butanol extract, a butanol extract with sugar, a butanol extract without sugar, an ethyl acetate extract, an ethanol extract, a 95% ethanol/5% hot water extract, or combinations thereof.
The extract of Acer tree may comprise an extract from concentrated Acer tree water issued from reverse osmosis, a concentrated Acer tree water issued from pre-boiling nanofiltration, a pasteurized Acer tree water, a sterilized Acer tree water, an Acer tree water issued from a high pressure processing, an Acer tree water sterilized by UV irradiation, an Acer tree water sterilized by microwave irradiation or combinations thereof.
The extract of Acer tree may comprise an Acer tree molecule.
The Acer tree molecule may comprise:
The residue of syrup or syrup-derived product production may comprise diatomaceous earth, celite, kieselguhr, silica, silicon dioxide, calcium, natural sugar sand, ground bones, slop, clay and the like.
The composition may be chosen from a nutraceutical composition, a cosmeceutical composition, a pharmaceutical composition and a functional food composition.
According to an embodiment, there is provided a method of prophylaxis and/or treatment of an ailment comprising administering to a subject in need thereof a composition according to the present invention.
The ailment may be a diabetes, a cancer, an arthritis, a micro-organism infection, a neurodegenerative disease, an inflammatory disease, an oxidative stress related disease, a heart disease, Alzheimer's diseases, a liver disorder a metabolic syndrome, a damaged hepatic function, a hepatic and liver dyslipidemia, a hepatitis, a liver cancer, an atherosclerosis, a hypertension, a skin disease, an eczema, and a psoriasis.
According to an embodiment, there is provided a use of a composition according to the present invention for the prophylaxis and/or treatment of an ailment.
The ailment may be a diabetes, a cancer, an arthritis, a micro-organism infection, a neurodegenerative disease, an inflammatory disease, an oxidative stress related disease, a heart disease, Alzheimer's diseases, a liver disorder a metabolic syndrome, a damaged hepatic function, a hepatic and liver dyslipidemia, a hepatitis, a liver cancer, an atherosclerosis, a hypertension, a skin disease, an eczema, and a psoriasis.
According to an embodiment, there is provided an ingredient composition comprising an extract of an Acer tree in association with an acceptable carrier.
The extract of Acer tree may be an extract from a non-concentrated or concentrated sap, a samara fruit, a samara seed, a stems of leaf, a stem of a samara, a twig, a root, a leaf, a bark, a heartwood, a sapwood, a whole branch, a bark of a branch, a wood of a branch, a sugar a syrup, a syrup extract, a syrup-derived product, a rejection of syrup or syrup-derived product production, a residue of syrup or syrup-derived product production, or combinations thereof.
The syrup derived product may comprise butter, granulated sugar, hardened sugar, soft sugar, taffy, flakes, an extract from lyophilisation of a sap, a maple concentrate or a maple syrup, an extract from drying of a sap, a maple concentrate or a maple syrup, an extract from crystallization of a sap, a maple concentrate or a maple syrup, an extract from pulverization of a sap, a maple concentrate or a maple syrup, an extract from atomization of a sap, a maple concentrate or a maple syrup, an extract from centrigugation of a sap, a maple concentrate or a maple syrup or combinations thereof.
The syrup extract may be chosen from a methanol extract, a butanol extract, a butanol extract with sugar, a butanol extract without sugar, an ethyl acetate extract, an ethanol extract, a 95% ethanol/5% hot water extract, or combinations thereof.
The extract of Acer tree may comprise an extract from concentrated Acer tree water issued from reverse osmosis, a concentrated Acer tree water issued from pre-boiling nanofiltration, a pasteurized Acer tree water, a sterilized Acer tree water, an Acer tree water issued from a high pressure processing, an Acer tree water sterilized by UV irradiation, an Acer tree water sterilized by microwave irradiation and combinations thereof.
The extract of Acer tree may comprise an Acer tree molecule.
The Acer tree molecule may comprise:
The residue of syrup or syrup-derived product production may comprise diatomaceous earth, celite, kieselguhr, silica, silicon dioxide, calcium, natural sugar sand, ground bones, slop, clay and the like.
The Acer tree may be chosen from Acer nigrum, Acer lanum, Acer acuminatum, Acer albopurpurascens, Acer argutum, Acer barbinerve, Acer buergerianum, Acer caesium, Acer campbellii, Acer campestre, Acer capillipes, Acer cappadocicum, Acer carpinifolium, Acer caudatifolium, Acer caudatum, Acer cinnamomifolium, Acer circinatum, Acer cissifolium, Acer crassum, Acer crataegifolium, Acer davidii, Acer decandrum, Acer diabolicum, Acer distylum, Acer divergens, Acer erianthum, Acer erythranthum, Acer fabri, Acer garrettii, Acer glabrum, Acer grandidentatum, Acer griseum, Acer heldreichii, Acer henryi, Acer hyrcanum, Acer ibericum, Acer japonicum, Acer kungshanense, Acer kweilinense, Acer laevigatum, Acer laurinum, Acer lobelii, Acer lucidum, Acer macrophyllum, Acer mandshuricum, Acer maximowiczianum, Acer miaoshanicum, Acer micranthum, Acer miyabei, Acer mono, Acer mono×Acer truncatum, Acer monspessulanum, Acer negundo, Acer ningpoense, Acer nipponicum, Acer oblongum, Acer obtusifolium, Acer oliverianum, Acer opalus, Acer palmatum, Acer paxii, Acer pectinatum, Acer pensylvanicum, Acer pentaphyllum, Acer pentapomicum, Acer pictum, Acer pilosum, Acer platanoides, Acer poliophyllum, Acer pseudoplatanus, Acer pseudosieboldianum, Acer pubinerve, Acer pycnanthum, Acer rubrum, Acer rufinerve, Acer saccharinum, Acer saccharum, Acer sempervirens, Acer shirasawanum, Acer sieboldianum, Acer sinopurpurescens, Acer spicatum, Acer stachyophyllum, Acer sterculiaceum, Acer takesimense, Acer tataricum, Acer tegmentosum, Acer tenuifolium, Acer tetramerum, Acer trautvetteri, Acer triflorum, Acer truncatum, Acer tschonoskii, Acer turcomanicum, Acer ukurunduense, Acer velutinum, Acer wardii, Acer x peronai, and Acer x pseudoheldreichii.
The Acer tree may be chosen from Acer Saccharum and Acer Rubrum L.
The ingredient composition may be a food ingredient composition, a non-food ingredient composition, or combination thereof.
According to an embodiment, there is provided a method of seasoning a food comprising administering to a food an ingredient composition according to the present invention.
According to an embodiment, there is provided an Acer tree essential oil composition comprising
a hydrophobic fraction extracted from an Acer tree biomass; and
a suitable solvent.
The Acer tree biomass may be from at least one of a samara fruit, a samara seed, a stem of leaf, a stem of a samara, a twig, a root, a leaf, a bark, a heartwood, a sapwood, a whole branch, a bark of a branch, a wood of a branch.
The hydrophobic fraction extracted from an Acer tree biomass may comprise an Acer tree molecule.
The Acer tree molecule may comprise:
The suitable solvent may be at least one of ethanol, polyethylene glycol, or a pharmaceutically acceptable carrier oil.
The pharmaceutically acceptable carrier oil may be at least one of sweet almond oil, kukui nut oil, apricot kernel oil, macadamia nut oil, avocado oil, meadowfoam oil, borage seed oil, olive oil, camellia seed oil, peanut oil, cranberry seed oil, pecan oil, evening primrose oil, pomegranate seed oil, fractionated coconut oil, rose hip oil, grapeseed oil, seabuckthorn berry oil, hazelnut oil, sesame oil, hemp seed oil, sunflower oil, jojoba, and watermelon seed oil.
According to an embodiement, there is provided a method of preparing a maple syrup comprising adding a hydrophobic fraction extracted from an Acer tree biomass before or during the preparation of a maple syrup.
According to an embodiement, there is provided a food composition comprising:
The Acer tree samara may be germinated.
The Acer tree samara may comprise a fruit.
The Acer tree samara may comprise a seed.
The Acer tree samara may be dried and/or fermented.
The Acer tree samara may be dessicated.
The germinated Acer tree samara may be marinated.
The food composition may further comprise a seasoning ingredient chosen from a salt, a pepper, a cheese, an oil, a vinegar, a salad sauce, and a vinaigrette.
The salt may be chosen from sodium chloride, a sea salt, and sodium acetate.
According to an embodiment, there is provided a solid sweetening composition for oral consumption comprising:
a Acer tree sugar extract;
at least one sweetener, and
The Acer tree sugar extract may be at least one of non-concentrated or concentrated sap, syrup, a syrup, a syrup extract, a syrup-derived product, a rejection of syrup or syrup-derived product production, a residue of syrup or syrup-derived product production, taffy, flakes, sugar, spread, an extract from lyophilisation of a sap, a maple concentrate or a maple syrup, an extract from drying of a sap, a maple concentrate or a maple syrup, an extract from crystallization of a sap, a maple concentrate or a maple syrup, an extract from pulverization of a sap, a maple concentrate or a maple syrup, an extract from atomization of a sap, a maple concentrate or a maple syrup, an extract from centrifugation of a sap, a maple concentrate or a maple syrup or combinations thereof.
The syrup derived products may comprise butter, granulated sugar, hardened sugar, soft sugar, taffy, flakes, or combinations thereof.
The syrup extracts may be chosen from a methanol extract, a butanol extract, a butanol extract with sugar, a butanol extract without sugar, an ethyl acetate extract, an ethanol extract, a 95% ethanol/5% hot water extract, or combinations thereof.
The Acer tree sugar extract may comprise an Acer tree molecule.
The Acer tree molecule may comprise:
The residue of syrup or syrup-derived product production may comprise diatomaceous earth, celite, kieselguhr, silica, silicon dioxide, calcium, natural sugar sand, ground bones, slop, clay and the like.
The at least one sweetener may be chosen from a nutritive sweetener and a non-nutritive sweetener.
The nutritive sweetener may be at least one of honey, birch syrup, pine syrup, hickory syrup, poplar syrup, palm syrup, sugar beet syrup, sorghum syrup, corn syrup, cane syrup, golden syrup, barley malt syrup, a molasse, brown rice syrup, agave nectar, yacon syrup, fructose, maltitol, brown sugar, Okinawa syrup or combinations thereof.
The non-nutritive sweetener may be at least one of adenosine monophosphate, acesulfame potassium, alitame, aspartame, anethole, cyclamate, glycyrrhizin, lo han guo, miraculin, neotame, perillartine, saccharin, selligueain A, a Stevia rebaudiana extract, sucralose, thaumatin, neohesperdine DC, thavmatin, brazzein and inulin.
The Stevia rebaudiana extract may comprise at least one of stevioside, rebaudioside A, rebaudioside B, and rebaudioside C.
According to an embodiement, there is provided an infusion composition for the preparation of a beverage comprising:
The infusion composition may further comprise a herbal component.
The herbal component may be at least one of a tea, and a herbal tea.
The tea may be at least one of Bai Hao Yinzhen tea, Bai Mu Dan tea, Pai Mu Tan tea, Gong Mei tea, Shou Mei tea, White Puerh tea, Ceylon White tea, Darjeeling White tea, Assam White tea, African White tea, Junshan Yinzhen tea, Huoshan Huangya tea, Meng Ding tea, Huangya tea, Da Ye Qing tea, Huang Tang tea, Junshan Yinzhen tea, Longjing tea, Hui Ming tea, Long Ding tea, Hua Ding tea, Qing Ding tea, Gunpowder tea, Bi Luo Chun tea, Rain Flower tea, Shui Xi Cui Bo tea, Camellia Sinensis tea, Yu Lu tea, Xin Yang Mao Jian tea, Chun Mee tea, Gou Gu Nao tea, Yun Wu tea, Da Fang tea, Huangshan Maofeng tea, Lu'An Guapian tea, Hou Kui tea, Tun Lu tea, Huo Qing tea, Wuliqing tea, Hyson tea, Zhu Ye Qing tea, Meng Ding Can Lu tea, Genmaicha tea, Gyokuro tea, Kabusecha tea, Sencha tea, Fukamushicha tea, Tamaryokucha tea, Bancha tea, Kamairicha tea, Kukicha tea, Mecha tea, Konacha tea, Matcha tea, Genmaicha tea, Bancha tea, Höjicha tea, Tencha tea, Aracha tea, Shincha tea, funmatsucha tea or combinations thereof.
