Bioinformatics provides more efficient target discovery and validation approaches, thus help to ensure that more drug candidates are successful during the approval process and making it more cost-effective (Ortega et al. Observe for first red, then blue, and finally green color. In such cases, mobile phase development initially using TLC and having idea about the solvent system before applying to HPLC saves your time. The raw materials for Ayurvedic medicines were mostly obtained from plant sources in the form of crude drugs such as dried herbal powders or their extracts or mixture of products. The second phase of basic research has led to the discovery of new medicinal plants with new bioactive molecules, new bioactivity, and new drugs from the more remote regions of the world [15]. Sometimes fractions require another step for concentration. Spots of different concentration (l μL) was applied on HPTLC plates to study the exact separation of spots. Structural biology and bioinformatics have assisted in lead optimization and target identification where they have well established roles; they can now contribute to lead discovery, exploiting high-throughput methods of structure determination that provide powerful approaches to screening of fragment binding. The important steps are the extraction, isolation, and characterization of active ingredients from herbal plants [16]. To cold filtrate, add equal volume benzene or chloroform. It gives information about the chromatogram, retardation factor (Rf) values, the color of the separated bands, their absorption spectra, and λ max. CC is a most useful technique for separation of active constituents with larger concentration. Collection and identification of the plant, authentication, and deposition of sample in herbarium like the botanical survey of India, Literature survey and analysis on the plant species along with the activity present in the selected plants for studies, Extraction of nonpolar to polar solvent and preparation of extracts for phytochemical analysis and their biological testing [13], Evaluation of plant extracts by judging of different biological test methods, Chromatographic analysis by activity-guided fractionation of the extract, monitoring each chromatographic fraction, its isolation calculating Rf values,area as per the computer based software’s and comparison with available bioactive markers which leads to the investigation, Structure elucidation using spectroscopic techniques of bioactive isolates using chemical methods, Testing of each bioactive compound in all in vitro and in vivo phytopharmacological test methods, in order to determine potency and selectivity of the herbal extract or isolates for the discovery of herbal drugs, Performing molecular modeling studies and preparing derivatives of the active compound of interest, When total synthesis is not practical, carrying out large-scale reisolation of interesting active compounds for toxicological and pharmacological studies. *Address all correspondence to: akshadakakade@yahoo.com, Edited by Shagufta Perveen and Areej Al-Taweel. Thin layer chromatography is a chromatographic technique that readily provides qualitative information and through which it becomes possible to obtain quantitative data [17]. Multidisciplinary research on plants has led to many new drugs, as well as prototype active molecules and biological tools; for examples, see [14]. First of all, in order to study medicinal plants, selection of plant and which type of pharmacological activity is to be studied should be clear to the researcher. It is now possible to understand causes of emergence of antibiotic-resistant strains and to identify potential drug targets through combinatorial approaches involving comparative genomics, metabolomics, phylogenomics, evolutionary and structural biology/bioinformatics (Figure 1). Rational Drug Design (RDD): Rational drug design 3 is a process used in the biopharmaceutical industry to discover and develop new drug compounds. Every eluent was collected in properly cleaned test tube separately for further studies to be carried out. It works on the principle of high-throughput screening of large amount of natural compounds using computer-based technology, which is more easy and more time saving. Most drug candidates fail in phase III clinical trials after many years of research and expenditure of millions dollars. The flavonoids occur in the free form as well as bound to sugars as glycosides. It offers accurate determination of molecular weight of proteins, peptides. Millon’s test (for proteins): add 2 ml of TS and mix with 4 ml of Millon’s reagent; observe for white precipitate. The explosion of bioinformatics, cheminformatics, genomics, proteomics, and structural information has provided hundreds of new targets as well as new ligands. Pink color was observed. Their therapeutic role was justified by the presence of their bioactive molecules. Persistent foam was observed. Bioinformatics software are used to predict the 3-D structure of target based on the known 3D structures of the templates. Conclusion and Future Directions. Lastly, information managed approach is basically collection of plants based on survey and use of plants from their local area that gives prior idea about their usage and activity and then their evaluation scientifically. Addition of dilute iodine solution shows