Drug Ingredients Research Articles

Development and validation of HPLC/DAD method for simultaneously determination of six prohibited substances in model matrices

The authorities have identified an emerging trend where over-the-counter products, represented as dietary supplements, contain hidden active ingredients that could be harmful. Consumers may unknowingly take products laced with varying quantities of approved prescription drug ingredients, controlled substances, and untested and unstudied pharmaceutically active ingredients. Hidden ingredients are increasingly becoming a problem in products promoted for sexual enhancement, weight loss, or bodybuilding. The tests have revealed the presence of some undesired substances like sildenafil, tadalafil, vardenafil, and their analogues in tainted sexual enhancement products. The content of these substances is usually around the daily curative dose. A simple high-performance liquid chromatography (HPLC) method for simultaneously determination of sildenafil, vardenafil, tadalafil, dapoxetine, yohimbine, and sibutramine was developed and validated. InfinityLab Poroshell 120 EC-C18 (150 '4.6 mm '4 μm particles) was used, as well as a diode-array detector (DAD) at 230 nm, and a gradient flow with 0.030 М ammonium acetate buffer and acetonitrile. The method is linear in the following range: 2.5–37.5 μg/mL for yohimbine, 2.06–30.9 μg/mL for vardenafil, 2.0–30.0 μg/mL for sildenafil, 3.1–46.5 μg/mL for tadalafil, 1.98–29.7 μg/mL for dapoxetine, and 2.2–66.0 μg/mL for sibutramine. The linearity coefficient is R2 = 1 for all substances. Model matrices were spiked, and the analytical recoveries for all substances are in the range 97.5%–99.5%. The method exhibited an upper hand compared with previously reported methods in terms of speed and simplicity. Additionally, the mobile phase (also used as extracting, column washing, and diluting solvent) was composed of only buffer and acetonitrile, which rendered the method much cheaper than others.

Network Pharmacology-Based Strategy for the Investigation of the Anti-Obesity Effects of an Ethanolic Extract of Zanthoxylum bungeanum Maxim.

Network pharmacology is considered as the next paradigm in drug discovery. In an era when obesity has become global epidemic, network pharmacology becomes an ideal tool to discover novel herbal-based therapeutics with effective anti-obesity effects. Zanthoxylum bungeanum Maxim (ZBM) is a medicinal herb. The mature pericarp of ZBM is used for disease treatments and as spice for cooking. Here, we used the network pharmacology approach to investigate whether ZBM possesses anti-obesity effects and reveal the underlying mechanism of action. We first built up drug–ingredient–gene symbol–disease network and protein–protein interaction network of the ZBM-related obesity targets, followed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. The results highlight apoptosis as a promising signaling pathway that mediates the anti-obesity effects of ZBM. Molecular docking also reveals quercetin, a compound in ZBM has the highest degree of connections in the compound-target network and has direct bindings with the apoptotic markers. Furthermore, the apoptotic effects of ZBM are further validated in 3T3-L1 adipocytes and in the high-fat diet–induced obesity mouse model. These findings not only suggest ZBM can be developed as potential anti-obesity therapeutics but also demonstrate the application of network pharmacology for the discovery of herbal-based therapeutics for disease treatments.

Complete Prevention of Contact Electrification by Molecular Engineering

Summary Electrostatic charges are easily generated on surfaces during contact electrification. Although these invisible charges have emerged as a new dimension in mediating the functions of surfaces, such as energy conversion, liquid transport, reactivity, and adsorbability, the accumulation of charges on surfaces can also pose many undesirable consequences. Despite notable progress, existing approaches in engineering antistatic surfaces suffer from limitations such as the need to modify bulk materials or for delicate control of patterning on surfaces that rely on the neutralization of generated charges. Herein, we report a general toolbox for designing antistatic coatings by leveraging on chemically heterogeneous components with electron-donating and electron-accepting functions, i.e., N-(2-aminoethyl)-3-aminopropyltrimethoxysilane and 1H,1H,2H,2H-perfluorooctyl trimethoxysilane, to molecularly engineer the surface potential to achieve an electrostatic homogeneity and completely prevent charge generation. Our approach is general, which allows the facile fabrication of antistatic coatings on various materials, even flexible and curved, with good re-writability and transparency.