The herbal tea may be at least one of anise tea, artichoke tea, roasted barley tea, bee balm tea, boldo tea, cannabis tea, catnip tea, Hex causue leaves tea, cinnamon tea, coffee leaves tea, coffee cherry tea, Cerasse tea, dried chamomile blossoms tea, chrysanthemum tea, citrus peel tea, bergamot tea, orange peel tea, dandelion tea, dill tea, echinacea tea, essiac tea, fennel tea, gentian tea, ginger root tea, ginseng tea, hawthorn tea, hibiscus tea, rose hip tea, honeybush tea, horehound tea, hydrangea tea, Jiaogulan tea, Kapor tea, Kava root tea, Ku Ding tea, Labrador tea, Lapacho tea, lemon balm tea, lemon grass tea, licorice root tea, lime blossom tea, yerba mate tea, mate de coca tea, mint tea, european mistletoe tea, neem leaf tea, nettle leaf tea, asiatic pennywort leaf tea, pennyroyal leaf tea, pine tea, red raspberry leaf tea, scorched rice tea, rooibos tea, roselle petals, rosemary memory herb tea, sage tea, skullcap tea, serendib tea, sobacha, spicebush leaf tea, spruce tea, staghorn sumac fruit tea, stevia tea, St. John's Wort tea, tulsi tea, uncaria tomentosa, valerian tea, Verbena tea, vetiver tea, roasted wheat tea, wax gourd tea, Wong Logat tea, woodruff tea, yarrow tea, yerba mate tea, yuen kut lam kam wo tea or combinations thereof.
According to an embodiment, there is provided a method of infusing an infusion composition according to the present invention, wherein said infusion composition is infused with a maple tree based matrix.
The maple tree based matrix may be chosen from a maple tree sap, a concentrated maple tree water, a maple tree syrup.
According to an embodiement, there is provided a compound of formula (I), or a pharmaceutically acceptable salt thereof:
wherein R1 and R4 are each independently chosen from
R2 and R3 are each independently chosen from
According to an embodiment, there is provided a molecule consisting of:
According to an embodiment, there is provided a method of treating an ailment comprising treating a subject with a therapeutically effective amount of a compound according to the present invention.
The ailment may be a diabetes, a cancer, an arthritis, a micro-organism infection, a neurodegenerative disease, an inflammatory disease, an oxidative stress related disease, a heart disease, Alzheimer's diseases, a liver disorder a metabolic syndrome, a damaged hepatic function, a hepatic and liver dyslipidemia, a hepatitis, a liver cancer, an atherosclerosis, a hypertension, a skin disease, an eczema, and a psoriasis.
According to an embodiment, there is provided a use of a compound according to the present invention for the preparation of a medicament for the treatment of an ailment.
According to the present invention, there is provided a use of a compound according to the present invention for the treatment of an ailment.
The ailment may be chosen from a diabetes, a cancer, an arthritis, a micro-organism infection, a neurodegenerative disease, an inflammatory disease, an oxidative stress related disease, a heart disease, Alzheimer's diseases, a liver disorder a metabolic syndrome, a damaged hepatic function, a hepatic and liver dyslipidemia, a hepatitis, a liver cancer, an atherosclerosis, a hypertension, a skin disease, an eczema, and a psoriasis.
The following terms are defined below.
The term “sugar plant” is intended to mean any plant used in the production of sugar. Such plants include, without limitation, maple tree, birch tree, sugar cane, sugar beet and agave, palm tree, among others.
The expressions “any variety of maple tree” or “an Acer tree” is intended to mean a maple tree of a species known to date, such as Acer nigrum, Acer lanum, Acer acuminatum, Acer albopurpurascens, Acer argutum, Acer barbinerve, Acer buergerianum, Acer caesium, Acer campbeffii, Acer campestre, Acer capillipes, Acer cappadocicum, Acer carpinifolium, Acer caudatifolium, Acer caudatum, Acer cinnamomifolium, Acer circinatum, Acer cissifolium, Acer crassum, Acer crataegifolium, Acer davidii, Acer decandrum, Acer diabolicum, Acer distylum, Acer divergens, Acer erianthum, Acer erythranthum, Acer fabri, Acer garrettii, Acer glabrum, Acer grandidentatum, Acer griseum, Acer heldreichii, Acer henryi, Acer hyrcanum, Acer ibericum, Acer japonicum, Acer kungshanense, Acer kweilinense, Acer laevigatum, Acer laurinum, Acer lobelii, Acer lucidum, Acer macrophyllum, Acer mandshuricum, Acer maximowiczianum, Acer miaoshanicum, Acer micranthum, Acer miyabei, Acer mono, Acer mono×Acer truncatum, Acer monspessulanum, Acer negundo, Acer ningpoense, Acer nipponicum, Acer oblongum, Acer obtusifolium, Acer oliverianum, Acer opalus, Acer palmatum, Acer paxii, Acer pectinatum, Acer pensylvanicum, Acer pentaphyllum, Acer pentapomicum, Acer pictum, Acer pilosum, Acer platanoides, Acer poliophyllum, Acer pseudoplatanus, Acer pseudosieboldianum, Acer pubinerve, Acer pycnanthum, Acer rubrum, Acer rufinerve, Acer saccharinum, Acer saccharum, Acer sempervirens, Acer shirasawanum, Acer sieboldianum, Acer sinopurpurescens, Acer spicatum, Acer stachyophyllum, Acer sterculiaceum, Acer takesimense, Acer tataricum, Acer tegmentosum, Acer tenuifolium, Acer tetramerum, Acer trautvetteri, Acer triflorum, Acer truncatum, Acer tschonoskii, Acer turcomanicum, Acer ukurunduense, Acer velutinum, Acer wardii, Acer x peronai, Acer x pseudoheldreichii or any new species not yet known.
The expression “maple-derived” or “maple tree-derived” is intended to mean that the product is derived from any parts (such as bark, leaves, branches, roots, fruits etc.) or any fluid (such as sap) of a member of the Acer genus, as well as extracts obtained from these parts or fluids.
The expression “high pressure processing” is intended to mean the use of physical pressure rather than heat, chemical or irradiation.
The term “extract” is intended to mean any substance made by extracting a part of a raw material (e.g. plant material and/or fluids as defined herein). The extraction method may be by using a solvent such as ethanol or water, or from pulverization, atomization, crystallization, lyophilization, centrifugation, etc of raw materials and/or fluids. Extracts may in solid (e.g. powder) form, semi-solid, semi-liquid, or liquid form. For example, extracts may be from non-concentrated or concentrated sap, a samara fruit, a samara seed, a stems of leaf, a stem of a samara, a twig, a root, a leaf, a bark, a heartwood, a sapwood, a whole branch, a bark of a branch, a wood of a branch, a sugar, a syrup, a syrup extract, a syrup-derived product, a rejection of syrup or syrup-derived product production, a residue of syrup or syrup-derived product production, or combinations thereof.
Features and advantages of the subject matter hereof will become more apparent in light of the following detailed description of selected embodiments, as illustrated in the accompanying figures. As will be realized, the subject matter disclosed and claimed is capable of modifications in various respects, all without departing from the scope of the claims. Accordingly, the drawings and the description are to be regarded as illustrative in nature, and not as restrictive and the full scope of the subject matter is set forth in the claims.
Further features and advantages of the present disclosure will become apparent from the following detailed description, taken in combination with the appended drawings, in which:
In embodiments there are disclosed nutraceutical, functional food, food ingredients and non-food ingredients (ingredient having no nutritional value, but having other effects) composition as well as pure total or partial extracts. Neutraceuticals and functional food are food or food product that provides health and medical benefits, including the prevention and treatment of disease. Products according to the present invention may range from isolated nutrients, dietary supplements and specific diets and herbal products, and processed foods such as cereals, soups, and beverages. The composition of the present invention contain extracts of an Acer tree from sap, samara (including the fruits, the seeds as well as the stem), leaves (including the stem), twigs roots, heartwood and sap wood and/or the bark of the tree, or even maple syrup or maple sugar or maple sap, maple concentrate. The composition of the present invention also encompass synthetic reconstructions of extracts from Acer Saccharum or any other variety of maple tree. The composition of the present invention may be consumed for the prophylaxis of ailments such as diabetes, such as diabetes melitus, cancer, arthritis, micro-organism infection such as bacterial infections, neurodegenerative diseases, inflammatory diseases, oxidative stress related diseases, and heart diseases, as an anti-oxidant, anticancer, neurodegenerative diseases, Alzheimer's disease, liver disorders (such as metabolic syndrome, damaged hepatic function, hepatic and liver dyslipidemia, hepatitis, liver cancer), atherosclerosis, hypertension, and skin diseases (such as eczema, psoriasis and the likes).
In embodiments there is disclosed an Acer tree essential oil composition. An essential oil is a concentrated, hydrophobic liquid containing volatile aroma compounds from plants. An oil is essential in the sense that it carries a distinctive scent, or essence, of the plant. Essential oils do not as a group need to have any specific chemical properties in common, beyond conveying characteristic fragrances. Essential oils of the present invention are generally extracted by distillation, but they may be extracted by expression (i.e. pressing), or solvent extraction. The essential oil of the present invention may be used in perfumes, cosmetics, soap and other products, and even for flavouring food and drink, and for scenting incense and household cleaning products, and for use as anti-foaming agents in the production of maple syrup.
Various essential oils have been used medicinally at different periods in history. The essential oil of the present invention may be used in medical application ranging from skin treatments to remedies for cancer, and as cosmeceutical products (products that provide cosmetic functions as well as pharmaceutical functions), such as exfolients, masks, ointments, lotions, gels, creams, and the like.
The essential oils of the present invention contain the hydrophobic fraction extracted from an Acer tree biomass (sap, samara (including the fruits, the seeds as well as the stem), leaves (including the stem), twigs, roots, heartwood and sap wood, the bark of the tree.). Because of their concentrated nature, the essential oils of the present invention generally should not be applied directly to the skin in their undiluted form. They should rather be diluted in a suitable solvent, such as ethanol, polyethylene glycol, or a pharmaceutically acceptable carrier oil. Carrier oils are used to dilute essential and other oils prior to application. They “carry” the essential oil onto the skin. Suitable carrier oils include but are not limited to sweet almond oil, kukui nut oil, apricot kernel oil, macadamia nut oil, avocado oil, meadowfoam oil, borage seed oil, olive oil, camellia seed oil, peanut oil, cranberry seed oil, pecan oil, evening primrose oil, pomegranate seed oil, fractionated coconut oil, rose hip oil, grapeseed oil, seabuckthorn berry oil, hazelnut oil, sesame oil, hemp seed oil, sunflower oil, jojoba, and watermelon seed oil.
According to an embodiment of the persent invention, the essential oils of the present invention may be used as anti-foaming agents in the production of maple syrup. Other foaming agents are usually used in the production of maple syrup (e.g. vegetal oils certified “biologic”, with the exception of oils from soya, peanuts, nuts, or sesame seeds, due to their known allergenic potential). Thus, according to the present invention, essential oils extracted from maple tree biomass may be employed to replace these vegetal oils.
In embodiments there is disclosed a food product which comprises germinated and/or fermented Acer tree samara (including the fruits and the seeds). The samara from an Acer tree is a fruit in which a flattened wing of fibrous, papery tissue develops from the ovary wall. The samara is comestible and may be germinated to yield germinated an Acer tree samara.
Acer tree samara are produced naturally by maple trees as part of their reproductive cycle each year. They are produced in large quantities, most of which simply fall to the ground and degrade, representing a significant missed economical opportunity for sugar bush operators. Therefore, according to the present invention, Acer tree samara may be incorporated into food product in their germinated form, in food product such as salads accompanied with high quality oils or vinegars, salad sauces or vinaigrettes. They may even be incorporated in stir fries with meat and other vegatables.
According to the present invention the samara may also be marinated prior to consumption in vinegar, for example, or in any other suitable marinating solution which may preserve the marinated samara for extended periods of time.
According to the present invention, the seeds of the samara may also be dessicated to serve as as healthy food. Furthermore, oil may also be extracted from samara to be employed as a food with healthy properties.
