Introduction
Introduction
Medicinal plants host a wide spectrum of microorganisms with various individual properties
and with considerable differences regarding qualitative and quantitative aspects.
In principle, the microbial load of plants is the result of a series of influences
caused by animate and inanimate sources, and microbial contaminants are easily transferred
via air- and soil-borne vectors (Fig. [1]). The persistence and resistance properties of the plant microflora are determined
by intrinsic as well as extrinsic factors which are due to natural, agricultural,
environmental and technological influences (Table [1]). Although bacterial endospores and fungal spores can be regarded as the two dominating
groups of contaminants associated with medicinal plants, a broad diversity of bacterial,
fungal cells and viruses can be found either in or on the plant material [1], [2], [3], [4]. Among these microorganisms, pathogens may also occur, and this fact particularly
limits the utilisation of these plants [1], [5], besides quality reduction caused by microbially induced spoilage. Moreover, in
analogy to the spices and herbs used in foods, it cannot be excluded that extraneous
matter and filth material originating from rodents, insects and inorganic sources
(e. g., stones) may be present in some preparations [3], [6], [7], [8].
According to experience, the degree of contamination usually depends on the distance
from the soil bottom the plant has been grown (for example see [9]). Certain plants (e. g., calamus, melissa, thyme, basil, fennel etc.) contain natural
barriers and antimicrobial substances which exert typical inhibitory effects on microbial
growth and stability. It has been estimated that around 1400 herbs and spices may
possess antimicrobial agents of different chemical nature such as oils [10], [11], peptides [12], liquid [13] and organic extracts [14]. Some of these biological activities have already been tapped for certain therapeutic
use [15], [16] and their specific healing properties can be even rooted in history. In addition,
it may also be expected that some antioxidants naturally occurring in plants may limit
some microbes as well [17], [18], [19]. However, medicinal plants are originally not germ-free and thus several hygiene
parameters have to be considered in routine control, especially when the plant is
to be applied for medical purposes. Following this fundamental need, the evaluation
of microbial contamination has increasingly become an integral part of HACCP concepts.
In this context, the microbial risk inherent to herbal plants may vary with regard
to the different stages of the production line and this has to be recognised in a
systematic strategy of quality assurance (Table [2]). Even in/at dried herbal products some microorganisms, in particular their spores,
may survive over long-term storage periods [20].
Taking into consideration the above described background situation, the intention
behind this review paper is to discuss the following items:
-
to give an extensive survey of published data regarding the microbial load associated
with herbal medicinal drugs;
-
to elucidate some microbiological criteria and methodological aspects which may be
useful to be further integrated in modern quality assurance of medicinal plants;
-
to consider the different modes of manipulation, which are usually applied for preparing
medicinal drugs such as treatment with boiling water, cold water extraction and ethanol
extraction, which usually affect the microbiological nature of the products thereby
obtained;
-
to use this survey as a basis for proposing tailor-made quality standards for herbal
medicinal plants which may form the basis for a possible re-consideration of chapter
5.1.4 of Ph.Eur. 2000 (21) in a constructive way.
Fig. 1 Possible pathways of microbial contamination of medicinal herbal drugs.
Table 1 Factors determining the microbiological quality of medicinal plants
|
Intrinsic
|
Nature of the plant and natural barriers
Structure of the plant
Plant composition (antimicrobial compounds and agents)
Intracellular microbial contaminations |
|
Extrinsic
|
Climate
Humidity
Location/position
Harvest method
Post-harvesting
Physical state (concis/toto)
Technological treatment
Packaging and storage conditions
Exogenous microbial contaminations |
Table 2 Evaluation of microbiological contamination risks at different stages during the production
of medicinal herbal drugs
| Production steps |
Risk level1
|
| Pre-cultivation |
(+) |
| Field cultivation |
++ |
| Harvest |
++ |
| Intermediate storage |
+ |
| Transportation |
(+) |
| Treatments (cleaning/cutting/drying/packaging) |
+ |
| Final product (packaged/stored) |
- |
|
1 Explanation of symbols: - usually no risk, (+) no to low risk, + low to medium risk,
++ high risk. |
Survey of Reports about Microbial Contaminants Associated with Medicinal Plants
Survey of Reports about Microbial Contaminants Associated with Medicinal Plants
Microbiological data about herbal medicinal plants documented in the literature were
collected. By generating three lists summarising the total aerobic mesophilic (Table
[3]), enterobacterial (Table [4]) and yeast and mould (Table [5]) counts, the heterogeneous data available in the literature were compiled in order
to provide a more concise survey. In these tables, an array of plants is listed along
with their corresponding ranges of viable counts. As far as permissible, medians were
calculated from the individual documents to allow a proper evaluation of the average
microbiological situation as attributed to each plant material. It is quite obvious
that pronounced differences in the total aerobic mesophilic counts (Table [3]) reflect the original and environmental criteria described above. For example, relatively
low bacterial counts found with some products (e. g., Fructus Myrtilli) may be due
to natural antimicrobials and probably to a generally good hygiene situation, whereas
high microbial counts (e. g., Herba Urticae) may indicate less favourable hygienic
conditions. Moreover, factors such as the distance of the plant from the soil and
the ratio between the size of the plant surface and the sample weight may play some
role. In analogy, this observation is also valid for the load with enterobacteria
(Table [4]), whereas not only the total enterobacterial counts were integrated in this list
but also their coliform sub-group, as far as provided by the authors. Although enterobacteria
can be ubiquitously found in nature, this family possesses some indicative value towards
faecal contamination. Together with the group of coliforms, it can be taken as an
indicator for undesired hygiene conditions, although this conclusion has to be related
to the magnitude of the viable count measured. With a few exceptions, spores of filamentous
fungi rather than yeasts seem to play an important role in/at herbal plants (Table
[5]).
A relatively limited number of reports exists about the presence of pathogenic microorganisms
in/on herbal plants. In general, contaminations with pathogens cannot be excluded.
Recently, Czech et al. [5] have screened a broad spectrum of pathogens and indicator germs. It was shown that
these microorganisms are relatively rarely found, with the exceptions of Bacillus cereus and Clostridium perfringens. However, these two spore-formers usually do not appear in magnitudes representing
a real toxicity potential. In the 1980’s, Leimbeck [31] detected E. coli and Pseudomonas aeruginosa in many of the samples and therefore suggested to treat the drugs with boiling water
for decontamination. In other studies, alternative treatments have been introduced
[22], [23], [26], [27], [32]. Since herbal plants frequently carry a considerable amount of moulds, the generation
of mycotoxins, especially as cold water maceration products are concerned, should
be taken into consideration [5]. Hitokoto et al. [33] have shown that moulds like Penicillium, Aspergillus, Rhizopus, Mucor, Cladosporium and Aureobasidium spp. can be found quite often in association with herbal drugs, but mycotoxin producers
were only present around the level of 2 %. On the contrary, Kumar and Roy [34] have detected considerable risk levels of aflatoxins in several herbal medicinal
samples of different taxa. From these findings we may conclude that environmental
conditions (climate, humidity, hygiene etc.) largely contribute to the mycotoxin problem.