Use of Near-Infrared-Mid-Infrared Dual-Wavelength Spectrometry to Obtain Two-Dimensional Difference Spectra of Sesame Oil as Inactive Drug Ingredient.

The present study has investigated the transformation of sesame oil kept at low temperature during a definite period of time for refinement (called winterization) as an inactive drug ingredient by using two-dimensional difference spectra (2D-DS) analysis of spectra collected using a near-infrared (NIR) and mid-infrared (MIR) dual-wavelength spectrometer (NIR-MIR-DWS). The NIR and MIR spectra were measured nearly simultaneously from samples of sesame oil before and after winterization. The difference spectrum analysis of the obtained NIR-MIR data elucidated that, after the winterization process, the absorbances at peaks attributed to C=O, C=C, and OH groups decrease while the absorbances arising from the main chain (CH2) increase. The result indicated the removal of lignan and the fatty acids with relatively short main chains. Moreover, sesame oil unwinterized was cooled from room temperature to near 1 ℃ and subsequently warmed to room temperature. And the cycle was repeated two times. Real-time monitoring during the cooling and warming processes were carried out using the NIR-MIR-DWS. The prediction results obtained from partial least square calibration model for the temperature suggests that there are subtle differences in the oil composition between the first cooling process and after the warming and cooling cycle. For the more detailed analysis, the 2D-DS method is proposed. The results of the analyses using 2D-DS revealed that the starting point of the transformation is around 15 ℃. It can be estimated that sesame oil is mainly transformed by the first cooling down. Moreover, it was implied that the structure of methylene (CH2) was significantly related to the modifications in sesame oil with temperature change. A series of experimental results elucidated that the winterization of sesame oil removed its impurities and stabilized its conditions. These results are probably the first report on the effect of the winterization process on sesame oil.

A Network Pharmacology Approach to Investigate the Anticancer Mechanism and Potential Active Ingredients of Rheum palmatum L. Against Lung Cancer via Induction of Apoptosis.

Rheum palmatum L. (RPL) is a known traditional herbal medicine with the functions of “heat-clearing and damp-drying” in traditional Chinese medicine. Its anti-cancer effect against lung cancer has been confirmed previously, but the related mechanisms and active substances for its action has been little studied. This study adopted the network pharmacology, built the network map of drug ingredients and disease targets (DDN), and discussed the effective components of RPL and its possible mechanisms. All constituents of RPL were collected through database search and literature mining, and the potential active constituents were screened. The inverse pharmacophore matching model was used to predict the targets of active ingredients, and the method was supplemented by database retrieval and literature mining. Compounds-target data were inputted into Cytoscape software to build the DDN of RPL, and functional annotation analysis and pathway enrichment analysis were carried out. Finally, 20 active compounds were screened, which acted on 817 targets. A total of 22,418 lung cancer-related targets were collected, and 761 overlapped with drug targets. By bioinformatics annotation of these overlapping genes, a total of 235 gene ontology (GO) functional annotation analyses and 46 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were obtained. It was found that the enrichment of GO and KEGG was associated with apoptosis, suggesting RPL plays an anti-lung cancer role via inducing cell apoptosis. Subsequent cell experiment results showed RPL and its active constituents inhibited the proliferation of A549 cells and reduced clone formation rate of A549 cells via induction of apoptosis. In this study, the pharmacodynamic basis and mechanism of RPL against lung cancer were studied from the perspective of systematic pharmacology, which would be beneficial for further elucidating the anticancer effect of RPL on lung cancer.

Mitsubishi names Jean-Marc Gilson its next CEO

Mitsubishi Chemical Holdings has named Jean-Marc Gilson, a Belgian-born chemical executive, its CEO effective April 1, 2021. He will succeed Hitoshi Ochi, who plans to retire. Currently the CEO of Roquette, a French food and drug ingredient company, Gilson has held executive positions at Dow Corning and Avantor Performance Materials. He worked for 5 years in Japan for Dow Corning. Gilson will be the first foreign CEO of Japan’s largest chemical company. His appointment comes as other Japanese chemical firms act to diversify their boards and management. JSR, for example, elevated the American executive Eric Johnson to CEO in 2018. In an Oct. 23 video press conference, Gilson declined to specify changes he would make to Mitsubishi’s diversified portfolio. But he cited DSM as a model for portfolio transformation, noting that the Dutch firm—a major competitor of Roquette’s—has shifted from chemicals to food, nutrition, and health products over the past