In embodiments there is also disclosed a solid sweetening composition for oral consumption containing an Acer tree sugar extract; and other sweeteners. The Acer tree Saccharum sugar extract may be from maple syrup, maple taffy, flakes, maple sugar, maple spread. The sweetening composition may also be prepared from any synthetic source of maple sugars or from synthetic compositions recapitulating natural maple-derived products.
The sweetener may be chosen from a nutritive sweetener and a non-nutritive sweetener. Examples of nutritive sweeteners include but are not limited to honey, birch syrup, pine syrup, hickory syrup, poplar syrup, palm syrup, sugar beet syrup, sorghum syrup, corn syrup, cane syrup, golden syrup, barley malt syrup, a molasse, brown rice syrup, agave nectar, yacon syrup, fructose, maltitol, brown sugar, Okinawa syrup or combinations thereof.
The non-nutritive sweeteners may include but are not limited to adenosine monophosphate, acesulfame potassium, alitame, aspartame, anethole, cyclamate, glycyrrhizin, lo han guo, miraculin, neotame, perillartine, saccharin, selligueain A, a Stevia rebaudiana extract, sucralose, thaumatin neohesperdine DC, thavmatin, brazzein, and inulin. The Stevia rebaudiana extract may include stevioside, rebaudioside A, rebaudioside B, and rebaudioside C.
In use, the selection of certain maple-sugar, for example a maple taffy with other sweetener (e.g. stevia extract) allows for unique organoleptic qualities to be combined into novel combinations of natural sugars. These may present advantageous nutritional and tasteful qualities in a synergistic manner, and may therefore stimulate the individuals in unexpected manners.
In embodiments there is also disclosed an infusion composition for the preparation of a beverage. The infusion composition is provided in a dried form comprising an extract of Acer tree leaves, bark, roots, twigs of leaves or stems of samara, samara (fruits/seeds) and mixture thereof. The composition may also include other herbal component such as tea, and/or a herbal tea. The infusion composition of the present invention may comprise the extract of Acer tree leaves in combination with a number tea such as the following non-limiting examples including Bai Hao Yinzhen tea, Bai Mu Dan tea, Pai Mu Tan tea, Gong Mei tea, Shou Mei tea, White Puerh tea, Ceylon White tea, Darjeeling White tea, Assam White tea, African White tea, Junshan Yinzhen tea, Huoshan Huangya tea, Meng Ding tea, Huangya tea, Da Ye Qing tea, Huang Tang tea, Junshan Yinzhen tea, Longjing tea, Hui Ming tea, Long Ding tea, Hua Ding tea, Qing Ding tea, Gunpowder tea, Bi Luo Chun tea, Rain Flower tea, Shui Xi Cui Bo tea, Camellia Sinensis tea, Yu Lu tea, Xin Yang Mao Jian tea, Chun Mee tea, Gou Gu Nao tea, Yun Wu tea, Da Fang tea, Huangshan Maofeng tea, Lu'An Guapian tea, Hou Kui tea, Tun Lu tea, Huo Qing tea, Wuliqing tea, Hyson tea, Zhu Ye Qing tea, Meng Ding Gan Lu tea, Genmaicha tea, Gyokuro tea, Kabusecha tea, Sencha tea, Fukamushicha tea, Tamaryokucha tea, Bancha tea, Kamairicha tea, Kukicha tea, Mecha tea, Konacha tea, Matcha tea, Genmaicha tea, Bancha tea, Höjicha tea, Tencha tea, Aracha tea, Shincha tea, funmatsucha tea or combinations thereof.
The infusion composition may also include herbal tea which include but are not limited to anise tea, artichoke tea, roasted barley tea, bee balm tea, boldo tea, cannabis tea, catnip tea, Ilex causue leaves tea, cinnamon tea, coffee leaves tea, coffee cherry tea, Cerasse tea, dried chamomile blossoms tea, chrysanthemum tea, citrus peel tea, bergamot tea, orange peel tea, dandelion tea, dill tea, echinacea tea, essiac tea, fennel tea, gentian tea, ginger root tea, ginseng tea, hawthorn tea, hibiscus tea, rose hip tea, honeybush tea, horehound tea, hydrangea tea, Jiaogulan tea, Kapor tea, Kava root tea, Ku Ding tea, Labrador tea, Lapacho tea, lemon balm tea, lemon grass tea, licorice root tea, lime blossom tea, yerba mate tea, mate de coca tea, mint tea, european mistletoe tea, neem leaf tea, nettle leaf tea, asiatic pennywort leaf tea, pennyroyal leaf tea, pine tea, red raspberry leaf tea, scorched rice tea, rooibos tea, roselle petals, rosemary memory herb tea, sage tea, skullcap tea, serendib tea, sobacha, spicebush leaf tea, spruce tea, staghorn sumac fruit tea, stevia tea, St. John's Wort tea, tulsi tea, uncaria tomentosa, valerian tea, Verbena tea, vetiver tea, roasted wheat tea, wax gourd tea, Wong Logat tea, woodruff tea, yarrow tea, yerba mate tea, yuen kut lam kam wo tea or combinations thereof.
According to an embodiment, the infusion composition according to the present invention may be infused with a maple tree based matrix, such as a maple tree sap, a concentrated maple tree water, a maple tree syrup. The maple tree based matrix is believed to act as a nutriprotective carrier.
In embodiments, there is also disclosed a compound of formula (I), or a pharmaceutically acceptable salt thereof:
where R1 and R4 are each independently chosen from
In embodiments, there is also disclosed molecules consisting of:
The compounds of formula (I) as well as the molecules are believed to be useful for the treatment of ailments, such as diabetes, cancers, arthritis, micro-organism infections, neurodegenerative diseases, inflammatory diseases, oxidative stress related diseases, heart diseases, Alzheimer's diseases, liver disorders, a metabolic syndromes, damaged hepatic functions, hepatic and liver dyslipidemias, hepatitis, liver cancers, atherosclerosis, hypertensions, skin diseases, eczema, and psoriasis.
The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.
The objective of the current example is to evaluate the ability of phenolic-enriched extracts of Canadian maple syrup, namely ethyl acetate (MS-EtOAc) and butanol (MS-BuOH), to inhibit carbohydrate hydrolyzing enzymes relevant to type 2 diabetes management.
Extracts are standardized to phenolic contents by the Folin-Ciocalteau method and assayed for yeast α-glucosidase inhibitory activities. On normalization to phenolic content, MS-BuOH exhibited higher inhibitory activity than MS-EtOAc (IC50=68.38 and 107.9 μg phenolics, respectively). The extracts are further assayed for inhibition of porcine α-amylase and rat α-glucosidase enzymes. MSBuOH exhibited higher rat α-glucosidase and porcine α-amylase inhibitory activities (IC50=135 and 103 μg phenolics, respectively) than MS-EtOAC extract (IC50>187 μg phenolics in both assays). These results suggest that maple syrup extracts have potential for phenolic-mediated type 2 diabetes management, with the MS-BuOH phenolic-enriched fraction having highest bioactivity.
Type 2 diabetes accounts for about 90-95% of all diagnosed cases of diabetes in adults (Centers for Disease Control and Prevention, 2010). Worldwide, at least 220 million people have diabetes and this figure is estimated to double by 2030 (World Health Organization, 2010). In the United States alone, in 2007, 23.7 million people (10% of American adults) had diabetes and this figure is expected to jump to 33% (i.e. one-third of all American adults) by 2050 (Centers for Disease Control and Prevention, 2010). Remarkably, the cost to manage diabetes by Americans in 2007 is $174 billion and this figure is expected to skyrocket based on the CDC's latest estimates (Centers for Disease Control and Prevention, 2010). Thus type 2 diabetes poses a major public health challenge with significant health care costs and burden.
The major source of blood glucose is hydrolyzed dietary carbohydrates such as starches. Dietary carbohydrates are hydrolyzed by pancreatic α-amylase with absorption aided by α-glucosidases in order to be absorbed by the small intestine (Elsenhans & Caspary, 1987). It is believed that inhibition of these enzymes can be an important strategy for management of type 2 diabetes (Krentz & Bailey, 2005).
Large polysaccharides (starch) are broken down by α-amylase which acts upon their internal bonds. Natural α-amylase inhibitors offer an attractive therapeutic approach to the treatment of postprandial hyperglycemia by ultimately decreasing glucose release from starch. The α-glucosidase enzyme catalyzes the final step of glucose absorption in the small intestine during the digestive process of carbohydrates, and hence α-glucosidase inhibitors could retard the rapid utilization of dietary carbohydrates and suppress postprandial hyperglycemia (Watanabe, Kawabata, Kurihara, & Niki, 1997). The clinical use of drug-inhibitors such as acarbose has been attempted for diabetic or obese patients. Acarbose has been shown to effectively reduce the intestinal absorption of sugars in humans (Cheng & Fantus, 2005; Jenkins et al., 1981). Recently, it has been shown that plant derived phenolics play a role in mediating α-glucosidase and α-amylase inhibition and thus have potential to contribute to the management of type 2 diabetes (Apostolidis, Kwon, & Shetty, 2006; Hogan et al., 2010; Kwon, Apostolidis, Kim, & Shetty, 2007; Kwon, Vattem, & Shetty, 2006).
Maple syrup is a natural sweetener and is the largest commercially available food product that is totally derived from the sap of deciduous trees. It is obtained by concentrating the sap collected from certain maple species including the sugar maple tree (Acer saccharum Marsh.) which is native to North America (Ball, 2007; Perkins & van der Berg, 2009). Maple syrup is produced primarily in North America with the vast majority of the world's supply coming from Canada (85%; primarily Quebec), followed by United States (Perkins & van der Berg, 2009). Previous reports have shown that maple syrup contains a wide variety of phenolic phytochemicals (Abou-Zaid, Nozzolillo, Tonon, Coppens, & Lombardo, 2008; Filipie & Underwood, 1964; Kermasha, Goetghebeur, & Dumont, 1995; Li & Seeram, 2010; Potter & Fagerson, 1992), which may have positive effects on human health. Recently, phenolic-enriched extracts of maple syrup are shown to have antioxidant, anti-mutagenic and human cancer cell anti-proliferative properties (Legault, Girard-Lalancette, Grenon, Dussault, & Pichette, 2010; Li & Seeram, 2010; Theriault, Caillet, Kermasha, & Lacroix, 2006). In addition, Honma, Koyama, and Yazawa (2010) reported that A. saccharum leaf extracts had phenolic-mediated potential for type 2 diabetes management via inhibition of the carbohydrate hydrolyzing enzyme-glucosidase.
A comprehensive evaluation of different sweeteners (including sugar cane, brown sugar, date sugar and corn syrup, among others) showed a phenolic-dependent α-glucosidase inhibitory activity, however, maple syrup is not evaluated (Ranilla, Kwon, Genovese, Lajolo, & Shetty, 2008). In addition, Ranilla et al. (2008) used crude water extracts, without previous removal of sugars that could possibly lead to substrate-related inhibition in the α-glucosidase bioassay. The objective of the current document is to evaluate the type 2 diabetes management potential, via inhibition of carbohydrate hydrolyzing enzymes, of phenolic-enriched extracts of maple syrup (namely, ethyl acetate and butanol) in which sugars are previously removed. For the identification of potential maple syrup compounds having type 2 diabetes management potential, it is important to determine the effects of different phenolic-enriched maple syrup extracts. This is because various organic solvents used for extraction of maple syrup will result in a different profile of phenolic compounds (Li & Seeram, 2010). Maple syrup is a plant-derived natural sweetener that contains a wide variety of natural phenolic compounds beyond its sugars (predominantly as sucrose). Thus, identification of the relevant maple syrup-derived compounds for type 2 diabetes management requires the evaluation of various maple syrup extracts that contains different phenolic profiles. In the present document, the potential of phenolic-standardized maple syrup extracts for type 2 diabetes management is reported for the first time. A clear knowledge of the relevant maple syrup compounds that contribute towards sugar absorption management in the gastrointestinal tract could potentially lead to the design of natural sweeteners with lower glycemic index.
Materials and Methods
Maple syrup (grade C) is provided by the Federation of Maple Syrup Producers of Quebec (Canada). The syrup is kept frozen in the laboratory until extraction. High performance liquid chromatography (HPLC) is performed on a Hitachi Elite LaChrom system consisting of a L2130 pump, L-2200 autosampler, and a L-2455 Diode Array Detector all operated by EZChrom Elite software. All solvents are of either ACS or HPLC grade and are purchased from Wilkem Scientific (Pawtucket, R.I.). α-Amylase (porcine pancreatic, EC 3.2.1.1), α-glucosidase (yeast, EC 3.2.1.20) and rat intestinal powder are purchased from Sigma-Aldrich (St. Louis, Mo.). Unless otherwise specified, all other chemicals are purchased from Sigma-Aldrich.