Table 3 Compilation of data (CFU/g) of total aerobic mesophilic counts analysed in medicinal
drugs by different researchers. ”Reported values” represent miscellaneous viable count
data, regardless whether mean values or single data were considered. From these data
medians were calculated based on all single and mean values as far as available
| Herbal Drugs |
Reported values |
Median |
Min |
Max |
References (n)1
|
| Cortex Frangulae |
3.0 x 105
|
|
|
|
3(1) |
| Cortex Frangulae |
9.8 x 103
|
|
|
|
22(1) |
| Cortex Hippocastani |
8.9 x 103
|
|
|
|
22(1) |
| Cortex Cinchonae |
6.7 x 103
|
|
|
|
23(3) |
| Cortex Cinnamomi |
1.0 x 105
|
|
|
|
3(2) |
| Cortex Rhois aromaticae |
2.0 x 102
|
|
|
|
3(1) |
| Flos Aurantii |
6.8 x 104
|
6.8 x 104
|
1.5 x 104
|
1.2 x 105
|
24 (2) |
| Flos Chamomillae |
1.7 x 106
|
3.2 x 105
|
4.5 x 103
|
7.1 x 106
|
3(16); 5(5); 23(3); 24(1); 25(1); 26(3) |
| Flos Hibisci |
4.2 x 104
|
4.2 x 104
|
1.8 x 104
|
6.6 x 104
|
3(10); 23(3) |
| Flos Malvae |
3.0 x 107
|
9.8 x 105
|
1.6 x 102
|
2.3 x 108
|
5(5); 23(3); 24(1); 27(6) |
| Flos Sambuci |
3.5 x 106
|
1.1 x 106
|
7.6 x 103
|
1.3 x 107
|
5(5) |
| Flos Tiliae |
9.5 x 104
|
4.1 x 104
|
3.0 x 104
|
3.4 x 105
|
5(5); 24(1) |
| Flos Verbasci |
2.2 x 105
|
1.8 x 105
|
1.0 x 104
|
4.5 x 105
|
5(5); 24(1); 26(1) |
| Flos Farfarae |
8.1 x 103
|
|
|
|
24(1) |
| Flos Symphyti |
5.8 x 104
|
|
|
|
3(2) |
| Flos Chamomillae romanae |
6.8 x 103
|
|
|
|
24(1) |
| Folium Betulae |
6.9 x 104
|
1.6 x 103
|
n. d.2
|
3.4 x 105
|
3(12); 23(3); 24(1); 26(1); 28(2) |
| Folium Crataegi cum flore |
8.9 x 105
|
1.8 x 105
|
3.3 x 104
|
2.8 x 106
|
5(4); 22(1); 23(3); 24(1); 27(6) |
| Folium Malvae |
3.0 x 106
|
3.5 x 106
|
2.0 x 104
|
3.5 x 106
|
5(5); 24(1); 29(2) |
| Folium Melissae |
1.7 x 105
|
1.5 x 105
|
7.1 x 103
|
3.7 x 105
|
5(5); 24(1) |
| Folium Menthae pip. |
1.7 x 107
|
8.6 x 105
|
1.9 x 104
|
1.7 x 108
|
3(9); 5(5); 23(3); 24(1); 25(1); 27(6); 29(2) |
| Folium Salviae |
2.1 x 106
|
2.6 x 105
|
4.7 x 103
|
1.8 x 107
|
3(3); 5(5); 24(1); 25(1); 28(1); 29(2) |
| Folium Sennae |
1.0 x 105
|
5.3 x 104
|
1.8 x 104
|
2.4 x 105
|
3(5); 5(4); 16(3) |
| Folium Uvae ursi |
1.1 x 104
|
3.2 x 103
|
9.5 x 102
|
5.7 x 104
|
3(12); 5(5); 22(1); 23(4); 24(1); 29(2) |
| Folium Althaeae |
4.2 x 107
|
|
|
|
27(6) |
| Folium Boldo conc. |
1.0 x 104
|
|
|
|
29(1) |
| Folium Boldo tot. |
2.9 x 103
|
|
|
|
29(1) |
| Folium Eucalypti conc. |
1.1 x 104
|
|
|
|
29(1) |
| Folium Eucalypti tot. |
9.4 x 103
|
|
|
|
29(1) |
| Folium Farfarae |
1.6 x 104
|
|
|
|
26(1) |
| Folium Hamamelidis |
2.8 x 105
|
|
|
|
22(1) |
| Folium Juglandis |
7.3 x 106
|
|
|
|
27(6) |
| Folium Orthosiphonis |
4.6 x 105
|
|
|
|
3(10) |
| Folium Plantaginis |
3.3 x 103
|
|
|
|
24(1) |
| Folium Ribis nigri |
1.5 x 105
|
|
|
|
3(8) |
| Folium Rubi idaei |
2.0 x 100
|
|
|
|
26(1) |
| Folium Symphyti (fresh) |
2.8 x 105
|
|
|
|
3(3) |
| Fructus Anisi |
3.5 x 105
|
9.8 x 104
|
1.7 x 103
|
1.1 x 106
|
5(5) |
| Fructus Carvi |
1.4 x 106
|
5.1 x 104
|
2.2 x 103
|
8.1 x 106
|
3(2); 5(5) |
| Fructus Foeniculi |
4.1 x 106
|
1.6 x 105
|
2.4 x 104
|
2.5 x 107
|
5(5); 23(3); 25(1) |
| Fructus Myrtilli |
3.2 x 103
|
2.4 x 103
|
2.2 x 102
|
9.9 x 103
|
5(4); 22(1); 23(3) |
| Fructus Sennae |
3.3 x 105
|
2.2 x 105
|
6.5 x 103
|
8.6 x 105
|
3(2); 5(3); 16 (3); 24(1); 30(1) |
| Fructus Cynosbati |
1.3 x 102
|
|
|
|
24(1) |
| Fructus Cynosbati powder |
1.1 x 105
|
|
|
|
3(10) |
| Fructus Juniperi |
2.4 x 102
|
|
|
|
23(3) |
| Herba Echinaceae |
1.4 x 106
|
1.9 x 106
|
2.9 x 105
|
1.9 x 106
|
5(3) |
| Herba Equiseti |
3.2 x 104
|
|
|
|
26(1) |
| Herba Hyperici |
6.4 x 106
|
2.3 x 105
|
1.8 x 104
|
3.6 x 107
|
5(5); 27(6) |
| Herba Passiflorae |
2.5 x 107
|
6.6 x 106
|
7.6 x 104
|
1.2 x 108
|
5(5); 24(1) |
| Herba Thymi |
7.9 x 106
|
3.3 x 106
|
3.3 x 106
|
7.9 x 106
|
5(5); 23(3) |
| Herba Urticae |
1.4 x 107
|
4.1 x 106
|
1.0 x 106
|
4.2 x 107
|
5(5); 23(3); 25(1); 26(1) |
| Herba Visci albi |
5.6 x 104
|
5.6 x 104
|
1.0 x 103
|
1.1 x 105
|
25(1); 28(2) |
| Herba Absinthii |
7.8 x 103
|
|
|
|
23(3) |
| Herba Anserinae |
4.0 x 105
|
|
|
|
26(1) |
| Herba Boraginis |
3.1 x 106
|
|
|
|
3(3) |
| Herba Millefolii |
1.7 x 105
|
|
|
|
25(1) |
| Herba Solidaginis |
1.8 x 106
|
|
|
|
3(8) |
| Herba Veronicae |
1.8 x 100
|
|
|
|
26(1) |
| Herba Violae tricoloris |
1.9 x 106
|
|
|
|
22(1) |
