pattern-oriented. The compound-oriented approach uses certain components with some chemical properties known as targets, while pattern-oriented uses all components that can be detected as targets. The compound-oriented approach is based on a marker compound concept with the target is certain chemical compounds with known molecular structures. A pattern-oriented approach based on a metabolite profile or fingerprinting or phytoequivalent concept with a target is all chemical compounds with structures known or unknown or that have partial chemical information eg. retention time, mass spectrum, and ultraviolet spectrum [55–57].
1.3.1 Marker Compound Concept
In phytopharmaceuticals, the resulting pharmacological activity comes from all the compounds in it. The active compound cannot be ascertained. Besides, the ingredients are very complex and varied, so that it becomes more challenging to characterize. Under these conditions, a marker is needed to represent phytopharmaceuticals’ characterization. These marker compounds are used to control quality but do not always represent the constituents associated with drug activity [1, 54, 58].
In pharmacopeia, phytopharmaceuticals are considered drugs so that the quality is based on a chemical marker, which is expected to ensure consistency. According to the European Medicines Agency (EMEA), chemical marker are constituents or constituent groups used for quality control purposes, have or do not have therapeutic activity. To be a marker of quality, ideally, a chemical marker has unique and specific characteristics and must contribute to the therapeutic effect of phytopharmaceuticals. Differences in the number of chemical markers can indicate differences in the phytopharmaceuticals pharmacological activity. Thus, the number of chemical markers can indicate the quality of phytopharmaceuticals [59, 60].
In the concept of marker compounds, the key first step in determining phytopharmaceuticals quality is the selection of marker compounds. There are three ways to choose or select a marker compound that will be discussed below as illustrated in Figure 1.2.
– Major or easy-to-get compound
The selection of marker compounds isn’t too easy because the therapeutic or bioactive components of phytopharmaceuticals are unknown and not yet clearly understood. Under such conditions, the marker compound can be selected from several components that are not specific but easily obtained. But, this has a weakness. The marker compound cannot ensure the effectiveness of phytopharmaceuticals [60]. This easy-to-get compound is usually a major compound. At least, because the levels of these compounds are large, they also have a major contribution to phytopharmaceuticals’ pharmacological activity.
Figure 1.2 Tree ways to select a marker compound.
An example of this case is ethyl-p-methoxycinnamate as a marker compound in the ethanolic extract of Kaempferia galanga rhizome [61]. The compound content is 14.54%. In the ethanolic extract of Andrographis paniculata, the Andrographolide level is 10.82%. This compound is used as a marker compound for products containing A. paniculate extract [62]. However, various research showed that the pharmacological activity of the extract is greater than its marker compound. This indicates that the selected marker compound is not therapeutically active. But the level of these compounds can be a marker of the quality of their pharmacological effects. These compounds can be called active marker compounds.
– In silico Virtual Screening
Marker compounds must have clear pharmacological activity so that their levels in the product are an indication of product quality. Bioassay screening is indispensable to obtain such compounds. The next process is to carry out bioassay-guided isolation. Then proceed with bioactivity confirmation using animal models, organ and tissue models, cellular models, or receptors and enzymatic. However, this method is time consuming, difficult, and has not revealed the effects of interactions with other compounds. Therefore requires an integrated new approach for predicting potential active constituents in herbs [60].
Recent developments in efforts to explain the pharmacological basis of medicinal plant functions have been carried out using in silico approaches such as virtual screening and tissue analysis. This approach can increase the discovery of active compounds and show the mechanism of action of medicinal plants, reduce costs, and improve the whole procedures’ efficiency. Some stages in the in silico approach are the target selection of medicinal plants, target plants’ database, ADME/T analysis (absorption, distribution, metabolism, excretion, toxicity), and in silico virtual screening. In silico virtual screening method is carried out with the following steps: virtual targets based on pharmacophore theory; double validation based on the theory of similarity in the form of small molecules; and analysis of target compounds based on docking. The initial half of implementing this method uses software technology, including the prediction of multiple activities and virtual screening. All are based on mass spectra data to provide accuracy. Compared to conventional screening methods, the in silico virtual screening method is superior because it is only required information on the structure of compounds. This method can also multi-target screening a large number of compounds in a relatively short period, thereby reducing costs and time [63].
The herbal formula for tuberculosis therapy in Indonesia uses several medicinal plants such as Curcuma xanthorrhiza Roxb., Tamarindus indica L., Citrus aurantifolia, and Zingiber officinale var. rubrum. The results of tests using in silico virtual screening successfully revealed 6 compounds: curcumin, demethoxycurcumin, 8-gingerol, phytol, oleic acid, and linoleic acid related to their activities in inhibiting cell growth and MTB (Mycobacterium tuberculosis) infection. These studies indicate that curcumin, gingerol, phytol can be used as a marker compound for C. xanthorrhiza Roxb., Z. officinale var rubrum, T. indica L., respectively [64].
– Combination of fingerprint and chemometric analysis
A fingerprint is a method that describes the characteristics of complex samples obtained from integrated chromatography or spectroscopy. The chemometric analysis is used to correlate chemical profile data obtained from the fingerprint method with pharmacological profile data from complex samples. Chemometric analysis with the help of mathematical, statistical, and computational sciences such as PCA, PLS-DA, hierarchical cluster analysis (HCA), K-nearest neighbour (KNN), and so on, can be used to obtain marker compound information in medicinal plants. The results of the correlation will show compounds that have certain activities. Using chemometric analysis, the HPLC profile data of L. japonica samples were correlated with bacteriostatic data. The results showed that 6 constituents are positively correlated with bacteriostatic activity. One such compound is chlorogenic acid which has been known to have bacteriostatic properties. These results indicate that chlorogenic acid can be used as a marker compound. The result also shows that the bacteriostatic effect of L. japonica is the result of many constituents, not only from chlorogenic acid [60].
Chemical marker is the main point of quality control for herbal medicines based on compound-oriented. To meet the objectives as parameters for quality, safety, and efficacy, the selection of compounds used as a chemical marker is very important. The chemical marker must be used at various stages of developing and manufacturing herbal medicines, from raw materials to finished products, from batch to batch production. Chemical marker has several requirements that must be fulfilled. The chemical structure is known, reference standards are available, and the levels in the sample can be measured using a reliable instrument.
1.3.2 Phytoequivalence Concept
One or a group of marker compounds cannot describe the composition of compounds in phytopharmaceuticals. Multicomponent