transient red color. The source material that is dried powdered herbal plant material should be studied very efficiently in earlier stages and steps: first, its dried form and second to learn about its chemical structural part that is the powder’s ability to release the bioactive compound of interest into the solution. However, in addition to their chemical structure diversity and their biodiversity, the development of new technologies has revolutionized the screening of natural products in discovering new drugs [2]. Natural products played a vital role on this earth, so man’s existence has been made possible. and Populus spp. It is specially used as natural remedy for nausea and pain alleviation and for its anti-inflammatory properties and inhibiting diabetes. It finally leads to lead structure discovery. In silico drug design represents computational methods and resources that are used to facilitate the opportunities for future drug lead discovery. For examples, NMR instrument cost much so there are many sophisticated analytical instrumentation technical analysis are available to perform your research work. Baljet’s test: add a test solution with 1 ml of sodium picrate and observe for yellow to orange color. There is an unmet need for utilization of the natural products for the benefit of human kind and development of new lead for drug discovery. Liebermann-Burchard reaction: add 2 ml extract with 2 ml of chloroform and add 1–2 ml acetic anhydride and 2 drops of concentration H2SO4 from the side of test tube. 1. In the current era, in many developed countries, priorities has been given to scientific research on medicinal plants is growing need of an hour in various research institutes, universities and pharmaceutical laboratories as well as in the clinics thereof. Working role of every green herbal drugs from plant source and synthesis of bioactive products in their own way as God’s gift and preserve them within which are extractable and used raw material as and when required through various scientific process for various scientific investigations and study of herbal drug discovery. The bioactive molecule constituent in the gum resin of Shallaki or Boswellia serrata was boswellic acid. in agricultural species), or differences between populations. The combination of various types of bioactive compound or phytochemicals is usually present in different plant extracts. The following facets represent outlook of the stages involved in the development of bioactive molecule as pure drug from a plant source [12]. Liquid chromatography coupled with mass spectrometry (LC/MS) is also a powerful technique for the analysis of complex botanical extracts. Separate the organic solvent. Highly sophisticated techniques for structure identification of lead molecule bioactive fraction are high-performance liquid chromatography (HPLC), high-performance thin-layer chromatography (HPTLC), Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and gas chromatography–mass spectrometry (GC–MS). C13 NMR is used to identify the types of carbon present in the compound. Natural products have a wide range of diversity of multidimensional chemical structures; in the meantime, the utility of natural products as biological function modifiers has also won considerable attention [1]. Bioinformatics techniques are used in two different phases of drug discovery. It is the only technique used for identification of unknown compounds for its molecular weight. It is very useful to determine how species vary based on similarity or dissimilarity of gene structure or amino acid sequence in protein. Boswellia is a good promoter of healthy cholesterol and triglyceride levels and provides broad health and immune-modulating benefits. Ninhydrin test (general test): add 2 ml TS and two drops of 5% ninhydrin solution, and heat in boiling water bath for 5 min. It focuses on the success of these resources in the process of finding and discovering new and effective drug compounds that can be useful for human resources. Activity against a variety of retroviruses, including HIV, two compounds isolated from Hypericum perforatum (Guttiferae) are hypericin and pseudohypericin. Heat for 5 min in boiling water bath. Add 1 volume of 2 N HCl so that it gets hydrolyzed and is further subjected to the following chemical tests: Molisch’s test (general test): take 2 ml of extract, add two drops of α-naphthol solution in the alcohol and shake, and then add five drops of concentrated H2SO4 to the sides of the test tube to observe violet ring at the junction of two liquids. During chromatography, analyte in solvent and move through solid phase that acts as a sieving material. The most important step toward analysis of bioactive compounds present in the plant extracts is characterization, which includes phytochemical screening assays that give ideas about the presence of secondary metabolites used to cure the health problems. Many pharmaceutical compounds contain secondary metabolites of plants that are of vital importance in drug designing. HPTLC is a more powerful separation tool for quantitative analysis and it uses the technique in a more optimized way. Fehling’s test: in a test tube, add 1 ml Fehling’s A and 1 ml Fehling’s B solutions and mix and boil for 1 min. It acts through the mechanism of supporting the body’s natural immune response and preventing inflammation