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Fringe tree (Chionanthus virginicus L.) stem bark offers a sustainable alternative to root bark.

Traditional preparations from the root bark of fringe tree (Chionanthus virginicus L.) have been used as a cholagogue and hepatic stimulant. Unfortunately harvesting of the root bark can result in significant damage and generally the tree is destroyed. This investigation has shown that the same phytochemically important compounds, lignans and secoiridoids, are present in the root and stem barks. While variations in the ratios of these constituents are evident, the stem bark offers a good substitute for the root bark. This new information should enable a more sustainable harvesting program for this natural resource, allowing for several years' harvesting of the biomass before natural death calls for replanting.

Key Words: Fringe tree, Chionanthus virginicus, herbal, phytoequivalence, Phytotherapy

Introduction

Fringe tree (Chionanthus virginicus L.) (Oleaceae) is a large shrub native to eastern North America. Native Americans used fringe tree root bark externally for cuts, bruises, wounds, toothache and internal pains. The herb was an official medicine in the National Formulary in the United States from 1914 to 1947 and was used as a tonic (Vogel 1970). Fringe tree is now used in Western herbal medicine as a cholagogue, choleretic, mild laxative, antiemetic and a depurative (Bone 2003).

The root bark contains five major lignans and three secoiridoids (Boyer et al 2005). The lignans are phillyrin, pinoresinol-[beta]-D-glucoside, pinoresinol-di-[beta]-D-glucoside, phillyrin-2-O-[beta]-D-glucoside and phillyrin-6-O-[beta]-D-glucoside, with the secoiridoids reported to be oleuropein, ligustroside and angustifolioside B. Structures of these compounds are given in Figure 1. The root bark is typically harvested by digging up the tree, peeling off the root bark and then generally the tree is destroyed and discarded. Careful root pruning while the tree is dormant can produce biomass without killing the tree, but even this approach usually results in substantial damage or death of the plants (Fletcher 2007).

The presence of phillyrin in the stem bark of fringe tree was reported by Steinegger in 1959. However there is no confirmation of the presence of the other known root bark compounds in the stem bark. The objective of the present study was to examine the stem bark of fringe tree by the LC/MS method of Boyer to determine if the same phytochemicals are present. This would then allow an assessment of the suitability of substituting the more environmentally sustainable stem bark. The stem bark can be peeled from pruned branches which actually encourages additional branching and regrowth when done properly. The use of the stem bark would encourage cultivation projects, because with proper management the trees can produce biomass for several years until natural death calls for replanting (Fletcher 2007).

Materials and methods

Plant material

A sample of dried stem bark of Chionanthus virginicus L. was obtained from and authenticated by B&K International, Virginia Beach, Virginia (specimen number 501114). The sample was collected in southeast USA, air dried and provided as shavings. A voucher specimen has been deposited at the Southern Cross University Herbarium, Ref No NCM-D-07-017. Samples of commercial root bark were obtained from MediHerb Pty Ltd, Warwick Australia, to be used as a secondary reference material to facilitate the identification of phytochemicals as reported by Boyer.

Oleuropein

Oleuropein was purchased from Extrasynthese, France, Cat. No. 0204.

LC/MS analysis

Analyses were carried out using a Shimadzu gradient HPLC system coupled to a quadrupole mass spectrometer (Shimadzu 2010-EV) operating in both positive and negative ion modes using APCI and ESI interfaces. Samples were analysed on an Agilent Zorbax Eclipse XDB-Phenyl 3.5[micro]m 150 x 3.00 mm column equipped with a Phenomenex Securityguard guard cartridge system. The mobile phase was acetonitrile:water (20:80) at a flow rate of 0.3 mL/min. Powdered bark samples were extracted with methanol and 4 [micro]L aliquots injected. The total run-time was 60 minutes.

Results

The LC-MS data for the root bark sample, Figure 2, allowed the identification of the 8 compounds documented by Boyer (2005). Pinoresinol-di-[beta]-Dglucoside (3) is the earliest eluting compound and is readily identifiable by its mass spectral fragmentations in negative ion mode (m/z 519,357 and 681). The HPLC chromatogram shown by Boyer was at 280 nm and this region is much cleaner than in the LC-MS. Pinoresinol-[beta]-Dglucoside (2) is readily identifiable as a strong peak in both the LC-MS (M-H=519) and the 280 nm traces (Figure 3). The resolution obtainable in the region of phillyrin-2-O-[beta]-D-glucoside (6), phillyrin-6-O-?-D-glucoside (7) and angustafolioside B (8) utilising a number of C-18 columns was not the same as that reported by Boyer, with the best separation enabled by the XDB Phenyl column. Despite peak broadening/overlap in this region of the MS trace, these three compounds are shown to be present via detection of ions that correlate to M-1 species at 695, 695 and 685 respectively. Oleuropein (4) was the major component of the root bark and was identified by comparison with authentic oleuropein and by a characteristic mass spectral fragmentation. Phillyrin (1) and ligustroside (5) were both easily identifiable in the root bark, being very well resolved from other material with M-1 peaks of 533 and 523 respectively. The characteristic mass spectral fragmentation patterns reported by Boyer (2005) have been confirmed for the compounds mentioned above.

These peak identities (using the same numbering system as Boyer for consistency) were then employed to determine if the same components were present in the stem bark sample, as well as the relative proportions in the two different plant parts. The HPLC-DAD traces at 280 nm for the two samples are given in Figure 3, with the traces normalised to reflect the actual concentrations present in the samples. Given the lower response of the secoiridoids at 280 nm, due to their lower absorptivity and the incomplete resolution of all of the peaks, the ratio of the M-H peaks for the compounds in the MS traces was used to assess the relative abundance of the individual compounds (with oleuropein set as 100). The total relative amount for the eight compounds was also compared, to determine if any dose adjustment would be required for the stem bark compared to the root bark. This information is provided in Table 1.

