Purification, Structural Characterization, and Anti-Inflammatory Effects of a Novel Polysaccharide Isolated from Orostachys fimbriata.

The present study elucidated the structural characteristics and anti-inflammatory activity of a novel polysaccharide isolated from Orostachys fimbriata, which is a traditional Chinese medicinal plant. O. fimbriata polysaccharide (OFP) was extracted and subsequently purified by chromatography using a DEAE cellulose-52 and Sephadex G-75 column. The molecular weight was determined as 6.2 kDa. HPGPC and monosaccharide composition analysis revealed a homogeneous polysaccharide containing only Glc. Chromatography and spectral analysis showed that the possible chemical structure consisted of →4)-α-Glcp-(1→ and a small quantity of →4,6)-ß-Glcp-(1→ in the main chain and →6)-ß-Glcp-(1→, α-Glcp-(1→, and ß-Glcp-(1→ in the side chain. Morphological analysis using scanning electron microscopy (SEM) and atomic force microscopy (AFM) indicated that OFP had a multi-branched structure, and the sugar chain molecules of polysaccharide appeared aggregated. OFP was found to exhibit anti-inflammatory activity by reducing the secretion of inflammatory factors in RAW264.7 cells and by decreasing the extent of xylene-induced ear swelling in mice.

Toxicological assessment of Opuntia dillenii (Ker Gawl.) Haw. cladode methanol extract, fractions and its alpha pyrones: Opuntiol and opuntioside.

ETHNOPHARMACOLOGICAL RELEVANCE: The edible plant Opuntia dillenii (Ker Gawl.) Haw. commonly known as Nagphana, belongs to the Cactaceae family. It is traditionally used to treat various ailments including inflammation, gastric ulcers, diabetes, hepatitis, asthma, whooping cough and intestinal spasm. AIM OF THE STUDY: Despite its traditional use in various countries, detailed toxicological studies of O. dillenii cladode are few. Thus in the current study, toxicity of O. dillenii cladode derived methanol extract, fractions and its α-pyrones: opuntiol and opuntioside have been addressed. METHODS: The test agents were assessed using both in vitro and in vivo toxicity assays. MTT on human embryonic kidney cell line (HEK-293), tryphan blue exclusion in rat neutrophils, Cytokinesis-B block micronucleus (CBMN) in human lymphocytes and genomic DNA fragmentation using agarose gel electrophoresis were performed. In acute toxicity test, mice orally received extract (5 g/kg) for 7 days followed by measurements of relative organ weight, biochemical (blood profile, liver and kidney function test) and histological studies (liver and kidney) were carried out. Rat bone marrow micronucleus genotoxicity assay was also conducted. RESULTS: O. dillenii derived test agents were non-cytotoxic and had no effect on the integrity of DNA. Methanol extract (5 g/kg) orally administered in mice did not cause any significant change in relative organ weights, biochemical parameters and liver and kidney histology as compared to vehicle control. In parallel, extract did not stimulate micronuclei formation in rat bone marrow polychromatic erythrocytes. CONCLUSION: These results led to conclude that edible O. dillenii extract is non-toxic via the oral route and appears to be non-cyto-, hepato-, nephro- or genotoxic, thereby supporting its safe traditional use against various ailments. Therefore, opuntiol and opuntioside may serve as lead compounds in designing new drug(s) derived from edible plants.

Separation of three chromones from Saposhnikovia divaricata using macroporous resins followed by preparative high-performance liquid chromatography.