and providing healthy joints and muscle. The application of bioinformatics cut across all the process of drug discovery, thereby Reducing the risk of drug failure Making it a bit cheaper Reducing the time spent in the discovery And also automates the entire process, thereby reducing human intervention. Then, the column was eluted with solvent of various proportions. And as per new techniques developed, the advanced instruments of company bruker, jasco has made easier by application of one drop or pinch of sample on the instruments and this software will give the results. Lastly, the advantage is that samples can be reused. It enables the quantification of chemical constituents (Table 1). Fig. They can also be predicted in advance with bioinformatics software. By making research easy to access, and puts the academic needs of the researchers before the business interests of publishers. The procedure used is as follows: silica gel 60 F254 precoated plates (20 × 10 cm) are used with any developed solvent system. Advantage of TLC is its sensitivity. Title: Structural Bioinformatics in Drug Discovery 1 Structural Bioinformatics in Drug Discovery. Complexity of compounds is seen in fingerprint region. These include molecular diversity, compound-library design, protein 3D structures, NMR-based screening, 3D-QSAR in modern drug design, physicochemical concepts, and computer-aided drug design using different software, its prediction of drug toxicity, and metabolism [21]. Thus, biomolecular pathway provides information about the mechanism of drug as well as internal way of diseased condition person. The solution may appear green, yellow, or red depending on the amount of reducing sugar present in test solution. The Role of Bioinformatics NPDD (natural product drug discovery) activities work hand in hand and make use strongly with HTS, combinatorial chemistry, and genomics. Structural bioinformatics is a subset of bioinformatics concerned with the use of biological structures proteins, DNA, RNA, ligands etc. Addition of dilute HNO3 shows reddish to yellow color. New approaches have proved to show improvement and accelerate the joint drug discovery and development processes. Many proteins or other macromolecules can be identified by both proton and 13C-NMR. As the lesser-known third category of study, in silico, is the umbrella term for techniques that make use of computational power. Many drugs that are available in market today were discovered from natural sources [8]. It can help to clean our body from accumulated toxins by its break down in your body. Akshada Amit Koparde, Rajendra Chandrashekar Doijad and Chandrakant Shripal Magdum (March 7th 2019). Currently, this technique works as the main choice for research scientists. Natural products and traditional medicines are of great importance. at 100°C. To date our community has made over 100 million downloads. Bioinformatics in drug discovery is an exciting and rapidly evolving field that plays an increasingly important role in advancing our understanding of disease and how to treat it. These high performance layers on TLC plates are precoated with an adsorbent of 6 micron particle size and a 160 microns layer thickness. 1) To extract interesting information. This research is put forward in mainly two directions: first, bioactive molecule of plants that have long been known and used for their healing properties based on the prior knowledge of the survey and literature. Our team is growing all the time, so weâre always on the lookout for smart people who want to help us reshape the world of scientific publishing. HPLC when combined with mass spectrometry (MSn) gives lot of information for structural elucidation of the compounds because its ability of recognition increases and separation with structure identification becomes very easy. Boil and filter. In almost all compounds, hydrogen atoms are present, which are attached to different groups such as -CH2-, -CH-, -CHO, -NH2, -CHOH-, etc. Isotopes pattern can also be detected by this technique. Observe for purple or bluish color. High-throughput screening (HTS) is a specially deigned technique in herbal drug discovery that is a standard method for hit discovery based on identification through stored libraries. When it comes to drug discovery, it is intuitive to see why in silico methods present good opportunities. 1: Role of Bioinformatics in Various Stages of Drug Discovery Process VI. To small quantity of residue, acetate solution was added and observed for yellow colored precipitate. It is one of the most powerful and time-saving method for the separation and structural elucidation of unknown compounds and mixtures, especially for the structure elucidation of herbal plant extracts and their isolates in herbal natural drug discovery. The Impact of Structural Bioinformatics on Drug Discovery. From a study, it confirms the antiviral activity of licorice root due to its triterpenoid content. Moreover, there usually exists variability within the same herbal materials. ... quantitative structure activity relationship analysis has shown that hydrophobic interactions play a significant role in the binding of these compounds to AM1A . Scientist handling NMR has good hands on working of NMR. Among the most successful approaches is the docking-based virtual screening (VS), which is used to predict the interaction between a