Method of preparing ginger extract, composition comprising said ginger extract and use thereof

Abstract

A method of preparing ginger extract by water and / or alcohol. Said ginger extract contains gingerols (e.g., 6-, 8-, 10-gingerol) and / or shogaols (e.g., 6-, 8-, 10-shogaol), and it can further contain Total Phenolic Contents (TPC) and / or GABA. The compositions containing said ginger extract are used in preparation of anti-inflammatory, antioxidant, antibacterial, analgesic or anti-cancer medicament, etc..

Description

[0001] METHOD OF PREPARING GINGER EXTRACT (Zingiber Officinale Roscoe; ZINGIBERACEAE FAMILY), COMPOSITION COMPRISING GINGER EXTRACT OBTAINED FROM SAID METHOD AND USE THEREOF

[0002] FIELD OF INVENTION

[0003] The present invention relates to a method of preparing ginger extract (Zingiber Officinale Roscoe; Zingiberaceae family), composition comprising ginger extract obtained from said method and use thereof.

[0004] BACKGROUND ART

[0005] Ginger, scientific name Zingiber officinale Roscoe belongs to the Zingiberaceae family, is an herb listed in National List of Essential Drugs. In traditional Thai medicine, ginger rhizomes are orally used to treat nausea and vomiting, flatulence or gassiness. In National List of Essential Drugs B.E. 2561, ginger medicine (powders, infusions, capsules) is in the herbal medicine group of the herbal medicine list (medicine for treating gastrointestinal syndromes).

[0006] Ginger is an herb that has spicy taste and pungent smell because it contains main active substances which are 6-gingerol, shogaol, diarylheptanoids, zingiberene, and contains essential oils (1). The substances in the gingerol group are, e.g., 6-gingerol, 8-gingerol, 10-gingerol; and the substances in the shogaol group are, e.g., 6-shogaol, 8-shogaol, 10-shogaol. Both groups of said substances have structural formulas are shown in Fig. 1.

[0007] In vitro studies have shown that active substances in ginger rhizomes have effects against cold and influenza virus (2). Ginger reduces the severity of lung inflammation and hyperoxia (3). Moreover, ginger has anti-inflammatory activity (4), reduces muscles and joints inflammation in patients with rheumatoid arthritis (5-7).

[0008] Furthermore, from experiences of the inventors of this application, it was found that use of a large amount of alcohol, e.g., ethanol etc. for extraction results in sticky extracts. This is because alcohol can extract a large amount of resin or gum from herbs. Particularly, ginger when the large amount of alcohol was used for extraction, essential oils are also obtained (or extracted), making the obtained extracts sticky and thus, it is difficult to make dried powders. The inventors of this application thus chose to use a small amount of ethanol for extraction and then evaporate the ethanol before drying it to obtain dried powders by spray-drying technique. From a search for patents / petty patents filed in Thailand which are related to ginger extract, 24 patents / petty patents were found while there are 2 petty patents on processes for preparing ginger extract. The others are ginger products.

[0009] Thai petty patent no. 10560 (filing date: June 27, 2014, Title of invention: Formulation of ginger extract, with controlled amount of 6-gingerol, containing components of ginger extract, microcellulose and silicon dioxide and processes for producing thereof). Said petty patent discloses the preparation of the extract using 90-95% alcohol to obtain ginger extract as oleoresin and has the amount of 6-gingerol equal to 15-20%. However, said petty patent does not disclose other substances, especially shogaol.

[0010] Thai petty patent no. 21444 (filing date: February 7, 2018, Title of invention: Processes of extracting gingerol and shogaol from ginger). Said petty patent discloses the preparation of ginger extract using supercritical carbon dioxide and 95% alcohol to obtain oleoresin extract as well.

[0011] In addition, from searching for patents / petty patents filed abroad, application no. KR20230086054A (patent application, filing date: 08.12.2021, Title of invention: Extraction method for increasing 6-gingerol in ginger using citric acid). Said patent application discloses an extraction method for increasing the amount of 6-gingerol of ginger using citric acid, use of the method for preparing a ginger extract using citric acid according to the present invention, an extract having an increased amounts of useful components can be prepared, and ginger extract with increased active ingredient content prepared. Said method can be used in the production of functional ginger processed products having various properties such as antioxidant, antiinflammatory, anti-cancer, antibacterial and immune enhancing.

[0012] Further, the invention of Korean patent application no. KR20230086054 A discloses a method of preparing ginger extract by extracting dried ginger using ethanol (preferably 70%) mixed with citric acid, and reflux extraction, i.e., ultrasonic treatment extract, enzyme treatment extract and citric acid complex treatment extract, was conducted. The increase in 2-fold of the amount of 6-gingerol can be obtained from said method which is different from that of this invention.

[0013] As can be seen, ginger, a household herb used for a long time, contains a variety of active substances with diverse activities, e.g., antioxidants, anti-inflammatory, antimicrobial etc. However, its use in the form of unprocessed ground ginger powders filled in capsules or in the form of ingested ginger juice or fresh ginger to relieve flatulence, heartburn, and nausea and vomiting, results in a high variability of the amounts of active substances and may be contaminated with heavy metals, pesticides and microorganisms. Therefore, the development of ginger extract preparations of this application can control specific amounts of active substances and can select extraction methods to obtain the desired group of active substances.

[0014] References

[0015] 1. Mao QQ, Xu XY, Cao SY, Gan RY, Corke H, Beta T, Li HB. (2019). Bioactive compounds and bioactivities of ginger (Zingiber officinale Roscoe). Foods. 8(6): 185.

