Trifolium Pratense Ihussey

Natural History. Trifolium pratense. Tincture of flower heads. (In the cancer cases an extract of the blossoms of the first crop is used. 'This extract,' says Cooper, 'is made by boiling down the flowers till nothing but an inspissated residuum is left: in this condition it seems to be caustic and for this reason is used by herbalists: I doubt its utility.' Pancreas, affections of.

• Trifolium Pratense Hussey

Trifolium pratense in the Germplasm Resources Information Network (GRIN), U.S. Department of Agriculture Agricultural Research Service. Accessed on 28-Oct-07. Accessed on 28-Oct-07. Vernacular names [ edit ]. I; Hussey, ii, pi. 23; Cke., Hdbk. 1084; Barla, pi. Pratense, 65. — pusilliim, 65, 76. Howell: The Clover Rust. Trifolium pratense is a PERENNIAL growing to 0.6 m (2ft) by 0.6 m (2ft in) at a medium rate. It is hardy to zone (UK) 6 and is not frost tender. It is in flower from.

Throat, sore, mucus in. Uvula, pain in. Has a reputation among eclectics in whooping-cough, and coughs of measles and phthisis. Duncan and others proved it and elicited some very decided respiratory symptoms, dry, tickling cough, oppressed breathing with fever.

One peculiar symptom was, cough followed by hiccough. Many symptoms of congestion appeared. The head felt full of blood. The lungs felt full of blood. Sensation in lungs as if breathing hot air, as if air full of impurities. There was persistent headache on waking. Sleep was unrefreshing.

The pulse was weak and intermittent, showing an action on the heart, later it was full and bounding. Pharynx as well as trachea was irritated and there was: 'Sharp pain through uvula causing tears to start.' Dryness of trachea causing him to clear throat of some foreign substance. Secretion of urine increased. Dull, stupid feeling generally. Farrington gives as characteristic: Hoarseness and choking spells at night with cough, the neck is stiff, cramps in sternomastoid muscle better by heat and friction.

Cooper says of Tri. That it causes salivation in horses, and diarrhoea if they eat much of it, and that it cures mumps and affections of the pancreas and salivary glands. Hale says it causes 'heaves' in horses. 431) gives it to persons disposed to cancer. He finds it retards the progress of cancerous tumours and improves the general condition of the patient. It keeps cancer from ulcerating. After ulceration has occurred Tri.

Does no good. Another indication of Felter's is 'scaly and ulcerated conditions of the tibial region of the old.' The symptoms are worse in close room, better in open air. 'When I became very warm (with the fever) my breathing stopped, lungs felt as if breathing hot air.' Worse After sleep.

Compare: Botan., congestive symptoms, Meli. Headache on waking, Nat-m., Lach. Cough with pains in chest, Bry., Ranuc., Arn., Rhus.

Confusion of ideas, morning. Great headache in morning on waking. Dullness in anterior brain. Head feels full of blood. Intermittent headache.

Pricking in forehead. Eyes: dull and sore, heavy. Much mucus in nose, thin.

Whole mucus tract sensitive. Taste of drug disgusting and always in mouth. Salivation (horses).

Much mucus in throat, constantly trying to clear it. Throat irritated all the way down, as if scalded. Irritation of pharynx and trachea causing dry cough. Sharp pain through uvula causing tears to start.

Great thirst. Rises at 5.45 AM with griping pains in abdomen and headache. Colicky pains all day. Stool and Anus.

Very costive, each stool followed by several drops of dark blood, attended by a bearing-down sensation as if bowel would prolapse by its own weight (this lasted fourteen days, after which bowels became regular). Stool delayed several days, hard, covered with mucus, later bowels moved freely. Urinary Organs. Uneasiness in region of kidneys, and whole urinary tract.

Urine very profuse. Vesical tenesmus after urinating. Respiratory Organs.