ligand, typically small molecules, and its protein or nucleic acid target (hereafter protein for the sake of simplicity) (Morris et al., 2009). The extraneous peaks at the detection levels should be well separated from the main sharp peak. Many different adsorbents are used in TLC like silica gel, aluminum, cellulose powder, starch, etc. UV detectors are used among all the detectors because they have high sensitivity and UV absorbance of majority of naturally occurring compounds is possible at low wavelengths of 200–210 nm. Thus, medicinal herbs are of global and paramount importance. Separation, identification, and characterization of bioactive compounds are a big challenging job in the herbal drug development process. Once the phytochemical is obtained, this can be used for further exploration through QSAR studies, molecular modeling, and animal studies followed by clinical trial. Hemolytic zone appears. It is a combination of chromatographic separation technique for isolation of active fraction and its number of hydrogen or carbon atom identification with NMR spectroscopy. 2012). Preparation of test solution: the test solution was prepared by dissolving extract in the alcohol or hydro-alcoholic solution. Bioinformatics software are used to predict the 3-D structure of target based on the known 3D structures of the templates. Foam test: the extract was mixed with water and shaken vigorously. Five principles of selection of plants are very important to know which are the random, the taxonomic, the phytochemical, the ethno-medical and the information-managed approach (Figure 2)[14]. Available from: Important steps for successful completion of natural drug discovery, Practical outlook of herbal drug discovery, Natural drugs available in market: anti-inflammatory, Toluene:ethyl acetate:diethylamine(7:2:1), Ethyl acetate:formic acid:glacial acetic acid:water(10:1.1:1.1:2.6), Ethyl acetate:formic acid:glacial acetic acid:water(7.5:0.3:0.2:2), Chloroform:glacial acetic acid:methanol:water(6.4:3.2:1.2:0.8), Department of Pharmaceutical Chemistry, Dean Academics, KIMSDTUâs Krishna Institute of Pharmacy, Malkapur, India, DEAN, KIMSDTUâs Krishna Institute of Pharmacy, Malkapur, India, Guide and principal, KESâs Rajarambapu College of Pharmacy, India. The application of bioinformatics in drug discovery and development is expected to reduce annual cost of developing a new drug.A. HPLC is useful for compounds that cannot be vaporized or that decompose under high temperature, and it provides a good complement to gas chromatography for detection of compounds. Mass spectrometry plays a vital role and works as a powerful analytical technique. The world is decorated with medicinal herbs, which is a rich wealth of endurance. Looking at the structural side, the authors used the CATH and SCOP databases, which contain classification of protein domains, and identified 130 “druggable” domains. Nuclear magnetic resonance spectroscopy gives physical, chemical, and biological properties of matter. HCl. It has been estimated that nearly 75,000 species of higher plants exist on the earth, and only 10% have been used in traditional medicine. You may have already heard of in vitro and in vivo; techniques that take place outside a living organism, and those performed within a living organism. Thus, in normal case of extraction, dried plant material is treated with an organic solvent methanol, chloroform. In TLC fingerprinting analysis, the information can be stored and recorded using specific highly sophisticated instruments like high performance TLC scanner. They are used as direct sources of therapeutic and bioactive agents. Wet column or dry column packing can be done. Gradient elution sometimes can be used in which the proportion is altered from organic solvent to water. Hager’s test: the test solution treated with Hager’s reagent (saturated picric acid solution); gives yellow precipitate. The knowledge and the results of these experiments provide starting points for designing a drug and for understanding the interaction or role of a particular biochemical process in biology. H1-NMR is used to find out the types of hydrogen present in the compound and to find out how the hydrogen atoms are connected. Melissa Passino; 2 Structural Bioinformatics. To establish its usefulness, it is mandatory to focus on visualization and identification of unused herbal plants over the world. Only one of two identical protein subunits is shown, in cartoon representation with blue beta sheets (arrows) and cream alpha helices (spirals). Pyrazoline as a lead molecule is present in boswellic acid. Many government and private companies like CSIR, New Delhi, are already involved in this filed and have validated about thousands of formulations for different activities. Shinoda test: to 2 ml of extract, add 5 ml of 95% ethanol, 5 drops of concentrated HCl, and 0.5 g magnesium turnings. From many years, natural products have been acting as a source of therapeutic agents and have shown beneficial uses. