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A critical survey of allelochemicals in action - the importance of total activity and the weed suppression equation.

Yoshiharu Fujii and Syuntaro Hiradate

Chemical Ecology Unit, Department of Biological Safety, National Institute for Agro-Environmental Sciences, 3-1-3 Kannondai, Tsukuba Science City, Ibaraki 305-8604, Japan, Email


In the search for natural chemicals useful as herbicides, compounds with high specific activity (biological activity per unit weight of the compound) should be considered, whereas for allelochemicals, compounds with high total activity (biological activity per unit weight of the organism) are important in natural ecosystems. The total activity is a function of the specific activity of natural chemicals and their content in the plant. Our recent finding of cyanamide from Vicia villosa was a result of this new strategy for “Total Activity Method”. L-DOPA from Mucuna pruriens, cis-cinnamic acid from Spiraea thunbergii and some other allelochemical candidates are discussed. For the practical evaluation of allelopathy in the field, the concept of “Weed Suppression Equation”, comparing nutritional and physical factors for competition, growth rate and allelopathic factor, is introduced.

Media summary

Total activity (biological activity per unit weight of the organism) is important for allelochemicals in action.

Key Words

Allelochemical, Total activity, Specific activity, hairy vetch, Vicia, Spiraea, Mucuna, Duranta


Allelopathy is an interaction between plants and other living organisms that is caused by specific chemicals (allelochemicals) released from plants (Molisch, 1937). There are many reports of the isolation of allelochemicals, and until now it is still not easy to evaluate the contribution of these reported compounds to the allelopathic phenomenon. This makes the concept of “Allelopathy” unclear for some scientists.

In the course of screening for allelochemicals in action in the field (in situ), we introduced a new strategy of the “Total Activity” concept according to the isolation method of enzymes in plants instead of the “Specific Activity” method used in many reports published. Although the concept of Total Activity is not completely new, and evaluated in some reports (Fujii et al. 1991, An et al, 2001), use of this concept for the isolation of allelochemicals has never been reported. In the search of natural agrochemicals to isolate new bioactive compounds with high specific activity, and use these chemicals as lead compounds for agrochemicals, specific activity is of course useful. But for the search for allelochemicals in action in the field, the “Total Activity Method” is more important to isolate such allelochemicals. By this strategy, we could isolate unique compounds and important allelochemicals in the field.

Concept and Strategy

As shown in Figure 1, Specific Activity means biological activity per unit weight of the compound, and total activity means biological activity per unit weight of the organism. Specific activity is easy to calculate, and in our case, we calculate EC50. For the isolation of allelochemical candidates from crude extract, each activity was measured on a “g” base, as shown in Figure 1 and Figure 3. For the comparison of Total Activity to other reports, an Index on a molar base was calculated after the isolation and identification of allelochemicals.



Index (mol base)

Direct use of

in action in situ

Total Activity
= 1/EC50 x Concentration in plants


1/(mol/L) x (mol/g fresh weight)
= (L/mol) x (mol/g) = L/g

Use of
natural Chemicals

Bioactive chemicals
with high specific activity

Specific Activity
= EC50 (50% inhibitory Concentration) mol/L

Figure 1. How to calculate Total Activity in comparison with Specific Activity.

In order to show the importance of Total Activity, Figure 2 explains and compares the explanatory value of Specific Activity and Total Activity. In this example, as for Specific Activity, compound A is ten times stronger than compound B. But concentration of compound B is hundred times higher. Thus, Total Activity of compound B is ten times stronger than compound A. In this example, we conclude that compound B is ten times important in its contribution to the allelopathic phenomenon in the field and thus more important as an allelochemical.


Specific Activity
(EC50, μM)


Total Activity
(no unit)

Compound A




Compound B




Specific Activity:

A> B (10 times)



B> A (100 times)

Total activity:

B> A (10 times)

(B is more important as allelochemicals)

Total activity= 1 / Specific Activity (EC50)×Concentration

Figure 2. Meaning of Specific Activity and Total Activity

In order to explain how to separate allelochemical candidates by “Total Activity” concept, Figure 3 explains the strategy. In each step of isolation, Specific Activity of each fraction (Fr. 1, 2, 3, …) was measured each time. Weight of each fraction was also measured and based on these data, Total Activity was calculated. From the standpoint of Total Activity, the Fraction with the highest Total Activity was selected for further fractionation.

Figure 3. Isolation scheme of allelochemicals based on Total Activity.


Figure 4. Isolation of L-DOPA from velvet bean.

Figure 5. Isolation of cis-cinnamic acid from Spiraea

Figure 4 shows the result of isolation of L-DOPA from velvet bean (Mucuna pruriens). The concentration of L-DOPA in the plant is extremely high and about 1 % of fresh weight in the leaves and roots (Fujii et al. 1991, 1992). Specific activity of L-DOPA is 4x10-4M, Total Activity is 200. This value is the highest before the isolation of cis-cinnamic acid from Spiraea thunbergii (Figure 5), with value of 1000. The Specific Activity of cis-cinnamic acid is 1000 times stronger than trans-cinnamic acid and about 3x10-6 M. This activity is equivalent to cis-ABA, one of the plant hormones (Hiradate et al. 2004, 2005).

Figure 6. Isolation of durantanin from Duranta.

Figure 7. Isolation of a cyanmide from hairy vetch.

By means of the Total Activity method, we have isolated potent natural chemicals from Duranta repens. As these compounds were new saponins, they are named as “durantanins”(Figure 6). These compounds were contained about 4 % in dry weight in Duranta (Hiradate et al. 1999).

Hairy vetch is a cover crop. We have studied the practical weed suppression activity of hairy vetch from 1990, and demonstrated its use in the field (Fujii 1999, 2000, 2001). As a result of our recommendation, hairy vetch is now cultivated as organic weed control in orchards and fallow of rice fields in Japan. Isolation of the plant growth inhibitory chemical from hairy vetch was very difficult and after ten years of research, we isolated cyanamide as a natural chemical (Figure 7) (Kamo et al. 2003). Cyanamide is a well- known synthetic fertilizer and herbicide synthesized about 100 years ago, but there has been no previous report of cynamide being a natural product before we reported. Cyanamide is a very simple compound with low molecular weight. As cyanamide has only one simple signal by NMR and MS, it is usually very difficult to identify it in the course of isolation. Without the strategy of Total Activity, it could have been difficult to isolate this compound.


Figure 8 summarises the tentative evaluation of famous compounds reported as allelochemicals by Total Activity. In the field, it is necessary to discriminate and evaluate the contribution of allelopathy with competition for light, nutrients and water. We are now developing the concept of the “Weed Suppression Equation” to discriminate between these factors.

Figure 8. Evaluation of allelochemicals by Total Activity


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Fujii Y, Shibuya T and Yasuda, T (1992) Allelopathy of velvetbean: Its discrimination and identification of L-DOPA as a candidate of allelopathic substances. Japan Agricultural Research Quartery 25, 238-247.

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Hiradate S, Morita S, Sugie H, Fujii Y and Harada J (2004) Phytotoxic cis-cinnamoyl glucosides from Spiraea thunbergii. Phytochemistry, 65 (6), 731-739.

Hiradate S (2004) Strategies for searching bioactive compounds: Total activity vs. specific activity. The 227th ACS National Meeting, Anaheim, CA, March 28 - April 1, 2004, AGFD7

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Kamo T, Hiradate S and Fujii Y (2003) First isolation of natural cyanamide as a possible allelochemical from hairy vetch Vicia villosa. J. Chem. Ecol. 29 (2), 275-284.

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