The preparation of UE-12 aqueous extract has been previously described (Kong et al., 2014 (link)). Briefly, Orthosiphon stamineus leaves were collected and taken through a series of stringent microbial tests as well as Atomic Absorption Spectrophotometry (AAS) to test for heavy metal presence. The leaves were then dried, ground into fine powder which was used in a water-based extraction. The microbial and trace metal element tests were repeated again on the extraction yield. The product was examined for the presence of functional groups/molecules by Fourier transform infrared spectroscopy (FTIR) and High-performance liquid chromatography (HPLC). Sinensetin (CAS no. 2306-27-6), eupatorin (CAS no. 855-96-9) and rosmarinic acid (CAS no. 20283-92-5) were purchased from ChromaDex®, Inc. (Irvine, CA). The extract and compounds were dissolved in dimethyl sulfoxide (DMSO) and filtered through a 0.2 µm membrane filter (Sartorius Stedim, Germany) followed by storage at −20°C in a desiccated storage container.
Rosmarinic acid
Manufactured by ChromaDex
Sourced in United States
Rosmarinic acid is a compound found in various plant species, including rosemary. It is a natural antioxidant with potential bioactive properties. The core function of rosmarinic acid is as a chemical standard for analytical testing and research purposes.
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Lab products found in correlation
Carvacrol, by Merck Group (5 mentions)
Methanol, by Carl Roth (4 mentions)
Sinensetin, by ChromaDex (3 mentions)
Acetic acid, by Merck Group (3 mentions)
Super purity water system, by Merck Group (3 mentions)
2 2 diphenyl 1 picrylhydrazyl (dpph), by Merck Group (2 mentions)
Formic acid, by Merck Group (2 mentions)
Eupatorin, by ChromaDex (2 mentions)
Phospholipon 90h pl 90h, by Lipoid (1 mentions)
Oleanolic acid, by Merck Group (1 mentions)
Triethanolamine, by Merck Group (1 mentions)
Galacturonic acid, by Merck Group (1 mentions)
Galactose, by Merck Group (1 mentions)
Rhamnose, by Merck Group (1 mentions)
Xylose, by Merck Group (1 mentions)
Arabinose, by Merck Group (1 mentions)
Alcl3, by Merck Group (1 mentions)
Hplc grade acetonitrile, by Thermo Fisher Scientific (1 mentions)
Sucrose, by Merck Group (1 mentions)
Tanshinone iia, by Merck Group (1 mentions)
12 protocols using rosmarinic acid
1
Extraction and characterization of Orthosiphon stamineus leaf bioactive compounds
2
Extraction and Analysis of Origanum onites
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Dried Origanum onites L. herb was obtained from “İnanTarım ECO DAB,” Turkey. Voucher specimens (No. L270711) are held by the Herbarium of the Department of Drug Technology and Social Pharmacy, Lithuanian University of Health Sciences. Ethanol (96%) as extraction solvent was purchased from Vilniaus degtinė (Vilnius, Lithuania). Phospholipon 90H (PL 90H) and 1,2-dimyristoyl-sn-glycero-3-phosphorylglycerol sodium salt (DMPG-Na) were graciously provided by Lipoid AG (Ludwigshafen, Germany). Purified water used for sample preparation and HPLC runs was produced using a Super Purity Water System (Millipore, USA). HPLC eluents: methanol (99.95%) was purchased from Carl Roth GmbH (Karlsruhe, Germany) and acetic acid (99.8%) from Sigma-Aldrich (St. Louis, MO, USA). Standards for HPLC analysis: carvacrol (>98%) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and rosmarinic acid (>98%) from ChromaDex (Santa Ana, TX, USA).
3
Bioactive Compounds from Rosemary, Flax, and Oats
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Dried rosemary (Rosmarinus officinalis L.) leaves were purchased from UAB “Sirdazole” (Vilnius, Lithuania). Linseed (Linum usitatissimum Linn.) was procured from UAB “Herba humana” (Vilnius, Lithuania), oat (Avena sativa L.) groats were purchased from UAB “Skaneja” (Vilnius, Lithuania).
