Evaluation of traditional medicines I: identification of PHELA using different chromatographic techniques

Abstract

PHELA is a herbal mixture of four African traditional medicinal plants that has been used for decades in wasting conditions and is now being developed by the Medical Research Council (MRC) as an immune booster for patients with compromised immune system. A chromatographic fingerprint of PHELA was needed for quality control purposes. Here, a comprehensive method for fingerprinting of PHELA using different chromatographic techniques is described. It involved extraction of the PHELA by either acidic or a simple 'salting-out' method, followed by Thin Layer Chromatography (TLC) analysis and/or preparative Column Chromatography (CC). The products were thereafter analyzed by High Performance Liquid Chromatography with UV-detector (HPLC-UV), HPLC with fluorescence-detector (HPLC-FL) and Gas-Chromatography with a Mass Selective Detector spectrometer (GC-MSD). The fingerprints were successfully used to differentiate PHELA from another common herbal product made from Hypericum perforatum (St. John's Wort), thereby illustrating its high potential for use in fingerprinting of PHELA and in differentiating it from other herbal medicines. By validating the different chromatographic techniques on the standardized extraction methods, this approach will enable wide application in quality control of PHELA using acceptable procedures, thereby promoting effective monitoring of the finished product in all countries where it will be used.

Keywords: Fingerprinting; PHELA; Quality Control; Traditional Herbal Medicine; chromatography.

Figure 1

Study design: chromatographic methods used for fingerprinting of PHE

Figure 2

(2a) = A TLC chromatogram of PHELA extract. Mobile phase was ethyl acetate: ammonia: methanol (17: 1: 2). Retention factors (RF) were: band 1 = 0.24, band 2 = 0.31 and band 3 = 0.92; (2b) = An HPLC-UV chromatogram of hexane extract of crude PHELA with marker peaks at 9.8 (Peak 1) and 24 (peak 2); (2c) = An HPLC-FLD chromatogram of salted extract of crude PHELA with marker peaks 12 (peak A), 22.81(peak B), 32.31(peak C) and 38.1 (peak D) minutes; (2d) = GC-MSD chromatogram of salted extract of crude PHELA with marker peaks at 6.3, 6.74, 7.25, 7.78 and 7.93 minutes

Figure 3

(3a) = HPLC-UV chromatograms of PHELA TLC-Band 2 with marker peaks at 4.27 and 9 minutes; (3b) = HPLC-FLD chromatograms of PHELA TLC-band 2 with a marker peak at 6.02 minutes ; (3c) = a GC/MSD chromatogram of PHELA TLC-band 2 with marker peaks at 2.26, 5.13, 5.89 and 8.01 minutes

Figure 4

(4a) = HPLC-UV chromatograms of PHELA TLC-Band 3 with marker peaks at 8.462, 17.894 and 23.131 minutes. (4b) = HPLC-FLD chromatograms of PHELA TLC-band 3 with marker peaks at 8, 20, and 33 minutes. (4c) = a GC/MSD chromatogram of PHELA TLC-band 3 with the marker peaks at 2.26, 5.88 and 8.01 minutes

Figure 5

(5a and 5b) = the UV-absorption spectra of the two marker peaks (peak 1 and peak 2) in HPLC-UV chromatogram of crude PHELA fig. 1b; and (5c and 5d) = GC-MSD chromatograms of the two marker peaks (peak 1 and peak 2) in fig. 4b.

Figure 6

(6a) = Column-chromatography (CC) of PHELA colored fraction 17 (CC-FR 17) eluting from column. (6b) = HPLC-UV chromatograms of PHELA CC-FR 17 with marker peaks at 4.3, 9.8, 18.7 and 25 minutes; (6c) = HPLC-FLD chromatograms of PHELA CC-FR 17 with marker peaks at 10 and 20 minutes. (6d) = a GC/MSD chromatogram of

Figure 7

(7a) = HPLC-UV chromatogram of the crude SJW extract; (7b) = GC/MSD chromatogram of the crude SJW extract; (7c) = HPLC-UV chromatogram of St John's Wort TLC-band A with marker peaks at 3.874, 4.275, 8.326 and 13.693 minutes; (7d) = HPLC-UV chromatogram of St John's Wort TLC-band B with marker peaks at 4.268 and 8.317 minutes; (7e) = HPLC-FLD chromatogram of St John's Wort TLC-band B; (7f) = GC/MSD chromatogram of TLC-Band B of SJW.