Analytical Techniques for Phytocannabinoid Profiling of Cannabis and Cannabis-Based Products-A Comprehensive Review.

Cannabis is gaining increasing attention due to the high pharmacological potential and updated legislation authorizing multiple uses. The development of time- and cost-efficient analytical methods is of crucial importance for phytocannabinoid profiling. This review aims to capture the versatility of analytical methods for phytocannabinoid profiling of cannabis and cannabis-based products in the past four decades (1980-2021). The thorough overview of more than 220 scientific papers reporting different analytical techniques for phytocannabinoid profiling points out their respective advantages and drawbacks in terms of their complexity, duration, selectivity, sensitivity and robustness for their specific application, along with the most widely used sample preparation strategies. In particular, chromatographic and spectroscopic methods, are presented and discussed. Acquired knowledge of phytocannabinoid profile became extremely relevant and further enhanced chemotaxonomic classification, cultivation set-ups examination, association of medical and adverse health effects with potency and/or interplay of certain phytocannabinoids and other active constituents, quality control (QC), and stability studies, as well as development and harmonization of global quality standards. Further improvement in phytocannabinoid profiling should be focused on untargeted analysis using orthogonal analytical methods, which, joined with cheminformatics approaches for compound identification and MSLs, would lead to the identification of a multitude of new phytocannabinoids.

Headspace screening: A novel approach for fast quality assessment of the essential oil from culinary sage.

Quality assessment of essential oil (EO) from culinary sage (Salvia officinalis L., Lamiaceae) is limited by the long pharmacopoeial procedure. The aim of this study was to employ headspace (HS) sampling in the quality assessment of sage EO. Different populations (30) of culinary sage were assessed using GC/FID/MS analysis of the hydrodistilled EO (pharmacopoeial method) and HS sampling directly from leaves. Compound profiles from both procedures were evaluated according to ISO 9909 and GDC standards for sage EO quality, revealing compliance for only 10 populations. Factors to convert HS values, for the target ISO and GDC components, into theoretical EO values were calculated. Statistical analysis revealed a significant relationship between HS and EO values for seven target components. Consequently, HS sampling could be used as a complementary extraction technique for rapid screening in quality assessment of sage EOs.

Essential oils and chemical diversity of southeast European populations of Salvia officinalis L.

The essential oils of 25 populations of Dalmatian sage (Salvia officinalis L.) from nine Balkan countries, including 17 indigenous populations (representing almost the entire native distribution area) and eight non-indigenous (cultivated or naturalized) populations were analyzed. Their essential-oil yield ranged from 0.25 to 3.48%. Within the total of 80 detected compounds, ten (ß-pinene, 1,8-cineole, cis-thujone, trans-thujone, camphor, borneol, trans-caryophyllene, α-humulene, viridiflorol, and manool) represented 42.60 to 85.70% of the components in the analyzed essential oils. Strong positive correlations were observed between the contents of trans-caryophyllene and α-humulene, α-humulene and viridiflorol, and viridiflorol and manool. Principal component analysis (PCA) on the basis of the contents of the ten main compounds showed that four principal components had an eigenvalue greater than 1 and explained 79.87% of the total variation. Performing cluster analysis (CA), the sage populations could be grouped into four distinct chemotypes (A-D). The essential oils of 14 out of the 25 populations of Dalmatian sage belonged to Chemotype A and were rich in cis-thujone and camphor, with low contents of trans-thujone. The correlation between the essential-oil composition and geographic variables of the indigenous populations was not significant; hence, the similarities in the essential-oil profile among populations could not be explained by the physical proximity of the populations. Additionally, the southeastern populations tended to have higher EO yields than the northwestern ones.

Flavonoids and Other Phenolic Compounds in Needles of Pinus peuce and Other Pine Species from the Macedonian Flora.

