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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 in phytopharmaceuticals does not constitute the sum of each components’ activities in it. Due to various synergistic effects, they form a unity that cannot be separated. In this way, multicomponents in phytopharmaceuticals are considered “an active ingredient”. This active ingredient has been clinically proven used as a standard reference for quality control. This concept is known as “phytoequivalence” developed in Germany to ensure the consistency of phytopharmaceuticals [65].
Multicomponent active ingredients in phytopharmaceuticals can be extracts or fractions of medicinal plants’ raw materials. So, this extract or fraction is treated as a single chemical entity in every possible way. However, some difficulties must be faced when applying Good Manufacturing Practices (cGMP) of medicinal products to phytopharmaceuticals. There are many element in the active ingredients of phytopharmaceuticals, including, active, inactive, unknown compound, and elements that are dietary rather than therapeutic. Another difficulty for this analysis is to provide a standard reference that is always available. The analytical method that meets these conditions is the chromatographic fingerprint. The use of chromatographic fingerprints for phytopharmaceuticals is carried out with a focus on compounds that can be detected by the type of chromatography used with or without knowing the compounds’ structure [66].
Chromatographic fingerprints are chromatographic patterns of multicomponent active ingredients that show some pharmacologically active chemical components and show chemical characteristics. The chemical components in phytopharmaceutical active ingredients are unknown and many are in low quantities. This makes it very difficult to obtain reliable chromatographic fingerprints representing pharmacologically active components and chemical characteristics. Chromatography (TLC, HPLC, and GC), which has a powerful separation ability, is an appropriate method to meet chemical fingerprinting desires. Furthermore, the application of hyphenated chromatography and spectrometry such as HPLC-DAD, GC-MS, CE-DAD, HPLC-MS, and HPLC-NMR, can provide additional spectral information, which is very helpful in determining the chemical structure of detected compounds. Chemical fingerprint combined with a chemometric approach will provide more accurate analysis results [65].
Several factors will influence in establishing good chromatographic fingerprints. These factors include sample preparation, instrument selection, measurement conditions, and analytical methods validation. These factors must bring up all compounds in the fingerprint profile so that they can represent the integrity of phytopharmaceuticals. In sample preparation, the critical point is the method of sample extraction. The selection of instruments must pay attention to the sensitivity, selectivity, ability of the detector, and the measurement conditions. The last thing to do is to validate the method [65].
Fingerprint analysis begins with the matrix construction of peak area data—retention time of the selected peak, known or unknown identity of the compound. The matrix is then analyzed to determine the similarity or difference compared to the standard fingerprint. The most important thing in this process is peak detection (integration) and peak selection, which becomes very difficult because of complex samples containing more than 100 peaks. The disadvantage of peak selection is the amount of peak data that will be rejected. For this reason, the most recent chemometric analysis uses all data points collected in the study. Next, chromatographic fingerprints are analyzed through comparisons to determine the similarity or dissimilarity of the sample with the reference standard, presented as a correlation coefficient or congruence coefficient. The fingerprint reference standard is derived from the standardized extract of phytopharmaceuticals, which has become a product prototype. Currently, chemometric analysis methods using pattern recognition have been highly developed with various methods such as K-nearest neighbors (KNN), soft independent modeling of class analogy (SIMCA), etc. [65].
1.4 Conclusion
The use of chemical fingerprints for quality control purposes only determines the similarities and/or differences. This objective can be applied from raw material authentication, in-process manufacturing control, and end process control of finished products. But it has not yet considered the complex relationship between chromatographic fingerprints and the efficacy of phytopharmaceuticals. The efficacy of phytopharmaceuticals has the characteristics of a complex mixture of chemical compounds found in herbs. For that reason, the evaluation method of their relationship is not a trivial task. It is not easy to find a suitable method for quality control of phytopharmaceutical. The variability of medicinal plants’ chemical content as the raw materials of phytopharmaceuticals is a challenge that must be conquered with the developments of chemical and chemometric analysis methods.
Acknowledgment