Discussion

The phytochemical profile of fringe tree root bark was confirmed to be as reported by Boyer (2005) with all 8 compounds identified by a combination of HPLC-DAD and LC/MS techniques. The ratio of the individual components was also similar to that reported, with pinoresinol-[beta]-D-glucoside (2) and oleuropein (4) being the major components. The data confirm that the secondary reference material used in this study was a typical example of the species, as reported by Boyer. When the HPLCDAD chromatographic profile of the stem bark sample is examined, it is shown to be very similar. Pinoresinol-[beta]-D-glucoside (2) is the major peak in the trace of both samples and all of the characteristic peaks found in the root bark sample are present in the stem bark sample, with identities being confirmed by the LC/MS fragmentation patterns. Full quantification of all the components was not performed in this comparison as reference materials were not commercially available, with the exception of oleuropein (4).

Within each plant part the relative amounts were shown to be very similar for most compounds. A major variation was in the proportion of angustifolioside (8). The relative amounts of some of the other components, most notably ligustroside (5) and pinoresinol di-[beta]-D-glucoside (3) are also somewhat different. However as is evident from the HPLC-DAD traces in Figure 3, the overall phytochemical fingerprint of these components is retained. It would be difficult to distinguish between the root and stem barks purely on the basis of phytochemical characteristics, as variation within the biomass of each plant part is likely to be as great or greater than any variation between the plant parts.

For the samples tested the total abundance of eight characteristic compounds in the stem bark was shown to be slightly lower than that of the root bark, with around 60% of the total amount being found. This could indicate that the therapeutic activity of the stem bark might be less than the root bark. However the testing of a wide variety of samples would be necessary to conclusively establish this quantitative difference. Even then its therapeutic relevance would be uncertain since the active components of fringe tree are not known.

Conclusion

The phytochemical profiles present in fringe tree root and stem bark have been compared by a combination of HPLC-DAD and LC/MS. The same 8 major compounds were found in the two plant parts. Although there are variations in the relative proportions of a number of the less predominant peaks, these are not considered to be sufficiently great to affect the overall safety and efficacy profile of this material. For the samples tested the relative amount of the 8 phytochemicals found in stem bark was around 60% of that of root bark and therefore an increase in the traditional dosage may be necessary. On the basis of these findings, the stem bark of fringe tree is recommended for consideration as a more environmentally sustainable option than the root bark.

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

References

Bone K. 2003. A Clinical Guide to Blending Liquid Herbs. St Louis Missouri: Churchill Livingstone.

Boyer L, Elias R, Taoubi K, Debrauwer L, Faure R, Baghdikian B, Balansard G. 2005. Lignans and Secoiridoids from the Root Bark of Chionanthus virginicus L.: Isolation, Identification and HPLC Analysis. Phytochem Anal 16;375-9.

Fletcher EJ. Personal communication 29 May 2007.

Steinegger E, Jacober H. 1959. Presence of phillyrin in the Oleaceae. Structure of the chionanthins. Pharm Acta Helv 34;585-92.

Vogel VJ. 1970. American Indian Medicine. Norman Oklahoma: University of Oklahoma Press.

KG Penman (a), KM Bone (a,b), RP Lehmann (a) *

(a) MediHerb Research Laboratories, 3/85 Brandl Street, Eight Mile Plains, Brisbane, Australia

(b) School of Health, University of New England, Armidale, 2031 NSW Australia

* Corresponding Author: R.P. Lehmann Mailing Address: MediHerb Research Laboratories, 3/85 Brandl Street, Eight Mile Plains, 4113, Brisbane, Australia Tel: +61 7 3423 6521 Fax: +61 7 3423 6599 E-mail: reg@mediherb.com.au
Table 1
Relative abundance of compounds 1-8 in fringe tree root and stem bark.

 Relative
 Abundance of
 [M-H]-(%)

 Elution ID Stem Root
 order no. Bark Bark

Phillyrin 7 1 0.6 0.6
Pinoresinol-[beta]-D-glucoside 2 2 12 9
Pinoresinol di-[beta]-D-glucoside 1 3 0.6 2.4
Oleuropein 6 4 100 100
Ligustroside 8 5 25 53
Phillyrin-2-O-[beta]-D-glucoside 3 6 1.6 3.9
Phillyrin-6-O-[beta]-D-glucoside 4 7 0.15 0.23
Angustifolioside B 5 8 3.8 20
Total Ion Count Abundance 60 100
of compounds 1-8

Figure 1
Structures of the five major lignans and three main secoiridoids
reported by Boyer (2005) from fringe tree root bark

 [R.- [R.- [R.- [R.-
ID Compound R sub.1] sub.2] sub.3] sub.4]

1 Phillyrin C[H.sub.3] H H
2 Pinoresinol- H H H
 [beta]-D-glucoside
3 Pinoresinol di- Glc H H
 [beta]-D-glucoside
4 Oleuropein OH H
5 Ligustroside H H
6 Phillyrin-2-O- C[H.sub.3] Glc H
 [beta]-D-glucoside
7 Phillyrin-6-O- C[H.sub.3] H Glc
 [beta]-D-glucoside
8 Angustifolioside B H Glc
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Author:Penman, K.G.; Bone, K.M.; Lehmann, R.P.
Publication:Australian Journal of Medical Herbalism
Geographic Code:8AUST
Date:Sep 22, 2008
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