Prim-O-glucosylcimifugin, cimifugin, and 5-O-methylvisamminoside are three major chromone derivatives of Saposhnikovia divaricata that have many pharmacological activities, such as anti-inflammatory and antitumor activities. In the present work, an effective method for the simultaneous separation of prim-O-glucosylcimifugin, cimifugin, and 5-O-methylvisamminoside with high purities was established using HPD-300 resin coupled with preparative high-performance liquid chromatography. The adsorption kinetics curves of the three compounds on the HPD-300 resin were studied and found to fit well according to the pseudo-second-order equation. The adsorption isotherm results indicated that the adsorption process of the three compounds was exothermic. After a one-run treatment with the resin, the contents of prim-O-glucosylcimifugin, cimifugin, and 5-O-methylvisamminoside increased from 0.29, 0.06, and 0.37% to 13.07, 2.83, and 16.91% with recovery yields of 76.38, 78.25, and 76.73%, respectively. Finally, the purities of the three compounds were found to reach more than 95% after further separation using preparative high-performance liquid chromatography. The method developed in this study was effective and could simultaneously separate three chromones from Saposhnikovia divaricate. The experimental results also showed that the HPD-300 resin is suitable for the separation of chromone derivatives.

Recovery of Anthocyanins and Monosaccharides from Grape Marc Extract by Nanofiltration Membranes.

Wastewaters and by-products generated in the winemaking process are important and inexpensive sources of value-added compounds that can be potentially reused for the development of new products of commercial interest (i.e., functional foods). This research was undertaken in order to evaluate the potential of nanofiltration (NF) membranes in the recovery of anthocyanins and monosaccharides from a clarified Carménère grape marc obtained through a combination of ultrasound-assisted extraction and microfiltration. Three different flat-sheet nanofiltration (NF) membranes, covering the range of molecular weight cut-off (MWCO) from 150 to 800 Da, were evaluated for their productivity as well as for their rejection towards anthocyanins (malvidin-3-O-glucoside, malvidin 3-(acetyl)-glucoside, and malvidin 3-(coumaroyl)-glucoside) and sugars (glucose and fructose) in selected operating conditions. The selected membranes showed differences in their performance in terms of permeate flux and rejection of target compounds. The NFX membrane, with the lowest MWCO (150-300 Da), showed a lower flux decay in comparison to the other investigated membranes. All the membranes showed rejection higher than 99.42% for the quantified anthocyanins. Regarding sugars rejection, the NFX membrane showed the highest rejection for glucose and fructose (100 and 92.60%, respectively), whereas the NFW membrane (MWCO 300-500 Da) was the one with the lowest rejection for these compounds (80.57 and 71.62%, respectively). As a general trend, the tested membranes did not show a preferential rejection of anthocyanins over sugars. Therefore, all tested membranes were suitable for concentration purposes.

Physicochemical and macromolecule properties of RG-I enriched pectin from citrus wastes by manosonication extraction.

The properties of pectin extracted from mandarin citrus peels by manosonication extraction (MSp) were systematically studied and compared with pectin obtained by the conventional maceration method (CMp). The yield of MSp (25.5%) was significantly higher than that of CMp (18.3%), while MSp exhibited two Mw fraction distributions. Monosaccharide analysis demonstrated that MSp had more branched RG-I regions (78.3 mol%) than CMp (36.6 mol%) with a high content of arabinose and galactose. The branched-chain morphological characteristics of samples were directly imaged by atomic force microscopy. MSp exhibited a significantly lower degree of methoxylation than CMp by FT-IR and NMR analysis, but X-ray diffraction analysis showed little difference in the level of crystallinity. Moreover, MSp and CMp showed non-Newtonian behaviour, and the increasing order of apparent viscosities was 1.0 w/v% MSp < 1.0 w/v% CMp < 2.0 w/v% CMp < 2.0 w/v% MSp. Thermal analysis and weight loss measurements indicated MSp exhibited greater thermal stability. The results also indicated that both MSp and CMp significantly enhanced the emulsion activity at high concentrations; the emulsions containing 1.5 w/v% pectin showed no phase separation over 21 days, suggesting that MSp could be a potential effective stabiliser in the food and beverage industry.

Isolation of an acidic polysaccharide from the flowers of Leucosceptrum canum Smith and its immunomodulatory activity evaluation.