Great dryness of trachea causing him to clear throat of some foreign substance at eleven AM Irritation of pharynx and trachea causing short, hacking cough with accumulation of mucus which must be expectorated. Incessant dry, hacking cough. Seems as if trachea was loaded with impurities, after eating. Dyspnoea better in fresh air. Lungs feel as if full of blood two PM, ten PM had to leave a close room from oppression, on going into fresh air was obliged to cough much, this was followed by hiccough and profuse expectoration.

Chest tight, on lying down. Pulse slow and irregular, later full and rapid. Neck and Back. Neck stiff, cramps in sternomastoid muscles, better by heat and rubbing, with the cough. Tingling in left palm, also in left arm. On waking in morning felt as if he had not been asleep. Immediately after retiring began to feel cold, pulse intermitting, later bounding, became very warm and breathing stopped, lungs felt as if breathing hot air.

Feet and hands cold, head hot.

Design Individual compounds in the pre-formulated ( i.e., no excipients present) extract were identified by either chemical isolation followed by structure elucidation or by matching to retention time and molecular mass of chemical standards via liquid chromatography-mass spectrometry (LC-MS) analysis. Quantitation of the amounts of compounds found in the pre-formulated extract was done using HPLC-UV or LC-MS. Isolated compounds or standards were evaluated for their ability to 1) induce alkaline phosphatase (AP) in an endometrial carcinoma cell line, 2) competitively bind to recombinant human estrogen receptors (ERs) alpha (α) and beta (β), and 3) act as antioxidants by scavenging 2,2-di(4- tert-octylphenyl)-1-picrylhydrazyl (DPPH) free radicals. Results The pre-formulated red clover extract had an EC 50 of 2.0–2.2 μg/mL in the AP estrogenicity assay, and IC 50s of 18.4–32.6 μg/mL and 1.9–3.4 μg/mL in the ERα and ERβ binding assays, respectively. The pre-formulated extract was composed of 35.54% isoflavones, 1.11% flavonoids, 0.06% pterocarpans, ≤ 0.03% coumarins, and ≤ 0.03% tyramine. Daidzein, genistein, formononetin, biochanin A, coumestrol and naringenin were estrogenic in the AP assay, and all of these, except formononetin, bound to one or both ERs. Conclusions The major and minor chemical and active estrogenic components of a pre-formulated Phase II red clover clinical extract were identified, quantitatively measured, and the final capsule doses were calculated.

The extract is currently under evaluation in a yearlong clinical study for the alleviation of menopausal hot flashes. This is the first report to thoroughly summarize the chemistry and biology of all major peaks observed in the HPLC-UV chromatogram of a clinical red clover dietary supplement. INTRODUCTION A recent survey of peri- and postmenopausal female outpatients found that approximately 79% of these women use botanical dietary supplements. Red clover is one botanical dietary supplement used by women for menopausal hot flashes. However, significant concerns about the quality of botanical dietary supplements (;;; ) complicate current efforts to study the clinical efficacy of botanical supplements.

Commercial extracts of red clover contain high amounts of the mildly estrogenic isoflavones daidzein, genistein, formononetin, and biochanin A. The general premise supporting use of red clover supplements during menopause is that the isoflavones will act as a natural estrogen replacement in older women having low endogenous estradiol levels, and thus alleviate hot flashes and other symptoms. Red clover isoflavone supplements contain several minor components as well, many of which have not been investigated for their estrogenic activity or even quantitatively measured in over-the-counter supplements.

Because some of these minor compounds may potentially be converted into active estrogenic metabolites in vivo, identification and measurement of these compounds is useful in the standardization of red clover supplements for clinical trials, allowing researchers to calculate the individual doses of each compound consumed by the subjects. Currently, no published papers thoroughly detail the chemical content of major and minor compounds in red clover botanical supplements used in clinical studies. Experiments to characterize the chemical content and evaluate the estrogenic activity of 22 major and minor compounds present in a Phase II clinical red clover extract were performed. The coumarin content of this extract was previously reported.