| Lichen Islandicus |
2.5 x 105
|
5.5 x 103
|
5.3 x 103
|
7.5 x 105
|
5(3) |
| Pericarpium Aurantii |
2.2 x 103
|
2.1 x 103
|
7.2 x 102
|
4.0 x 103
|
5(4) |
| Radix Althaeae |
1.6 x 105
|
2.5 x 104
|
7.4 x 102
|
1.0 x 106
|
5(5); 23(3); 24(1); 28(1) |
| Radix Liquiritiae |
3.5 x 105
|
3.4 105
|
2.4 x 104
|
8.6 x 105
|
3(8); 5(4) |
| Radix Primulae |
6.4 x 104
|
4.3 x 104
|
2.2 x 104
|
1.5 x 105
|
5(4) |
| Radix Valerianae |
1.1 x 105
|
9.1 x 104
|
2.8 x 104
|
2.2 x 105
|
5(5); 25(1) |
| Radix Gentianae |
4.6 x 102
|
|
|
|
23(3) |
| Radix Ginseng |
1.7 x 106
|
|
|
|
22(1) |
| Radix Harpagophyti |
6.9 x 105
|
|
|
|
22(1) |
| Radix Ipecacuanhae |
1.3 x 103
|
|
|
|
23(4) |
| Radix Ratanhiae |
1.1 x 103
|
|
|
|
23(3) |
| Radix Rhei |
7.2 x 104
|
|
|
|
28(1) |
| Radix Symphyti |
8.2 x 105
|
|
|
|
3(14) |
| Rhizoma Curcumae xanthorrhizae |
1.6 x 106
|
|
|
|
23(3) |
| Rhizoma Graminis |
2.8 x 104
|
|
|
|
3(10) |
| Rhizoma Zingiberis |
1.1 x 104
|
|
|
|
3(3) |
| Semen Lini |
2.6 x 107
|
1.3 x 105
|
5.0 x 101
|
2.3 x 108
|
3(502); 5(5); 23(3); 25(1) |
| Semen Psyllii |
5.4 x 104
|
1.0 x 104
|
1.8 x 103
|
2.3 x 105
|
5(5); 24(1) |
| Semen Hippocastani |
2.4 x 102
|
|
|
|
23(3) |
|
1 (n), number of samples examined in the corresponding study. |
|
2 n. d., non-detectable. |
Table 4 Compilation of data (CFU/g) of Enterobacteriaceae and coliform counts analysed in
medicinal drugs by different researchers. ”Reported values” represent miscellaneous
viable count data, regardless whether mean values or single data were considered.
From these data medians were calculated based on all single and mean values as far
as available
| Herbal Drugs |
Reported values
(Enterobacteriaceae) |
Median |
Min |
Max |
Reported values
(Coliforms) |
Median |
Min |
Max |
References (n)1
|
| Cortex Frangulae |
|
|
|
|
2.0 x 103
|
|
|
|
3(1) |
| Cortex Cinchonae |
5.0 x 101
|
|
|
|
|
|
|
|
23(3) |
| Cortex Cinnamomi |
|
|
|
|
1.5 x 103
|
|
|
|
3(2) |
| Cortex Rhois aromaticae |
|
|
|
|
1.0 x 102
|
|
|
|
3(1) |
| Flos Aurantii |
2.5 x 101
|
2.5 x 101
|
n. d.2
|
2.5 x 101
|
|
|
|
|
24(2) |
| Flos Chamomillae |
2.6 x 105
|
8.0 x 104
|
n. d. |
1.0 x 106
|
2.9 x 105
|
1.8 x 105
|
1.0 x 104
|
9.7 x 105
|
3(16); 5(5); 23(3); 24(1); 26(3) |
| Flos Hibisci |
n. d. |
|
|
|
5.0 x 102
|
|
|
|
3(10); 23(3) |
| Flos Malvae |
3.4 x 105
|
1.3 x 104
|
n. d. |
1.7 x 106
|
3.1 x 105
|
1.2 x 105
|
5.0 x 101
|
1.0 x 106
|
5(5); 23(3); 24(1); 27(6) |
| Flos Sambuci |
5.3 x 105
|
8.9 x 104
|
5.0 x 101
|
2.4 x 106
|
5.2 x 105
|
8.9 x 104
|
5.0 x 101
|
2.4 x 106
|
5(5) |
| Flos Tiliae |
2.2 x 103
|
9.5 x 102
|
5.0 x 101
|
7.1 x 103
|
2.2 x 103
|
1.1 x 103
|
1.0 x 102
|
5.6 x 103
|
5(5); 24(1) |
| Flos Verbasci |
2.2 x 104
|
7.1 x 102
|
5.0 x 100
|
1.3 x 105
|
2.7 x 104
|
1.0 x 103
|
2.0 x 102
|
1.3 x 105
|
5(5); 24(1); 26(1) |
| Flos Farfarae |
n. d. |
|
|
|
|
|
|
|
24(1) |
| Flos Symphyti |
|
|
|
|
2.4 x 103
|
|
|
|
3(2) |
| Flos Chamomillae romanae |
n. d. |
|
|
|
|
|
|
|
24(1) |
| Folium Betulae |
n. d. |
|
|
|
3.8 x 103
|
|
|
|
3(12); 23(3); 24(1) |
| Folium Crataegi cum flore |
1.5 x 105
|
3.9 x 104
|
n. d. |
7.1 x 105
|
1.8 x 105
|
6.9 x 104
|
2.2 x 104
|
5.7 x 105
|
5(4); 23(3); 24(1) |
| Folium Malvae |
5.4 x 105
|
4.8 x 104
|
5.0 x 101
|
3.0 x 106
|
6.2 x 105
|
2.7 x 104
|
1.6 x 104
|
3.0 x 106
|
5(5); 24(1) |
| Folium Melissae |
4.4 x 104
|
3.2 x 104
|
n. d. |
1.1 x 105
|
4.5 x 104
|
5.0 x 104
|
5.0 x 101
|
1.1 x 105
|
5(5); 24(1) |
| Folium Menthae pip. |
2.1 x 104
|
5.0 x 102
|
5.0 x 101
|
9.3 x 104
|
2.6 x 104
|
5.8 x 103
|
5.0 x 101
|
9.3 x 104
|
3(9); 5(5); 23(3); 24(1); 25(1) |
| Folium Salviae |
2.3 x 105
|
1.3 x 103
|
5.0 x 101
|
1.4 x 106
|
2.0 x 105
|
1.8 x 104
|
5.0 x 101
|
1.1 x 106
|
3(3); 5(5); 24(1); 25(1) |
| Folium Sennae |
2.9 x 104
|
1.5 x 104
|
5.0 x 101
|
9.6 x 104
|
2.6 x 104
|
1.0 x 104
|
5.0 x 101
|
9.6 x 104
|
3(5); 5(4); 16(3) |
| Folium Uvae ursi |
3.6 x 101
|
5.0 x 101
|
n. d. |
5.0 x 101
|
2.8 x 102
|
5.0 x 101
|
5.0 x 101
|
1.4 x 103
|
3(12); 5(5); 23(4); 24(1) |
| Folium Orthosiphonis |
|
|
|
|
5.3 x 103
|
|
|
|
3(10) |
| Folium Plantaginis |
5.0 x 100
|
|
|
|
|
|
|
|
24(1) |
| Folium Ribis nigri |
|
|
|
|
2.4 x 103
|
|
|
|
3(8) |
| Folium Symphyti (fresh) |
|
|
|
|
1.4 x 104
|
|
|
|
3(3) |
| Fructus Anisi |
2.0 x 105
|
3.6 x 103
|
5.0 x 101
|
9.6 x 105
|
1.1 x 105
|
3.6 x 103
|
5.0 x 101
|
5.1 x 105