A water-soluble polysaccharide (LCP-05) was isolated from the flowers of Leucosceptrum canum Smith. LCP-05 was an acidic polysaccharide with a molecular weight of approximately 8.9 kDa. Monosaccharide composition analysis indicated that LCP-05 was composed of Man, Rha, GlcA, GalA, Glc, Gal and Ara in a molar ratio of 0.83:1.68:0.33:2.15:1.00:1.45:1.22. The framework of LCP-05 was speculated to be a branched rhamnogalacturonan with the backbone consisting of α-1,2,4-linked Rhap and α-1,4-linked GalAp, and bearing branches at the O-4 position of the Rha residues. The side chains are terminated primarily with the Araf and Glcp residues. LCP-05 was found to be able to significantly induce the production of NO, IL-6, and TNF-α in RAW 264.7 cells, and to induce RAW 264.7 cell's suppressive effect on both cell growth and cell migration of 4 T1 mammary breast cancer cells.

Screening active fractions from Pinus massoniana pollen for inhibiting ALV-J replication and their structure activity relationship investigation.

The objective was to identify the active fractions of polysaccharide against replication of ALV-J and elucidate their structure activity relationship. The optimal extraction conditions were extracting temperature 90℃, pH 9 and the ratio of liquid to solid 30:1. Under these conditions, extraction yield of total polysaccharide was 6.5 % ± 0.19 %. Total polysaccharide was then purified by DEAE-52 cellulose and Sephadex G-200 gel. Three fractions, PPP-1, PPP-2, and PPP-3, were identified with molecular weight of 463.70, 99.41, and 26.97 kDa, respectively. Three polysaccharide fractions were all composed of 10 monosaccharides in different proportions. Compared with PPP-1, which was mainly composed of glucose, PPP-2 and PPP-3 contained a higher proportion of galactose, glucuronic acid and galacturonic acid. The Congo red assay indicated that the PPP-2 may have a triple helical structure, while PPP-1 and PPP-3 were absent. In vitro assay showed that there was no significant cytotoxicity among the polysaccharide fractions under the concentration of 800 µg mL-1 (P > 0.05). The antiviral test showed that PPP-2 had the strongest activity, indicating PPP-2 was the major antiviral component. The structure-activity relationship showed that the antiviral activities of polysaccharide fractions were affected by their monosaccharide composition, molecular weight, and triple helical structure, which was a result of a combination of multiple molecular structural factors. These results showed that the PPP-2 could be exploited as a valued product for replacing synthetic antiviral drugs, and provided support for future applications of polysaccharide from Pinus massoniana pollen as a useful source for antiviral agent.

Composition, physicochemical properties, and anti-fatigue activity of water-soluble okra (Abelmoschus esculentus) stem pectins.

Okra, Abelmoschus esculentus (L.) Moench, an annual herbaceous plant, is widely distributed in tropical and subtropical regions. Water-soluble pectic hydrocolloids from okra stems (HOS) were extracted and purified using polydivinylbenzene HP-20 resins. The sugar composition of the purified HOS with an weight-average molecular weight of 178.4 ± 2.1 kDa and a polydispersity index of 1.02 ± 0.02 contained galacturonic acid (34%), galactose (31%), rhamnose (21%), arabinose (4.2%), glucuronic acid (2.5%), xylose (1.2%), and other monosaccharides (6.1%) by weight. Its favorable rheological behaviors were evident on relatively higher concentrations (20, 25, and 30 mg/mL) and moderately lower pH levels (3 and 5) of HOS. The anti-fatigue experiments in vivo demonstrated that a high dose of HOS (450 mg/kg feed) prolonged the exhaustive swimming time of mice, significantly induced an increase in blood glucose and glycogen, and decreased lactic acid and serum urea nitrogen levels. HOS digestion in vivo was fairly conducive to the improvement of energy storage capacity and renal function for physically induced fatigue, compared with the conventional herbal supplement Panax quinquefolium. Accordingly, HOS exhibits potential for reutilization of okra stem waste.