Dottikon set to boost drug ingredient manufacturing

Dottikon set to boost drug ingredient manufacturing

Research of water extraction from xenoderm as an active pharmaceutical ingredient in drugs

Today, burns are one of the most common types of injuries in the home and at work around the world. Therefore, the issue of treatment of burns remains relevant today for medicine and pharmacy in particular.
 In Ukraine, the method of treatment of burns using xenodermoimplants from porcine skin is used, and therefore the crushed substrate of cryolyophilized porcine skin (xenoderm) is a promising active ingredient in the technology of various drug forms.
 The aim of the work was to study the structural and mechanical properties of water extract from the crushed substrate of the xenoderm and to determine its amino acid composition by using physicochemical analysis, namely using high performance liquid chromatography (HPLC).
 A glass pycnometer and a Heppler BH 2 MLW drop ball viscometer were used to determine the density and viscosity of the water extract from the xenoderm. The density and viscosity of the water extract were studied at different temperatures.
 The dependence of the density and viscosity of the water extract from the xenoderm on temperature was studied and it was found that with increasing temperature the dynamic viscosity decreases and the density changes slightly.
 A glass pycnometer and a viscometer with falling ball were used to determine the density and viscosity of the xenoderm water extract. Chromatographic separation of amino acids was performed on a liquid chromatograph Agilent 1200 with a fluorescent detector.
 Chromatographic determination of amino acids was performed on a liquid chromatograph Agilent 1200 (USA) with a fluorescent detector G1315A (USA) and an autosampler 1313A.
 Using the HPLC method, 16 amino acids were identified (essential – 6; conditionally – 2; nonessential – 8). Identified amino acids are almost in a bound state (1.5%), the largest amount is glutamic acid (0.23%), glycine (0.19%), aspartic acid (0.18%), proline (0.17%) and arginine (0.17%). In unbound form, the content of glutamic acid (0.09%) and glycine (0.06%) is the highest.
 Based on the results of research, you can choose quality indicators, to determine the appropriate criteria that can be proposed for the standardization of water extract from the crushed substrate of the xenoderm.

Kefiran ameliorates malfunctions in primary and functional immune cells caused by lipopolysaccharides

Kefiran is a water-soluble polysaccharide well recognized as a bioactive ingredient to enhance nutritional and health-promoting features. Also, some therapeutic properties have made this macromolecule an active ingredient in ointments and oral anti-inflammatory drugs. However, the details of the molecular and cellular aspects of these effects have not been addressed. In this study, lipopolysaccharides (LPS)-induced monocytes, lymphocytes, and monocyte-derived dendritic cells (MDDCs) as representative cells for both innate and adaptive immunity were treated with kefiran for 2 h. Kefiran had an anti-inflammatory effect on monocytes to reduce pro-inflammatory cytokines, interleukin 1 β (IL-1β) & tumor necrosis factor α (TNF-α), as well as nuclear factor kappa b (NF-kb). However, it did not affect lymphocytes. Overexpression of Toll-like receptor 4 (TLR4) in LPS-induced cells was not reduced after kefiran treatment. Kefiran balanced MDDCs secretion of pro/anti-inflammatory cytokines by reducing and enhancing the expression of IL-1β and interleukin 10 (IL-10), respectively. Also, kefiran decreased the number of apoptotic immature MDDCs and promoted dose-dependent phagocytosis capacity of MDDCs. According to the results of the current study, it may be concluded that the immunomodulatory effects of kefiran are due to antagonist against innate immune receptors especially TLR4. The results of this study can be used as a guide to developing kefiran-based non-aggressive anti-inflammatory drugs. Furthermore, understanding the immunobiological effects of kefiran on monocytes and lymphocytes was another outcome of this study.

The best of times in the worst of times

Last fall, when the pharmaceutical services sector converged on Frankfurt, Germany, for the CPhI Worldwide drug ingredient trade show, the refrain had become rather monotonous. It was —the same story that attendees had been telling since the sector’s recovery from the 2008 recession. That story might have been expected to take a turn this year because of a pandemic that led to the cancellation of CPhI Worldwide, scheduled for Milan next month. One would think that disruptions in business—including supply chain jolts, delays and cancellations of clinical trials for drugs in development, and the cessation of manufacturing-site visits and regulatory audits—would knock the sector off course. But contract development and manufacturing organization (CDMO) executives and industry consultants say the surge continues, propelled only in part by the raft of COVID-19 therapies in development and the race to bring forward a vaccine for the disease. Contractors offering both active pharmaceutical ingredient

Techniques and Strategies for Potential Protein Target Discovery and Active Pharmaceutical Molecule Screening in a Pandemic.