|
5(5) |
| Fructus Carvi |
1.1 x 105
|
6.5 x 104
|
4.0 x 103
|
4.0 x 105
|
7.1 x 104
|
3.9 x 104
|
8.0 x 102
|
2.7 x 105
|
3(2); 5(5) |
| Fructus Foeniculi |
9.2 x 104
|
1.3 x 104
|
5.0 x 101
|
5.8 x 105
|
9.8 x 104
|
1.5 x 104
|
5.0 x 101
|
4.5 x 105
|
5(5); 23(3); 25(1) |
| Fructus Myrtilli |
4.0 x 101
|
5.0 x 101
|
n. d. |
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
5(4); 23(3) |
| Fructus Sennae |
1.9 x 104
|
1.3 x 103
|
5.0 x 101
|
5.0 x 104
|
5.6 x 103
|
2.6 x 103
|
5.0 x 101
|
1.7 x 104
|
3(2); 5(3); 16(3); 24(1) |
| Fructus Cynosbati |
n. d. |
|
|
|
|
|
|
|
24(1) |
| Fructus Cynosbati powder |
|
|
|
|
6.1 x 103
|
|
|
|
3(10) |
| Fructus Juniperi |
n. d. |
|
|
|
|
|
|
|
23(3) |
| Herba Echinaceae |
3.7 x 105
|
1.1 x 104
|
8.0 x 102
|
1.1 x 106
|
3.0 x 105
|
7.4 x 103
|
6.0 x 102
|
8.9 x 105
|
5(3) |
| Herba Equiseti |
5.0 x 102
|
|
|
|
|
|
|
|
25(1) |
| Herba Hyperici |
5.1 x 104
|
6.7 x 104
|
4.3 x 103
|
9.5 x 104
|
4.2 x 104
|
4.0 x 104
|
3.4 x 103
|
9.5 x 104
|
5(5) |
| Herba Passiflorae |
4.2 x 104
|
4.8 x 102
|
5.0 x 101
|
2.0 x 105
|
7.2 x 103
|
5.0 x 101
|
5.0 x 101
|
3.5 x 104
|
5(5); 24(1) |
| Herba Thymi |
4.4 x 105
|
1.8 x 105
|
5.0 x 101
|
1.8 x 106
|
4.1 x 105
|
2.5 x 105
|
1.5 x 104
|
1.4 x 106
|
5(5); 23(3) |
| Herba Urticae |
7.8 x 105
|
5.3 x 104
|
n. d. |
3.2 x 106
|
8.5 x 105
|
1.9 x 105
|
2.0 x 103
|
2.3 x 106
|
5(5); 23(3); 25(1) |
| Herba Visci albi |
5.0 x 102
|
|
|
|
|
|
|
|
25(1) |
| Herba Absinthii |
n. d. |
|
|
|
|
|
|
|
23(3) |
| Herba Boraginis |
|
|
|
|
7.0 x 103
|
|
|
|
3(3) |
| Herba Millefolii |
5.0 x 102
|
|
|
|
|
|
|
|
25(1) |
| Herba Solidaginis |
|
|
|
|
1.1 x 104
|
|
|
|
3(8) |
| Lichen Islandicus |
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
5(3) |
| Pericarpium Aurantii |
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
5(4) |
| Radix Althaeae |
4.1 x 104
|
5.0 x 101
|
n. d. |
2.4 x 105
|
5.1 x 104
|
1.5 x 103
|
5.0 x 101
|
2.1 x 105
|
5(5); 23(3); 24(1) |
| Radix Liquiritiae |
3.6 x 103
|
3.9 x 103
|
1.7 x 103
|
5.0 x 103
|
3.1 x 103
|
2.9 x 103
|
1.0 x 103
|
4.9 x 103
|
3(8); 5(4) |
| Radix Primulae |
1.7 x 104
|
1.4 x 103
|
1.0 x 102
|
6.6 x 104
|
1.7 x 104
|
1.2 x 103
|
1.0 x 102
|
6.6 x 104
|
5(4) |
| Radix Valerianae |
6.3 x 103
|
2.8 x 102
|
5.0 x 101
|
3.1 x 104
|
6.7 x 103
|
5.0 x 101
|
5.0 x 101
|
2.7 x 104
|
5(5); 26(1) |
| Radix Gentianae |
n. d. |
|
|
|
|
|
|
|
23(3) |
| Radix Ipecacuanhae |
n. d. |
|
|
|
|
|
|
|
23(4) |
| Radix Ratanhiae |
n. d. |
|
|
|
|
|
|
|
23(3) |
| Radix Symphyti |
|
|
|
|
2.9 x 104
|
|
|
|
3(14) |
| Rhizoma Curcumae xanthorrhizae |
5.0 x 101
|
|
|
|
|
|
|
|
23(3) |
| Rhizoma Graminis |
|
|
|
|
1.5 x 103
|
|
|
|
3(10) |
| Rhizoma Zingiberis |
|
|
|
|
5.0 x 102
|
|
|
|
3(3) |
| Semen Lini |
1.1 x 105
|
2.0 x 102
|
n. d. |
4.9 x 105
|
1.1 x 105
|
7.1 x 104
|
5.0 x 101
|
4.9 x 105
|
3(502); 5(5); 23(3); 25(1) |
| Semen Psyllii |
5.1 x 103
|
2.8 x 102
|
5.0 x 101
|
2.9 x 104
|
2.8 x 103
|
5.0 x 101
|
5.0 x 101
|
1.3 x 104
|
5(5); 24(1) |
|
1 n, number of samples examined in the corresponding study. |
|
2 n. d., non-detectable. |
Table 5 Compilation of data (CFU/g) of yeast and mould counts analysed in medicinal drugs
by different researchers. ”Reported values” represent miscellaneous viable count data,
regardless whether mean values or single data were considered. From these data medians
were calculated based on all single and mean values as far as available
| Herbal Drugs |
Reported values (Yeasts) |
Median |
Min |
Max |
Reported values (Moulds) |
Median |
Min |
Max |
References (n)1
|
| Cortex Frangulae |
|
|
|
|
5.8 x 103
|
|
|
|
22(1) |
| Cortex Hippocastani |
|
|
|
|
4.5 x 103
|
|
|
|
22(1) |
| Cortex Cinchonae2
|
|
|
|
|
2.1 x 103
|
|
|
|
23(3) |
| Flos Aurantii2
|
|
|
|
|
9.8 x 103
|
9.8 x 103
|
1.5 x 103
|
1.8 x 104
|
24(2) |
| Flos Chamomillae |
1.4 x 105
|
4.3 x 104
|
2.8 x 103
|
5.2 x 105
|
2.5 x 103
|
1.1 x 103
|
3.2 x 102
|
1.0 x 104
|
5(5); 26(3) |
| Flos Chamomillae2
|
|
|
|
|
5.5 x 102
|
5.5 x 102
|
1.7 x 102
|
9.2 x 102
|
23(3); 24(1) |
| Flos Hibisci2
|
|
|
|
|
4.7 x 101
|
|
|
|
23(3) |
| Flos Malvae |
3.4 x 102
|
5.0 x 101
|
5.0 x 101
|
1.2 x 103
|
2.4 x 106
|
2.0 x 104
|
1.0 x 102
|
1.2 x 107
|
5(4); 27(6) |
| Flos Malvae2
|
|
|
|
|
1.0 x 103
|
1.0 x 103
|
n. d. |
2.0 x 103
|
23(3); 24(1) |
| Flos Sambuci |
8.3 x 104
|
4.0 x 103
|
5.0 x 101
|
2.6 x 105