The heteropolysaccharide of Mangifera indica fruit: Isolation, chemical profile, complexation with ß-lactoglobulin and antioxidant activity.

A 91 kDa heteropolysaccharide (F2) was isolated from Mangifera indica fruit via extraction with H2O, purification by C2H5OH, starch removal and ion exchange chromatography. This polymer was made up mostly of Ara, Gal, Glc, Rha, Xyl, and GalA in a 37: 29: 9:3:2:19 molar proportion. It inherited a small backbone containing GalpA and Rhap units substituted with very large side chains containing differently linked Ara and Gal units plus esterified gallic acid (GA) residue. Several enzymes generated oligosaccharides including (i) Ara2-10Ac6-22, (ii) Gal1-8Ac5-26 and (iii) GA1Gal1Ac7 were characterized. This polysaccharide, which showed dose dependent antioxidant activity, exhibited synergism with gallic acid, and formed a complex (K = 1.2 × 106 M-1) with ß-lactoglobulin. Accordingly, H2O treatment produces a polysaccharide with desired biochemical properties; this could be effective in designing innovative functional food with flexible makeup.

Studies on chemical composition, rheological and antioxidant properties of pectin isolated from Riang (Parkia timoriana (DC.) Merr.) pod.

Although synthetic antioxidant food additives are widely used in a variety of food products, some of them are suspected of having a noxious effect on human health. As a consequence, much research attention has been focused on developing natural antioxidant compounds from plants. Riang (Parkia timoriana (DC.) Merr.) is known as a traditional medicinal plant in which its various parts have been reported to exhibit antioxidant and numerous biological activities. In this study, pectins from Riang pod husk and pod powder were extracted, and their physico-chemical, rheological, and antioxidant properties were characterized. The extracted pectins showed high uronic acid content (> 65%) and high molecular weight (200-250 kDa) and the yields were approximately 15 and 36%w/w (dry basis), for Riang husk pectin (RHP) and Riang pod powder pectin (RPP), respectively. Furthermore, both pectins were classified as a high methoxyl with their DE of ~66%. Rheological measurements revealed a pseudoplastic behavior above 2% w/v. RHP contained higher content of total phenolics, flavonoids and tannin, compared with RPP. Antioxidant activities of RHP were consequently higher than RPP in all studied assays. The highest antioxidant activities of RHP and RPP, obtained from ABTS assay, were 0.95 and 0.24 mmol Trolox equivalents/g, respectively.

In Silico Identification of Potential Natural Product Inhibitors of Human Proteases Key to SARS-CoV-2 Infection.

Presently, there are no approved drugs or vaccines to treat COVID-19, which has spread to over 200 countries and at the time of writing was responsible for over 650,000 deaths worldwide. Recent studies have shown that two human proteases, TMPRSS2 and cathepsin L, play a key role in host cell entry of SARS-CoV-2. Importantly, inhibitors of these proteases were shown to block SARS-CoV-2 infection. Here, we perform virtual screening of 14,011 phytochemicals produced by Indian medicinal plants to identify natural product inhibitors of TMPRSS2 and cathepsin L. AutoDock Vina was used to perform molecular docking of phytochemicals against TMPRSS2 and cathepsin L. Potential phytochemical inhibitors were filtered by comparing their docked binding energies with those of known inhibitors of TMPRSS2 and cathepsin L. Further, the ligand binding site residues and non-covalent interactions between protein and ligand were used as an additional filter to identify phytochemical inhibitors that either bind to or form interactions with residues important for the specificity of the target proteases. This led to the identification of 96 inhibitors of TMPRSS2 and 9 inhibitors of cathepsin L among phytochemicals of Indian medicinal plants. Further, we have performed molecular dynamics (MD) simulations to analyze the stability of the protein-ligand complexes for the three top inhibitors of TMPRSS2 namely, qingdainone, edgeworoside C and adlumidine, and of cathepsin L namely, ararobinol, (+)-oxoturkiyenine and 3α,17α-cinchophylline. Interestingly, several herbal sources of identified phytochemical inhibitors have antiviral or anti-inflammatory use in traditional medicine. Further in vitro and in vivo testing is needed before clinical trials of the promising phytochemical inhibitors identified here.