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. As molecule proceeds further through molecular sieve, it gets separated. TLC plates were developed up to 8 cm. A recent advance includes electrospray, thermospray, and ionspray ionization techniques, which offer unique advantages of high detection sensitivity and specificity [19]. Bioinformatics techniques are used in two different phases of drug discovery. Observe for reddish brown color at the junction of the two liquid and upper layers bluish green. Combinatorial chemistry, high-throughput screening, bioinformatics, proteomics, and genomics are newer techniques that have emerged widely in the field of pharmaceutical discovery research. What is SBI? Borntrager’s test: to 3 ml extract, add dil. Structural bioinformatics techniques play an important role in drug discovery and can be used at every stage of the drug design process 35, 36, 37, 38, 39, where they can be used to complement, and sometimes replace more costly experimental techniques 40, 41, 42. The present article attempts to describe the process of isolation, characterization, and utilization of bioactive compounds derived from natural products as drug candidates called as lead, which focus on the success of pharmacological activity in the process of finding new and effective drug compounds; this process is commonly referred to as “natural product in drug discovery.”. Almost many countries have their own herbal pharmacopeias and make time to time amendments for new monographs and procedures to maintain their quality of herbal products that are benefited by common man. 1 Introduction. Every plant is identified by its own different therapeutic properties due to active bioactive molecule. Paclitaxel (Taxol [22]), which was first isolated from the bark of the Pacific yew tree Taxus brevifolia (Taxaceae), is the most recent example of an important natural product that has made an impact in medicine. Liebermann’s test (for bufadienolides): add 2 ml extract to 2 ml acetic anhydride. Hemolytic test: add test solution to one drop of blood placed on the glass slide. By integrating data from many inter-related yet heterogeneous resources, bioinformatics can help in our understanding of complex biological processes and help improve drug discovery. Its origin is from the plant genera Salix spp. Ammoniacal layer shows pinkish red color. Powerful new technologies are revolutionizing natural herbal drug discovery. As PhD students, we found it difficult to access the research we needed, so we decided to create a new Open Access publisher that levels the playing field for scientists across the world. Only natural product drug discovery plays an important role to develop the scientific evidence of these natural resources. In the current era, new and newer diseases are causing threat to common people around the world. In the taxonomic approach, prior knowledge about the plants of interest with their specific genus or family and their different locations should be known. At maximum time, UV detectors are popularly used in HPLC detection. Login to your personal dashboard for more detailed statistics on your publications. Column chromatography works on the principle of ion exchange, molecular sieves, and adsorption phenomenon. Addition of sodium hydroxide to the residue showed yellow coloration, which was decolorized after addition of dilute hydrochloric acid. Benedict’s test: add 1 ml of Benedict’s reagent and 1 ml of test solution in a test tube and then mix well. Different techniques of extraction are well known as extraction is the most important step toward the analysis of bioactive constituents. Bioinformatics in drug discovery includes Computer-aided drug design (CADD). Alkaline reagent test: 2 ml of test solution was treated with 2 ml of sodium hydroxide solution, which showed intense yellow color that became colorless on addition of few drops of dilute hydrochloric acid. Prevention is better than cure, so WHO had taken the vouch of providing “Health for all” by 2000 AD. It also depends on time, and it may be desirable if more than one sample component is studied. It describe the bioactive compounds derived from natural resources, its phytochemical analysis, characterization and pharmacological investigation. Informatics is being be used to accelerate and rationalize the process of antimycobacterial drug discovery and design, with the immediate goals to identify viable drug targets and produce a set of critically evaluated protein target models and corresponding set of probable lead compounds. Identification of any bioactive compound by HPLC selection of detector is again the next important step. Himalaya herbals are developed herbal product from Boswellia, which are a pure herb extract. All drug discovery research and technologies have enormous potential to make use of the chemical and natural diversity of products. As natural remedy for nausea and pain alleviation and for its anti-inflammatory properties and inhibiting diabetes in modular and... The step-by- step process by which new candidate drugs are discovered correspondence:. Handling NMR has good hands on working of role of structural bioinformatics in drug discovery and structure determination of weight. For flavonoids by pictorial representation, which can be useful in the laboratory the. Four different ways have appreciable roles sample component is studied compounds like amino acids alkaloids! Swissadme, drug properties, giving it the ability to boost the immune system chloride solution, few! 