|
1.0 x 104
|
4.0 x 103
|
5.0 x 102
|
4.0 x 104
|
5(5) |
| Flos Tiliae |
7.0 x 102
|
5.0 x 101
|
5.0 x 101
|
2.0 x 103
|
4.5 x 104
|
3.0 x 103
|
3.0 x 103
|
1.3 x 105
|
5(3) |
| Flos Verbasci |
4.5 x 103
|
4.0 x 103
|
3.0 x 102
|
1.2 x 104
|
9.0 x 103
|
8.5 x 103
|
1.0 x 102
|
1.8 x 104
|
5(5); 26(1) |
| Folium Betulae |
|
|
|
|
4.1 x 103
|
4.1 x 103
|
2.0 x 103
|
6.1 x 103
|
26(1); 28(2) |
| Folium Crataegi cum flore |
3.1 x 103
|
3.0 x 103
|
5.0 x 101
|
6.3 x 103
|
8.6 x 106
|
1.2 x 104
|
9.1 x 103
|
4.3 x 107
|
5(3); 22(1); 27(6) |
| Folium Malvae |
1.7 x 104
|
5.3 x 102
|
5.0 x 101
|
7.0 x 104
|
3.5 x 104
|
4.2 x 103
|
8.2 x 102
|
1.3 x 105
|
5(4) |
| Folium Malvae2
|
|
|
|
|
1.6 x 105
|
1.0 x 104
|
2.0 x 103
|
4.7 x 105
|
24(1); 29(2) |
| Folium Melissae |
6.5 x 102
|
5.0 x 101
|
5.0 x 101
|
2.0 x 103
|
1.0 x 104
|
1.2 x 104
|
9.0 x 102
|
2.0 x 104
|
5(5) |
| Folium Menthae pip. |
3.1 x 104
|
1.2 x 104
|
5.0 x 101
|
1.0 x 105
|
2.0 x 106
|
1.3 x 105
|
1.0 x 102
|
1.1 x 107
|
5(5); 27(6) |
| Folium Menthae pip.2
|
|
|
|
|
9.0 x 103
|
8.5 x 103
|
1.0 x 103
|
2.0 x 104
|
23(3); 24(1); 25(1); 29(2) |
| Folium Salviae |
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
4.7 x 104
|
3.0 x 104
|
1.9 x 103
|
1.3 x 105
|
5(4); 28(1) |
| Folium Salviae 2
|
|
|
|
|
3.8 x 104
|
2.5 x 104
|
3.0 x 103
|
1.0 x 105
|
24(1); 25(1); 29(2) |
| Folium Sennae |
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
6.6 x 103
|
2.0 x 103
|
9.1 x 102
|
1.7 x 104
|
5(3) |
| Folium Uvae ursi |
6.5 x 102
|
5.0 x 101
|
5.0 x 101
|
3.0 x 103
|
9.2 x 103
|
2.0 x 103
|
5.0 x 101
|
3.2 x 104
|
5(5); 22(1) |
| Folium Uvae ursi2
|
|
|
|
|
3.4 x 102
|
2.8 x 102
|
1.0 x 101
|
8.0 x 102
|
23(4); 24(1); 29(2) |
| Folium Althaeae |
|
|
|
|
3.5 x 107
|
|
|
|
27(6) |
| Folium Boldo conc.2
|
|
|
|
|
1.7 x 103
|
|
|
|
29(1) |
| Folium Boldo tot.2
|
|
|
|
|
1.3 x 102
|
|
|
|
29(1) |
| Folium Eucalypti conc.2
|
|
|
|
|
6.0 x 103
|
|
|
|
29(1) |
| Folium Eucalypti tot.2
|
|
|
|
|
1.0 x 102
|
|
|
|
29(1) |
| Folium Farfarae |
|
|
|
|
2.2 x 103
|
|
|
|
26(1) |
| Folium Hamamelidis |
|
|
|
|
2.9 x 104
|
|
|
|
22(1) |
| Folium Juglandis |
|
|
|
|
2.0 x 107
|
|
|
|
27(6) |
| Folium Plantaginis2
|
|
|
|
|
1.3 x 103
|
|
|
|
24(1) |
| Folium Rubi idaei |
|
|
|
|
6.3 x 102
|
|
|
|
26(1) |
| Fructus Anisi |
1.7 x 104
|
1.7 x 104
|
5.0 x 101
|
8.2 x 104
|
1.1 x 103
|
2.0 x 102
|
5.0 x 101
|
3.5 x 103
|
5(5) |
| Fructus Carvi |
1.6 x 104
|
5.0 x 101
|
5.0 x 101
|
7.7 x 104
|
3.0 x 103
|
5.0 x 101
|
5.0 x 101
|
1.5 x 104
|
5(5) |
| Fructus Foeniculi |
1.4 x 102
|
5.0 x 101
|
5.0 x 101
|
5.0 x 102
|
7.3 x 102
|
4.0 x 102
|
5.0 x 101
|
2.2 x 103
|
5(5) |
| Fructus Myrtilli |
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
3.6 x 103
|
2.3 x 102
|
1.0 x 102
|
1.4 x 104
|
5(3); 22(1) |
| Fructus Sennae |
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
2.2 x 103
|
4.0 x 102
|
1.0 x 102
|
6.0 x 103
|
5(3) |
| Fructus Sennae2
|
|
|
|
|
2.5 x 104
|
5.0 x 103
|
9.0 x 102
|
7.0 x 104
|
16(3); 24(1); 30(1) |
| Fructus Cynosbati 2
|
|
|
|
|
3.0 x 101
|
|
|
|
24(1) |
| Fructus Juniperi |
|
|
|
|
n. d. |
|
|
|
23(3) |
| Herba Echinaceae |
1.0 x 102
|
5.0 x 101
|
5.0 x 101
|
2.0 x 102
|
3.3 x 103
|
9.1 x 102
|
9.0 x 102
|
8.2 x 103
|
5(3) |
| Herba Equiseti |
|
|
|
|
1.1 x 102
|
|
|
|
26(1) |
| Herba Hyperici |
6.3 x 104
|
2.0 x 104
|
5.0 x 101
|
2.1 x 105
|
4.7 x 106
|
1.6 x 105
|
4.0 x 104
|
2.3 x 107
|
5(4); 27(6) |
| Herba Passiflorae |
8.0 x 101
|
5.0 x 101
|
5.0 x 101
|
2.0 x 102
|
2.9 x 103
|
2.0 x 102
|
1.0 x 102
|
1.4 x 104
|
5(5) |
| Herba Thymi |
4.8 x 103
|
4.6 x 103
|
5.0 x 101
|
1.0 x 104
|
5.5 x 104
|
3.2 x 104
|
5.0 x 103
|
1.5 x 105
|
5(4) |
| Herba Urticae |
5.0 x 103
|
1.1 x 103
|
5.0 x 101
|
2.0 x 104
|
1.3 x 105
|
5.3 x 104
|
6.3 x 102
|
5.3 x 105
|
5(5); 26(1) |
| Herba Visci albi |
|
|
|
|
8.9 x 103
|
|
|
|
28(2) |
| Herba Absinthii2
|
|
|
|
|
2.0 x 101
|
|
|
|
23(3) |
| Herba Anserinae |
|
|
|
|
1.8 x 100
|
|
|
|
26(1) |
| Herba Millefolii |
|
|
|
|
4.0 x 102
|
|
|
|
25(1) |
| Herba Veronicae |
|
|
|
|
1.6 x 102
|
|
|
|
26(1) |
| Herba Violae tricoloris |
|
|
|
|
3.2 x 103
|
|
|
|
22(1) |
| Lichen Islandicus |
3.6 x 103
|
6.0 x 102
|
5.0 x 101
|
1.0 x 104
|
2.1 x 104
|
1.4 x 103
|
3.0 x 102
|
6.0 x 104
|
5(3) |
| Pericarpium Aurantii |
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
1.4 x 102