3,4,5-Tri-O-caffeoylquinic acid methyl ester isolated from Lonicera japonica Thunb. Flower buds facilitates hepatitis B virus replication in HepG2.2.15 cells.

Caffeoylquinic acids are well known for their prominent antiviral activities. Beyond our expectations, we initially found 3,4,5-Tri-O-caffeoylquinic acid methyl ester (3,4,5-CQME) from L. japonica can facilitate HBV DNA and antigens secretion. This study aimed to investigate its underlying molecular mechanism. The results indicate that 3,4,5-CQME signally increased intracellular and secreted HBsAg levels by more than two times in HepG2.2.15 cells and HepAD38 cells. Furthermore, levels of HBeAg, HBV DNA and RNA were significantly enhanced by 3-day 3,4,5-CQME treatment; it didn't directly affect intracellular cccDNA amount, although it slightly increased cccDNA accumulation as a HBV DNA replication feedback. In addition, treatment with 3,4,5-CQME significantly induced HBx protein expression for viral replication. We utilized a phospho-antibody assay to profile the signal transduction change by 3,4,5-CQME to illuminate its molecular mechanism. The results indicate that treatment with 3,4,5-CQME activated AKT/mTOR, MAPK and NF-κB pathways verified by immunoblot. Moreover, 3,4,5-CQME upregulated the expression of nuclear transcriptional factors PGC1α and PPARα. In short, 3,4,5-CQME promotes HBV transcription and replication by upregulating HBx expression and activating HBV transcriptional regulation-related signals. As caffeoylquinic acids are widely present in traditional Chinese medicines, the risk of intaking caffeoylquinic acids-containing herbs for hepatitis B treatment requires more evaluation and further research.

Antioxidant and antihyperlipidemic activities of high sulfate content purified polysaccharide from Ulva pertusa.

In this study, purification of polysaccharide ulvan by anion exchange chromatography was prepared, and the major polysaccharide fraction (FU) was collected at 1.0 M NaCl elute by anion exchange chromatography, then high sulfate content purified ulvan (HFU) was prepared with sulfur trioxide/N,N-dimethylformamide (SO3-DMF) in formamide. The antioxidant activity and the antihyperlipidemic activity of HFU in mice were determined. The results showed that treatment with HFU could improve the antioxidant and antihyperlipidemic activity. Compared with the hyperlipidemic group, the antihyperlipidemic activity of HFU (125 mg/kg) was the strongest, TC concentrations were significantly decreased by 26.7% (P < .01), significantly reduced LDL-C (32.6%, P < .01), significantly increased HDL-C (19.6%, P < .01), and HFU-treated group (250 mg/kg) exhibited optimal effects on TG (29.0%, P < .01); the HFU groups at the doses of 125 mg/kg could significantly decrease the MDA (29.9%, P < .01); the HFU groups at the doses of 500 mg/kg could increase the activities of GSH-Px obviously (19.9%, P < .01).

Arsenosugar standards extracted from algae: Isolation, characterization and use for identification and quantification purposes.