Flavonoids and other phenolic compounds in young needles of four pine species, Pinus peuce, P. nigra, P. mugo and P. sylvestris from the Macedonian flora were investigated. The amount of total phenols and total flavonoids were determined using Folin-Ciocalteau and aluminum chloride assay, respectively. The obtained results revealed that the total phenolic content (TPC) and total flavonoids content (TFC) varied among different pine species ranging from 9.8 to 14.0 mg GAE/g and from 3.3 to 7.2 mg CE/g of dried plant material, respectively. Qualitative analysis of flavonoids and other phenolic components was made by a LC-DAD/ESI-MS(n) optimized chromatographic method. A total of 17 phenolic components were identified and classified as: acids (2), procyanidins (2) and flavonoid glycosides (13). The most prevalent components were flavonoid glycosides, especially flavonols and methylated flavonols (9). Additionally, 3 components were found as acylated flavonol glycosides with ferulic and p-coumaric acid. The last one was found not only in esterified form but also in the free form. Only one flavone-apigenin glycoside was detected. Procyanidins were identified as catechin derivatives, both dimers and trimers.

Fingerprinting of morphine using chromatographic purity profiling and multivariate data analysis.

Chromatographic purity profiling (CPP) is the common name of a group of analytical and chemometric applications for detection, identification and quantitative determination of related substances and other impurities in active pharmaceutical ingredients (APIs) and finished dosage forms (FDFs). CPP is used for fingerprinting and discriminating between samples, thus representing a core activity in modern drug analysis. The worldwide demand for morphine and its congeners is tremendous and depends entirely on the supply of natural opiates. The aim of this research was to develop a methodology that enables identification of a source of morphine, thus revealing falsification of the substance. The characteristic and reproducible features of impurity profiles for 28 samples of morphine (6 morphine sulfate, 9 morphine hydrochloride and 13 morphine base) were captured by a new LC/MS method for impurity profiling of morphine. The impurity profile encompasses the related substances specified in relevant Ph.Eur. monographs, as well as the other morphinane like impurities, including the naturally occurring co-extracted alkaloids. Different pattern recognition techniques (unsupervised and supervised) were used to reveal the differentiation features of the morphine fingerprints for classification and authentication purposes. The results described in this research open the possibility of using the chromatographic purity profile combined with multivariate data analysis for fingerprinting of morphine samples.

Chemical composition and antimicrobial activity of essential oils of Juniperus excelsa Bieb. (Cupressaceae) grown in R. Macedonia.

BACKGROUND: There are no information of the yield, chemical composition and antimicrobial activity of essential oils of berries (EOB) or leaves (EOL) of Juniperus excelsa Bieb. (Cupressaceae) growing wild in R. Macedonia. MATERIALS AND METHODS: Plant material was collected from two localities during two seasons. Essential oil composition was analyzed by gas chromatography/flame ionization detector/mass spectrometry (GC/FID/MS) and antimicrobial screening was made by disc diffusion and broth dilution method. RESULTS AND DISCUSSION: EOB yield ranged from 1.6-9.4 ml/kg and from 8.9-13.9 ml/kg for EOL. Two chemotypes of essential oil were differentiated, α-pinene-type (with 70.81% α-pinene in EOB and 33.83% in EOL), also containing limonene, ß-pinene and ß-myrcene while the sabinene-type (with 58.85-62.58% sabinene in EOB and 28.52-29.49% in EOL), was rich in α-pinene, ß-myrcene, limonene, cis-thujone, terpinolene and α-thujene. The most sensitive bacteria to the antimicrobial activity of EOB was Haemophilus influenzae (MIC = 31 µl/ml). EOL have showed high activity towards: Staphylococcus aureus, Streptococcus pyogenes and Haemophilus influenzae (MIC = 125 µl/ml). The pinene-type of essential oil showed moderate activity against Streptococcus pneumoniae, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pyogenes, Corynebacterium spp. and Campylobacter jejuni (MIC >50%). The sabinene-type of the oil showed moderate activity to Streptococcus pyogenes, Haemopilus influenzae, Campylobacter jejuni and Escherichia coli (MIC >50%). No activity was observed toward Candida albicans. CONCLUSION: The analysis of EOB and EOL revealed two chemotypes (α-pinene and sabinene type) clearly depended on the geographical origin of the Macedonian Juniperus excelsa which also affected the antimicrobial activity of these oils.

Chemical composition and antimicrobial activity of the essential oils of Pinus peuce (Pinaceae) growing wild in R. Macedonia.