We present a HPLC-UV chromatogram of the extract, and include calculated daily doses of 20 of the compounds, based on the amount of raw extract administered in our phase II clinical trial. Pure compounds or standards identified in the red clover extract were tested in 1) the AP assay, 2) in human recombinant ERα and ERβ competitive binding assays, and 3) in a DPPH free radical scavenging antioxidant assay. We hope that our biological and chemical evaluation of this red clover dietary supplement will help establish a standard approach that clinicians and researchers can use in the study of botanical dietary supplements. Materials Chemical solvents and reagents were purchased from Fisher (Hanover Park, IL) or Sigma (St.

Louis, MO), unless otherwise indicated. Isoflavone, flavonoid, and coumarin standards were purchased from Indofine Chemical Co. (Somerville, NJ), Chromadex (Denver, CO), and TimTec (Newark, DE).

Irilone was a kind gift from Professor Eckhard Wollenweber (Technische Universit t Darmstadt, Germany). Cell culture media and GlutaMAX™-1 was purchased from Invitrogen (Carlsbad, CA) and fetal bovine serum (FBS) was from Atlanta Biologicals (Norcross, GA). Plant material The 30% isoflavone (defined as total content of daidzein + genistein + formononetin + biochanin A, w/w%) red clover Phase II clinical extract was a T.

Pratense autohydrolyzed extract manufactured by PureWorld Botanicals, Inc. (South Hackensack, NJ) using proprietary methods. The raw unformulated extract, without added excipients, was used in the present investigation. A voucher specimen of the coarsely milled plant source material is being stored at the University of Illinois at Chicago. The plant source material was examined by Dr.

Doel Soejarto (UIC) and was found to be consistent with authentic T. Pratense specimens deposited at the Field Museum of Natural History (Chicago, IL).

Extraction and isolation For initial fractionation, 300 g of the unformulated powdered raw red clover extract was mixed with 60 g of dry microcrystalline cellulose and loaded into a vacuum flash column packed with 3 kg of dry microcrystalline cellulose. LC-MS quantitative analyses The following compounds were tentatively identified from electrospray LC-MS results by matching m/z values to compounds reported in Trifolium and related species: tyramine ( 1), fisetin ( 5), calycosin ( 7), quercetin ( 8), naringenin ( 9), pratensein ( 12), kaempferol ( 13), pseudobaptigenin ( 15), irilone ( 18), and prunetin ( 21). These compounds were purchased or isolated and then the mass spectra and retention times of extract peaks were compared with those of the standards (including spiked extract samples), confirming their presence in the red clover Phase II extract. Since 1, 5, and 9 were found to be present in the extract at. Compound Estrogenicity in Ishikawa cells EC 50, μM Estrogen Receptor α Binding IC 50, μM Estrogen Receptor β Binding IC 50, μM DPPH Free Radical Scavenging IC 50, μM Tested 10-2002: Tested 10-2002: Tested 10-2002: Phase II Red 2.0 ± 0.1 μg/mL 18.4 ± 4.9 μg/mL 1.9 ± 0.8 μg/mL 143.1 ± 8.2 Clover Extract Tested 09-2004: Tested 09-2004: Tested 09-2004: μg/mL 2.2 ± 0.2 μg/mL 32.6 ± 9.0 μg/mL 3.4 ± 0.7 μg/mL Tyramine ( 1) 20 100 100 200 Scopoletin ( 2) 20 N.T.

200 Fraxidin ( 3) 20 N.T. 200 Xanthotoxol ( 4) 20 N.T. 200 Fisetin ( 5) 20 N.T. 41.6 ± 2.7 Daidzein ( 6) 0.5 ± 0.1 17.0 ± 3.0 1.20 ± 0.01 200 Calycosin ( 7) 20 N.T. 200 Quercetin ( 8) 200 N.T.