Sample Preparation
Preparation of phenolic-enriched extracts of maple syrup has been previously reported (Li & Seeram, 2010). Briefly, maple syrup (20 L) is subjected to liquid-liquid partitioning with ethyl acetate (10 L×3) and n-butanol (10 L×3) successively. The combined ethyl acetate extracts are dried under reduced pressure and accurate weights are obtained as 4.71 g (MS-EtOAc). After concentration, remaining sugars are removed from the combined n-butanol extracts (108 g) by further extraction with methanol (100 mL×3) at room temperature to afford 57 g (MS-BuOH). All samples are standardized to solid content of 125 mg/mL for further assaying.
Standardization of Maple Syrup Extracts
Standardization based on total phenolic content. Total phenolic content is determined according to the Folin-Ciocalteu's method and are measured as gallic acid equivalents (GAEs) as previously reported (Singleton & Esau, 1969). Briefly, the extracts are appropriately diluted with methanol/H2O (1:1, v/v), and 200 μL of sample is incubated with 3 mL of methanol/H2O (1:1, v/v) and 200 μL of Folin-Ciocalteau reagent for 10 min at 25° C. After this, 600 μL of 20% Na2CO3 solution is added to each tube and vortexed. Tubes are further incubated for 20 min at 40° C. After incubation, samples are immediately cooled in an ice bath to room temperature. Samples and standards (gallic acid) are processed identically and all tests are performed in triplicate. The absorbance is read at 755 nm, and the total phenolic content is calculated from the standard curve obtained from a Spectramax plate reader (Molecular Devices, Sunnyvale, Calif., USA).
Standardization Based on HPLC-UV Analyses.
The HPLC-UV analyses are carried out as previously reported (Li & Seeram, 2010). Briefly, a Luna C18 column (250×4.6 mm i.d., 5 μM; Phenomenex) with a flow rate at 0.75 ml/min and injection volume of 20 μL for both extracts is used. The extracts are dissolved in dimethylsulphoxide (DMSO) and analyzed at equivalent phenolic contents. A gradient solvent system consisting of solvent A (0.1% aqueous trifluoroacetic acid) and solvent B (methanol, MeOH) is used as follows: 0-10 min, from 10% to 15% B; 10-20 min, 15% B; 20-40 min, from 15% to 30% B; 40-55 min, from 30% to 35% B; 55-65 min, 35% B; 65-85 min, from 35% to 60% B; 85-90 min, from 60% to 100% B; 90-93 min, 100% B; 93-94 min, from 100% to 10% B; 94-104 min, 10% B.
Antioxidant Activity by 1,1-diphenyl-2-picrylhydrazyl (DPPH) Radical Inhibition Assay
The antioxidant potentials of MS-EtOAC and MS-BuOH are determined on the basis of the ability to scavenge the DPPH radicals as previously described (Nanjo et al., 1996). The DPPH radical scavenging activity of ascorbic acid (vitamin C) and the synthetic commercial antioxidant, butylated hydroxytoluene (BHT), are also assayed as positive controls. The assay is conducted in a 96-well format using serial dilutions of 100 μL aliquots of test compounds (ranging from 2500 to 26 μg/mL), ascorbic acid (1000-10.4 μg/mL), and BHT (250,000-250 μg/mL). Then DPPH (150 μL) is added to each well to give a final DPPH concentration of 137 Absorbance is read after 30 min at 515 nm, and the scavenging capacity (SC) is calculated as SC %=[(A0−A1/A0]×100, where A0 is the absorbance of the reagent blank and A1 is the absorbance with test samples. All tests are performed in triplicate. IC50 values denote the concentration of sample required to scavenge 50% DPPH radicals.
Carbohydrate Hydrolysis Enzyme Inhibition Assays
Since phenolic phytochemicals have been extensively shown to have α-glucosidase inhibitory activity (Apostolidis & Lee, 2010; Apostolidis et al., 2006; Hogan et al. 2010; Honma et al., 2010; Khan, Tiwari, Ahmad, Srivastava, & Tripathi, 2004; Kwon et al., 2006, 2007), the extracts are standardized to a phenolic content of 3.75 mg GAE/mL to be evaluated on the same basis using the assay below.
Yeast α-glucosidase Inhibition Assay.
A mixture of 50 μL of extract and 100 μL of 0.1M phosphate buffer (pH 6.9) containing yeast α-glucosidase solution (1.0 U/ml) is incubated in 96 well plates at 25° C. for 10 min. After pre-incubation, 50 μL of 5 mM p-nitrophenyl-α-D-glucopyranoside solution in 0.1M phosphate buffer (pH 6.9) is added to each well at timed intervals. The reaction mixtures is incubated at 25° C. for 5 min. Before and after incubation, absorbance is recorded at 405 nm by a micro-plate reader (VMax, Molecular Device Co., Sunnyvale, Calif., USA) and compared to that of the control which had 50 μL buffer solution in place of the extract. The α-glucosidase inhibitory activity is expressed as inhibition % and is calculated as follows:
Rat α-glucosidase Inhibition Assay.
To validate the yeast α-glucosidase inhibition results, the rat α-glucosidase assay with the fractions that resulted at the highest inhibition is used Rat intestinal α-glucosidase assay is referred to the method of Kwon et al. (2007) with a slight modification. A total of 1 g of rat-intestinal acetone powder is suspended in 10 mL of 0.9% saline, and the suspension is sonicated twelve times for 30 s at 4° C. After centrifugation (10,000 g, 30 min, 4° C.), the resulting supernatant is used for the assay. Sample solution (50 μL) and 0.1M phosphate buffer (pH 6.9, 100 μL) containing α-glucosidase solution is incubated at 25° C. for 10 min. After preincubation, 5 mM p-nitrophenyl-α-D-glucopyranoside solution (50 μL) in 0.1M phosphate buffer (pH 6.9) is added to each well at timed intervals. The reaction mixtures are incubated at 25° C. for 30 min and readings are recovered every 5 min. Before and after incubation, absorbance is read at 405 nm and compared to a control which has 50 μL of buffer solution in place of the extract by micro-plate reader. The α-glucosidase inhibitory activity is expressed as inhibition % and is calculated as follows:
Porcine α-amylase Inhibition Assay.
A mixture of 50 μL of extract or acarbose and 50 μL 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M sodium chloride) containing α-amylase solution (13 U/mL) is incubated at 25° C. for 10 min using an 1.5 mL Eppendorf tube. After pre-incubation, 50 μL 1% soluble starch solution in 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) is added to each well at timed intervals. The reaction mixtures are then incubated at 25° C. for 10 min followed by addition of 1 mL dinitrosalicylic acid colour reagent. The test tubes are then placed in a boiling water bath for 10 min to stop the reaction and cooled to room temperature. The reaction mixture is then diluted with 1 mL distilled water and absorbance is read at 540 nm using a 96-well mircoplate reader.
Statistical Analysis
All experiments are performed twice and analysis for each experiment is carried out in triplicate. Means, standard deviations, the degree of significance (p<0.05 —One way ANOVA and t-test) are determined using Microsoft Excel XP. Inhibition concentration (IC50) values are calculated using ED50plus vol. 1 developed by Vargas
Results and Discussion
Phenolic Content of Maple Syrup Extracts and their Antioxidant Activity
On a dry weight (DW) basis, the MS-EtOAc extract has the highest total phenolic content (340 mg/g DW) followed by the MS-BuOH (30 mg/g DW) extract (Table 1). Similarly, for the antioxidant activity as measured by the DPPH radical scavenging assay, the MS-EtOAc extract exhibits higher antioxidant activity (IC50=77.5 ppm) compared to the MS-BuOH fractions (IC50>1000 ppm) (Table 1).
The HPLC-UV chromatograms of MS-EtOAc and MS-BuOH (shown in
It is previously reported that the ethyl acetate extract of maple syrup contains a wide variety of phenolic phytochemicals including small phenolic molecules and flavonoids, predominantly as flavonols and flavanols (Abou-Zaid et al., 2008; Kermasha et al., 1995). Recently, it has been reported that the butanol extract of maple syrup (MS-BuOH) predominantly contained lignans, coumarins, and a stilbene, along with several previously reported small phenolic compounds (Li & Seeram, 2010). It should be noted that similar to other food matrices, the utilization of different organic solvents for extraction of maple syrup yields extracts with differing phenolic profiles. While both MS-EtOAc and MS-BuOH contain predominantly phenolic compounds, their individual phenolic constituents are quite different as previously reported (Abou-Zaid et al., 2008; Li & Seeram, 2010).
Yeast/Rat α-Glucosidase and Porcine α-Amylase Inhibition Assay
The extracts are standardized to phenolic content of 3.75 mg GAE/mL and assayed for yeast α-glucosidase inhibition. Both extracts have a dose-dependent α-glucosidase inhibitory activity with the MS-BuOH having highest (82% at highest dose, IC50 68.38 μg phenolics) followed by MS-EtOAc (67% at highest dose, IC50 107.9 μg phenolics) (
The above findings indicate that when the extracts are evaluated at equivalent phenolic content, the MS-BuOH exhibited higher α-glucosidase inhibition potential in the yeast based assay (
The phenolic standardized MS-BuOH and MS-EtOAc extracts are further assayed for α-amylase inhibition in a porcine based assay. At the test concentrations, the MS-EtOAc extract has no inhibitory activity (IC50>187 μg) while the MS-BuOH extract has α-amylase inhibition with IC50=103 μg phenolics (
Phenolic compounds are secondary metabolites of plant origin which constitute one of the most abundant and ubiquitous groups of natural metabolites and form an important part of both human and animal diets (Bravo, 1998; Crozier et al., 2000; Vattem, Ghaedian, & Shetty, 2005). Many studies have shown that phenolic phytochemicals have high antioxidant activity and other biological properties (Al-Farsi, Alsalvar, Morris, Baron, & Shahidi, 2005; Seeram et al., 2005; Shahidi & Ho, 2005; Yahia, 2010). Various researchers have identified the phenolic constituents of maple syrup in different extracts (Abou-Zaid et al., 2008; Filipie & Underwood, 1964; Kermasha et al., 1995; Li & Seeram, 2010; Potter & Fagerson, 1992) and are further related to antioxidant (Legault et al., 2010; Li & Seeram, 2010; Theriault et al., 2006; Yosikawa, Kawahara, Arihara, & Hashimoto, 2010) human cancer cell antiproliferative (Legault et al., 2010; Theriault et al., 2006) and anti-inflammatory properties (Legault et al., 2010). The present document show that maple syrup phenolic-enriched extracts have type 2 diabetes management potential, via inhibition of carbohydrate hydrolyzing enzymes, with the MSBuOH fraction having the highest bioactivity. There are therefore potential bioactivities unique to the MS-BuOH phenolic phytochemicals in relation to type 2 diabetes management.
During the production of maple syrup, apart from natural phenolic constituents, other unique phenolic and non-phenolic compounds are formed during the intensive heating involved in transforming sap into syrup (Li & Seeram, 2010). For example, a novel process-derived phenolic compound in MS-BuOH has recently been identified (Li & Seeram, 2011). Thus these process-derived compounds may impart additional biological effects to maple syrup, and therefore contribute to the observed health benefits and biological activities of maple syrup.
The present document is the first report of the type 2 diabetes management potential of maple syrup. These findings indicate that compared to MS-EtOAC, the MS-BuOH is the most active extract and it has a particular phenolic profile and related bioactivities. The understanding of the mechanism of action and identification of compounds responsible for the observed α-glucosidase and α-amylase inhibitory activities coupled with animal and clinical trials could lead to the development of a maple syrup sweetener with lower glycemic index designed for type 2 diabetes prevention.
The objective of the current example is to evaluate In Vitro Phenolic-Mediated Anti-hyperglycemic Properties of Sugar and Red Maple Leaf Extracts.