Viruses remain a major challenge in the fierce fight against diseases. There have been many pandemics caused by various viruses throughout the world over the years. Recently, the global outbreak of COVID-19 has had a catastrophic impact on human health and the world economy. Antiviral drug treatment has become another essential means to overcome pandemics in addition to vaccine development. How to quickly find effective drugs that can control the development of a pandemic is a hot issue that still needs to be resolved in medical research today. To accelerate the development of drugs, it is necessary to target the key target proteins in the development of the pandemic, screen active molecules, and develop reliable methods for the identification and characterization of target proteins based on the active ingredients of drugs. This article discusses key target proteins and their biological mechanisms in the progression of COVID-19 and other major epidemics. We propose a model based on these foundations, which includes identifying potential core targets, screening potential active molecules of core targets, and verifying active molecules. This article summarizes the related innovative technologies and methods. We hope to provide a reference for the screening of drugs related to pandemics and the development of new drugs.

Can bacteria clean your kitchen counter?

It’s a good time to be a symbiotic microbe. Global sales of probiotics will rise to $55 billion this year from $30 billion in 2013, according to the food and drug ingredient firm Nutralliance. And ...

Possible Use of Ganoderma Mushroom Extracts for the Treatment of Some Chronic Risk Factors Associated with Severe Covid-19

The novel coronavirus (SARS-CoV-2) emerged in Wuhan, China in December 2019 causing the coronavirus disease 2019 (COVID-19). The disease has caused a pandemic that had disrupted social and economic life globally. Intense research is ongoing to understand the nature of the virus and pathogenesis as well as the development of drugs and vaccines. Ample evidence suggests that the elderly people, those with pre-existing chronic medical conditions, such as lung disease, heart disease, diabetes, obesity, chronic kidney disease, compromised immunity and even men may be at higher risk of serious illness and intensive care. The virus tends to trigger the over-production of pro-inflammatory cytokines, which results in multiorgan damage, resulting in death. Following this review, we proposed the use of Ganoderma mushroom extracts, which have certain properties relevant to the management of COVID-19 complications including anti-inflammatory, antiviral, immune modulatory, and antioxidant, anti-aging and multiorgan protection. Most of this research has been done on rodent models with few clinical trials. Since, the mushroom is generally regarded as safe (GRAS) and have been used for centuries in Asian traditional medicine, we recommend the use of this mushroom in the management of the disease including as active ingredients in COVID-19 drugs, and alternatives to synthetic drugs, health supplements, adjuvant therapy or functional foods.

Rapid quantification of artemisinin derivatives in antimalarial drugs with dipstick immunoassays

Substandard antimalarial drugs will result in unsatisfied therapeutic efficacy and increase the risk of resistance development. The point-of-care, qualitative, or semi-quantitative dipstick immunoassays cannot differentiate the substandard drugs with confidence. A rapid and quantitative analytical method that can be used under field conditions is needed. Here, three lateral flow immunoassays (LFIAs) based on colloidal gold nanobeads (CGN) as labels were developed for quantification of artemether, dihydroartemisinin and artesunate contents in antimalarial drugs with the aid of a portable optical scanner. Also, time-resolved fluorescent nanobeads (TRFN)-LFIA, coupled with a portable fluorescent lateral flow reader, was developed for quantification of artesunate. Commercial antimalarial drugs were used to validate these LFIAs with comparison to the gold standard high-performance liquid chromatography (HPLC) method. The drug contents estimated with these CGN-LFIAs were in the range of 85.5–109.3% of the contents determined by HPLC with a coefficient of variation (CV) of 4.5–13.0%. The TRFN-LFIA results were in the range of 93.7–108.4% of contents determined by HPLC with a CV of 5.2–8.9%. There were no significant differences between the results of CGN-LFIA and TRFN-LIFA (P = 0.5277, t-test). Both types of LFIAs with portable readers may be used for quantitation of active ingredients in antimalarial drugs and for screening substandard antimalarial drugs in resource-limiting settings.