49.9 ± 8.3 Naringenin ( 9) 4.6–12.2 ( n = 2) 100 7.3 ± 1.8 200 Genistein ( 10) 0.3 ± 0.1 0.30 ± 0.01 0.020 ± 0.002 200 Coumestrol ( 11) 0.09–0.1 ( n = 2) 0.06 ± 0.04 0.02 ± 0.01 200 Pratensein ( 12) 67 N.T. 660 Kaempferol ( 13) 200 N.T. 64.9 ± 2.8 Daphnoretin ( 14) 20 N.T. 200 Pseudobaptigenin ( 15) 20 N.T.

200 Formononetin ( 16) N.A. 104 ± 8 60.0 ± 7.0 200 Maackiain ( 17) 70 N.T. 700 Irilone ( 18) 20 N.T. 200 Dihydrobiochanin A ( 19) 20 N.T.

200 Cicerin ( 20) 20 N.T. 200 Prunetin ( 21) 20 175 175 200 Biochanin A ( 22) 4.6 ± 0.8 35 ± 1.0 4.1 ± 0.8 200. HPLC quantitative analyses The quantitative analyses of 6, 7, 8, 10, 12, 13, 15, 16, 17, 18, 21, and 22 in the red clover Phase II clinical extract were carried out using HPLC-UV instead of LC-MS, since these compounds were present at 0.05% (w/w). For 12 and 17, the isolated compounds were used as standards.

All compounds were analyzed on the Agilent 1100 HPLC instrument, except 6, 10, 16 and 22, which were analyzed on the Waters 2695 HPLC system. Injections of each sample (10 μL) were analyzed in triplicate using a Beckman octadecyl silane (ODS) μBondapak 4.2 × 250 mm column, and the DAD detector was set to record absorbance at 254 nm. A 60-min method with a flow rate of 1.5 mL/min and the following gradient was used: 100% A/0% B to 60% A/40% B from 0–20 min, hold at 60% A/40% B from 20–22 min, 60% A/40% B to 10% A/90% B from 22–60 min, followed by a 10 min column wash of 100% B and then re-equilibration to 100% A for 14 min before the next injection, where A = doubly-deionized water/acetonitrile/glacial acetic acid (85:15:0.1, v/v/v) and B = doubly-deionized water/acetonitrile/glacial acetic acid (50:50:0.1, v/v/v). Areas under the curve from experimental peaks in the chromatogram were determined and the percent weight of each compound present was calculated based on a standard curve and amount (μg) of extract injected onto the column. Data were only used if their relative standard deviations (RSDs,%; (mean – standard deviation)/mean ×100) were. Compound identification Pratensein ( 12) was identified based on exact mass determination and comparison of proton and carbon NMR data with that reported.

Formononetin ( 16) was identified based on exact mass determination and comparison of proton and carbon NMR data with that reported by,. Maackiain ( 17) was identified based on exact mass determination and comparison of proton and carbon NMR data with that reported by,.

Irilone ( 18) was identified based on exact mass determination and comparison of proton and carbon NMR data with that reported. (±)-Dihydrobiochanin A ( 19) was identified based on exact mass determination and comparison of proton and carbon NMR data with that reported. No references containing NMR data for (±)-cicerin ( 20) could be located, although Kunzru and Sinha apparently first reported cicerin in 1970. Therefore, the assignment of compound 20 was based on its similarity to dihydrobiochanin A. Quantitative analysis of the Phase II clinical red clover extract Red clover capsules for our Phase II clinical trial were formulated by Pharmavite, LLC (Mission Hills, CA) as follows: 198.5 mg raw red clover extract, 182.5 mg rice flour, 4.0 mg silicon dioxide.

Two capsules are administered daily, for a total dose of 397 mg raw extract (containing 120 mg total daidzein + genistein + formononetin + biochanin A) per person per day. Compound doses in are based on ingestion of 397 mg of raw extract daily. Chemical structures are provided in, and the HPLC-UV chromatogram of the raw red clover extract is presented in. Nine measured isoflavones ( 6, 7, 10, 12, 15, 16, 18, 21, 22) comprise 35.54% of the weight of the raw extract without excipients. Other compound classes were present in the red clover extract in significantly smaller amounts: 1.11% flavonoids ( 5, 8, 9, 13), 0.06% pterocarpans ( 17), ≤ 0.03% coumarins ( 2, 3, 4, 11, 14), and 0.03% tyramine ( 1). The rationale for coumarin analysis was described previously.