Red maple and sugar maple (Acer rubrum and Acer saccharum, respectively) leaves are collected in the summer and fall of 2010 from Canada and are evaluated for seasonal variation in terms of phenolic contents, antioxidant activities, and α-glucosidase and α-amylase inhibitory activities, relevant to type 2 diabetes management. Dried leaves are extracted in methanol, dried under vacuum and suspended in DMSO. The phenolic contents of summer red maple leaves (RML-S) and summer sugar maple leaves (SML-S) are higher than red and sugar maple leaves collected in the fall (RML-F and SML-F, respectively). The extracts are also assayed for α-glucosidase inhibitory activities with SML-S extracts having the highest inhibitory activity (IC50=21 μg/mL). The α-glucosidase inhibitory activities are dependent on both phenolic content and phenolic profile. When the α-amylase inhibitory activity is evaluated, a non-phenolic dependent seasonal variation is observed only with red maple leaves, with RML-F having the highest inhibitory activity (IC50 7.3 mg/mL). These results show that sugar and red maple leaf extracts have potential for phenolic-mediated α-glucosidase inhibition, relevant to type 2 diabetes management, with SML-S extract having the highest bioactivity, which could be related to unique phenolic compounds identified in this research.
Introduction
Non-insulin dependent diabetes mellitus, a common disorder of glucose and fat metabolism, is strongly associated with diets high in calories and linked to changes in dietary pattern towards high calorie sweetened foods with disaccharides such as maltose and sucrose (Garg et al. 1994). Worldwide, at least 220 million people have diabetes and this figure is estimated to double by 2030 (World Health Organization 2011). In the United States alone, in 2007, 23.7 million people (10% of American adults) had diabetes and this figure is expected to jump to 33% (i.e. one-third of all American adults) by 2050 (Center for Disease Control 2011).
Hyperglycemia is a condition characterized by a rapid rise in blood glucose levels subsequent to hydrolysis of starch by pancreatic α-amylase and intestinal α-glucosidase-mediated absorption of glucose in the small intestine. One of the therapeutic approaches for decreasing postprandial hyperglycemia is to retard absorption of glucose by the inhibition of carbohydrate hydrolyzing enzymes, α-amylase and α-glucosidase, in the digestive organs (Deshpande et al. 2009). Therefore, inhibition of these enzymes can significantly decrease the postprandial hyperglycemia after a mixed carbohydrate diet and can be a key strategy in the control of diabetes mellitus (Hirsh et al. 1997). Recent research findings have shown that plant-derived phenolics play a role in mediating α-glucosidase and α-amylase inhibition and thus have potential to contribute to the management of type-2 diabetes (Hogan et al. 2010; Apostolidis et al. 2011a; Apostolidis et al. 2011b; Apostolidis and Lee 2010; Kwon et al. 2007).
Maple syrup is a natural sweetener and is the largest commercially available food product that is totally derived from the sap of deciduous trees. It is obtained by concentrating the sap collected from certain maple species including the sugar maple (Acer saccharum Marsh.) and red maple (Acer rubrum L.) trees which are both native to North America (Ball 2007; Van Den Berg and Perkins 2007). Maple syrup is produced primarily in North America with the vast majority of the world's supply coming from Canada (85%; primarily Quebec), followed by United States (Perkins and Van Der Berg 2009). Previous reports have shown that maple syrup contains a wide variety of phenolic phytochemicals (Li and Seeram 2011; Li and Seeram 2010; Abou-Zaid et al. 2008), which may have positive effects on human health. Recently, phenolic-enriched extracts of maple syrup are shown to have antioxidant, anti-mutagenic and human cancer cell anti-proliferative properties (Li and Seeram 2011; Li and Seeram 2010; Legault et al. 2010; Theriault et al. 2006). In addition, Apostolidis et al. (2011a) reported that maple syrup extracts had phenolic-mediated potential for type 2 diabetes management via inhibition of the carbohydrate hydrolyzing enzyme α-glucosidase.
The sugar maple and red maple species are native to Northeastern American forests and their leaves are responsible for most of the red and orange autumn coloration of these forests. The variation in color pigmentation occurs due to changes in three plant pigments among the trees (Schaberg et al. 2008). Two of these classes of pigments, chlorophylls that appear green and carotenoids that appear yellow, are synthesized during the growing season to enable or protect photosynthetic light capture (Schaberg et al. 2008). In contrast, anthocyanin pigments that give leaves a red or purple color are often synthesized toward the end of the leaf's lifespan (Field et al. 2001; Matile 2000). Anthocyanins are a non-functional by-product of leaf senescence (Archetti 2000; Matile 2000). Their biosynthesis is induced due to exposure to a wide variety of stresses such as UV-B radiation (Mendez at al. 1999), osmotic stress (Kaliamoorthy and Rao 1994), drought (Balakumar at al. 1993), low temperatures (Krol et al. 1995), nutrient deficiencies (Rajendran et al. 1992), wounding (Ferreres et al. 1997), pathogen infection (Dixon et al. 1994) and exposure to ozone (Foot et al. 1996). The observed anthocyanin buildup following exposure to stress raises the possibility that anthocyanins may function, in part, to prevent stress-induced damage. The phenolic variation in sugar maple leaves harvested in fall (autumn) and summer is evaluated by Baldwin et al. (1987) and the results showed that during fall, sugar maple leaves have higher phenolic contents when compared to summer.
Although sugar and red maple belong to the same family (Aceraceae) of trees, their leaf phenolic profile has certain differences and similarities. Both red and sugar maple leaves contain small amounts of methyl gallate (Abou-Zaid et al. 2009), while only red maple contains a rare galloyl sugar, galloyl rhamnose (Abou-Zaid and Nozzolillo 1995). In addition, red maple is highly resistant to forest tent caterpillar, in contrast to sugar maple, due to the presence of ethyl-m-digallate at high amount (Abou-Zaid et al. 2001). Recently, Honma et al (2010) reported that Sugar maple leaf extracts had phenolic-mediated potential for type 2 diabetes management via inhibition of the carbohydrate hydrolyzing enzyme α-glucosidase and identified acertanin (ginnalin A) as the active compound. On the other hand, Japanese red maple (Acer pycnanthum K. Koch) appeared to have similar effect against type 2 diabetes, but the active compounds for the observed effect are identified as ginnalins B and C (Honma et al. 2011).
The phenolic polymorphism of red and sugar maple leaves and the potential health benefits that derive from them have significant applications. The aim of this document is to evaluate the differences between sugar and red maple leaves, collected in summer and fall in terms of phenolic contents, antioxidant activities, and type 2 diabetes management via inhibition of the carbohydrate hydrolysis enzymes, α-glucosidase and α-amylase.
Materials and Methods
General Experimental Procedures.
High performance liquid chromatography (HPLC) is performed on a Hitachi Elite LaChrom system consisting of a L2130 pump, L-2200 autosampler, and a L-2455 Diode Array Detector all operated by EZChrom Elite software. All solvents are of either ACS or HPLC grade and are purchased from Wilkem Scientific (Pawtucket, R.I.). α-Amylase (porcine pancreatic, EC 3.2.1.1), α-glucosidase (yeast, EC 3.2.1.20) and rat intestinal powder are purchased from Sigma-Aldrich (St. Louis, Mo.). Unless otherwise specified, all other chemicals of analytical grade are purchased from Sigma-Aldrich.
Sample Preparation and HPLC Phenolic Profiling.
Sugar maple leaves (SML) and red maple leaves (RML) are collected in Canada in 2010 during summer (SML-S and RML-S) and fall (SML-F and RML-F) and are to the laboratory as previously reported (González-Sarrias et al. 2011). Voucher specimens are deposited at the Bioactive Botanical Research Laboratory in the University of Rhode Island, R.I., USA. Leaves are kept frozen in the laboratory until extraction. Air-dried leaves of red maple and sugar maple (8.5 g) are extracted by sonication with methanol (150 mL) at room temperature for 40 min. The methanol extracts are dried under reduced vacuum. The SML and RML leaf extracts (10 mg/mL in DMSO) are injected into HPLC system with a Luna C18 column (250×4.6 mm i.d., 5 μM; Phenomenex) and 15 μL injection volume. A gradient solvent system consisting of solvent A (0.1% aqueous trifluoroacetic acid) and solvent B (methanol, MeOH) is used at a flow rate of 0.75 mL/min as follows: 0-30 min, 10% to 60% B; 30-35 min, 60% to 100% B; 35-40 min, 100% B; 40-41 min, 100% to 10% B; 41-51 min, 100% B. A linear standard curve between ginnalin A concentration and UV absorbance area at 280 nm is constructed for quantification and standardization purposes (r2=0.9942).
Total Phenolic Content.
The total phenolics are determined following the procedure modified from Shetty et al (1995). Briefly, 1 mL extract is transferred into a test tube and mixed with 1 mL 95% ethanol and 5 mL distilled water. To each sample, 0.5 mL 50% (v/v) Folin-Ciocalteu reagent is added and vortex mixed. After 5 min, 1 mL 5% Na2CO3 is added to the reaction mixture and allowed to stand for 60 min. The absorbance is read at 725 nm using a Thermo Scientific Genesys 10uv spectrophotometer (Madison, Wis.). The absorbance values are converted to total phenolics and are expressed in mg gallic acid/g sample dry weight (DW). A standard curve is established using varying concentrations of gallic acid in ethanol.
Antioxidant Assay.
The antioxidant potential of the extracts is determined on the basis of the ability to scavenge the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals as previously described (Nanjo et al. 1996). The DPPH radical scavenging activity of ascorbic acid (vitamin C) and the synthetic commercial antioxidant, butylated hydroxytoluene (BHT), are also assayed as positive controls. The assay is conducted in a 96-well format using serial dilutions of 100 μL aliquots of test compounds (ranging from 2500 to 26 μg/mL), ascorbic acid (1000-10.4 μg/mL), and BHT (250,000-250 μg/mL). Then DPPH (150 μL) is added to each well to give a final DPPH concentration of 137 μM. Absorbance is read after 30 min at 515 nm, and the scavenging capacity (SC) is calculated as SC %=(A0−A1/A0)×100, where A0 is the absorbance of the reagent blank and A1 is the absorbance of test samples. All tests are performed in triplicate. IC50 value denotes the concentration of sample required to scavenge 50% of DPPH radicals.
Yeast α-glucosidase Inhibition Assay.
A mixture of 50 μL of extract and 100 μl of 0.1 M phosphate buffer (pH 6.9) containing yeast α-glucosidase solution (1.0 U/ml) is incubated in 96 well plates at 25° C. for 10 min. After pre-incubation, 50 μl of 5 mM p-nitrophenyl-α-D-glucopyranoside solution in 0.1M phosphate buffer (pH 6.9) is added to each well at timed intervals. The reaction mixtures are incubated at 25° C. for 5 min. Before and after incubation, absorbance is recorded at 405 nm by a micro-plate reader (VMax, Molecular Device Co., Sunnyvale, Calif., USA) and compared to that of the control which had 50 μL buffer solution in place of the extract. The α-glucosidase inhibitory activity is expressed as % inhibition and is calculated as follows:
Rat α-glucosidase Inhibition Assay.
The rat intestinal α-glucosidase assay is conducted according to the method of Kwon et al (2007) with a slight modification. A total of 1 g of rat-intestinal acetone powder is suspended in 10 mL of 0.9% saline, and the suspension is sonicated twelve times for 30 sec at 4° C. After centrifugation (10000×g, 30 min, 4° C.), the resulting supernatant is used for the assay. Sample solution (50 μL) and 0.1 M phosphate buffer (pH 6.9, 100 μL) containing α-glucosidase solution is incubated at 25° C. for 10 min. After preincubation, 5 mM p-nitrophenyl-α-D-glucopyranoside solution (50 μL) in 0.1M phosphate buffer (pH 6.9) using a multi-channel pipette. The reaction mixtures are incubated at 25° C. Before and after incubation, absorbance is read at 405 nm and compared to a control which had 50 μL of buffer solution in place of the extract by micro-plate reader. The α-glucosidase inhibitory activity is expressed as % inhibition and is calculated as follows:
Porcine α-amylase Inhibition Assay.
A mixture of 50 μL of extract and 50 μL 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M sodium chloride) containing α-amylase solution (13 U/mL) is incubated at 25° C. for 10 min using an 1.5 mL Eppendorf tube. After pre-incubation, 50 μL of 1% soluble starch solution in 0.02 M sodium phosphate buffer (pH 6.9 with 0.006 M NaCl) is added to each well at timed intervals. The reaction mixtures are then incubated at 25° C. for 10 min followed by addition of 1 mL dinitrosalicylic acid color reagent. The test tubes are then placed in a boiling water bath for 10 min to stop the reaction and cooled to room temperature. The reaction mixture is then diluted with 1 mL distilled water and absorbance is read at 540 nm using a 96-well mircoplate reader.
