Technique Used in Detection of Herbal Drugs

Hemi Gayakwad^*
*Correspondence: Hemi Gayakwad, Department of Forensic Science and Technology, Rajendra Institute of Medical Sciences, Ranchi, Jharkhand, India, Tel: 7667787217, Email:

Keywords

Determination techniques • Quality assessment • Caffeine • Herbal drugs • Chemical fingerprinting

Introduction

Plant based medications and cosmetics, as well as over the counter health food products and prescription drugs, are becoming more popular in non-allopathic healthcare systems. While plant derived chemical mixtures are widely used in healthcare, their effectiveness is limited by their low oral absorption in both developed and developing nations. According to WHO estimates, almost 80% of the world's population still relies on herbs and other traditional medicines to meet their basic health care needs. As a therapy for diabetes, arthritics, and liver disease, they have been widely accepted by the medical community. The World Health Organization classifies all forms of herbal medicine, including raw plant material, processed plant material, and therapeutic herb products. If a final designated herbal medicine comprises active components such as aerial or subterranean sections of plants or other plant material or combination thereof, it is an herbal medication. Herbal medicines. People throughout the globe are increasingly turning to herbal medicines as a result of this widespread trend. There are a variety of herbal medicine items on the market, including herbal tea, herbal supplements in tablet and pill form as well as fresh and dried plant extracts. Traditionally, herbals have been seen as safe, and more and more individuals are taking them without a prescription. But some may create health issues, be ineffective, or interfere with other medications. Drugs may only be evaluated on the basis of active ingredient concentration if herbal formulations are standardized.Industry and other organizations working with Ayurvedic, and herbal goods have a basic need for quality assessment of herbal preparations [1].

Traditional knowledge and current scientific procedures must be properly integrated in order to produce herbal medications that are safe and effective, according to herbal drug technology.

In a safe method of reverse pharmacology based on traditional knowledge database, herbal medications may be generated that are scientifically confirmed and technologically standardized. In drug discovery, development, and therapy, this might be critical [2].

Botanical materials may be tested for quality and conformance to pharmacopoeias and regulatory standards using micro and macro analyses. There are two methods for determining the presence of small amounts of contaminants: Thin Layer Chromatography (TLC) and High Performance Thin Layer Chromatography (HPTLC). Quality control and standardization commonly employ a wide range of analytical methods such as volumetric analysis, gravimetric measurements, gas chromatography, column chromatography, HPLC, and other advanced techniques [3].

As a result, every herb must be thoroughly inspected to ensure that it meets quality standards and consistently delivers on its promises. Product quality, effectiveness, performance, and safety may all be assured by standardization.

Materials and Methods

Analytical evaluation technique in herbal drugs

Three key definitions from the pharmacopoeia serve as a foundation for quality control: Are you sure this plant belongs to you?

Is there anything else in there that shouldn't be there, such other herbs? Assay: Is the active components content within the prescribed limits of assay [4].

Since the active ingredients in the majority of herbal medications are unknown, evaluating the content is obviously the most challenging task. Chemically defined components may be utilized as markers, regardless of any therapeutic action, for control reasons. Qualities such as the kind of preparation, sensory qualities, physical properties, adulteration, pollutants, moisture, ash content, and solvent residues have to be tested to confirm the product's identification and purity. Quality control for herbal medicines relies on the identification of the raw herbal material or the botanical quality [5].

Macro and microscopy may be used to determine a person's identity. Voucher specimens are trustworthy sources of information. When diseases spread among plants, the look of the plant might alter, making it difficult to accurately identify the affected plants.

With regards to the safe use of pharmaceuticals, purity deals with aspects like ash values, contaminants (such other plants), and heavy metals. Modern purity assessment, on the other hand, takes into account microbiological contamination, aflatoxins, radioactivity, and pesticide residues thanks to enhanced analytical technologies. The consistent composition of herbal remedies may be established using analytical procedures such as photometric analysis (UV, IR, MS and NMR), Thin Layer Hromatography (TLC), High Performance Liquid Chromatography (HPLC) and Gas Chromatography (GC).

Chromatography and chemical fingerprints of herbal medicines

An example of chromatography is the separation and identification of components, compounds, and mixtures by utilizing stationary and mobile phases, respectively.