Sulfate (SO4-sug) and sulfonate (SO3-sug) arsenosugar standard solutions were obtained using preparative liquid chromatography. Several commercial algae samples were characterized (total contents and speciation) to select the most appropriate in relation to their arsenosugar contents. Water extracts from the selected sample (Fucus vesiculosus) were fractionated using a Hamilton PRP-X100 preparative column, and the presence of arsenic species in the isolated fractions was ascertained by IC-ICP-MS. Two of the fractions successfully presented only one arsenic species corresponding to sulfate and sulfonate arsenosugars at suitable concentrations. To unequivocally confirm the presence of both compounds, high-resolution mass spectrometry (ESI-TOF/MS) was used and the exact mass determined with errors lower than 0.5 ppm. The standard solutions obtained were successfully used to identify and quantify SO4-sug and SO3-sug in several edible algae samples purchased in local market. Total arsenic content for analyzed samples ranged from 34 to 57 mg kg-1, concentration values found for SO3-sug ranged from 5 to 36 mg As kg-1 and SO4-sug was only found in fucus with a concentration of 9.3 mg As kg-1.

Microwave-Assisted Extraction, Purification, Partial Characterization, and Bioactivity of Polysaccharides from Panax ginseng.

Polysaccharides are a main active substance in Panax ginseng; however, microwave-assisted extraction used to prepare P. ginseng polysaccharides (MPPG) has rarely been reported, and knowledge of the bactericidal activity of P. ginseng polysaccharides remains low. Thus, this study was designed to investigate the extraction of P. ginseng polysaccharides by using two methods-hot water extraction and microwave-assisted extraction-and compare their chemical composition and structure. In addition, their antibacterial and antioxidant activities were also determined. The data implied that P. ginseng polysaccharides extracted by microwave-assisted extraction possessed a higher extraction yield than hot water extraction (WPPG) under optimized conditions, and the actual yields were 41.6% ± 0.09% and 28.5% ± 1.62%, respectively. Moreover, the preliminary characterization of polysaccharides was identified after purification. The WPPG with the molecular weight (Mw) of 2.07 × 105 Da was composed of Man, Rib, Rha, GalA, Glu, Gal, and Arab, and the typical characteristics of polysaccharides were determined by IR spectra. Compared with WPPG, MPPG had a higher Mw, uronic acid content, and Glu content. More importantly, the antioxidant activity of MPPG was higher than WPPG, which was probably ascribed to its highly Mw and abundant uronic acid content. Besides, both of them exhibited high bactericidal activity. These results demonstrate that microwave-assisted extraction is an effective method for obtaining P. ginseng polysaccharides, and MPPG could be applied as an antioxidant and antibacterial agent.

Chemical and bioactivity screening of subcritical water extracts of chokeberry (Aronia melanocarpa) stems.

Subcritical water extracts of chokeberry (Aronia melanocarpa) stems were chemically and biologically characterised. Chemical profile was defined by GC-MS analysis whereas anti-oxidant, anti-diabetic and tyrosinase-inhibitory activities of the extracts were investigated by in vitro assays. Antioxidant activity assays revealed strong activity against DPPH radical (IC50 = 0.1 mg/mL) and reducing power (IC50 = 0.25 mg/mL). The extracts demonstrated remarkable amylase (0.59 mmol ACAE/g) and glucosidase (7.50 mmol ACAE/g) inhibitory effects. Anti-tyrosinase activity of aronia stem extracts obtained by subcritical water was calculated to be 15.87 mg KAE/g extract. GC-MS analysis of chokeberry stem subcritical water extracts revealed the presence of different chemical classes. The compounds present in the highest concentrations were polyols arabitol (13.7%), xylitol (3.5%), and glycerol (1.96%), as well as sugars such as fructose (3.04%), ribose (1.99%) and xylulose (1.18%).

Relationship between structural properties and antitumor activity of Astragalus polysaccharides extracted with different temperatures.