|
1.0 x 102
|
1.0 x 102
|
2.7 x 102
|
5(1) |
| Radix Althaeae |
2.5 x 102
|
5.0 x 101
|
5.0 x 101
|
1.0 x 103
|
2.7 x 103
|
1.7 x 103
|
3.0 x 101
|
7.3 x 103
|
5(5); 28(1) |
| Radix Althaeae2
|
|
|
|
|
1.2 x 106
|
2.5 x 102
|
1.5 x 102
|
3.5 x 102
|
23(3); 24(1) |
| Radix Liquiritiae |
1.3 x 103
|
5.0 x 101
|
5.0 x 101
|
3.8 x 103
|
2.3 x 105
|
2.5 x 104
|
3.2 x 103
|
6.6 x 105
|
5(3) |
| Radix Primulae |
3.8 x 102
|
1.0 x 102
|
5.0 x 101
|
1.3 x 103
|
2.1 x 103
|
6.0 x 102
|
1.0 x 102
|
7.2 x 103
|
5(4) |
| Radix Valerianae |
1.4 x 103
|
5.0 x 101
|
5.0 x 101
|
5.5 x 103
|
1.2 x 103
|
6.0 x 102
|
1.0 x 102
|
3.6 x 103
|
5(4) |
| Radix Gentianae2
|
|
|
|
|
4.0 x 100
|
|
|
|
24(3) |
| Radix Ginseng |
|
|
|
|
2.9 x 103
|
|
|
|
22(1) |
| Radix Harpagophyti |
|
|
|
|
1.2 x 105
|
|
|
|
22(1) |
| Radix Ipecacuanhae2
|
|
|
|
|
1.1 x 101
|
|
|
|
23(4) |
| Radix Ratanhiae2
|
|
|
|
|
9.4 x 101
|
|
|
|
26(3) |
| Radix Rhei |
|
|
|
|
1.0 x 104
|
|
|
|
28(1) |
| Rhizoma Curcumae xanthorrhizae2
|
|
|
|
|
8.7 x 105
|
|
|
|
26(3) |
| Semen Lini |
8.0 x 101
|
5.0 x 101
|
5.0 x 101
|
2.0 x 102
|
1.8 x 102
|
2.0 x 102
|
5.0 x 101
|
3.0 x 102
|
5(5) |
| Semen Psyllii |
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
5.0 x 101
|
7.2 x 102
|
1.0 x 102
|
5.0 x 101
|
2.3 x 103
|
5(5) |
| Semen Hippocastani2
|
|
|
|
|
9.3 x 102
|
|
|
|
23(3)
|
|
1 n, number of samples examined in the corresponding study. |
|
2 Yeasts and moulds were not specified separately. |
Microbial Quality Standards, Class Plans and Sampling Guidelines
Microbial Quality Standards, Class Plans and Sampling Guidelines
The Ph.Eur. 2000 [21] provides different guidances and tolerance levels which can be applied for evaluating
the microbiological quality of herbal medicinal plants. In this version, fundamental
distinctions are made by categories, depending on the purpose of medical application
and the preparation technique. Herbal products can be assigned mainly to categories
3B, 4A and 4B. In contrast to other chapters outlined in the Ph.Eur. 2000, these evaluation
criteria do not form a mandatory part, but are used as a recommendation for target
levels. Unfortunately, this chapter neither contains any advice regarding sampling
plans (e. g., answers to the question ”How many samples have to be drawn from a lot
?”) nor does it suggest typical attributive class plans, which are frequently applied
in modern food quality assessment.
The European Herbal Infusion Association (EHIA) basically follows the ISO guidances,
which comprise sampling plans and standardized protocols for aerobic mesophilic bacteria,
yeasts and moulds, E. coli and Salmonella [35]. In principle, those methods established for the examination of foods are used to
assess the microbiological quality of herbal infusions. The EHIA usually tolerates
higher microbiological threshold levels [35] than given in the Ph. Eur. 2000.
The examination of the microbiological quality is commonly based on general and specific
evaluation criteria, each of them having a more or less pronounced indicative meaning,
which further enables practical conclusions (Table [6]). In principle, most quality aspects of medicinal plants can be compared with those
considered in the area of food microbiology, since spices and herbs, tea, vegetables,
cereals may exhibit similar microbiological tendencies. Differences in the guidelines
proposed by different associations were presented in detail by Kolb [35]. However, medicinal drugs possess several important differences to the food area:
besides their content of specific compounds of particular pharmaceutical and medical
relevance with dose-dependent properties, these products are necessarily not consumed
for ”conventional purposes” because they do not primarily fulfil a nutritive or relishing
function. Moreover, the consumers of medicinal plants are usually not healthy, but
rather people who undergo some form of therapeutic treatment. This means that on the
one hand toxicological factors and on the other hand higher risk levels and hazard
classes have to be considered. Furthermore, diverse physical processing techniques
performed during the pharmaceutical technology have to be taken into account. For
example, treatment with boiling water is some kind of extraction procedure for relevant
compounds but also should aim at microbial decontamination.