Statistical Analyses.
All experiments are performed twice and analyses for each experiment are carried out in triplicate. Means, standard deviations, the degree of significance (p<0.05—One Way ANOVA and t-Test) are determined using Microsoft Excel XP. Inhibition concentration (IC50) is calculated using ED50plus vol. 1 developed by Vargas
Results and Discussion
Total Phenolic Content, Phenolic Profile and Antioxidant Activity.
The total phenolic contents in SML and RML collected in summer and fall are evaluated. Seasonal variation between leaves collected in the summer and fall are observed both in SML and RML (
Previous reports have shown that fall (autumn) sugar maple leaves have higher phenolic contents than summer (Baldwin et al. 1987). In addition, phenolic biosynthesis has been shown to depend on a variety of environmental factors such as UV-B radiation (Mendez et al. 1999), osmotic stress (Kaliamoorthy and Rao 1994), drought (Balakumar et al. 1993), low temperatures (Krol et al. 1995), nutrient deficiencies (Rajendran et al. 1992), wounding (Ferreres et al. 1997), pathogen infection (Dixon et al. 1994) and exposure to ozone (Foot et al. 1996). The present results show that SML and RML are most probably under certain stress during the summer months, which significantly affects their phenolic biosynthesis. After a brief temperature survey for the temperature fluctuation in the summer and fall months in Canada for years 2007-2010 (Weather Underground 2011), it is noted that in July 2010 a high mean temperature is observed (81° F. compared to 76°, 76° and 73° F. observed for 2007, 2008 and 2009 respectively). This discrepancy could be a factor contributing to the higher phenolic content observed in the summer specimens (
The phenolic profiles of the tested extracts are obtained by determining the phenolic constituents of SML and RML and a seasonal effect on the observed compounds are also studied (
The antioxidant activity is evaluated based on the DPPH free-radical scavenging activity. The results show that both RML and SML summer leaves have higher antioxidant activities than the fall leaves (Table 1). More specifically, RML-S has an IC50 of 8.5 ppm while the IC50 for RML-F is 15.3 ppm (Table 2), while SML-S has a higher antioxidant activity than SML-F (15 and 19.1 ppm, respectively) (Table 2).
These results correlate with the observed total phenolic contents since the summer months in both SML and RML have both higher phenolic contents and DPPH free-radical scavenging activities.
Yeast and Rat Intestine α-glucosidase Inhibition.
The α-glucosidase inhibitory activities of the collected samples are evaluated using both yeast and rat intestinal enzyme sources. Due to absorbance reading being interfered by the dark color of the samples, the maximum concentration tested in this assay is 12.5 mg/mL. At the tested doses, the observed inhibitory activities are not sufficient to give an accurate IC50 value (
Based on previous observations, natural compounds tend to have higher yeast α-glucosidase inhibitory activities, than the rat intestinal α-glucosidase activities (Apostolidis et al. 2011a). In order to observe a better dose-dependent inhibitory effect and estimate the accurate IC50 values, the yeast α-glucosidase inhibitory activities of the samples are determined (Table 3). Similarly to rat α-glucosidase inhibition results, SML-S has the highest inhibitory potential (IC50 21 μg/mL). In addition, both summer SML and RML samples have higher inhibitory activities than the fall samples (IC50: SML-F-39 μg/mL, RML-S-115 μg/mL, RML-F-128 μg/mL) (Table 3).
In terms of seasonal variation all summer samples have higher phenolic contents (
Porcine α-amylase Inhibition.
The effect of the extracts on the inhibition of porcine α-amylase are evaluated and all samples appear to have a dose-dependent inhibitory activity. More specifically, no significant difference is observed between SML-S and SML-F (IC50 10.4 and 10.8 mg/mL, respectively) (
The observed results show that there is seasonal variation in α-amylase inhibitory activity only in RML and that this variation is not phenolic-dependent (
Conclusions
The present document reports the seasonal variation in sugar and red maple leaves harvested in summer and fall, in terms of total phenolic contents and corresponding antioxidant and carbohydrate hydrolyzing enzyme inhibitory activities. Based on the above-mentioned observations, sugar maple leaves collected in the summer of 2010 have superior potential for α-glucosidase inhibition, relevant to type 2 diabetes management. Additionally, this effect is dependent on both the phenolic contents and the individual phenolic profiles. The understanding of the mechanism of action and identification of compounds responsible for the observed α-glucosidase and α-amylase inhibitory activities coupled with animal and clinical trials could lead to the development of maple tree leaf ingredients designed for type-2 diabetes prevention.
General Experimental Procedures
Nuclear Magnetic Resonance (NMR) spectra for all compounds are recorded on a Bruker 400 MHz Biospin spectrometer (1H: 400 MHz, 13C: 100 MHz) using deuterated methanol (methanol-d4) as solvent. Mass Spectral (MS) data are carried out on a Q-Star Elite (Applied Biosystems MDS) mass spectrometer equipped with a Turbo Ionspray source and are obtained by direct infusion of pure compounds. High performance liquid chromatography (HPLC) are performed on a Hitachi Elite LaChrom system consisting of a L2130 pump, L-2200 autosampler, and a L-2455 Diode Array Detector all operated by EZChrom Elite software. All solvents are either ACS or HPLC grade and are obtained from through Wilkem Scientific (Pawcatuck, R.I.). Unless otherwise stated, all reagents including the MTS salt [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfenyl)-2H-tetrazolium salt], gallic acid, Folin-Ciocalteu reagent and etoposide standards are obtained from Sigma-Aldrich.
Plant Materials
Plant materials are collected in summer of 2009 by the Federation of Maple Syrup Producers of Quebec (Quebec, Canada), shipped to our laboratory in August 2009, and identified by Mr. J. Peter Morgan (Senior Gardener, College of Pharmacy, University of Rhode Island). Voucher specimens for all plant materials are assigned unique identification codes and are deposited in the Heber Youngken Medicinal Garden and Greenhouse (College of Pharmacy, University of Rhode Island). Voucher specimen codes for the Sugar maple tree parts are: leaves JPMCL1; twigs/stem, JPMCS1; bark JPMCB1; sapwood/heartwood, JPMCH1, and fruit JPMCF2. Voucher specimen codes for the Red-leaf maple tree parts are: leaves, JPMCL2; stem/twigs, JPMCS2; bark JPMCB2; sapwood/heartwood JPMCH2 and fruit JPMCF2.
Preparation of Extracts
Briefly, all plant extracts are prepared using dried and pulverized parts of harvested plants. For each dried and ground maple plant material (ca. 10.0 g), extractions are performed using methanol (3×100 ml) to afford a dried methanol extract, after solvent removal in vacuo. The dried weights of the extracts obtained from the Sugar and Red-leaf maple species are: leaves=0.7 g and 3.3 g; twigs/stem=0.3 g and 0.69 g, bark=0.85 g and 0.80 g; sapwood/heartwood=0.05 g and 0.13 g, fruit 0.6 g and 1.8 g respectively.
Determination of Total Phenolic Content
The total phenolic contents of the maple extracts are determined according to the Folin-Ciocalteu method and is measured as gallic acid equivalents (GAEs) as previously reported by our laboratory (Li et al., 2009). Briefly, the extracts are diluted 1:100, or as appropriate, with methanol/H2O (1:1, v/v), and 200 μl of sample is incubated with 3 ml of methanol/H2O (1:1, v/v) and 200 μl of Folin-Ciocalteau reagent for 10 min at 25° C. After this, 600 μl of 20% Na2CO3 solution is added to each tube and vortexed. Tubes are further incubated for 20 min at 40° C. After incubation, samples are immediately cooled in an ice bath to room temperature. Samples and standards (gallic acid) are processed identically. The absorbance is determined at 755 nm, and final results are calculated from the standard curve obtained from a Spectramax plate reader.
Analytical HPL-UV Analyses of the Maple Extracts
A Luna C18 column (250×4.6 mm i.d., 5 μM; Phenomenex) with a flow rate at 0.75 ml/min and injection volume of 20 μl for all samples (extracts and ginnalin-A) is used. A gradient solvent system consisting of solvent A (0.1% aqueous trifluoroacetic acid) and solvent B (methanol, MeOH) is used as follows: 0-10 min, from 10 to 15% B; 10-20 min, 15% B; 20-40 min, from 15 to 30% B; 40-55 min, from 30 to 35% B; 55-65 min, 35% B; 65-85 min, from 35 to 60% B; 85-90 min, from 60 to 100% B; 90-93 min, 100% B; 93-94 min, from 100 to 10% B; 94-104 min, 10% B.
HPLC-UV Standardization of Maple Extracts to Ginnalin-A Content A stock solution of 1 mg/ml of a pure standard of ginnalin A (isolated as described below) is prepared in DMSO and then serially diluted to afford samples of 0.5, 0.25, 0.125, 0.0625, 0.03125 mg/ml concentrations, respectively. Each sample is injected in triplicate and a linear six-point calibration curve (r2=0.9997) is constructed by plotting the mean peak area percentage against concentration. Plant extracts are prepared at stock solutions of 2.2 mg/ml in DMSO. All HPLC-UV analyses are carried out with 20 μl injection volumes on a Luna C18 column (250×4.6 mm i.d., 5 μM; Phenomenex) and monitored at a wavelength of 280 nm. A gradient solvent system consisting of solvent A (0.1% aqueous trifluoroacetic acid) and solvent B (methanol, MeOH) is used with a flow rate at 0.75 ml/min as follows: 0-30 min, 10% to 60% B; 30-35 min, 60% to 100% B; 35-40 min, 100% B; 40-41 min, 100% to 10% B; 41-51 min, 100% B. The ginnalin-A concentrations of the maple extracts are quantified based on the standard curve.
Isolation and Identification of Ginnalins A, B and C.
Air-dried and ground twigs/stems (547 g) of the Red-leaf maple species are extracted with methanol (700 ml×3) at room temperature to yield 37 g of dried extract after solvent removal in vacuo. A portion of the dried methanol extract (35 g) is reconstituted in water and subjected to liquid-liquid partitioning sequentially with varying solvents, hexane (500 ml×3), ethyl acetate (500 ml×3) and butanol (500 ml×3). The combined butanol extract, after solvent removal in vacuo, yielded 16.1 g of dried extract. A portion of the dried butanol extract (4 g) is chromatographed on a Sephadex-LH-20 column (4.5×64 cm), eluting with a gradient system of methanol/water (7/3 v/v to 100/0 v/v), and then with acetone/water (7/3 v/v). On the basis of analytical HPLC-UV profiles, fourteen combined fractions (Fr. 1-14) are obtained. Ginnalin-A (also known as acertannin, aceritannin, or 2,6-di-O-galloyl-1,5-anhydro-D-glucitol) (1, 306 mg, brown solid) is obtained from Fr. 5 and identified by NMR (1H and 13C) and mass spectral data which corresponded with literature reports (Song et al., 1982; Honma et al., 2010). Similarly Fr. 2 (1.55 g), which contained a mixture of ginnalins B and C is further purified by semipreparative HPLC-UV. Briefly a portion of Fr. 2 (60 mg) is purified on a Waters Sunfire Prep C18 column (250×19 mm i.d., 5 μm) with a gradient solvent system of MeOH/H2O and flow rate of 2 ml/min. Both ginnalin-B (2, 17 mg, brown solid) and ginnalin-C (3, 15.7 mg, brown solid) are identified by their by 1H and 13C-NMR data which corresponded with literature (Song et al., 1982).