This study investigated the effects of different temperatures on structural characterization and antitumor activity of polysaccharides from Astragalus membranaceus. APS4 and APS90 were extracted at 4°C and 90°C, respectively, and purified by Sephadex G-200 column. APS4-90 were obtained from APS4 after treatment at 90°C for 6h. MTT results showed that APS4 possessed the highest inhibitory effects on MGC-803, A549 and HepG2 cells. HPGPC analysis showed that the average molecular weights of these polysaccharides were approximately 1.5×106Da, while the asymmetrical peak of APS4-90 suggested heat degradation and configuration changes of APS4. GC, NMR and methylation results showed that these three polysaccharides had similar monosaccharide components (mainly contain glucose), and their backbones were composed of (1→2)­α­d­Glcp. However, APS4 showed higher content of (1→2,6)­α­d­Glcp compared to APS4-90 and APS90, which indicated that higher branched degree would be responsible for the stronger in vitro antitumor activity in APS4. These results were also confirmed by specific rotation and SEM analysis. Our study suggested that APS4 had the potential application for cancer treatment.

Phenolic constituents of the aerial parts of Impatiens glandulifera Royle (Balsaminaceae) and their antioxidant activities.

As a continuation of investigating Impatiens L. genus, eight flavonoids, eriodyctiol, eriodyctiol 7-O-ß-ᴅ-glucoside, kaempferol 3-O-ß-ᴅ-glucoside, kaempferol 3-O-ß-ᴅ-galactoside, kaempferol 3-rhamnosyl-di-glucoside, kaempferol 3-O-ß-ᴅ-rutinoside, quercetin 3-O-ß-ᴅ-glucoside and quercetin 3-O-ß-ᴅ-galactoside, two phenolic acids - p-hydroxybenzoic acid and protocatechuic acid, and 2-methoxynaphthalene-1,4-dione were isolated from the aerial parts of I. glandulifera collected in Poland. The structures of the compounds were established by analysis of their spectroscopic (1H and 13C NMR) and spectrometric (MS) data, as well as by comparison of these with those reported in the literature. Quercetin 3-O-ß-ᴅ-glucoside, kaempferol 3-O-ß-ᴅ-galactoside and kaempferol 3-O-ß-ᴅ-rutinoside were isolated for the first time from the investigated taxon. In addition, the antioxidant activities in different tests of all obtained compounds were evaluated. The results clearly showed that among analyzed constituents, quercetin 3-O-ß-ᴅ-glucoside exhibited antioxidant activity comparable or better than ascorbic acid and Trolox which were used as a positive control.

Quantitative ¹H-NMR Spectroscopy for Profiling Primary Metabolites in Mulberry Leaves.

The primary metabolites in aqueous extract of mulberry (Morus alba L.) leaves were characterized by using proton nuclear magnetic resonance (¹H-NMR) spectroscopy. With the convenience of resonance assignment, GABA together with the other 10 primary metabolites was simultaneously identified and quantified in one ¹H-NMR spectrum. In this study, external calibration curves for metabolites were employed to calculate the concentrations of interests. The proposed quantitative approach was demonstrated with good linearity (r² ranged in the interval of 0.9965-0.9999), precision, repeatability, stability (RSD values in the ranges of 0.35-4.89%, 0.77-7.13% and 0.28-2.33%, respectively) and accuracy (recovery rates from 89.2% to 118.5%). The established ¹H-NMR method was then successfully applied to quantify 11 primary metabolites in mulberry leaves from different geographical regions within a rapid analysis time and a simple sample preparation procedure.

[Quality Evaluation of Saposhnikoviae Radix (Differences between Wild-type and Cultivated Products)].

　Saposhnikoviae Radix ("Boufu") is an important crude drug used in Kampo formulation. It is extracted from wild-type plants. However, recently, extraction has become difficult because of a decrease in wild-type plants. Therefore, cultivated plants account for the majority of the market, from which the crude drug is extracted. However, the cultivation techniques used are not sufficient to obtain the desirable extracts. In this study, we compared the contents of the extract and the quantitative values of characteristic constituents obtained from wild-type and cultivated plants, and found a remarkable difference. Therefore, it is considered that these indicators play an important role in the establishment of better cultivation technology.