The chemical composition and antimicrobial activity of the essential oils isolated from twigs with needles (T+N) and from twigs without needles (T-N) from wild Pinus peuce Griseb. (Pinaceae), from three different locations in R. Macedonia, were investigated. Essential oil yields of T+N ranged from 7.5 mL/kg to 12.5 mL/kg and for T-N from 13.8 mL/kg to 17.3 mL/kg. GC/FID/MS analysis of the essential oils revealed eighty-four components, representing 93.7-95.7% and 91.2-92.0% of the T+N and T-N oils, respectively. The major components in T+N and T-N oils were monoterpenes: α-pinene (23.8-39.9%, 21.2-23.3%), camphene (2.2-5.5%, 0.7-2.0%), ß-pinene (10.1-17.1%, 8.2-16.4%), myrcene (1.2-1.41%, 1.6-2.5%), limonene+ß-phellandrene (6.8-14.0%, 8.8-23.6%) and bornyl acetate (2.3-6.9%, 1.1-3.4%), followed by the sesquiterpenes: trans-(E)-caryophyllene (3.6-4.3%, 3.2-7.3%), germacrene D (7.1-9.5%, 5.0-10.3%) and δ-cadinene (2.1-3.1%, 3.3-4.2%, respectively). Antimicrobial screening of the essential oils was made by disk diffusion and broth dilution methods against 13 bacterial isolates of Gram-positive and Gram-negative bacteria and one strain of Candida albicans. T-N essential oils showed antimicrobial activity toward Streptococcus pneumoniae, Staphylococcus aureus, S. epidermidis and Candida albicans as well as Streptococcus agalactiae, Acinetobacter spp. and Haemophilus influenzae. The antimicrobial activity of T+N essential oils was greater, especially against Streptococcus agalactiae, S. pyogenes, Enterococcus and Candida albicans, followed by Haemophilus influenzae, Acinetobacter spp., Escherichia coli, Salmonella enteritidis, Staphylococcus aureus and S. epidermidis. Minimal inhibitory concentrations (MICs) of all tested essential oils ranged from 15-125 µL/mL. Summarizing the obtained results, the antimicrobial activity of Pinus peuce T+N and T-N essential oils collected from different localities in R. Macedonia varied considerably. These alterations in the antimicrobial activity can be attributed to the differences in the quantitative composition and percentage amounts of the components present in the respective essential oils, although it was evident that there were no differences in the qualitative composition of the essential oils, regardless of the locality of collection, or the type of plant material (T+N or T-N).

Polyphenols in representative Teucrium species in the flora of R. Macedonia: LC/DAD/ESI-MS(n) profile and content.

In the present work, the polyphenolic profile and content of four Teucrium species (T. chamaedrys L., T. montanum L., T. polium L., T. scordium L.) from the Macedonian flora were examined. A LC/DAD/ESI-MS(n) chromatographic method was optimized and 31 phenolic compounds were identified, quantified and classified into four groups: hydroxycinnamic acid derivatives (2), phenylethanoid glycosides (12), flavonoid glycosides (11) and flavonoid aglycones (6). The total phenolic content (mg/g dry herb) ranged from 28.2 (T. montanum), 30.9 (T. scordium), 35.1 (T. polium) to 52.1 (T. chamaedrys). Phenylethanoid glycosides were the predominant group ofpolyphenols in the studied samples contributing 60% of the total phenolic content for T. polium and T. scordium and around 90% for T. montanum and T. chamaedrys. The systematic analysis for identification and quantification of all present phenolic compounds contributes to the chemotaxonomy of the investigated Teucrium species and to the valorization based on their phenolic profiles and content.

Chemical characterization of Centaurium erythrea L. and its effects on carbohydrate and lipid metabolism in experimental diabetes.