[0016] 2. Sun Y, Yu CL, Yan YL, Zhang FL, Chen J, Hu ZY, He J, Meng XY, Wu QF. Inhibitory effects and related molecular mechanisms of Huanglian-Ganjiang Combination against H1N1 influenza virus. Rev Bras Farmacogn. 2023; 33(3):514-522.

[0017] 3. ifci A, Tayman C, Yakut HI, Halil H, akir E, akir U, Aydemir S. Ginger (Zingiber officinale) prevents severe damage to the lungs due to hyperoxia and inflammation. Turk J Med Sci. 2018; 48(4): 892-900.

[0018] 4. Ozkur M, Benlier N, Takan I, Vasileiou C, Georgakilas AG, Pavlopoulou A, Cetin Z, Saygili El. Ginger for healthy ageing: A systematic review on current evidence of its antioxidant, anti-inflammatory, and anticancer properties. Oxid Med Cell Longev. 2022; 2022:4748447.

[0019] 5. Zhang YM, Shen J, Zhao JM, Guan J, Wei XR, Miao DY, Li W, Xie YC, Zhao YQ. cedrol from ginger ameliorates rheumatoid arthritis via reducing inflammation and selectively inhibiting JAK3 phosphorylation. J Agric Food Chem. 2021; 69( 18):5332-5343.

[0020] 6. Szymczak J, Grygiel-Gdrniak B, Cielecka-Piontek J. Zingiber Officinale Roscoe: The antiarthritic potential of a popular spice-preclinical and clinical evidence. Nutrients. 2024; 16(5):741.

[0021] 7. Aryaeian N, Shahram F, Mahmoudi M, Tavakoli H, Yousefi B, Arablou T, Jafari Karegar S. The effect of ginger supplementation on some immunity and inflammation intermediate genes expression in patients with active Rheumatoid Arthritis. Gene. 2019; 698: 179-185.

[0022] SUMMARY OF THE INVENTION

[0023] The present invention relates to a method of preparing ginger extract to obtain the extract which contains active substances for medical use, involving various post-harvest ginger processing and the use of solvents of both water and alcohol for extraction. This is to obtain ginger extract containing 2 groups of active substances, i.e., gingerol, e.g., 6-, 8-, 10-gingerol, and shogaol, e.g., 6-, 8-, 10-shogaol, including total phenolic content (TPC) as desired.

[0024] Moreover, the present invention relates to all ginger extracts obtained from the methods of this invention that are used as ingredients in herbal products, supplements, foods, cosmetics or are used as pharmaceutical compositions or formulations for use in the treatment of diseases, e.g., analgesic, anti-inflammatory, antioxidant creams, oral sprays for reducing bacteria / reducing halitosis, etc. Said ginger extracts can further contain gamma-aminobutyric acid (GABA), and its formulation can include one or more other plant extracts in a synergistic manner. Said pharmaceutical products, compositions or formulations have the properties of treating diseases, anti-inflammatory, analgesic, antioxidants, antimicrobial etc.

[0025] DETAILED DESCRIPTION

[0026] The inventors have conducted research to obtain a method of preparing ginger extract comprising active substances that have pattern shown in Figs. 2A and 2B. The ginger suitable for this method is ginger rhizomes that are of about 8 to about 10 months or more, preferably about 10 months. Said method comprises the steps as follows: i. Processing ginger to obtain processed ginger; and drying obtained processed ginger to give dried processed ginger, or drying ginger to obtain dried ginger.

[0027] Said processing step is selected from:

[0028] Reducing the size of fresh ginger and immersing it in water at room temperature for about 20 to about 24 hours, preferably about 24 hours,

[0029] Reducing the size of fresh ginger and freezing it at temperature of less than about 0 °C for about 7 days to about 30 days, or

[0030] Reducing the size of fresh ginger and acid pickling it, wherein acid used for pickling, e.g., commercially available vinegar comprising 5 % v / v of acetic acid, 5 % v / v of acetic acid, for about 24 hours to about 48 hours (preferably about 24 hours), wherein reducing the size of fresh ginger is making ginger into pieces or slices.

[0031] Moreover, drying ginger of step i. as above is heat-drying including oven-drying, air-drying, evaporation, dehydration or the like, preferably oven-drying. Said drying step is conducted at temperature of about 80 to about 90 °C, preferably about 85 °C, for about 20 to about 24 hours, preferably about 24 hours, ii. Reducing the size of dried processed ginger or dried ginger of step i. to obtain smallsized dried processed ginger or small-sized dried ginger, wherein said reducing the size of dried processed ginger or dried ginger of step i. is grinding or homogenizing, preferably homogenizing, iii. Extracting small- sized dried processed ginger or small- sized dried ginger of step ii. with solvents including water, alcohol or the mixture thereof, preferably extracting small-sized dried processed ginger or small-sized dried ginger of step ii. with the mixture of solvents or with water to obtain ginger extract, wherein the extraction with the mixture of water and alcohol is the extraction with the mixture of water and ethanol at a ratio of water: ethanol of about 20 to about 70% by volume, preferably about 50% by volume and optional:

[0032] Extracting by adding small-sized dried processed ginger or small-sized dried ginger of step ii. with the mixture of water and alcohol or with water at room temperature for from about 0.5 hour to about 1 hour, repeated twice, or

[0033] Extracting by using small-sized dried processed ginger or small-sized dried ginger of step ii. to the mixture of water and alcohol at a ratio of about 1 part to about 5 to 20 parts, preferably about 1 : 10, repeated twice.