It is only possible to make high quality herbal medicines if rigorous guidelines are followed. Other techniques that may aid in precise botanical identification include photochemical screening and standardization. Many steps must be taken to ensure the quality and uniformity of herbal medications. Proper agricultural techniques, quality raw materials, and manufacturing processes are all critical components in herbal medicine quality control [6].

Depending on the stage of harvest and the parts of the plants gathered, herbal products may include a broad variety of chemical components. As a result, the majority of phytochemical ingredients of herbal products must be determined so that pharmacological research can be validated, the bioactivities of active compounds can be understood, and product quality control can be improved. You may use HPLC and GC, as well as capillary electrophoresis, to assess the product's quality. This is achievable thanks to chromatographic procedures like Capillary Electrophoresis (CE). Phyto equivalency is a term used by German scientists to describe herbal products. It is important to compare the chemical fingerprint of a herbal product to a clinically proven reference product.

Chromatographic fingerprint of herbal medicine is an extract of common chemical components of pharmacological action and/or chemical properties. To appropriately represent the herbal medication's chemical makeup, this chromatographic profile should contain the terms "integrity" and "fuzziness." Confidentiality can be maintained even when different samples of the drug have different amounts and/or concentration levels of the compound's characteristic constituents; chromatographic fingerprints can demonstrate both "sameness" and differences between various samples, thus allowing for the authentication and identification of herbal medicines (hence the "fuzziness"). The quality of herbal medicinal extracts should thus be evaluated by looking at various constituents in the herbal drug extracts, rather than just one or two marker components. Any herbal medication or extract, on the other hand, has hundreds of unidentified constituents, many of which are present in minute amounts. Furthermore, there is frequently a wide range of variation in the same botanical ingredients. 'That's why developing dependable, chemically distinctive fingerprints for pharmaceutically active compounds isn't a simple task. Fortunately, chromatography has a great capacity to separate complicated chemical components in herbal extracts into several simple sub-fractions [7].

Thin layer chromatogram

For herbal analysis prior to the development of instrumental chromatography techniques like GC and HPLC, TLC was the most prevalent, adaptable method of choice While other pharmacopoeias, such as the American Herbal Pharmacopoeia (AHP), British Pharmacopoeia (BP), United States Pharmacopoeia (USP), European Pharmacopoeia (UP), Chinese drug monographs and analyses, and the Pharmacopoeia of the People's Republic of China, etc., no longer use TLC for their herbal medicine analysis, it is still widely used today. We utilize it in conjunction with other chromatographic procedures as a simpler kind of first screening with a semi-quantitative evaluation since it changes less than instrumental chromatography. A solute is distributed between two phases in thinlayer chromatography: a stationary phase acting through sorption and a liquid mobile phase. Applied to a glass, plastic, or metal sheet or plate is a thin, homogeneous coating of dried finely powdered substance. Plates made out of glass are the most frequent. Depending on the kind of support, its preparation, and the solvents used, partitioning or adsorption may also be used to produce separation [8].

It is possible to make identification by comparing the Rf values and relative sizes of spots obtained from a chromatographic run of an unknown and a reference sample on the same plate. Semiquantitative estimates are often made by comparing the spots' sizes and intensities visually.

The HPTLC technique is one of the most advanced TLC based analytical methods. It is the most adaptable, dependable, and budget-friendly method of separation. As a strong analytical instrument for chromatographic information of complicated mixtures of pharmaceuticals, natural products and clinical samples, it has the advantages of automated scanning, comprehensive optimization and so on. Using TLC/HPTLC to create herbal medicine fingerprints has several benefits, including its ease of use and adaptability, speed, high sensitivity, and ease of sample preparation. A simple way of testing herbal goods for quality and probable adulteration is TLC. New TLC methods including Forced Flow Planar Chromatography (FFPC), Rotation Planar Chromatography (RPC), Over Pressured Layer Chromatography (OPLC), and Electro Planar Chromatography (EPLC) are also being updated (EPC). In a simple but efficient preparative forced flow method, hydrostatic pressure is used to boost mobile phase velocity. Parallel and serially coupled layers may be used to evaluate a large number of samples (up to 216) simultaneously, enabling for high throughput screening and the study of complex matrices [9].