Extra virgin olive oil was purchased from UAB “Anira” (Kaunas, Lithuania). The emulsifier/rheology modifier ViscOptima™ SE (Sodium Polyacrylate, Ethylhexyl Cocoate, PPG-3 Benzyl Ether Myristate and Polysorbate 20) was provided by CRODA (Goole, UK).
HPLC and TLC standards of triterpenic acids (ursolic acid (>98%), oleanolic acid (>97%), galacturonic acid (>97%), xylose (>99%), galactose (>99%), rhamnose (>99%) and arabinose (>98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rosmarinic acid was obtained from ChromaDex (Santa Ana, CA, USA). Triethanolamine (>99%) was procured from Sigma-Aldrich.n -1-(-1-naphtyl)-ethylenediamine dihydrochloride (>98%) was purchased from Tokyo Chemical Industry (Tokyo, Japan).
The EpiDerm™ (EPI-200) reconstructed human skin models and MTT kit to test cell viability was purchased from MatTek (Bratislava, Slovakia). The Quantikine® human IL-1α/IL-1F immunoassay was from R&D Systems (Minneapolis, MN, USA).
Extra virgin olive oil was purchased from UAB “Anira” (Kaunas, Lithuania). The emulsifier/rheology modifier ViscOptima™ SE (Sodium Polyacrylate, Ethylhexyl Cocoate, PPG-3 Benzyl Ether Myristate and Polysorbate 20) was provided by CRODA (Goole, UK).
HPLC and TLC standards of triterpenic acids (ursolic acid (>98%), oleanolic acid (>97%), galacturonic acid (>97%), xylose (>99%), galactose (>99%), rhamnose (>99%) and arabinose (>98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rosmarinic acid was obtained from ChromaDex (Santa Ana, CA, USA). Triethanolamine (>99%) was procured from Sigma-Aldrich.
The EpiDerm™ (EPI-200) reconstructed human skin models and MTT kit to test cell viability was purchased from MatTek (Bratislava, Slovakia). The Quantikine® human IL-1α/IL-1F immunoassay was from R&D Systems (Minneapolis, MN, USA).
4
Rosmarinic Acid and Carvacrol Synthesis
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Rosmarinic acid (>98%) was purchased from ChromaDex (Santa Ana, TX, USA). All other chemicals, including carvacrol (>98%) and AlCl3 (>99.9%), were from Sigma-Aldrich (St. Louis, MO, USA).
5
Phytochemical Extraction and Identification
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Gamborgs B-5 basal medium, sucrose, 5-azacytidine and tanshinone IIA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tanshinone I, dihydrotanshinone I, and salvianolic acid B were purchased from Wuhan ChemFaces Biochemical Co., Ltd. (WUHAN, HUBEI, China). Rosmarinic acid was purchased from ChromaDex (Los Angeles, CA, USA). Cryptotanshinone was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). HPLC grade ethanol was purchased from Honeywell Chemicals (Seelze, Germany). HPLC grade acetonitrile was purchased from Fisher Scientific (Seoul, Korea).
6
Phytochemical Screening and Antioxidant Assays
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Methanol and ethanol (analytical grade), acetonitrile and formic acid (HPLC grade) were purchased from Merck, Petaling Jaya, (Malaysia). 2,2-diphenyl-1-picrylhydrazyl, quercetin, gallic acid, aluminium chloride, bovine serum albumin, copper sulphate, folin-ciocalteu’s reagent, anthrone, sodium carbonate and potassium tartrate were purchased from Sigma-Aldrich, (Germany). The reference compounds rosmarinic acid, 3′-hydroxy-5,6,7,4′-tetramethoxyflavone, sinensetin and eupatorin were purchased from ChromaDex, (USA). Deionised water for HPLC was prepared using ultra pure water purifier system Thermo Scientific, (USA). The reverse phase Acclaim Polar Advantage II C18 column (3 μm, 3 × 150 mm) was purchased from Dionex, Thermo scientific, (USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, potassium chloride, potassium dihydrogen phosphate, dimethyl sulfoxide and sodium chloride were purchased from Sigma-Aldrich, (USA). Disodium hydrogen phosphate was purchased from Fluka, (Switzerland).