Cell Lines and Culture Conditions
The extracts are solubilized in DMSO and normalized based on their phenolic content to evaluate their antiproliferative activities against the colon cell lines. Human colon cancer cell lines Caco-2 (adenocarcinoma), HT-29 (adenocarcinoma) and HCT-116 (carcinoma) and the normal colon cells CCD-18Co are obtained from American Type Culture Collection (Rockville, USA). Caco-2 cells are grown in EMEM medium supplemented with 10% v/v fetal bovine serum, 1% v/v nonessential amino acids, 1% v/v L-glutamine and 1% v/v antibiotic solution (Sigma). HT-29 and HCT-116 cells are grown in McCoy's 5a medium supplemented with 10% v/v fetal bovine serum, 1% v/v nonessential amino acids, 2% v/v HEPES and 1% v/v antibiotic solution. CCD-18Co cells are grown in EMEM medium supplemented with 10% v/v fetal bovine serum, 1% v/v nonessential amino acids, 1% v/v L-glutamine and 1% v/v antibiotic solution and are used from PDL between 26 to 35 for all experiments. Cells are maintained at 37° C. in an incubator under a 5% CO2/95% air atmosphere at constant humidity. The pH of the culture medium is determined using pH indicator paper (pHydrion™ Brilliant, pH 5.5-9.0, Micro Essential Laboratory, NY, USA) inside the incubator. Cells are counted using a hemacytometer and are plated at 3,000-5,000 cells per well, in a 96-well format for 24 or 48 h prior to sample treatment depending on the cell line. All of the test samples are solubilized in DMSO (<0.5% in the culture medium) by sonication and are filter sterilised (0.2 μm) prior to addition to the culture media. Control cells are also run in parallel and subjected to the same changes in medium with 0.5% DMSO.
Cell Proliferation and Viability Tests (Trypan Blue Exclusion and MTS Assays)
At the end of either 48 or 72 h of sample treatment, trypsinised cells (2.5 g/l trypsin, 0.2 g/l EDTA) are suspended in cell culture medium, counted using a Neubauer haemacytometer (Bad Mergentheim, Germany) and viability measured using Trypan blue dye exclusion. Results of proliferation and viability in extract-treated cells are expressed as percentage of those values obtained for control (0.5% DMSO) cells. All experiments are performed in triplicate.
The MTS assay is carried out as described previously (Li et al., 2009) with modifications. At the end of 48 or 72 h of treatment with serially diluted test samples, 20 μl of the MTS reagent, in combination with the electron coupling agent, phenazine methosulfate, is added to the wells and cells are incubated at 37° C. in a humidified incubator for 3 h. Absorbance at 490 nm (OD490) is monitored with a spectrophotometer (SpectraMax M2, Molecular Devices Corp., operated by SoftmaxPro v.4.6 software, Sunnyvale, Calif., USA), to obtain the number of cells relative to control populations. 20 μl of etoposide 4 mg/ml (Sigma) is assayed as a negative control of proliferation. The results are expressed as the concentration that inhibit growth of cell by 50% vs. control cells (control medium used as negative control), IC50. Data are presented as the mean±S.D. of three separated experiments on each cell line. Etoposide provided consistent IC50 values of 10-20 μM (HT29, HCT116 and Caco-2) and 30-40 μM for the CCD-18Co cells.
Flow Cytometry Analysis of Cell Cycle
Cells (2×105) are collected after the corresponding experimental periods, fixed in ice-cold ethanol:PBS (70:30) for 30 min at 4° C., further resuspended in PBS with 100 μg/ml RNAse and 40 μg/ml propidium iodide, and incubated at 37° C. for 30 min. DNA content (10,000 cells) is analysed using a FACS Calibur instrument equipped with FACStation running FACS Calibur software (BD Biosciences, San Diego, Calif., USA). The analyses of cell cycle distribution are performed in triplicate for each treatment. The coefficient of variation, according to the ModFit LT Version 2 acquisition software package (Verity Software House, Topsham, Me., USA), is always less than 5%.
Morphological Evaluation of Apoptosis
Cells (2.5×104/ml) are treated for 48 and 72 h and fixed with methanol: acetic acid (3:1, v/v) and stained with 50 mg/ml Hoechst 33242 dye at 37° C. for 20 min. Afterwards, the cells are examined under a Nikon Eclipse TE2000-E inverted microscope (Nikon, N.Y., USA). Etoposide (Sigma) 20 μM is assayed as a standard inducer of apoptosis. Morphological evaluation of apoptosis is carried twice for each sample.
Statistical Analysis
Two-tailed unpaired student's t-test is used for statistical analysis of the data. A p value <0.05 is considered significant.
Results
Standardization of Maple Plant Part Extracts
In the current document, various plant parts of the two maple species are subjected to established extraction protocols to enrich them in phenolic contents (Li et al. 2009). The total phenolic content of all of the extracts are evaluated by the Folin-Ciocalteu method and is measured as gallic acid equivalents (GAEs) which ranged from 28.65 to 63.73 mg/L (Table 4). The extracts are further standardized to ginnalin-A (1), ginnalin-B (2) and ginnalin-C (3) contents (chemical structures shown in
The HPLC-UV chromatograms of the extracts from the different plant parts of the Red-leaf maple and Sugar maple are shown in
Antiproliferative Activity on Cancer Colon Cells by Extracts
The extracts are normalized to deliver equivalent amount of phenolics (50% dry weight) in the antiproliferative assays. All of the maple extracts inhibited the proliferation of HCT-116, Caco-2 and HT-29 cell lines in a time-dependent manner but did not have the similar effect on the normal colon CCD-18Co cells (Table 5). Overall, among the extracts, the leaves and stem extracts showed greater effects than the bark, fruit and sapwood extracts. Also, between the two maple species, extracts of the Red-leaf maple tree showed greater antiproliferative activity than from the Sugar maple tree. In all cases, cell viability is always above 90% at tested doses so extracts are not cytotoxic (results not shown).
After 72 h, the highest antiproliferative effects against the colon cancer cell lines are observed from the leaves and stem extracts of the Red-leaf maple with IC50 values ranging from 35-91 μg/ml and from 55-111 μg/ml, respectively. On the other hand, the IC50 values after treatment with the bark extracts from the Red and Sugar maple tree ranged from 52-91 and from 59-92 μg/ml, respectively. Moderate activity is found in the leaves and stem extracts from the Sugar maple tree (IC50=87-134 and 101-146 μg/I, respectively). Finally, extracts from heartwood and fruits of both species of maple tree showed IC50 values ranging from 127-183) (Table 5).
Among the colon cancer cells, the HCT-116 cells are most sensitive to all of the maple extract treatments compared to the Caco-2 and HT-29 cell lines (Table 5). There is a significant difference between the IC50 values of the extracts against the colon cancer cells compared to the CCD-18Co normal cells (over 2-fold). These results indicate a possible selectivity of the extracts towards colon cancer cells suggesting that these extracts may have potential as colon cancer chemopreventive agents. However further studies would be required to confirm this.
Antiproliferative Activity on Cancer Colon Cells by Ginnalins
Table 6 shows the antiproliferative activities of ginnalins-A, B and C on the colon cancer and normal colon cells. Among the three purified compounds, ginnalin A showed the best activity with IC50 values ranging from 16-24 μg/ml. Among the cell lines, the HCT-116 colon cancer cells are most sensitive to this compound. All ginnalins showed selective activity towards the colon cancer cells than the normal colon cells similar to the maple extracts.
Cell Cycle Distribution Analysis
Inhibition of proliferation is further examined by measuring cell cycle distribution. At 48 h of the experiment, HCT-116, Caco-2 and HT-29 control cells are distributed as follows: 58.7±3.6% in G0/G1 phase, 30.8±1.7% in S phase and 10.5±2.0% in G2/M phase; 56.2±2.1% in G0/G1 phase, 31.0±2.4% in S phase and 12.8±0.40% in G2/M phase; and 59.0±1.1% in G0/G1 phase, 31.1±0.9% in S phase and 9.9±0.5% in G2/M phase, respectively (data not shown). At 72 h of the experiment, the proportion of these control cells in the G0/G1 phase increased to 66.3-70.9% whereas cells in the S and G2/M phases decreased to 18.2-23.2% and to 7.2-9.7%, respectively (
At 48 h treatment with the maple extracts (at doses corresponding to their IC50 values) an increase of cells in S phase (p<0.05) concomitant with a decrease in G0/G1 (p<0.05) and a slight increase in G2/M phase are observed. In accordance with the HCT116 cells being most sensitive among the cell lines in terms of reduced cell growth, changes observed in cell cycle distribution are more pronounced in these HCT-116 cells, with a clear arrest in the S-phase with a range of 45.8-55% (p<0.05). This increase is maintained during the 72 h of sample treatment to 48.6-57.3% (p<0.05), a 150% increase when compared to control cells, in the S phase accompanied by a decrease of cells in G0/G1 phase (range 34.6-42.2%) (p<0.05) whereas no significant changes of the G2/M ratio are observed (
It should be noted that incubation of the normal colon CCD-18Co cells with the various maple plant part extracts for 48 and 72 h did not cause significant changes in cell cycle when compared with control cells (69.3±1.1% in G0/G1 phase, 17.6±0.9% in S phase and 13.1±1.0% in G2/M phase; 76.5±2.0% in G0/G1 phase, 15.2±0.9% in S phase and 8.3±1.1% in G2/M phase, respectively), except with the incubation of etoposide (50 μM) used as a positive control (
These results indicate that the compounds present in the maple tree extracts, at subtoxic levels, can inhibit the proliferation of colon cancer cells by blocking the progression of cell cycle at S-phase.
Apoptosis Assessment
Another possible mechanism related to the antiproliferative activity derived from the maple plant part extracts in the colon cancer cells could be mediated by the induction of apoptosis. Therefore, we carried out the morphological evaluation of apoptosis by monitoring for changes in nuclear chromatin distribution that can be stained by the DNA-binding fluorochrome Hoechst 33242 dye. Incubation of the colon cancer cells and normal colon cells with extracts mirrored the pattern followed by untreated cells, thus indicating the absence of apoptosis (data not shown).
a IC50 (in μg/ml) is defined as the concentration required to achieve 50% inhibition over control cells (DMSO 0.5%); IC50 values are shown as mean ± S.D. from three independent experiments.
aIC50 (in μg/ml) is defined as the concentration required to achieve 50% inhibition over control cells (DMSO 0.5%); IC50 values are shown as mean ± S.D. from three independent experiments.
The Red maple extracts show promising α-glucosidase inhibitory activities with IC50 values ranging from 4-10 μg/mL. This example shows the isolation and structural elucidation of five new gallotannins (compounds RMS 4, RMS 5, RMS 9, RMS 7, RMS 24), assigned the common name maplexins A-E, along with eight other known gallic acid derivatives (
The dried stems of Red maple are extracted with methanol and fractionated with hexane, EtOAc and n-butanol. From the EtOAc extracts five new gallotannin compounds, along with eight known gallic acid derivatives are isolated by using a combination of chromatographic column separations.
Briefly, the dried stems of Red maple (500 g, dry) are ground and extracted exhaustively with methanol. The combined dried methanol extract is re-suspended in water and partitioned successively with n-hexane, EtOAc and n-butanol. The EtOAc fraction (18 g) is subjected to a silica gel chromatography column (CHCl3/MeOH) to yield three fractions (A1-A3). Fraction A3 (8 g) is chromatographed on a Sephadex LH-20 column and eluted with MeOH to give seven sub-fractions (B1-B7). Fraction B4 is chromatographed on a C18MPLC column eluting with a gradient system of MeOH/H2O (9:1 to 3:7, v/v) to afford 14 sub-fractions (C1-C14). Fraction C2 is separated by semi-preparative HPLC eluted with MeOH/H2O (20/80 v/v 3.2 mL/min) to yield compounds RMS2 (2.8 mg), RMS3 (2.5 mg) and gallic acid (460 mg). Fraction C3 is separated by semi-preparative HPLC eluted with MeOH/H2O (25/75 v/v 3.2 mL/min) to yield ginnalins B (18 mg) and C (9.2 mg). Fraction C5 is separated by semi-preparative HPLC eluted with MeOH/H2O (30/70 v/v 3.2 mL/min) to yield compound RMS5 (5.3 mg) and methyl gallate (7.7 mg). Fraction C6 is separated by semi-preparative HPLC eluted with MeOH/H2O (27/73 v/v 3.2 mL/min) to yield compound RMS6 (25 mg). Fraction C9 is separated by semi-preparative HPLC eluted with MeOH/H2O (25/75 v/v 3.2 mL/min) to yield ginnalin A (13 mg) and 3,4-dihydroxy-5-methoxybenzoic acid methyl ester (4.6 mg). Fraction C12 is separated by semi-preparative HPLC eluted with MeOH/H2O (41/59 v/v 3.2 mL/min) to yield methyl syringate (0.8 mg). Fraction B6 is chromatographed on a C18MPLC column eluting with a gradient system of MeOH/H2O (8:2 to 3:7, v/v) to afford 10 sub-fractions (D1-D10). Fraction D1 is separated by semi-preparative HPLC eluted with MeOH/H2O (30/70 v/v 3.2 mL/min) to yield 3,6-di-O-galloyl-1,5-anhydro-D-glucitol (1.4 mg). Fraction D8 is separated by semi-preparative HPLC eluted with MeOH/H2O (35/65 v/v 3.2 mL/min) to yield compound RMS9 (5 mg). Their structures are characterized using physicochemical and spectroscopic methods.