ETHNOPHARMACOLOGICAL RELEVANCE: Centaurium erythrea L. fam. Gentianaceae (CE) has been traditionally used for centuries in folk medicine of Balkans as a bitter medicinal herb for digestive complications and for treating febrile conditions and diabetes. The aim of this study was to gain insight into the chemical composition and underlying biochemical mechanism of action of the antihyperglycemic and antilipidemic activities of the dry extract of Centaurium erythrea L., wildly growing and traditionally used medicinal plant in the Republic of Macedonia. MATERIALS AND METHODS: An ultrasonic methanol maceration of the aerial parts of the dried plant was performed and the extract was freeze-dried. HPLC-DAD-ESI-MS(n) was carried out on 150 mm × 4.6mm, 5 µm RP-18 Eclipse XDB column, at 40 °C. Mobile phase: water with 1% formic acid (A) and methanol (B) with linear gradient starting with 10% B was used to reach 15% at 5 min, 40% B at 25 min, 55% of B at 50 min and 100% at 60 min, with flow rate of 0.4 mL min(-1). Normal and streptozotocin (STZ) hyperglycemic Wistar rats were used for assessment of the antihyperglycemic and antilipidemic activity by measurement of the key carbohydrate-related enzymes and substrates, as well as lipid state of the organism. RESULTS: HPLC-DAD-ESI-MS(n) analyses revealed presence of four different secoiridoids, seven flavonoid glycosides and seven xanthones in the freeze-dried extract of CE representing 53%, 25% and 22% of all compounds, respectively. The short-term (12 days) treatment of the STZ-diabetic rats with CE-extracts resulted in a 74% reduction of the produced hyperglycemia, which is only 6% less than the reduction caused by glibeclamide (GLB, positive control). The CE-extract had a significant impact on the hepatic carbohydrate metabolism enhancing the direct synthesis of glycogen, normalizing phosphorylase a activity and reducing the activity of glucose-6-phosphatase, which further causes reduction in production of blood glucose level. The long-term (45 days) treatment showed that the HbA1c in CE-treated group of animals was even lower than in the GLB-treated groups. The antilipidemic assessment of the CE-extract revealed decrease of total cholesterol, triglycerides, HDL and LDL level in the blood of the normal and STZ-hyperglycemic rats. CONCLUSION: The obtained results indicate that treatment with CE extract in STZ-diabetic rats regulates the elevated level of blood glucose and carbohydrate-related disturbances slightly better than the effect of glibenclamide. There was also regulation of the serum lipid status in diabetic rats. Identified groups of bitter compounds in the extract (flavonoides, iridoids and xanthones) probably have influence on the expressed antihyperglycaemic effect.

Aroma compounds of mountain tea (Sideritis scardica and S. raeseri) from western Balkan.

The composition of the volatile aroma components was defined in the dried and fresh aerial parts of Sideritis scardica Griseb. from R. Macedonia and S. raeseri Boiss. & Heldr. from R. Macedonia, Albania and Greece. Analysis was made by gas chromatography (GC/FID/MS) equipped with a headspace (HS) sampler. Thirty-two components (15 monoterpenes representing 33.2- 62.8% and 17 sesquiterpenes representing 25.2- 51.2% of the entire volatiles) were identified as aroma components of dried plant material of S. scardica. Thirty components {14 monoterpenes (19.3-74.2%), 2 alcohols (6.2- 38.4%) and 14 sesquiterpenes (18.2- 33.5%)} were identified as aroma components in the fresh aerial parts of S. scardica. The predominant components were trans-caryophyllene, ß-pinene, α-pinene and 1-octen-3-ol, which were found only in the fresh samples. In the aerial parts of S. rteseri, 43 components were identified in the dried samples {22 monoterpenes (65.7-94.3%) and 21 sesquiterpenes (5.4- 27.8%)} and 29 components {15 monoterpenes (77.3-90.7%) and 14 sesquiterpenes (6.3- 18.2%)} in the respective fresh samples. Prevailing components in all tested samples of S. raeseri were ß-pinene, α-pinene, α-copaene, sabinene and limonene. Only minor differences were revealed in the qualitative composition of the aroma volatiles between the dried and fresh plant material of both species. Furthermore there was almost no difference in the chemical profiles of the aroma compounds between S. scardica and S. raeseri, except for 1- octen-3-ol, which was present only in fresh S. scardica.

Development and validation of a reversed-phase HPLC method for determination of alkaloids from Papaver somniferum L. (Papaveraceae).