[0034] Preferably, single extraction is conducted at a ratio of small-sized dried processed ginger or small-sized dried ginger of step ii. to the mixture of water and alcohol of about 1 :20.

[0035] In addition, the extraction with water is:

[0036] Extracting with water at temperature from about 80 to 87 °C, preferably about 85 °C, or

[0037] Adding water to small-sized dried processed ginger or small-sized dried ginger of step ii. and heating water at temperature from about 80 to about 87 °C, preferably about 85 °C, for about 2 to about 5 hours, preferably about 3 hours.

[0038] The extraction of small-sized dried processed ginger or small-sized dried ginger of step iii. can optionally further comprise the following steps: a. Evaporating the mixture of water and alcohol or water from ginger extract of step iii. to reduce its volume by at least half by evaporating the mixture of water and alcohol or water from ginger extract at temperature of not more than 60 °C to obtain concentrated ginger extracts, b. Drying concentrated ginger extract of step a. by spray-drying at the condition of inlet of 140 °C / outlet of 80 °C.

[0039] Alternatively, a carrier may be added prior to step b), wherein said carrier is selected from lactose, maltodextrin, gum arabic, methylcellulose, hydroxypropyl methylcellulose, povidone, pregelatinized starch, carboxymethylcellulose, ethyl cellulose, polyvinyl alcohol, hydroxypropyl cellulose, or the like, preferably lactose, maltodextrin, or gum arabic, optionally wherein the addition of maltodextrin is in the amount of about 1 to about 5 % w / w, preferably 2 % w / w, most preferably maltodextrin is optionally added in the amount of about 2 % w / w.

[0040] Furthermore, the present invention relates to ginger extract obtained from the above method, wherein said ginger extract comprises active substances selected from the group comprising of gingerol and / or shogaol groups, wherein structural formulas of said active substances are as shown in Fig. 1, wherein active substances comprise the group of gingerol which is selected from the group consisting of 6-gingerol, 8-gingerol, and 10-gingerol in the amount of no more than about 10 mg / g, which contain 6-gingerol in the range of about 10 to about 50 mg / g.

[0041] Moreover, said active substances also comprise shogaol group which is selected from the group consisting of 6-shogaol, 8-shogaol, and 10-shogaol in the amount of no less than about 1 mg / g.

[0042] Said extract also contains the amount of Total Phenolic Content of not less than about 20 mg gallic acid equivalent / g and optionally further contain gamma-aminobutyric acid (GABA) in the range of about 100 to about 500 pg / g.

[0043] In addition, the present invention relates to a pharmaceutical composition comprising the above ginger extract and pharmaceutical acceptable additives, wherein said composition has pattern of active substances of ginger extract as shown in Fig. 3, wherein said pharmaceutical composition optionally further contains one or more other extracts, e.g., the extract from plants belong to Acanthaceae family, such as Kariyat (Andrographis paniculata Wall ex Ness.), Cucurbitaceae family, such as Miracle grass or Jiaogulan (Gynostemma pentaphyllum (Thunb.) Makino), Anacardiaceae family, such as mango (Mangifera indica Linn.), Lauraceae family, such as Cinnamon (Cinnainoinuin verum J. Presl. or synonym: C. zeylanicum Nees.), Chinese Cinnamon (Cinnamoimim aromaticum Nees or C. cassia Blume.), Thai Cinnamon (Cinnamomum bejolghotha (Buch-Ham.) Sweet) or a combination thereof.

[0044] The pharmaceutical acceptable additives present in the pharmaceutical composition of this invention may optionally be selected from the group consisting of preservatives, pH adjusters, sweeteners, flavors, emulsifiers, lubricants, stabilizers, humectants.

[0045] The present invention also relates to a formulation comprising the above pharmaceutical composition, as an active ingredient, wherein said formulation can optionally be in the form of pellets, tablets, effervescent tablets, granules, capsules, lotions, serums, creams, gels and emulgels, ointments, sprays, patches, powders or is present in any one of devices, e.g., rollers or the like, or said formulation can be used in foods, cosmetics, supplements or pharmaceuticals.

[0046] Moreover, the present invention relates to a method of anti-inflammatory, antioxidant, antibacterial, e.g. inhibition of oral bacteria, treating cancer, e.g., hepatocellular carcinoma or colon cancer, or analgesic, which comprises administering therapeutically effective amount of the composition or the formulation as stated above to patient in need thereof, including use of the composition or the formulation as mentioned above for the manufacture of a medicament for anti-inflammatory, antioxidant, antibacterial, e.g. inhibition of oral bacteria, treating cancer, e.g., hepatocellular carcinoma or colon cancer, or analgesic.

[0047] EXAMPLES

[0048] The inventors have developed a method of preparing the ginger extract starting from ginger raw material processing step and selecting solvents for extraction to obtain the ginger extracts which contain desired groups of active substances that have the desired patterns. Said ginger extract preparation can be divided into 2 steps as follows:

[0049] 1. Ginger raw material processing

[0050] Raw material: mature ginger, 10 months old a) Drying ginger rhizomes: Clean fresh ginger rhizomes, then cut them into pieces or slices and heat-dried them, e.g. oven-drying, air-drying, evaporation, dehydration or the like, preferably oven-drying:

[0051] Drying temperature may be selected from about 80 to about 90 °C, preferably 85 °C,

[0052] Drying time is about 20 to about 24 hours, preferably 24 hours, b) Immersing fresh ginger that cut into pieces or slices in water for about 20 to about 24 hours, preferably 24 hours, then oven-drying it as stated in step a), or c) Freezing fresh ginger that cut into pieces or slices at temperature of less than 0 °C for about 7 days or 1 week or 30 days, then oven-drying it as stated in step a).