Several approaches have been made to optimise the design of sampling plans, that form
the basis for a reliable evaluation of samples and for making decisions whether a
produced or packaged lot should be accepted or rejected [36], [37], [38]. According to modern hygiene standards, so-called ”attributive class plans” usually
prescribe a uniform strategy for the examination and evaluation of a defined number
of samples with a certain mass [39]. These plans allow attributive conclusions (”good” vs.”bad” or ”lower than” vs.
”higher than”, ”acceptable” vs. ”non-acceptable”) and should be based on a risk assessment
which allows one to clearly discriminate among accepted and rejected lots. In the
case of medicinal plants, the classical pathogens such as Salmonellae or Listeriae
and, according to recent experiences, also EHEC and Campylobacter, are not tolerated at all in any sample. For that reason usually portions of 10 or
25 grams or larger amounts (depending on the risk dimension) should be examined following
standardised methodologies. The only acceptable result thereby obtained should be
”not detectable in ... grams”. Hence, these so-called 2-class plans follow a very
strict strategy. In addition, so-called 3-class plans offer some kind of compromise
within product evaluation, since the triple distinction between ”acceptable”, ”still
acceptable or tolerated” and ”non-acceptable” samples is facilitated. In this context,
the following criteria have to be regarded : n... total number of examined samples;
c...number of tolerated samples exceeding the tolerance level m but not the maximum
level M, which must not be exceeded by any of the samples. Although these plans may
lack some clarity [38], they are sensitive tools, if general quality parameters (e. g., total aerobic mesophilic,
coliform, endospore counts, etc.) are considered. They also intend to recognise the
accuracy of the used method as affected by possible variations of the measured values.
Nevertheless, not only the ”non-accepted” samples but also the number of samples which
can be ”still accepted or tolerated” have to be based on profound experiences.
Under practical conditions, most methodological instructions usually prescribe sampling
plans which follow some correlation between the product lot size and the number of
samples, which have to be drawn for testing. This relationship has led to the understanding
that bigger lots have to be examined using a higher number of samples than small-lot
size productions. Nevertheless, other key factors such as the targeted product safety,
methodological selectivity and possible methodical variations are not considered in
a sufficient way. By outlining this difficulty, Hildebrandt [40] has described a simplified function to demonstrate that the overall quality of an
examination can only be efficiently enhanced by exponentially increasing the number
of samples (n): n = safety × selectivity × variation. From this function we may conclude that sample
numbers over five do not offer some marked advantage, unless enhanced safety factors
and hazard potentials have to be taken into account. Busse [41] has also critically illuminated the problematic situation of applying attributive
class plans for screening a defined number of samples for pathogens in bigger lots
and also the rationale behind defining variables of 3-class plans.
Table 6 Indicative meaning of groups of microorganisms usually examined in microbiological
routine control of medicinal herbal plants
| Group |
Meaning |
| Total aerobic mesophilic count |
General hygiene and quality parameter |
| Enterobacteria |
General hygiene and quality parameter, indicator for faecal contamination, but also
ubiquitously present |
| Coliforms |
Indicator for faecal contamination, but also ubiquitously present |
| Enterococci |
Indicator for faecal contamination, but also ubiquitously present |
| Pseudo- and aeromonades |
Indicator for enhanced spoilage potential, mainly from water-borne sources |
| Coagulase-positive staphylococci |
Indicator for pathogens of human origin |
| Aerobic and anaerobic sporeforming bacteria |
bacteria, typical soil microflora |
| Lactobacilli |
Possible indicator for spoiled plant material |
| Yeasts and moulds |
Ubiquitously present microorganisms, in part indicator for possible mycotoxigenic
potential |
| Pathogens |
Occurrence bears high health risk, to be avoided (Salmonella, Listeria monocytogenes, E. coli, EHEC1,
Campylobacter, Yersinia, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus cereus,
Clostridium perfringens, mycotoxin producers) |
|
1 EHEC...enterohaemorrhagic strains of E.coli.
|
Methodological Aspects
Methodological Aspects
In the Ph.Eur. 2000 [21], most media and diluents used in microbiological routine examination are specified.
They can be applied for diverse materials and products. However, in the light of the
above described situation, recent methodological developments and additionally emerged
microbial pathogens have induced some need for re-considering the methodological principles
and testing criteria. In their screening paper, Czech et al. [5] have introduced an array of methods that may be used to assess the microbiological
quality of medicinal plants. As far as possible, these authors followed the basic
instructions outlined in the Ph.Eur. 2000, but some aspects were also derived from
current food examination protocols or from methods used in medical routine examination.
A general flow diagram of a microbiological quality control program for plant samples
is presented in Fig. [2].
Fig. 2 General structure of a protocol for the microbiological examination of medicinal herbal
drugs. P/A Presence/Absence testing.
Influence of Different Preparation Techniques on the
Microbiological Quality
Influence of Different Preparation Techniques on the
Microbiological Quality
Medicinal herbal drugs are applied in different forms, and manipulation and processing
factors largely determine the microbiological quality of the final products [2], [3]. The application of hot water extraction (herbal infusion, herbal tea) usually compensates
for microbiological contaminations, since it can be expected that boiling water markedly
reduces the viable counts by several log units and also inactivates possible pathogens
[28]. However, those drugs which are subjected to cold water extraction (herbal maceration)
may host a considerable amount of microbes, and the extraction procedure carried out
at ambient temperature usually enables microbial multiplication. Herbal tinctures,
which are made by ethanol extraction should, in general, provide good hygiene conditions,
but the result depends on the alcoholic concentration applied.
With these presumptions, a series of tests were performed to examine the influence
of cold water maceration on the microbiological situation of a selection of herbal
medicinal drugs. For this purpose, we prepared a 1 % (w/v) mixture of the plant in
sterilised tap water, followed by an extraction (standing) period for 24 hours at
22 °C. After this treatment the contents were mixed by shaking and the maceration
process was stopped by applying a cooking step for 10 seconds. The whole procedure
was accompanied by a microbiological monitoring of the supernatant samples (Fig. [3]). It is evident from the distribution of the viable counts that during the extraction
procedure at 22 °C a pronounced microbial propagation takes place, so that even extremely
high populations can be measured in the macerations before the heating step. During
this period, the total aerobic count and also the enterobacterial counts developed
towards a magnitude which would not permit the direct use of this product. The spore
counts differed with regard to their basic load in the individual samples. As expected,
the final boiling procedure largely improved the hygiene situation: enterobacteria
decreased to the detection limit (non-detectable in 0.1 ml), and total areobic counts
as well as spore counts yielded results lower than 104 CFU per ml of the maceration liquid. Based on this finding we would strongly recommend
the application of a final heat treatment as an obligatory step. However, possible
changes in the pharmacological properties during heat treatment may play some role
and have therefore to be considered.
Another test series with selected plants was carried out to study the particular microbiological
situation in herbal ethanolic tinctures. Tinctures were prepared by maceration of
20 grams of drug samples with 80 ml ethanolic solutions at different concentrations,
according to the Ph.Eur. 2000. All samples were examined 6 days after preparation.
