CHEMICAL STANDARDIZATION AND PREPARATION OF BIOSUBSTANCES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: EVANS-ALLEN
• Reporting Frequency: Annual
• Accession No.: 0187444
• Project Start Date: Feb 1, 2001
• Project End Date: Feb 1, 2006
• Program Code: [(N/A)]- (N/A)

Recipient Organization
SOUTH CAROLINA STATE UNIVERSITY
(N/A)
ORANGEBURG,SC 29117

Performing Department
ENGINEERING TECHNOLOGIES

Non Technical Summary
Chemical standardization of biosubstances in Echinacea preparations and all herbs will contribute to the safe use of herbs for medicinal purposes by consumers. This project studies the chemical standardization of Echinacea and its biosubstances for medicinal use and development in commercial health products.

Animal Health Component

20%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
202	2235	2000	100%

Knowledge Area
202 - Plant Genetic Resources;

Subject Of Investigation
2235 - Herbs and spices;

Field Of Science
2000 - Chemistry;

Keywords

standardization

echinacea

plant chemistry

natural substances

cell culture

herbicides

pesticides

herbs

chemical composition

plant genetics

plant extracts

roots

leaves

flowers

harvesting date

age

greenhouse production

chemical analysis

quantitative analysis

hplc (chromatography)

gas chromatography

liquid chromatography

mass spectrometry

biological activity

performance evaluation

purification

infrared spectroscopy

nuclear magnetic resonance

comparative analysis

analytical chemistry

Goals / Objectives
(1) Preparation of freeze dried extracts of the root, leaf and flower of Echinacea plants of different age, grown in greenhouse conditions and/or by farmers, harvested at different times of the year, with water and 80% alcohol;(2) Determine the presence and the amount of alkamides, ferulic acid, quercetin, niacin, verbacoside, rutin, rutoside, chlorogenic acid, isochlorogenic acid, luteolin, luteolin-7-glucoside, apigenin, ascorbic acid, beta-carotene, beta-sitostero, caffeic acid, cichoric acid, echinocoside, echinolone, limonene, myrcene, and inulin chemicals with HPLC, GC-MS or LC-MS and minerals by AAS; (3) Determine the presence of commom herbicides and pesticides in these extracts; (4) Determine the above chemicals form commercial Echinacea products for medicinal use; (5) Isolate and characterize the bioactive chemicals from cell culture of Echinacea.

Project Methods
Three species of Echinacea will be analyzed: (1)Echinacea Angustflora, (2) Echinacea purpurea, (3) Echinacea Pallida. These plant species will be procured from reputed growers as well as be grown in a controlled greenhouse environment. The root, leaves, flowers and the fruits of these species will be separated and dried at room temperature at reduced pressure and then extracted with water and 80% ethanol. These extracts will be freeze dried, stored and refrigerated for further studies. Pure samples of alkamides, ferulic acid, quercetin, niacin, verbacoside, rutin, rutoside, chlorogenic acid, isochlorognic acid, luteolin, luteolin-7-glucoside, apigenin, absorbic acid, beta-carotene, beta-sitosterol, caffeic acid cichoric acid, echinacoside, echinolone, limonene, myrcene, and inulin will be purchased from commercial suppliers or from other researchers within the USA or abroad or isolated from the solid preparations mentioned in objective One. Preparative LC (HPLC) will achieve isolation of all biosubstances in small quantity. The chemical identification of the biosubstances will be carried out by UV-VIS spectroscopy, IR spectroscopy, emission spectroscopy, mass spectroscopy, and NMR spectroscopy. An MS data bank will be created for all of the biosubstances. GC-MS and LC-MS data will be carefully analyzed to identify the presence of common herbicides and pesticides. Chemical profiles for biosubstances of major commercial Echinacea preparations purchased from drug stores will be made using LC-MS. thes data willbe compared with the biosubstance profile of the three Echinacea species.

Progress 02/01/01 to 02/01/06

Outputs
Herbal remedies are becoming increasingly popular in USA. Perhaps the most popular of all are preparations from Echinacea, many species of which grow wild and now farmers are growing them. Echinacea products are claimed to raise opsonic index, the antibody that renders bacteria and other cells susceptible to phagocytosis, thus demonstrating antiviral and antibiotic properties. In addition, it is thought that these preparations are side reactions free. However, there are problems with the Echinacea preparations displayed on the shelves of the drugstores. The labels on the bottles do not provide the names and the amounts of bioactive materials and do not list possible side effects. Obviously, chemical standardization of biosubstances in Echinacea preparations should be done for the benefit of the consumers, growers, processors, and medical practitioners. In this project three species of Echinacea (popular in USA and also investigated by some researchers) will be grown under controlled conditions. Biosubstances will be isolated from various parts of the plant. Standardization method for the biosubstances were developed using the state of the art instrumentation. In addition, the project has developed conditions for growing 2 to 4 liters scale Echinacea cell culture, isolate biosubstances from the cells, and standardize them. Isolation of biosubstances form cell culture has fewer complexities and the final products having consistent composition. Information on cultivation of the Echinacea will be disseminated to farmers.