An HPLC method for the separation of six target alkaloids from Papaver somniferum L. (morphine, codeine, oripavine, thebaine, papaverine, and noscapine) was developed, optimized, and validated. The chromatographic behavior of these alkaloids was investigated using a reversed-phase chromatography at acidic and alkaline pH. The effects of ion-pairing agents, pH value of the mobile phase, concentration of the buffer components, mobile phase organic modifier, and column temperature were studied. Regardless of the large differences in their pKa values, all alkaloids were separated within a close retention window, and good peak shape was achieved for each of the six alkaloids. The proposed method has adequate selectivity, linearity, accuracy, precision, and reproducibility and is applicable for poppy straw.

Dietary burden of phenolics per serving of "Mountain tea" (Sideritis) from Macedonia and correlation to antioxidant activity.

This work was afforded from 2 points of view, phytochemical evaluation and relation to antioxidant activity and dietary burden of phenolics of a cup of "Mountain tea", a drink obtained by domestic infusion of Sideritis. Phytochemically, two extraction protocols using water and methanol as solvent were used for comparison. Methanol and boiling water extracts (by domestic infusion procedure) showed that extracts were rich in bound forms of phenolics such as hydroxycinnamic acids, phenylethanoid glycosides and flavonoid glycosides. The total phenolic content for Sideritis species ranged around 190 mg per serving (2 g infusion bag) for methanol extracts and around 72 mg per serving in water extracts. Among the two different Macedonian Sideritis species, Sideritis raeseri (wild growing) showed the highest phenolics content in both extracts (212 mg and 89 mg per serving, respectively). Concerning the phenolic content in the different aerial parts, leaf was the richest plant organ in phenolics followed by flower and stem with the lowest amount. The methanol extract from Sideritis raeseri (wild growing) showed the highest antioxidant capacity as shown by DPPH, ABTS and FRAP assays. The antioxidant capacity was linearly correlated with phenolic content. Nutritionally, the dietary burden of phenolics of a "Mountain tea" bag for domestic infusion (serving size) was established at 89 mg for an homogeneous and equal distribution of the different aerial parts (leaf, flower and stem). However, and according to our results a rate of 60% leaf and 40% flower would increase the content of bioavailable phenolics and also the total phenolics content of a serving bag of "Mountain tea".

Effects of Teucrium polium spp. capitatum flavonoids on the lipid and carbohydrate metabolism in rats.

CONTEXT: The main objective of the study was to investigate the biochemical mechanism of the antidiabetic activities of the dry extracts of Teucrium polium L. ssp. capitatum (L.) Arcangeli (Lamiaceae), from Republic of Macedonia, traditionally used to treat diabetes. MATERIALS AND METHODS: Aerial parts of the plant were extracted in alcohol and freeze- or spray-dried, analyzed by high performance liquid chromatography (HPLC) and examined for insulinotropic effect in INS-1E cells in vitro. Their effect on blood glucose, lipids and carbohydrate-related enzymes was tested in normo- and streptozotocin hyperglycemic rats. RESULTS AND DISCUSSION: HPLC analyses revealed several flavonoids: luteolin, apigenin, cirsiliol, diosmetin, cirsimaritin and cirsilineol as both free aglycons and glycosides. The extract and mixture of commercial flavonoids showed a distinct insulinotropic effect on INS-1E cells at 500 µg/ml. Intragastric (i.g.) administration of identical doses of the extract (125 mg/kg) in both normo- and hyperglycemic rats was more efficient in lowering the blood glucose than intraperitoneal injection (35% vs. 24% reduction) with highest effect (50% reduction) 8 h after administration. After 10 days of treatment, the magnitude of the effect was comparable to i.g. administration of 2.5 mg/kg of glibenclamide (38% reduction). No effect was seen on blood lipid profiles. In OGTT (oral glucose tolerance test), the extract lowered blood glucose levels by ~35%. The treatment reduced hepatic glycogen and tended to normalize the activity of gluconeogenic enzymes. CONCLUSION: The results demonstrate that examined plant extracts contain flavonoids with insulinotropic and antihyperglycemic effects.

Phenolic compounds of mountain tea from the Balkans: LC/DAD/ESI/MSn profile and content.