[0053] Extraction method for analyzing the amounts of active substances in ginger

[0054] Drying processed ginger in various methods and then fine grinding, weighing 0.5 g., then extracting it with 70 % 5.0 ml of ethanol by rotary mixer, rotation speed of 50 rpm for 30 min, then centrifugated at 3500 rpm for 5 min. Pour supernatant into a 10 ml volumetric flask. After that, the remaining residue was re-extracted with 70% 5.0 ml of ethanol, then supernatant was combined, and the volume was adjusted to 10 ml and filtered. Then, 6 active substances were analysed by using HPLC-DAD technique at a wavelength of 227 nm, to give the pattern of active substance as shown in Fig. 2B compared to reference standards (Fig. 2A).

[0055] From the analysis data, it was found that frozen and dried fresh ginger contains an increase in the amounts of active substances, especially gingerol group, e.g. 6- gingerol, 8-gingerol, 10-gingerol higher than those of normal dried fresh ginger. In addition, the amount of 6-shogaol is also increased. (Table 1)

[0056] Table 1 The amounts of active substances in various processed gingers in various methods.

[0057] Examples code:

[0058] 1. GGCP031: dried ginger, which is fresh mature ginger, 10 months old (GGCP030) that was cut into thin slices and oven-dried at 85 °C for 24 hours.

[0059] 2. GGCP032: dried ginger immersed in water, which is fresh mature ginger, 10 months old (GGCP030) that was cut into thin slices and immersed in RO water for 24 hours and oven-dried at 85 °C for 24 hours.

[0060] 3. GGCPO33: Dried frozen ginger, which is fresh mature ginger, 10 months old (GGCP030), that was cut into thin slices and frozen for 1 week, then oven-dried at 85 °C for 24 hours.

[0061] 2. Ginger extracts preparation

[0062] Raw material: mature ginger, 10 months old, which is fresh or dried ginger from step 1

[0063] Reducing the size of fresh or dried ginger to be smaller, wherein a method of said reducing the size of fresh or dried ginger, e.g., grinding or homogenizing, preferably homogenizing in blender. a) Preparing alcohol extract of ginger

[0064] Homogenizing fresh or dried ginger and immersing it into 50% ethanol solvent at a ratio of ginger powders to solvent of 1 :5-20 (preferably 1 : 10), leaving it at room temperature for about 0.5 hour to 1 hour. Repeating the extraction for another round, then combining the two extracts. Alternatively, if single extraction is conducted, the ratio of ginger powders to solvent will be 1 :20 and then spray- dried it at the condition of inlet of 140 °C / outlet of 80 °C. Moreover, at least half of solvent can be removed (by evaporating the obtained extract to reduce solvent volume by at least half using the temperature of not more than 60 °C). Before spray-drying, a carrier is added (optional), wherein selected carrier is, e.g., lactose or maltodextrin, in the amount of 1-5% w / w (preferably 2% w / w; here, maltodextrin 2% w / w is used) to give ginger extract in the form of dried powders. When comparing the addition of lactose or maltodextrin with not adding same, it was found that the addition of lactose or maltodextrin diluted the amounts of active substances to a lesser extent. More of these additives are added, the less active substances in ginger extract are obtained while the yield increases. (Table 2). For adjusting tests of the amount of lactose or maltodextrin (from 1% w / w to 2% w / w), it was found that the percentage yield was increased from about 27% to 32-33% while the amounts of active substances were decreased, especially 6- gingerol (decreased by about 25%) (Table 3).

[0065] Consequently, the addition of lactose or maltodextrin into the extract to increase bulk content in the dried powders can be done in the range of 1-5% w / w (preferably 2% w / w).

[0066] Table 2 Comparison of the amounts of active substances in ginger extract between the addition of carrier which is maltodextrin and without adding carrier.

[0067] Example code GGCP035: Dried ginger, which is fresh ginger, 10 months old, from Nan province (GGCP034), cut into thin slices and oven-dried at temperature 85 °C for 24 hours.

[0068] Table 3 Comparison the amounts of active substances in ginger extract when evaporating at least half solvent and adding carrier which is maltodextrin of 2% w / w before spray-drying (repeated experiment 3 times)

[0069] Example code

[0070] GGCP044: Dried ginger, which is mature fresh ginger, 10 months old (GGCP043), cut into thin slices and oven-dried at temperature 85 °C for 24 hours. b) Preparing hot water extract of ginger Extracting ground dried ginger with hot water at temperature from about 80 to 87 °C, preferable temperature of 85 °C, at a ratio of ginger powders to solvent is of 1 :5-20 (preferably 1 : 10), left it at room temperature for about 0.5 hour to 1 hour, repeated another extraction. The obtained extract was combined, and then drying it to be dried powders by spray-drying at the condition of inlet of 140 °C / outlet of 80 °C. Moreover, at least half of solvent can be removed (by evaporating the obtained extract to reduce solvent volume by at least half using the temperature of not more than 60 °C). Before spray-drying, a carrier is added (optional), wherein selected carrier is, e.g., lactose or maltodextrin, in the amount of 1-5% w / w (preferably 2% w / w; here, maltodextrin 2% w / w is used) to give ginger extract in the form of dried powders. c) Preparation of hot water extract of ginger by boiling

[0071] Ground dried ginger was extracted by boiling it in water, the ratio of ginger powders to solvent is 1 :5-20 (preferably 1 :20), boiled it at temperature of from about 80 to 87 °C, preferably at 85 °C, for 2-5 hours (preferably 3 hours) to evaporate water from the extract to approximately half of the total volume and then drying to be dried powders by spray-drying at the condition of inlet of 140 °C / outlet of 80 °C. Before spray drying, a carrier is added (optional), wherein selected carrier is, e.g., lactose or maltodextrin, in the amount of 1-5% w / w (preferably 2% w / w; here, maltodextrin 2% w / w is used) to obtain ginger extract in the form of dried powders.