Impacts
Small farmers were able to grow Echinacea and sell them to companies that manufacture herbal preparations at much higher prices than they earn by selling traditional produces. Instead of corn or soybean they are able to raise several herbal plants that will be useful for consumption in the United States, but also exportable to other nations. This project provided label information for herbal products so that they can eventually be used as prescription drugs with none or little side effects and perhaps less habit forming to users. With proper content information available meaningful clinical research can be conducted for future development of herbal medicine.

Publications

• None. (2006)

Progress 01/01/04 to 12/31/04

Outputs
Herbal remedies are becoming increasingly popular in USA. Perhaps the most popular of all are preparations from Echinacea, many species of which grow wild and now farmers are growing them. Echinacea products are claimed to raise opsonic index, the antibody that renders bacteria and other cells susceptible to phagocytosis, thus demonstrating antiviral and antibiotic properties. In addition, it is thought that these preparations are side reactions free. However, there are problems with the Echinacea preparations displayed on the shelves of the drugstores. The labels on the bottles do not provide the names and the amounts of bioactive materials and do not list possible side effects. Obviously, chemical standardization of biosubstances in Echinacea preparations should be done for the benefit of the consumers, growers, processors and medical practitioners. In this project three species of Echinacea (popular in USA and also investigated by some researchers) will be grown under controlled conditions. Biosubstances will be isolated from various parts of the plant. Standardization method for the biosubstances will be developed using the state-of-the-art instrumentation. In addition, the project will develop conditions for growing 2-4 liters scale Echinacea cell culture, isolate biosubstances from the cells and standardize them. Isolation of biosubstances form cell culture will have fewer complexities, the final products having consistent composition. Information for cultivation of the Echinacea will be disseminated to farmers.

Impacts
(1) Small farmers will be able to grow Echinacea and sell them to companies that manufacture herbal preparations at much higher prices than they earn by selling traditional produces. Instead of corn or soybean they will be able to raise many herbal plants that will be useful for consumption at home but also exportable to other nations. (2) This project will also provide label information for herbal products so that they can eventually be used as prescription drugs with none or lot fewer side effects and perhaps less habit forming to users. With proper content information available meaningful clinical research can be conducted for future development of herbal medicine.

Publications

• None (2004)

Progress 01/01/03 to 12/31/03

Outputs
Echinacea is one of the most widely used herb in the US for immunostimulation and preparations are available over-the-counter. Unfortunately, the labels do not list the names of the active constituents with amounts. Most researchers think that the phenolic acid compounds are regarded as the active compounds in the preparations; others include volatile constituents and echinamides. We have developed a quantitative analytical method using HPLC for eleven phenolic compounds in including caffeic acid, chlorogenic acid, 4-coumaric acid, protocatechuic acid, syringic acid, vanillic acid and 4-hydroxybenzoic acid.

Impacts
The project has several potentials: (1) Development of standardization procedure that will be eventually incorporated into United States Pharmacopoeia required for FDA approval of Echinacea products as prescription drugs. (2) Provide demonstration to farmers for growing high quality Echinacea plants as alternative crop in lieu of traditional crop for higher profit.

Publications

• X. Yao, K. Watson and N. Datta-Gupta, 2002. GC/MS Analysis of volatile constituents of Echinacea species. Presented at the 29th Annual Meeting Federation of Analytical Chemistry & Spectroscopic Societies, Providence, Rhode Island, October 13-17, 2002
• X. Yao and N. Datta-Gupta, GC/MS derivatization of free phenolic acids in Echinacea species with TMS derivatization. Presented at the ACS 54th Southeast Meeting, Charleston, SC, November 13-17, 2002

Progress 10/01/01 to 09/30/02

Outputs
Echinacea is one of the most widely used herb in the US for immunostimulation and preparations are available over-the-counter. Unfortunately, the labels do not list the names of the active constituents with amounts. Most researchers think that the phenolic acid compounds are regarded as the active compounds in the preparations; others include volatile constituents and echinamides. We have analyzed the volatile constituents in three species of Echinacea (E. angustifolia, E. pallida and E. purpurea). We have also used a combination of solid phase extraction and GC/MS instrumentation to quantitatively determine seven (7) phenolic acids (caffeic acid, chlorogenic acid, 4-coumaric acid, protocatechuic acid, syringic acid, vanillic acid and 4-hydroxybenzoic acid. The method is good but time consuming. We have also grown the three species of Echinacea in an experimental plot

Impacts
The project has several potentials: (1) Development of standardization procedure that will be eventually incorporated into United States Pharmacopoeia required for FDA approval of Echinacea products as prescription drugs. (2) Provide demonstration to farmers for growing high quality Echinacea plants as alternative crop in lieu of traditional crop for higher profit.

Publications

• X. Yao, K. Watson and N. Datta-Gupta. 2002. "GC/MS Analysis of volatile constituents of Echinacea species" Presented at the 29th Annual Meeting Federation of Analytical Chemistry & Spectroscopic Societies, Providence, Rhode Island, October 13-17, 2002
• X. Yao and N. Datta-Gupta. 2002 "GC/MS derivatization of free phenolic acids in Echinacea species with TMS derivatization " Presented at the ACS 54th Southeast Meeting, Charleston, SC, November 13-17, 2002