Gas chromatography and volatile components in herbal medicines and GC–MS and herbal medicines

Many of the active fixings in herbal remedies are volatile chemical molecules, which is well-known. A gas chromatography examination of volatile chemicals in herbal medicines therefore plays a significant role. Numerous benefits accrue to GC testing of volatile oils. To begin, GC analysis of the volatile oil provides a reliable "fingerprint" that may be used to locate the source of the substance. Each plant's volatile oil has a distinct composition and concentration ratio of organic components, and contaminants in the volatile oil may easily be identified. It is very easy to extract and standardize the volatile oil, and the components may be identified via GC-MS analysis. It is possible to track or evaluate specific properties of herbal remedies based on the relative amounts of their constituents. Signs of oxidation, enzymatic alteration, or microbiological fermentation may be found in changes to the volatile oil's chemical makeup [10].

The exceptional sensitivity of GC's detection for practically all volatile chemical substances is certainly a benefit. Particularly for FID detection and GC-MS, this is true. It is also possible to separate a large amount of volatile molecules at once due to capillary columns' great selectivity. Since then, GC has been a popular and helpful analytical method in the study of herbal medicines. It is also possible to get trustworthy information on the compounds' identities using the hyphenated GC-MS apparatus. However, the most important drawback of GC is its inability to analyze tests of polar and nonvolatile chemicals. To do this, time consuming sample work-up, including possible derivatization, is required. Our full examination of herbal remedies now requires the use of the liquid chromatography as well.

High-performance liquid chromatography and HPLC–DAD, HPLC–MS and others

In HPLC, or high pressure liquid chromatography, the stationary phase is tiny particle (3 nm-50 nm) packing housed in a column with a small diameter (2 nm-5 mm), one end of which is coupled to a source of pressured eluent, and the eluent is injected into the column by a nozzle (mobile phase). Ion exchange, partition, and adsorption are the three most used methods of high performance liquid chromatography. For the examination of herbal remedies, HPLC is a preferred approach due to its ease of use and the fact that it is not restricted by the volatile or stable nature of the sample substance. HPLC may be used to examine almost any herbal medicine ingredient. As a result, HPLC has been the most extensively utilized technique for the testing of herbal remedies for the past few decades.One of the most widely used analytical separation procedures for medicinal herbs is Reversed Phase (RP) columns.

The pH adjustment, pump pressures, and mobile phase compositions are only a few of the factors that go into creating the ideal HPLC separation conditions. Because of this, it seems that a sound experimental technique is needed in order to determine the proper separation.

In the subject of liquid chromatography research, certain novel approaches have recently been created in order to achieve greater separation. Counter Current Chromatography (HSCCC), limited Size Exclusion Chromatography (SEC), reversed phase ion pairing HPLC, and strong anion exchange HPLC are all examples of this kind of chromatographic method (SAX-HPLC). They'll make it possible to properly separate herbal medicine extracts for the first time. With the use of a single wavelength UV detector, HPLC's adaptability for the examination of chemical components in herbal medicines seems to be limited since many herbal remedies comprise non-chromophores. This has led to an increase in the use of HPLC analysis with ELSD (evaporative light scattering detection), showing that ELSD is an effective detection method for non-chromophoric compounds. It is feasible to do direct HPLC analysis of several active components in herbal medicines since the ELSD detector's reaction is not reliant on the analytic structure or chromospheres of analytes, and so is the case with UV detectors. Herbal drugs' fingerprints may be generated this way as an example. Furthermore, HPLC-IR, HPLC-MS, and HPLC-NMR need hyphenated HPLC to perform qualitative analysis or structural elucidation of the chemical components in herbal medications.

HPLC-DAD is currently a standard procedure in the vast majority of chemistry labs across the globe. Because of the added UV spectral information, it is now much simpler to do qualitative analyses on the more complicated materials found in herbal medicines. A good example would be to look at the peak purity and compare it to the standard spectrum of a known molecule in the sample under study. The development of electrospray mass spectrometry, which combines liquid chromatography with mass spectrometry, has opened up new avenues for the investigation of herbal remedies. Online qualitative analysis may be conducted using HPLC fingerprints to record the complete herbal extracts with additional information.