[0072] There is only 1 active substance analyzed from hot water extract of dried ginger, e.g., 6-gingerol (Table 4) (in the amount of 5-8 mg / g) because hot water cannot extract gingerol and shogaol groups well.

[0073] When ginger residue remains from hot water extraction is extracted with water mixed with alcohol (70% ethanol, according to the extraction methods for analyzing active substances), it was found that there are high amounts of 6 active substances remaining in ginger residue (Table 5).

[0074] When comparing ginger residue remaining from hot water extraction of ginger to ginger residue remaining from the 50% ethanol extraction of ginger (according to Item 2(a)), it was found that ginger residue after 50% ethanol extraction had lower amounts of 6 active substances than those of ginger residue after hot water extraction, indicating that 50% ethanol could extract active substances better (Table 5).

[0075] Table 4 The amounts of active substances in water extract of ginger by immersing and boiling ginger and then spray-drying it to be dried powders

[0076] (without adding carrier).

[0077] Table 5 The amounts of active substances in ginger residue after extracting it with alcohol and hot water. d) Preparing the extract of dried pickled ginger Preparing pickled ginger with vinegar

[0078] 1. The vinegar used is commercially available vinegar (5 % acetic acid) or 5 % acetic acid. pH of said vinegar (1000 ml.) is of 2.44 ± 0.1.

[0079] 2. Cut 1 kg of fresh ginger into thin slices and pouring it into a prepared container.

[0080] 3. Adding vinegar to said fresh ginger in said prepared container, closing lid and immersing it for 24-48 hours (preferably 24 hours).

[0081] 4. Drying pickled ginger at temperature 85 °C for 24 hours (according to ginger raw material processing).

[0082] 5. Grinding dried pickled ginger (pickled at 24-48 hours) and extracting it with 50 % ethanol at a ratio of ginger powders to solvent at 1 :5-20 (preferably

[0083] 1 : 10), leaving it at room temperature for about 0.5 hour to 1 hour with stirring it periodically. Extraction was performed 2 times, and the obtained extract was poured through a sieve. The extracts obtained twice were combined, and spray-dried to be dried powders at the condition of inlet of 140 °C / outlet of 80 °C. Alternatively, if single extraction is conducted, the ratio of ginger powders to solvent will be 1 :20 (single extraction and filter it) and then drying it to be dry powders by spray-drying. Moreover, at least half of solvent can be removed (by evaporating the obtained extract to reduce solvent volume by at least half using the temperature of not more than 60 °C). Before spray-drying, a carrier is added (optional), wherein selected carrier is, e.g., lactose or maltodextrin, in the amount of 1-5% w / w (preferably 2% w / w; here, maltodextrin 2% w / w is used) to give pickled ginger extract in the form of dried powders.

[0084] From the analysis results, it was found that the extract of pickled ginger (without adding maltodextrin or lactose) contained the increase in gingerol and shogaol groups including total phenolic content when comparing with the extract of dried ginger (Table 6) and when adding carrier, e.g., lactose or maltodextrin of 2 % w / w, 2-4 folds amounts of active substances were decreased, while approximately 2-fold percentage yield was increase. Table 6 The amounts of active substances in alcohol extract of dried pickled ginger.

[0085] The solvents used in this step are, e.g., hot water, alcohol, the mixture of water and alcohol which is in various proportions, ethyl acetate, butanol or propylene glycol, preferably hot water, at temperature 85 °C. The extraction was performed with said solvents at least twice (ratio of 1: 10) or single immersion extraction was performed (ratio of 1:20).

[0086] The addition of carrier in the amount of 1-5% w / v was performed to obtain the concentrated extract of dried ginger powders, wherein suitable amount of carrier is about 1-5% w / v (preferably 2% w / v), wherein suitable samples of said carrier can be selected from lactose, maltodextrin, gum arabic, methylcellulose, hydroxypropyl methylcellulose, povidone, pregelatinized starch, carboxymethylcellulose, ethyl cellulose, polyvinyl alcohol, hydroxypropyl cellulose, or the like, preferably lactose, maltodextrin or gum arabic.

[0087] Powder-drying said concentrated extract of ginger was performed, said powder-drying can be selected from spray-drying, freeze-drying, heat-drying, or the like, preferably spray-drying. Suitable conditions for spray-drying are in the range of about 140 / 80 °C (inlet / outlet) to obtain dried ginger extract which are in the form of dried powders, dried flakes, dried granules, and dried slices.

[0088] Pharmacological study of ginger extract

[0089] 1. Study of scavenging activity [2,2-Diphenyl-l-picrylhydrazyl radical scavenging activity]

[0090] Test method (Reference: Rangkadilok N, Sitthimonchai S, Worasuttayangkum L, Mahidol C, Ruchirawat M, Satayavivad J. Evaluation of free radical scavenging and antityrosinase activities of standardized longan fruit extract. Food Chem Toxicol. 2007; 45(2): 328-36)

[0091] DPPH method (2,2-diphenyl-l-picrylhydrazyl) is a method of DPPH radical scavenging assay using an antioxidant agent which is DPPH* that is synthetic agent in the form of stable free radical, soluble in methanol and is purple. When DPPH* reacts with the extract at various concentrations, its purple color will fade to be yellow. The incubation was conducted at 37 °C for 30 min. and absorbance was measured at 517 nm. After that, the obtained absorbance values were used to calculate the percentage of scavenger activity. If samples or test extracts had high scavenging activity, the intensity of purple agent would decrease. The test results were reported as effective concentration 50% (ECso) that means the amounts of antioxidants that cause the concentration of DPPH* to be reduced by half. Vitamin C or ascorbic acid was used as reference standards.