Compound RMS4, 3-O-galloyl-1,5-anhydro-D-glucitol: colorless amorphous solid; [α]20D +25 (c 0.280, MeOH); UV (MeOH) λmax (log ε): 276 (4.10), 216 (4.41) nm; IR νmax 1693, 1610 cm−1; for 1H NMR and 13C NMR data, see Table 7 and Table 8; HREIMS at m/z 315.0717 [M-H]− (calcd for C13H15O9, 315.0716) is a colorless amorphous solid, has a molecular formula of C13H16O9 determined by HRESIMS at m/z 315.0717 [M-H]− (calcd for C13H15O9, 315.0716). Its IR absorptions implies the presence of ester carbonyl (1693) and aromatic ring (1610). The analysis of 1H-NMR (Table 7) and 13C-NMR (Table 8) spectra showed typical galloyl signals at δH 7.14 (s, 2H), δC 167.1, 145.0 (2×C), 140.5, 120.5, 108.9 (2×C). Eight proton signals at δH 3.27-5.04 indicates the presence of a substructure similar to that of a sugar moiety. Apart from the galloyl carbon signals, six oxygenated carbon signals at δC 81.1, 79.8, 69.5, 68.6, 68.5 and 61.3 are observed in the 13C-NMR spectrum, which also supportes the presence of a sugar substructure. Further combined analysis of 1H-1H COSY, HSQC and HMBC spectrum allows the establishment of the structure of RMS4. The HSQC spectrum allows the assignment of all the protons to their bonding carbons. From the 1H-1H COSY spectrum, a 1-deoxysugar moiety (C-1 to C-6), drawn with bold bond in
Compound RMS5, 4-O-galloyl-1,5-anhydro-D-glucitol: colorless amorphous solid; [α]20D +15 (c 0.060, MeOH); UV (MeOH) λmax (log ε): 276 (4.10), 216 (4.41) nm; IR νmax 1690, 1608 cm−1; for 1H NMR and 13C NMR data, see Table 7 and Table 8; HREIMS at m/z 315.0719 [M-H]− (calcd for C13H15O9, 315.0716) shows the same molecular formula as compound RMS2 (i.e. C13H16O9 as per HRESIMS data) as well as similar UV and IR data. The 1H- and 13C-NMR spectra indicates the presence of similar galloyl and 1,5-anhydro-glucitol substructures as RMS4. Further analysis of the 1H-1H COSY, HSQC and HMBC data found that the only difference between RMS4 and RMS5 is the linkage position connecting the galloyl and the 1,5-anhydro-glucitol moiety. The galloyl is eventually deduced to be attached at C-4 of the glucitol by the HMBC correlations from H-4 to C-7′. The D-configuration of the glucitol is determined by the similar acid hydrolysis method as for RMS4. Compound RMS5 is thus determined as 4-O-galloyl-1,5-anhydro-D-glucitol assigned the common name maplexin B.
Compound RMS9, 2,3-di-O-galloyl-1,5-anhydro-D-glucitol: colorless amorphous solid; [α]20D +13 (c 0.120, MeOH); UV (MeOH) λmax (log ε): 276 (4.10), 216 (4.41) nm; IR νmax 1705, 1600 cm−1; for 1H NMR and 13C NMR data, see Table 7 and Table 8; HREIMS at m/z 467.0826 [M-HT (calcd for C20H19O13, 467.0826) is obtained as a colorless amorphous solid, shows the molecular formula of C20H20O13 as determined by HRESIMS at m/z 467.0826 μM-H]− (calcd for C20H19O13, 467.0826). The 1H- and 13C-NMR data shows were similar to those of compounds RMS4 and RMS5, indicating that the structures of both compounds are closely related, and the only difference is likely the presence of an additional galloyl moiety in RMS9. Further analysis of the 2D NMR data allows the establishment of the structure of RMS9. In the HMBC spectrum, the correlations from H-2 to C-7′, from H-3 to C-7″ indicated that the two galloyl groups are linked at C-2 and C-3 of 1,5-anhydro-glucitol, respectively. The D-configuration of the glucitol is determined by the same method as for compound RMS4. Compound RMS9 is therefore elucidated as 2,3-di-O-galloyl-1,5-anhydro-D-glucitol assigned the common name maplexin C.
Compound RMS7, 2,4-di-O-galloyl-1,5-anhydro-D-glucitol: colorless amorphous solid; [α]20D +6 (c 0.170, MeOH); UV (MeOH) λmax (log ε): 276 (4.10), 216 (4.41) nm; IR νmax 1703, 1601 cm−1; for 1H NMR and 13C NMR data, see Table 7 and Table 8; HREIMS at m/z 467.0821 [M-H]− (calcd for C20H19O13, 467.0826) has the same molecular formula (I.e. C20H20O13) as compound RMS5 based on the HRESIMS at m/z 467.0821 [M-H]− (calcd for C20H19O13, 467.0826). The IR and UV spectrum are also similar to RMS5. Initial analyses the 1H- and 13C-NMR data revealed the presence of two galloyl groups and a 1,5-anhydro-glucitol moiety. The difference between RMS7 and RMS9 is the linkage position of the galloyl with 1,5-anhydro-glucitol. The two galloyl groups are finally assigned to attachment at C-2 and C-4 of the 1,5-anhydro-glucitol on the basis of the HMBC correlations from H-2 to C-7′ and from H-4 to C-7″, respectively. The D-configuration of the glucitol is determined similar to that of compound RMS4. Compound RMS7 is thus elucidated as 2,4-di-O-galloyl-1,5-anhydro-
Compound 24, 2,4,6-tri-O-galloyl-1,5-anhydro-D-glucitol colorless amorphous solid; [α]20D +10 (c 0.130, MeOH); UV (MeOH) λmax (log ε): 276 (4.10), 216 (4.41) nm; IR νmax 1710, 1598 cm−1; for 1H NMR and 13C NMR data, see Table 1 and Table 2; HREIMS at m/z 619.0916 [M-H]− (calcd for C27H23O17, 619.0935) is obtained as colorless amorphous solid, shows the molecular formula of C27H24O17 as determined by HRESIMS at m/z 619.0916 [M-H]− (calcd for C27H23O17, 619.0935). In the 1H and 13C-NMR spectrum (Table 7 and Table 8, respectively), three sets of signals for galloyl moieties, eight proton signals at δH 3.46-5.22 and six oxygenated carbon signals at 6c 76.8, 73.5, 71.8, 71.1, 66.6 and 62.7 are observed. The aforementioned spectral data suggests that compound RMS24 is similar to the above compounds, the only difference being the presence of three galloyl groups attached to the 1,5-anhydro-glucitol moiety. The HMBC correlations from H-2 to C-7′, from H-4 to C-7″, and from H2-6 to C-7′″ indicates that the three galloyl groups are linked at C-2, C-4 and C-6 of the 1,5-anhydro-glucitol, respectively. The D-configuration of the glucitol is determined similar to that of RMS4. Compound RMS24 is thus elucidated as 2,4,6-tri-O-galloyl-1,5-anhydro-
Maplexins A-E, i.e. compounds RMS4, RMS 5, RMS 9, RMS 7 and RMS 24 (each 2 mg) are added to a mixture of concentrated HCl (0.5 mL), H2O (2 mL) and dioxane (3 mL) and refluxed for 2 h, respectively. After completion of the reaction (monitored by TLC), the mixture is evaporated to dryness. The dry reaction mixture is partitioned between CHCl3 and H2O (3×5 mL). The aqueous layer is neutralized with Na2CO3 and then concentrated to dryness. The concentrate is dissolved in methanol and purified by Sephadex LH-20 chromatography to give 1,5-anhydro-D-glucitol, which is identified by co-TLC and specific rotation with the standard (Rf=0.43, CHCl3-MeOH 10:1, positive value for optical rotation). The ESI-MS and 13C NMR spectrum (See supporting information) further supported the results.
Apart from the maplexins described herein, eight known compounds are identified as ginnalins B (RMS12), (Song, C. et al. 1982), C (RMS27), (Song, C. et al. 1982) and A (RMS26) (Bock, K, et al, 1980) 3,6-di-O-galloyl-1,5-anhydro-
1H-NMR [δ, (Multiplicity, JHH in Hertz)] Spectroscopic Data for
aData were measured in CD3OD at 500 MHz.
13C-NMR (δ Values) Spectroscopic Data for Compounds RMS4,
aData were measured in CD3OD at 125 MHz.
The α-glucosidase inhibitory properties and the structure-activity relationship (SAR) of all 13 compounds isolated from red maple stems is then investigated. Compounds RMS9, 7, 26, 18, 24 and RMS17 are found to be inhibitors of α-glucosidase enzyme in a concentration-dependent manner (Table 9). Compounds RMS12, 4, 5, and 27, which possess one galloyl group each, do not show any activity in this assay while compounds RMS9, 7, 26, and 18, which possess two galloyl groups each, shows moderate α-glucosidase inhibitory activity. Remarkably, Maplexin E, compound RMS24 which has three galloyl groups shows powerful α-glucosidase inhibitory activity in this assay. Maplexin E is 20 fold more potent than the known α-glucosidase inhibitory drug, acarbose (IC50 8.26 and 161.38 respectively).
a IC50 values are shown as mean ± S.D. from three independent experiments.
b Positive control.
The α-glucosidase inhibitory activities of compounds RMS9, 7, 26 and 18, with two galloyl groups each, also show significant differences in effects with IC50 values of 1745.78, 1221.84, 95.38 and 88.42 μM, respectively. Compounds RMS26 and RMS18 showed stronger activities than compounds RMS9 and RMS7, which suggested that the α-glucosidase inhibitory activities of these gallotannins are influenced by both the number and positions of the galloyl groups. Thus, it is apparent that a galloyl group attached at the C-6 position of the glucitol moiety increased activity.
The antioxidant activities of 13 compounds are evaluated in the DPPH free radical scavenging assay. (Li and Seeram, 2011) All of isolates except RMS11 and RMS15 show better DPPH free radical scavenging activities than Vitamin C and BHT, the standard antioxidant materials (table 9). The IC50 values of compounds RMS12, 4, 5, 27 are from 30.49 to 47.99 μM, compounds RMS9, 7, 26, and 18 are from 17.74 to 18.80 μM, and compound RMS24 is 13.06 μM. These results suggest that the antioxidant activity of gallotannins are influenced mainly by the number of the galloyl groups, while location of the galloyl group on the 1,5-anhydro-D-glucitol moiety had less influence on the antioxidant activity.
Thus, the identified new compounds from the Red maple species with α-glucosidase inhibitory potential include maplexin E (24), a natural agent that showed in vitro α-glucosidase inhibitory activity far superior to acarbose, a clinically available drug. Interestingly, our SAR study also indicates that these compounds may be synthetically manipulated with regards to the numbers and location of the galloyl groups on the 1,5-anhydro-α-glucitol moiety to enhance activity.
erythro
threo
Catechin
Epicatechin
Icariside E4
New
Dihydrodehydrodiconiferyl alcohol
Vanillic acid
Scopoletin
New
Cleomiscosin C
Cleomiscosin A
Cleomiscosin B
Cleomiscosin D
Koaburside
While preferred embodiments have been described above and illustrated in the accompanying drawings, it will be evident to those skilled in the art that modifications may be made without departing from this disclosure. Such modifications are considered as possible variants comprised in the scope of the disclosure.
This application claims priority from U.S. provisional patent applications 61/406,290, filed Oct. 25, 2011, and 61/449,333, filed Mar. 4, 2011, the specifications of which is hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA2011/001164 | 10/14/2011 | WO | 00 | 8/7/2013 |
| Number | Date | Country | |
|---|---|---|---|
| 61406290 | Oct 2010 | US | |
| 61449333 | Mar 2011 | US |