Astragaloside IV: An Effective Drug for the Treatment of Cardiovascular Diseases.

Cardiovascular disease (CVD), the number one cause of death worldwide, has always been the focus of clinical and scientific research. Due to the high number of deaths each year, it is essential to find alternative therapies that are safe and effective with minimal side effects. Traditional Chinese medicine (TCM) has a long history of significant impact on the treatment of CVDs. The mode of action of natural active ingredients of drugs and the development of new drugs are currently hot topics in research on TCM. Astragalus membranaceus is a commonly used Chinese medicinal herb. Previous studies have shown that Astragalus membranaceus has anti-tumor properties and can regulate metabolism, enhance immunity, and strengthen the heart. Astragaloside IV (AS-IV) is the active ingredient of Astragalus membranaceus, which has a prominent role in cardiovascular diseases. AS-IV can protect against ischemic and hypoxic myocardial cell injury, inhibit myocardial hypertrophy and myocardial fibrosis, enhance myocardial contractility, improve diastolic dysfunction, alleviate vascular endothelial dysfunction, and promote angiogenesis. It can also regulate blood glucose and blood lipid levels and reduce the risk of cardiovascular diseases. In this paper, the mechanism of AS-IV intervention in cardiovascular diseases in recent years is reviewed in order to provide a reference for future research and new drug development.

In silico evaluation of antimicrobial, antihyaluronidase and bioavailability parameters of rosmarinic acid in Perilla frutescens leaf extracts

The present study reports ultrasound-assisted extraction of rosmarinic acid extract using the leaves of Perilla frutescens L. by using ethanol solvent. P. frutescens rosmarinic acid extract (PF-RAE) exhibited antimicrobial activity against bacteria and fungi. PF-RAE exhibited excellent antioxidant activity against DPPH and ABTS radicals. PF-RAE exhibited significant in vitro hyaluronidase inhibitory activity with maximum inhibition of 44.35%. HPLC analysis showed that rosmarinic acid is the major component of PF-RAE and it is responsible for antimicrobial, antioxidant and hyaluronidase inhibitory activities. Further, we report some of the plausible mechanisms of multiple biological functions of rosmarinic acid by in silico studies. Molecular docking studies reported that rosmarinic acid strongly inhibits bacterial co-/posttranslation enzyme peptide deformylase (promising antibacterial target) as well as fungi posttranslation enzyme N-myristoyltransferase (promising antifungal target) with binding affinities of 7.9 and 7.1 Kcal/mol, respectively. Further, rosmarinic acid exhibited strong affinity with human hyaluronidase protein by H-bonding interactions. Rosmarinic acid satisfactorily follows the Lipinski’s rule of five with ‘0’ violations, showed excellent hydrophilicity, oral bioavailability, skin permeability and gastrointestinal permeability and showed no toxicity at all. We concluded that rosmarinic acid can be developed as an excellent pharmaceutical and nutraceutical drug ingredient.

Possible inhibition mechanism of dobutamine hydrochloride as potent inhibitor for human glucose-6-phosphate dehydrogenase enzyme

Glucose-6-phosphate dehydrogenase (G6PD) is the first rate-limiting enzyme in the pentose phosphate pathway. One of the enzyme’s most important functions is the production of a reducing agent that is essential for preserving the level of reduced glutathione (GSH). However, some chemicals, such as industrial waste and the active ingredients of several drugs, can cause reduction or blockage in this enzyme’s activity. This case causes the occurrence of anemia by damaging erythrocytes. In this study, the G6PD enzyme was purified 21,981 fold with affinity chromatography and the effects of the active ingredients of some antiarrhythmic drugs on enzyme activity were investigated with in vitro and in silico methods. We found that dobutamine hydrochloride significantly decreased enzyme activity and its inhibitory constant (Ki ) value was calculated as 19.02 ± 4.83 mM. The in vitro study results also show that dobutamine hydrochloride is a potent inhibitor of enzyme activity. We also found that dobutamine hydrochloride inhibits the hG6PD enzyme’s activity by causing structural alterations in substrate and coenzyme binding sites. Communicated by Ramaswamy H. Sarma

On-line dissolution analysis of multiple drugs encapsulated in electrospun nanofibers.