[0092] The test results showed that the extract of dried ginger and the extract of dried pickled ginger contain the same active substances, gingerol and shogaol groups, but in different amounts (Fig. 3 and Table 7), while hot water extract of ginger contains only 1 active substance which is 6-gingerol. When 3 types of ginger extract were tested for DPPH assay, it was found that alcohol extract of pickled ginger has the best scavenging activity (IC50 value = 82.8±1.9 pg / ml.), followed by alcohol extract of dried ginger (IC50 value = 184.4±6.9 pg / ml.), and hot water extract of dried ginger at a concentration of 200 pg / ml shows only 32% scavenging activity (Table 8).

[0093] For scavenging activity test in leukemia cells (HL-60 Assay), it was found that at low concentration (40 pg / ml.), all 3 types of said extracts have no cytotoxicity (% Cell viability >80%) and no scavenging activity. However, at high concentration (400 pg / ml.), alcohol extract of dried ginger and hot water extract of dried ginger have scavenging activity in leukemia cells (HL-60) at 60% and 82%, respectively, while alcohol extract of dried pickled ginger does not have such activity (6%).

[0094] Moreover, it was found that alcohol extract of ginger has cytotoxicity of HL-60 at high concentration, while hot water extract did not show any cytotoxic activity even at high concentration.

[0095] When considering active substances, it was found that alcohol extract of ginger contains both gingerol and shogaol groups, and the extract of pickled ginger contains higher active substances than the extract of dried ginger, especially 6-gingerol, 8-gingerol, 10-gingerol, and 6- shogaol, thus it shows better scavenging activity, while hot water extract contains only 1 active substance which is 6-gingerol, it thus has the least scavenging activity.

[0096] Table 7 The amounts of active substances in various ginger extracts tested for pharmacological activity Table 8 Scavenging activity of ginger extracts

[0097] Remarks:

[0098] *Cell viability <50%: samples have cytotoxicity to cells used in the tests.

[0099] **The concentration of DMSO used in reaction is 10% (Normally used at 1%).

[0100] Regarding the test results of in vitro scavenging activity of ginger extract, said extract, especially alcohol extract of dried ginger or alcohol extract of dried pickle ginger of this invention, can be used for the manufacture of a pharmaceutical composition or cosmetics which are in various forms, for example, capsules or tablets, lotions, serums, creams, gels and emulgels, sprays, patches or are present in any one of devices, e.g., rollers etc.

[0101] Product Examples

[0102] Formulations comprising pharmaceutical composition of this invention which comprises ginger extract of this invention The pharmaceutical composition of this invention comprising ginger extracts of this invention was formulated as an oral spray, said formulation was shown in Table 9 using the pharmaceutical composition of this invention containing dried powders of ginger extract which is of about 0.5-5% w / w (preferably 1% w / w) or water extract (liquid) of ginger which is of about 2-8% w / w (preferably 5% w / w).

[0103] Oral antimicrobial activity tests (Antimicrobial activity of ginger mouthwash against Streptococcus mutans)

[0104] From oral antibacterial activity, it was found that an oral spray comprising the pharmaceutical composition of this invention which comprises ginger extracts of the present invention as a main component in combination with other herbal extracts as stated above, for example, longan extract, kariyat extract, mango extract, cinnamon extract or miracle grass extract, have inhibition effect against S. mutans (Fig. 4). The formulation of Table 9 is the use of ginger extract in combination with longan extract of 0.2-2% w / w (preferably 0.5% w / w).

[0105] It was found that ginger extract in both dried powders and water forms in the amount used in said formulation has the same S. mutans inhibitory effects.

[0106] Table 9 The formulation of an oral spray comprising the pharmaceutical composition of the present invention which comprises ginger extract of this invention.

[0107] The study of the inhibition effect on colon cancer and hepatocellular carcinoma cells (ability of inhibiting proliferation of cancer cells)

[0108] Alcohol extract of dried ginger (Fig. 3A) was tested on colon cancer cells (H508) and hepatocellular carcinoma cells (HepG2) by the total testing time of 72 hours and then was reacted with thiazolyl blue tetrazolium bromide (MTT) and was dissolved in dimethyl sulfoxide (DMSO) for measuring absorbance.

[0109] The test results of ginger extract on colon cancer cells H508 show that said extract has inhibitory effect on colon cancer cells with the LD50 value of 390.4775 p.g / ml, and the test results of ginger extract on hepatocellular carcinoma cells (HepG2) show that said extract has inhibitory effect on hepatocellular carcinoma cells with the LD50 value of 160.0631 .g / ml (Fig. 5).

[0110] BRIEF DESCRIPTION OF DRAWINGS

[0111] Fig. 1 shows structural formulas of all six active substances which are gingerol group, e.g. 6-gingerol, 8-gingerol, and 10-gingerol, and shogaol group, e.g., 6-shogaol, 8-shogaol, 10- shogaol in ginger.