Twenty-one samples of Sideritis species (S. scardica, S. raeseri, S. taurica, S. syriaca and S. perfoliata) from various locations on the Balkan Peninsula were evaluated for their chemical constituents. Chemical analyses were focused on secondary metabolites, particularly phenolic compounds, which have several roles in the plant physiological processes and have demonstrated significant health beneficial effects. The occurrence of hydroxycinnamic acids, phenylethanoid glycosides and flavonoids has been investigated in taxonomically related taxa of the genus Sideritis. A systematic method for phenolic compounds identification was developed using tandem mass spectrometry coupled to high performance liquid chromatography with diode array detection. Scanning for precursor ions of commonly found phenolics in Sideritis species using LC/MS11 with an ion trap instrument permitted the specific determination of hydroxycinnamic acid derivatives, and phenylethanoid and flavonoid glycosides. Further characterization of each phenolic compound was performed using MS/MS product-ion analysis and common-neutral-loss analysis. This on-line technique allowed identification of three hydroxycinnamic acid derivatives, eight phenylethanoid glycosides, and twenty-four flavonoid glycosides. All the taxa analysed produced very similar phenolic patterns characterized by the presence of 5-caffeoylquinic acid, lavandulifolioside, verbascoside, hypolaetin 7-O-[6'''-O-acetyl]-allosyl(1-->2)glucoside, apigenin 7-(4"-p-coumaroylglucoside), 4'-O-methylisoscutellarein 7-O-[6'''-O-acetyl]-allosyl(1-->2)glucoside, and minor amounts of isoverbascoside, apigenin 7-O-allosyl(1-->2)glucoside, isoscutellarein 7-O-allosyl-(1-->2)-[6"-O-acetyl]-glucoside, hypolaetin 7-O-allosyl-(1-->2)-[6"-O-acetyl]-glucoside and 4'-O-methylhypolaetin 7-O-[6'''-O-acetyl]-allosyl-(1-->2)-[6"-O-acetyl]-glucoside. These results show that the investigated species are systematically very closely related. Phenylethanoid glycosides and flavonoid acetylglycosides are dominant and constitute 90% of the total phenolic compounds compared with hydroxycinnamic acid and flavonoid 7-O-glycosides. Principal component analysis (PCA) was performed for the nature and content of the different compounds to be correlated to the particular Sideritis species and also to the locations.

Hepatoprotective effect of the ethyl acetate extract of Teucrium polium L. against carbontetrachloride-induced hepatic injury in rats.

Hepatoprotective activity of the ethyl acetate extract of Teucrium polium L. was investigated using rats with CCl4-induced liver damage. Specific biochemical parameters (glutathione peroxidase, superoxide dismutase, reduced glutathione and total antioxidative status) were estimated in blood and in liver homogenate. Lipid peroxidation in CCl4-intoxicated rats was evidenced by a marked increment in the levels of thiobarbituric acid reactive substances. Histopatological examinations of the liver were undertaken to monitor the liver status. Silymarin was used as a standard to compare the hepatoprotective activity of the extract. Some biochemical parameters in groups treated with the Teucrium polium extract at a dose of 25 mg kg(-1), showed significantly different values than that of the CCl4--treated group. The liver biopsy of all experimental rat groups treated with the Teucrium polium ethyl acetate extract showed significant restoration of the normal histomorphological pattern of liver cells. The study substantiates the potential hepatoprotective activity of the ethyl acetate extract of Teucrium polium L.

In vitro antioxidant activity of some Teucrium species (Lamiaceae).

The chemical composition and antioxidant activity of different extracts (diethyl ether, ethyl acetate and n-butanol) obtained from Teucrium species (T. chamaedrys, T. montanum, T. polium) were investigated in this work. Phytochemical screening of the plant extracts proved the presence of flavonoids luteolin, apigenin and/or diosmetin. The chemical composition of extracts was evaluated by HPLC and spectrophotometry. Antioxidant activities of the extracts were evaluated using three complementary in vitro assays: inhibition of DPPH (1,1-diphenyl-2-picrylhydrazyl) radical, inhibition of hydroxyl radicals and protection of beta-carotene-linoleic acid model system. In the first two assays, strong inhibitory activity was shown by T. montanum and T. chamaedrys extracts. In the beta-carotene-linoleic acid model system, extracts from T. polium showed remarkable activity. These findings demonstrated that Teucrium species possess free radical and hydroxyl radical scavenging activity as well as antioxidant activity in vitro.