Some correlation between ethanol concentration and total aerobic mesophilic counts
was observed, although only the higher ethanol concentrations (60, 70, 80 %) led to
marked decontaminating effects (Fig. [4]). Only that product which basically had a high bacterial endospore count (Herba
Passiflorae) showed relatively high residual counts. Thus, products rich in spores
should not be used for the preparation of tinctures.
In addition to these procedures, alternative methods such as treatment with ethylene
oxide or radiation with ionic rays lead to decontamination effects [42]. These methods can be seen as a compromise between ensuring the microbiological
safety of the product and avoiding consumer’¿s risk and special legal permissions
are required in many countries.
Fig. 3 Changes of microbiological quality parameters (TAMC... total aerobic mesophilic count,
sporeformer count, enterobacterial count) during the maceration procedure of selected
herbal drugs with cold water. A...initial counts of the maceration, B...counts determined
after storage of the maceration for 24 hours at 22 °C, C... residual counts determined
after a final boiling procedure.
Fig. 4 Total aerobic mesophilic counts (TAMC) of tinctures of selected herbal drugs at different
ethanolic concentrations, 6 days after preparation.
Conclusions and Future Perspectives
Conclusions and Future Perspectives
Taking into account the pronounced variability within the hygiene situation of herbal
medicinal plants, it is difficult to propose uniform criteria for the evaluation of
microbiological quality parameters. However, in front of the growing importance of
thresholds, tolerance and target levels as well as specifications in daily business,
the guidances outlined in the Ph.Eur. 2000 should be carefully re-considered, since
there is an urgent need for having available standards for allowing legal decisions.
These guidelines should be applicable not only for conventionally but also for biologically
produced plants. Especially the latter ones are known to be treated with natural fertilizers
(e. g., stinging nettle broth) containing considerably higher microbial levels and
also preferred by many insects, which often carry contaminants.
Although the establishment of 3-class plans may bear problems, this strategy offers
the advantage of giving a more detailed insight into sample quality. By considering
the currently available literature and also the experience with influences of different
preparation procedures on the microbiological situation, the following conclusions
can be pointed out:
-
In general, excellent hygiene quality as indicated by the absence of pathogens in
at least 10 grams of product and a low level of microbial contamination (i. e., low
total aerobic mesophilic, endospore and enterobacterial counts) should be the main
goal for each producer of medicinal plants. This also forms the optimum prerequisite
for providing herbal plants of high quality and with desired therapeutic benefits.
This requirement is of particular importance for those products and preparations which
are assigned to category 3 of Ph.Eur. 2000.
-
In agreement with the Pharmacopoeia, a clear distinction should be made in terms of
considering the impact of different preparation techniques of the drugs. Agreement
should be obtained among expert panels if a product with a certain microbiological
load can be used for certain applications, or if it would be necessary to obligatorily
carry out some treatment with boiling water or with alternative methods. Heating and
ethanolic extraction do not yield sterile drugs, but usually products with relatively
low viable counts (expectedly, of approximately < 104 per ml). The residual microflora thereby obtained is represented mainly by bacterial
spores, enterobacteria usually do not survive these conditions.
-
Standardised methodological protocols describing in detail the techniques of microbiological
examination should be approved and validated, so that comparable laboratory conditions
ensure the agreement between different assessors.
-
Following the above described targets, an attempt was made to propose some alternative/additional
criteria and revised threshold levels of the microbiological guidances as given in
the Ph.Eur. 2000 (Table [7]). The overview gained from the literature available has been taken as a basis for
generating this classification. We suggest that this evaluation scheme be treated
as a recommendation and a basis for discussion. Being aware that following this proposal
would cause additional work load and costs, it should also be taken into consideration
that this effort has some preventive importance and helps to avoid costs arising from
quality problems and consumer complaints. Alternatively, the strategy of a two-step
examination could be followed: 1) analysis of a bulk sample proportionally drawn from
all (n) samples, followed by 2) analysis of the individual samples, if the bulk sample
has indicated results ranging around or over the proposed thresholds. However, the
decision whether this strategy is acceptable or not depends on the homogeneity of
the material to be considered and should be carefully evaluated.
Table 7 Proposed microbiological examination and evaluation criteria for relevant categories
of herbal medicinal drugs according to the Ph. Eur. 2000 (only brief descriptions
of preparations/categories are given)
| Categories |
Parameter1
|
Criteria2
|
|
Category 3
|
|
|
A Preparations for oral and rectal application
B Preparations for oral application, antimicrobial treatment
impossible or TAMC > 103 per ml or g tolerated |
Salmonella
E. coli
Staph. aureus
Enterobacteria
TAMC
Yeasts and moulds |
This sub-category is of limited relevance for herbal medicinal drugs
2-CP, sample size: 10 g, n = 5, c = 0, m = M = 0
2-CP, sample size: 1 g, n = 5, c = 0, m = M = 0
2-CP, sample size: 1 g, n = 5, c = 0, m = M = 0
3-CP, n = 5, c = 1, m = 102/g, M = 103/g
3-CP, n = 5, c = 2, m = 103/g, M = 104/g
3-CP, n = 5, c = 2, m = 102/g, M = 103/g |
|
Category 4
|
|
|
A Preparations including treatment with boiling water
B Preparations not including treatment with boiling water |
Salmonella
E. coli
Enterobacteria
TAMC
Yeasts and moulds
Salmonella
E. coli
Staph. aureus
Enterobacteria
TAMC
Yeasts and moulds |
2-CP, sample size 10 g, n = 5, c = 0, m = M = 0
3-CP, n = 5, c = 1, m = 10, M = 102/g
3-CP, n = 5, c = 2, m = 105/g, M = 106/g
3-CP, n = 5, c = 3, m = 106/g, M = 107/g
3-CP, n = 5, c = 3, m = 104/g, M = 105/g
2-CP, sample size: 10 g, n = 5, c = 0, m = M = 0
2-CP, sample size: 1 g, n = 5, c = 0, m = M = 0
2-CP, sample size: 1 g, n = 5, c = 0, m = M = 0
3-CP, n = 5, c = 1, m = 102/g, M = 103/g
3-CP, n = 5, c = 2, m = 104/g, M = 105/g
3-CP, n = 5, c = 2, m = 103/g, M = 104/g |
| Explanation of superscripts: |
|
1 Parameters: Detection of (presumptive) pathogens (Salmonella, E.coli, Staphylococcus
aureus) according to standardised protocols (enrichment, selective plating, presumptive
detection); examination of microbiological quality parameters (enterobacteria, TAMC
= total aerobic mesophilic count, yeasts an moulds) according to cultural plate count
techniques of serially diluted samples. |
|
2 Criteria: 2-CP: 2-class attributive plans; 3-CP: 3-class attributive plans; n: total
number of samples examined, c: number of tolerated samples exceeding lower threshold
level m, M: upper threshold level, which must not be exceeded by any of the samples;
viable counts are CFU/g. |
Acknowledgements
Acknowledgements
We gratefully acknowledge the helpful comments of Gudrun Abel (Neumarkt), C. Erdelmeier
(Karlsruhe), D. Flamme (Neumarkt), R. Franke (Bruckmühl), C. Franz (Vienna), G. Franz
(Regensburg), and K. Rahn (Karlsruhe).