Electrospun nanofiber is a very attractive material which can be used as the support to form multiple-drug dosage. Understanding the dissolution process of different active drug ingredients released from electrospun fibers is of great importance to control and evaluate the quality of medicated nanofibers. Here we present the first study of on-line automatic analysis of dissolution of multiple drugs in electrospun fiber mats. Single-needle electrospinning technology is utilized to combine polymers, hydrophilic polyvinylpyrrolidone (PVP) and hydrophobic polycaprolactone (PCL) as carrier to load three poorly water-soluble non-steroidal anti-inflammatory drugs (paracetamol, nimesulide, and ibuprofen). The loading of the drugs in PVP/PCL electrospun fibers are characterized by various techniques, including scanning electron microscopy, X-ray diffraction, Fourier infrared spectroscopy and differential scanning calorimetry. The in vitro dissolution is investigated by our home-made portable analyzer, which can simultaneously on-line determine multiple drugs released from the nanofibers by a single step. The analysis shows a wide linear detection range of the drugs with limit-of-detection (LOD) down to μg/mL-level. The dissolution profiles of three ingredients in nanofibers can be monitored every thirty seconds from the beginning to the end in the entire dissolution process from only one HSCE run. The kinetic information of the dissolution, including the dissolution curve, characteristic dissolution time and dissolution efficiency, is obtained and evaluated for different dissolution media, drug loading content and the ratio of PVP/PCL. Our study provides a promising method for rapid and accurate dissolution testing of nanofiber-based drugs, and would extend the applications of separation techniques in pharmaceutical analysis.

Diaryl Ether: A Privileged Scaffold for Drug and Agrochemical Discovery.

Diaryl ether (DE) is a functional scaffold existing widely both in natural products (NPs) and synthetic organic compounds. Statistically, DE is the second most popular and enduring scaffold within the numerous medicinal chemistry and agrochemical reports. Given its unique physicochemical properties and potential biological activities, DE nucleus is recognized as a fundamental element of medicinal and agrochemical agents aimed at different biological targets. Its drug-like derivatives have been extensively synthesized with interesting biological features including anticancer, anti-inflammatory, antiviral, antibacterial, antimalarial, herbicidal, fungicidal, insecticidal, and so on. In this review, we highlight the medicinal and agrochemical versatility of the DE motif according to the published information in the past decade and comprehensively give a summary of the target recognition, structure-activity relationship (SAR), and mechanism of action of its analogues. It is expected that this profile may provide valuable guidance for the discovery of new active ingredients both in drug and pesticide research.

Genotyping-by-sequencing to determine the genetic structure of a Tibetan medicinal plant Swertia mussotii Franch.

Swertia mussotii Franch. has been widely used in Tibetan folk medicine as an important drug ingredient for the treatment of hepatitis and hepatobiliary disease and for homeopathic purposes. But the lacking natural resources of S. mussotii is becoming more obvious with increasing market demands. The main objective of this work was to examine genetic differentiation among S. mussotii populations from different geographical locations and use the knowledge gained to facilitate future selection of the species suitable for large-scale cultivation to meet the increasing demand for medicinal use of this plant. In this study, 68 S. mussotii Franch samples collected from Zhongda (16), Chengduo (16), and Xiewu (8) County in Qinghai Province, Xiaojin (14) and Jinchuan (14) County in Sichuan Province, China and 4 samples from related species, were genotyped using genotyping-by-sequencing (GBS) technique. GBS scored 4095 SNPs with a minor-allele frequency > 0.25 and an average read depth < 200 based on the 72 samples examined. Analyses of all SNPs provided evidence of genetic differentiation among populations. Structural and phylogenetic topological analyses supported the genetic differentiations among populations. Gene ontology annotation results revealed that genetic differentiations often occurred in genes known or hypothesized to be involved in various metabolic processes such as catalytic and transferase activity, cellular component, nucleus, biological, cellular and metabolic process. The present results also revealed a significant positive correlation between the genetic similarity and geographical distance (P = 0.032). Moreover, the adaptability of S. mussotii to the environment had a strong effect on its chemical composition and phenotypic changes. Therefore, we hypothesize that the population structure is strongly influenced by local adaptive pressure. The adaptive evolution of S. mussotii species in heterogeneous environments may play an important role in inducing genetic differentiation among different geographical populations. This is evidenced by the observed changes in phenotypic traits and chemical compounds in S. mussotii plants from different geographic locations.