For decades, LC-MS and HPLC-DAD have been more often used for the analysis of herbal medications. Plants and herbal treatments may be examined using HPLC, particularly hyphenated HPLC techniques, which have received several good appraisals. Chromatography is used for separation, and DAD and MS are used in conjunction with HPLC-DAD-MS methods to identify the samples. Using DAD and MS, a chromatogram may be studied in real time for each peak. With this hyphenation, LC-DAD-MS has become a strong technique for the quick identification of phytochemicals in botanical extracts and may be utilized to avoid the time-consuming exclusion of all compounds to be identified. Using HPLC/NMR hyphenation for the analysis of herbal medicines in the future may be a useful and interesting analytical approach. According to current trends for chemical compound structure determination, the utilization of UV, Fourier Transformation Infrared Spectrum (FTIR), MS and NMR for chromatography is becoming more prevalent.

Evaluation of chemical fingerprints of herbal medicines

It was typical practice in early chemo metric studies to first convert chromatographic data to a retention time-peak area data matrix, regardless of whether the peaks' identities were known or not. In order to analyze the data, the fingerprints were compared to each other and the principal component analysis was utilized to determine the degree of similarity or dissimilarity (Figure 1).

Nielsen explains, "The quality of the data (even simple retention time-peak area data) hinges on peak detection (integration) and how the peaks are selected for the data analysis." For chromatograms obtained from complex samples with more than 100 peaks, determining the appropriate integration parameters might be difficult. Selection and extraction of data analysis peaks is thus difficult and subjective, and huge amounts of chromatogram data are missed. A subjective peak selection may be avoided when all data points are included in chemometric analysis, which eliminates the need for peak identification and integration. In accordance with Nielsen's recommendation, the full chromatographic profiles were used to conduct direct chemometric analysis. If required, data compression methods like wavelet or Fourier processing might be used to aid in the analysis. The peak shape may be used in data analysis while performing examination of fingerprints, making pre-treatment of overlapping peaks considerably simpler when the full chromatographic profile is used for direct chemometric analysis. Prior to performing fingerprint examination and chemometric analysis, the chromatographic profiles must be appropriately matched to adjust for slight shifts in retention durations.

Results and Discussion

Unlike Western medicine, traditional Chinese medicine is grounded on a completely distinct set of beliefs. As a result, this isn't just a case of using cutting-edge technology to check on the quality of things that was in use for decades. According to this research, the quality control of herbal medications has only begun. For herbal medication quality control, the use of chromatographic fingerprints has been suggested. Only by using chemical fingerprints for quality control can we assess the integrated sameness and/or variation of presently available herbal products and regulate their stability. However, the most critical aspect of herbal medicine quality control (QRFE) has yet to be considered: the complicated relationship between chromatographic fingerprints and efficacy (QRFE). Because the efficiency of traditional herbal treatments is well-known to have a complex combination of chemical substances found in the plants, it is clearly not a straightforward effort to assess appropriately their connection. Natural diversity in herbs and sophisticated chemical compositions make THMs a far more difficult problem to solve. Herbal medicines' effectiveness cannot be determined only by their chemical composition. Here, biochemistry, molecular biology and cell biology play a critical role in developing reliable tests. These biological tests might be connected to chemical fingerprints to ensure their efficiency and uniformity. However, to our knowledge, there is not enough study on this topic to reach the necessary standards. Research into the link between chromatographic fingerprints and herbal medication effectiveness is thus a pressing need for herbal medicine quality control. Studies on probable contaminations of herbal goods, such as pesticides that are too concentrated or prohibited, microbiological contaminants, heavy metals, and toxic chemicals, should also take place in parallel. In actuality, a broad number of disciplines are involved in quality control research for herbal medicines. Herbal medicine quality control and innovative therapy discoveries involving various chemical ingredients need interdisciplinary collaboration across chemistry, pharmacology, medicine and even statistics.

Conclusion

Chemical fingerprints of herbal items may be assessed using a variety of chemometric techniques. Chemical pattern recognition, similarity estimation, and spectral correlation chromatography are all examples of information to since the time of the Saints and Munis, herbal therapy has been utilized to treat a broad variety of ailments. Herbal formulations cannot be successfully manufactured without an in-depth knowledge of the active components. Unlike Western medicine, traditional Chinese medicine is grounded on a completely distinct set of beliefs. As a result, this isn't just a case of using cutting-edge technology to check on the quality of things that was in use for decades. According to this research, the quality control of herbal medications has only begun.

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