[0112] Fig. 2 shows chromatogram; Fig. 2A is chromatogram of reference standards, and Fig. 2B is chromatogram of dried ginger extract which comprises (1) 6-gingerol, (2) 8-gingerol, (3) 6-shogaol, (4) 10-gingerol, (5) 8-shogaol, (6) 10-shogaol.

[0113] Fig. 3 shows HPLC chromatogram of active substances in ginger extract which was used for testing scavenging activity; (1) 6-gingerol, (2) 8-gingerol, (3) 6-shogaol, (4) 10-gingerol, (5) 8-shogaol, (6) 10-shogaol; Fig. 3 A is HPLC chromatogram of the extract of dried ginger (extracted with 50 % ethanol) and added 2% maltodextrin (GGEXP025); Fig. 3B is HPLC chromatogram of the extract of dried pickled ginger (extracted with 50 % ethanol) and added 2% maltodextrin (GGEXP026); and Fig. 3C is HPLC chromatogram of the extract of dried ginger (extracted with hot water) and added 2 % maltodextrin (GGEXP027).

[0114] Fig. 4 shows test results of inhibitory effects on the growth of oral bacteria Streptococcus mutans of an oral spray containing dried powders of ginger extract (Lot 12-03) and liquid ginger extract (Lot 13-02); Fig. 4A shows inhibition zones for 12-03 (the concentration of the extract at 1%), 13-02 (the concentration of the extract at 5%) and MQ (milliQ water); and Fig. 4B shows inhibition zones for 12-03 (the concentration of the extract at 0.5%), 13-02 (the concentration of the extract at 2.5%) and MQ (milliQ water). Fig. 5 shows test results of ginger extract (50% ethanol); Fig. 5A shows test results on hepatocellular carcinoma cells (HepG2); and Fig. 5B shows test results on colon cancer cells, (H508).

Claims

1. CLAIMS1. A method of preparing ginger extract which comprises active substances that have the pattern shown in Figs. 2A and 2B, said method comprising the steps as follows: i. Processing ginger to obtain processed ginger; and drying obtained processed ginger to give dried processed ginger, or drying ginger to obtain dried ginger, ii. Reducing the size of dried processed ginger or dried ginger of step i. to obtain smallsized dried processed ginger or small-sized dried ginger, iii. Extracting small- sized dried processed ginger or small- sized dried ginger of step ii. with solvents, e.g., water, alcohol or the mixture thereof, preferably extracting small-sized dried processed ginger or small-sized dried ginger of step ii. with the mixture of solvents or with water to obtain ginger extract.

2. The method according to claim 1, wherein the suitable ginger is ginger rhizomes that are of about 8 to about 10 months or more, preferably about 10 months.

3. The method according to claims 1 or 2, wherein the processing of step i. is selected from:Reducing the size of fresh ginger and immersing it in water, Reducing the size of fresh ginger and freezing it, or Reducing the size of fresh ginger and acid pickling it, wherein reducing the size of fresh ginger is making ginger into pieces or slices.

4. The method according to any one of claims 1 to 3, wherein, in ginger processing step i., immersing fresh ginger in water is optionally at room temperature for about 20 to about 24 hours, preferably of about 24 hours.

5. The method according to any one of claims 1 to 3, wherein, in ginger processing step i., freezing fresh ginger is optionally at temperature of at least about 0 °C for about 7 days to about 30 days.

6. The method according to any one of claims 1 to 3, wherein, in ginger processing step i., pickling fresh ginger is optional with acid, wherein acid used for pickling is, e.g., commercially available vinegar comprising 5 % v / v of acetic acid, 5 % v / v of acetic acid, for about 24 hours to about 48 hours (preferably about 24 hours).

7. The method according to any one of claims 1 to 6, wherein drying of step i. is heat-drying e.g. oven-drying, air-drying, evaporation, dehydration or the like, preferably oven-drying.

8. The method according to any one of claims 1 to 7, wherein drying of step i. is conducted at temperature of about 80 to about 90 °C, preferably about 85 °C, for about 20 to about 24 hours, preferably about 24 hours.

9. The method according to any one of claims 1 to 9, wherein reducing the size of step ii. is grinding or homogenizing, preferably homogenizing.

10. The method according to any one of claims 1 to 9, wherein the extraction with the mixture of water and alcohol is the extraction with the mixture of water and ethanol at a ratio of water: ethanol of about 20 to about 70% by volume, preferably about 50% by volume.

11. The method according to any one of claims 1 to 10, optional:Extracting by adding small-sized dried processed ginger or small-sized dried ginger of step ii. with the mixture of water and alcohol or with water at room temperature for about 0.5 hour to about 1 hour, repeated twice, orExtracting by using small-sized dried processed ginger or small-sized dried ginger of step ii. to the mixture of water and alcohol at a ratio of about 1 part to about 5 to 20 parts, preferably about 1 : 10, repeated twice, preferably single extracting at a ratio of small-sized dried processed ginger or small-sized dried ginger of step ii. to the mixture of water and alcohol of about 1 :20.

12. The method according to any one of claims 1 to 10, wherein the extraction with water is:Extracting with water at temperature from about 80 to 87 °C, preferably about 85 °C, or Adding water to small-sized dried processed ginger or small-sized dried ginger of step ii. and heating water at a temperature from about 80 to about 87 °C, preferably about 85 °C, for about 2 to about 5 hours, preferably about 3 hours.

13. The method according to any one of claims 1 to 12, wherein the extraction of step iii. further comprises the following steps: a. Evaporating the mixture of water and alcohol or water from ginger extract of step iii. to reduce its volume by at least half to obtain evaporated ginger extract, b. Drying evaporated ginger extract of step a. by spray-drying.