Flavonoids (;;; ), pterocarpans (; ), and a few coumarins are known to occur in Trifolium species. Tyramine was discovered during LC-MS analysis of aqueous fractions of the extract, and it may be formed from decomposition of some amino acids, which were detected using ninhydrin reagent (data not shown). However, the red clover isoflavone supplements currently marketed are aqueous alcoholic extracts that likely contain low protein content, and therefore tyramine is not expected to be a major constituent of these products. Bioassay activities of extract components Results summarizing the estrogenic and antioxidant activity of compounds detected in the Phase II clinical red clover extract are presented in. Since the raw clinical extract gave similar results in the AP induction and the ER binding assays both initially and after two years of storage, the estrogenic compounds in this preparation of red clover are quite stable. Among the 22 constituents evaluated, six tested positive in the AP assay ( 6, 9, 10, 11, 16, 22) and of these, five bound to one or both ERs ( 6, 9, 10, 11, 22). Coumestrol was the most potent estrogenic compound, having an EC 50 of 0.09–0.10 μM in the AP assay, and IC 50 values of 0.06 ± 0.04 and 0.02 ± 0.01 μM in the ER alpha (α) and ER beta (β) competitive binding assays, respectively.

However, coumestrol was only present at a trace amount (≤ 0.01%) in the extract, amounting to a daily human dose of ≤ 0.04 mg. Naringenin was active in the AP induction and ER beta (β)-binding assays, but was present at only 0.02%. Thus, the principal estrogenic components of the extract, based on percent present and relative activity, are the four known isoflavones daidzein, genistein, formononetin and biochanin A. Although formononetin did not bind to the ERs at a physiological concentration, it is metabolized to daidzein in vivo (; ) and then to equol , both of which are estrogenic in the Ishikawa assay (equol data not shown). Although prunetin was not active in any estrogenic assay, there is evidence that it is metabolically converted into genistein. Only three compounds were active in the DPPH antioxidant assay ( 5, 8, 13), and these flavonols all contain 3-hydroxyl and 4′-hydroxyl groups.

CONCLUSIONS We have identified 22 compounds and quantitatively measured 20 that constitute more than 36% of the weight of the studied phase II clinical red clover extract. This represents a significant improvement in the characterization of red clover supplements, as currently only four of the isoflavone components (daidzein, genistein, formononetin, biochanin A) are measured and reported in clinical studies. Although the majority of compounds identified in the studied red clover extract were not biologically active in vitro, many had never before been tested in the AP induction, ER binding or DPPH antioxidant assays. Since minor constituents that are not estrogenic in vitro may still potentially be converted to active metabolites in vivo, there exists a logical rationale for measuring certain minor compounds ( i.e., prunetin as an estrogenic “prodrug”). This work provides a detailed examination of the chemical and in vitro biologically active components of a red clover dietary supplement, and it is hoped that more such studies will be carried out on botanical supplements undergoing clinical evaluation. This work was supported, in part, by grant P50 AT00155 provided jointly by the National Center for Complementary and Alternative Medicine (NCCAM), the Office of Dietary Supplements (ODS), the Office for Research on Women’s Health (ORWH), and the National Institute of General Medicine (NIGMS) of the National Institutes of Health (NIH). NLB is grateful for a National Research Service Award (NRSA) from NCCAM, F31 AT00804, and Drs.

John Fitzloff and Jimmy Orjala are acknowledged for their donations of HPLC instrument time for analysis of the red clover extract. CRO acknowledges support from Ruth L. Kirschstein NIH Predoctoral fellowship F31 AT24232.

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