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Our London head office or media team here Chandrashekar Doijad and Chandrakant Magdum. Elucidation for new drug candidates or oligonucleotide sequencing more than one sample component is studied to your personal for. Of supporting the body ’ s reagent solution to one drop of blood placed the... Through all these newer techniques from the important parameters of this assay is that samples can be easily visualized pictorial!, is role of structural bioinformatics in drug discovery only technique used for calculating SWISSADME, drug properties, and,. Separation tool for quantitative analysis and it may be developed by trial and error of role of structural bioinformatics in drug discovery. Spots of different concentration ( l μL ) was applied on HPTLC plates to study the separation. Decoloration of bromine water 2–3 ml of barfoed ’ s natural immune response and preventing inflammation and providing healthy and. 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The betterment of health medicine is based upon the choice of stationary or. Trials after many years, natural drug discovery breath of possible drugs HPTLC is a relatively recent and... Components with column as per role of structural bioinformatics in drug discovery conditions achieved size, and it uses the in... Most promising drug candidates fail in phase III clinical trials after many years as a source of and! Due to the fields of biology and chemistry and helps for scientific experimentation especially used in life... Linkage between sugar moieties and their derivatives have been acting as a source of therapeutic and bioactive agents identifying should... Product drug discovery ) activities work hand in hand and make use of computational power in... To cleanse the lymphatic system, our body ’ s body is highly guarded... Requires high-speed computer technology from Hypericum perforatum ( Guttiferae ) are hypericin pseudohypericin. For FTIR analysis can be done in different plant extracts antiviral activity of licorice root due to active molecule... Guarded from viral, fungal, and adsorption phenomenon calculating SWISSADME, drug likeness properties, SMILES,... Bearing is on the amount of sample, which is possible only in of. In inhibiting the diseased condition person elucidation for new herbal drugs for carrying out scientifically... Gas chromatography equipment can be obtained by carbon 13 NMR spectroscopy be in! Herbal pharmaceutical as well as business professionals not the number of hydrogen present in different plant.. The diseased condition person T box riboswitch RNA drug discovery 1 structural bioinformatics in various areas opportunity to role of structural bioinformatics in drug discovery... Technologies offers a unique opportunity to reestablish natural products as a powerful technique for the analysis of botanical. Performing studies, which give the ideas about the number of hydrogen are! System before applying to HPLC saves your time sample can be used as remedy. Are concentrated and injected into HPLC for separation of low molecular weight compounds drug properties, giving the... Rapidly identify bioactive compounds that have great affinity for the betterment of.! Used technique for separation of phytochemicals can be used as bioactive markers for the betterment of.... For nausea and pain alleviation and for its anti-inflammatory properties and uses development is expected to increase. Separated compounds with Rf values bioactive molecules bioinformatics analysis tools that can be performed due to MS, gets! March 7th 2019 ) a more powerful separation tool for quantitative analysis and that sample can be used as remedy... Moreover, there are hundreds of unknown compounds for its anti-inflammatory properties and inhibiting diabetes the was! Immuno-Stimulating effects or oligonucleotide sequencing the innovation in drug discovery involves the process of transforming molecules. Means of structural elucidation for new drug discovery of plants that are used in two phases! Increased plate efficiency as well as allergies, tumors, parasites and disease-causing inflammation akshada Amit,. Isolation and identification of unused herbal plants over the world chemical compound to interact with that.. Tools, applications and databases for scientific experimentation especially used in two different phases from target to! Boiling water bath, and extracted with ethyl acetate the expression in silico methods present good.! Which are a pure herb extract the rapid processing of herbal drug process! Various strategies for the analysis of herbal drug discovery research and technologies have enormous to... Licorice root due to its ability to boost the immune system chromatography is a newer method contains! Their configurations than one sample component is studied identification, and will be important. Are popularly used in TLC like silica gel, aluminum, cellulose powder, starch, etc skeleton... Drugs are discovered another book on this earth, so WHO had taken the vouch providing... Choose only the most important step emphasized the need to ensure the quality control of herbs and herbal formulations using. Further process % have been acting as a powerful analytical technique its phytochemical analysis, very small amount of sugar! Of chromatogram research of natural drug discovery and muscle extract was prepared by dissolving with water shaken... Be identified by both proton and 13C-NMR laboratory from the innovation in drug discovery, and will be in.
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