7
Origanum onites L. Herb Extraction Analysis
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Dried Origanum onites L. herb was obtained from “İnanTarım ECO DAB’’, Antalya, Turkey. The Herbarium of the Department of Drug Technology and Social Pharmacy, Lithuanian University of Health Sciences holds the voucher specimens (No. L170711). Ethanol (96%) for extraction was purchased from Vilniaus degtinė (Vilnius, Lithuania). EE100 was supplied from Evonik (Essen, North Rhine-Westphalia, Germany). The water used in HPLC and for sample preparation was produced with a Super Purity Water System (Millipore, Bedford, MA, USA). HPLC eluents: methanol (99.95%) was purchased from Carl Roth GmbH (Karlsruhe, Germany) and acetic acid (99.8%) from Sigma-Aldrich (St. Louis, MO, USA). Standards for HPLC analysis: carvacrol (>98%) was purchased from Sigma-Aldrich (St. Louis, MO, USA) and rosmarinic acid (>98%) was from ChromaDex (Santa Ana, TX, USA).
8
Analytical UHPLC of Botanical Compounds
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Formic acid, methanol and LCMS grade acetonitrile were purchased from MERCK (Malaysia). HPLC grade water was prepared from distilled water using a Milli-Q-system (Millipore, MA,USA) and was used during analytical UHPLC analysis. Standards used were rosmarinic acid (Chromadex, CA, USA), caffeic acid, chlorogenic acid, eupatorin and sinensetin (Extrasynthase, Genay, France). All of other solvents and chemicals used in this study were analytical grade. Stock and working standards were prepared by dissolving these analytes in 100% methanol. The standard solutions stored at 4°C were stable for at least 3 months.
9
HPLC Standards and Chemicals for Phytochemical Analysis
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Acetonitrile (ACN) gradient grade for liquid chromatography LiChrosolv® Reag. Ph Eur; phosphoric acid suitable for HPLC, LiChropur™, 85%; and 1,1-diphenyl-2-picrylhydrazyl (97%) were purchased from Merck (Poznań, Poland). Water for HPLC was produced in the laboratory using a water purifier that provides high-purity deionised water for laboratory use WCA R03 DP ECO from Cobrabid Aqua (Warsaw, Poland). Standards of chlorogenic acid (3-O-Caffeoylquinic acid), prim-O-glucosylcimifugin (cimifugin β-D-glucopyranoside), rosmarinic acid, cimifugin, 4′-O-β-D-glucosyl-5-O-methylvisamminol, imperatorin (pentosalen), caffeic acid (3,4-Dihydroxycinnamic acid) and astragalosides I-IV were purchased from ChromaDex® (Irvine, CA, USA).
10
Extraction and Formulation of Origanum onites
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Dried Origanum onites L. herb was obtained from “İnanTarım ECO DAB’’, Turkey. Voucher specimens (No. L170711) have been deposited at the Herbarium of the Department of Drug Technology and Social Pharmacy, Lithuanian University of Health Sciences. Carvacrol (>98%), HPLC eluent acetic acid (99.8%), as well as wall material gum arabic and β-Cyclodextrin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tween 20 was purchased from Scharlau Chemie S.A. (Barcelona, Spain) and bovine Gelatin bloom value 100 was purschased from Gelita AG. (Eberbach, Germany). HPLC eluent methanol was from Carl Roth GmbH (Karlsruhe, Germany) and rosmarinic acid (>98%) was from ChromaDex (Santa Ana, TX, USA). Extraction solvent ethanol (96% v/v) was purchased from Vilniaus degtinė (Vilnius, Lithuania). The purified water used in HPLC and for sample preparation was produced with a Millipore Super Purity Water System (Sigma-Aldrich Corp., St.Louis, MO, USA).
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