14. The method according to claim 13, wherein evaporating the mixture of water and alcohol or water from ginger extract is conducted at temperature of not more than 60 °C and spraydrying is conducted at the condition of inlet of 140 °C / outlet of 80 °C.

15. The method according to any one of claims 1 to 14, wherein a carrier is optionally added prior to step b.

16. The method according to any one of claims 1 to 15, wherein said carrier is selected from lactose, maltodextrin, gum arabic, methylcellulose, hydroxypropyl methylcellulose, povidone, pregelatinized starch, carboxymethylcellulose, ethyl cellulose, polyvinyl alcohol, hydroxypropyl cellulose, or the like, preferably lactose, maltodextrin, or gum arabic.

17. The method according to any one of claims 1 to 16, wherein said carrier is optionally added in the amount of about 1 to about 5 % w / w, preferably 2 %w / w, most preferably optional adding maltodextrin in the amount of about 2 % w / w.

18. The ginger extract obtained from the method according to any one of claims 1 to 17.

19. The ginger extract according to claim 18 which comprising active substances selected from the group comprising of gingerol and / or shogaol groups, wherein structural formulas of said active substances are as shown in Fig. 1.

20. The ginger extract according to claim 18 or 19, wherein active substances comprise the group of gingerols which are selected from the group consisting of 6-gingerol, 8-gingerol and 10- gingerol.

21. The ginger extract according to any one of claims 18 to 20, wherein active substances comprise the group of gingerols in the amounts of not less than about 10 mg / g.

22. The ginger extract according to any one of claims 18 to 21, which contain 6-gingerol in the range of about 10 to about 50 mg / g.

23. The ginger extract according to any one of claims 18 to 22, wherein active substances comprise shogaol group which is selected from the group comprising 6-shogaol, 8-shogaol, and 10-shogaol.

24. The ginger extract according to any one of claims 18 to 23, wherein active substances comprise shogaol group in the amount of not less than about 1 mg / g.

25. The ginger extract according to any one of claims 18 to 24, wherein said extract contains the amount of Total Phenolic Content of not less than about 20 mg gallic acid equivalent / g and optionally further contains Gamma-Aminobutyric acid (GABA) in the range of about 100 to about 500 |4g / g.

26. A pharmaceutical composition comprising the ginger extract according to any one of claims 18 to 25, and pharmaceutical acceptable additives.

27. The pharmaceutical composition according to claim 26, wherein said composition has the pattern of active substances of the ginger extract as shown in Fig. 3.

28. The pharmaceutical composition according to claim 26 or 27, which optionally further contains one or more other extracts, e.g., the extract from plants belong to Acanthaceae family, such as Kariyat (Andrographis paniculata Wall ex Ness.), Cucurbitaceae family, such as Miracle grass or Jiaogulan (Gynostemma pentaphyllum (Thunb.) Makino), Anacardiaceae family, such as mango (Mangifera indica Linn.), Lauraceae family, such as Cinnamon (Cinnamomum verum J. Presl. or synonym: C. zeylanicum Nees.), Chinese Cinnamon (Cinnamomum aromaticum Nees or C. cassia Blume.), Thai Cinnamon (Cinnamomum bejolghotha (Buch-Ham.) Sweet) or a combination thereof.

29. The pharmaceutical composition according to any one of claims 26 to 28, wherein said additives are selected from the group consisting of preservatives, pH adjusters, sweeteners, flavors, emulsifiers, lubricants, stabilizers, humectants.

30. A formulation comprising the pharmaceutical composition according to any one of claims 26 to 29 as an active ingredient.

31. The formulation according to claim 30 which is optionally in the form of pellets, tablets, effervescent tablets, granules, capsules, lotions, serums, creams, gels and emulgels, ointments, sprays, patches, powders or is present in any one of devices, e.g., rollers.

32. The formulation according to claim 30 or 31 which is used in foods, cosmetics, supplements or pharmaceuticals.

33. A method of anti-inflammatory and / or antioxidants, which comprises administering therapeutically effective amount of the composition according to any one of claims 26 to 29, or the formulation according to any one of claims 30 to 32 to patient in need thereof.

34. A method of antibacterial, which comprises administering therapeutically effective amount of the composition according to any one of claims 26 to 29, or the formulation according to any one of claims 30 to 32 to patient in need thereof.

35. The method of claim 34 which is inhibition of oral bacteria.

36. A method of treating cancer, which comprises administering therapeutically effective amount of the composition according to any one of claims 26 to 29, or the formulation according to any one of claims 30 to 32 to patient in need thereof.

37. The method of claim 36, wherein cancer is hepatocellular carcinoma or colon cancer.

38. A method of analgesic, which comprises administering therapeutically effective amount of the composition according to any one of claims 26 to 29, or the formulation according to any one of claims 30 to 32 to patient in need thereof.

39. Use of the composition according to any one of claims 26 to 29, or the formulation according to any one of claims 30 to 32 in the manufacture of a medicament for anti-inflammatory and / or antioxidants.

40. Use of the composition according to any one of claims 26 to 29, or the formulation according to any one of claims 30 to 32 in the manufacture of a medicament for antibacterial.

41. The use of claim 40 wherein antibacterial is for oral bacteria.

42. Use of the composition according to any one of claims 26 to 29, or the formulation according to any one of claims 30 to 32 in the manufacture of a medicament for treating cancer.

43. The use of claim 42, wherein cancer is hepatocellular carcinoma or colon cancer.

44. Use of the composition according to any one of claims 26 to 29, or the formulation according to any one of claims 30 to 32 in the manufacture of a medicament for analgesic.

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