Targeted delivery to macrophages and dendritic cells by chemically modified mannose ligand-conjugated siRNA.

Extrahepatic delivery of small interfering RNAs (siRNAs) may have applications in the development of novel therapeutic approaches. However, reports on such approaches are limited, and the scarcity of reports concerning the systemically targeted delivery of siRNAs with effective gene silencing activity presents a challenge. We herein report for the first time the targeted delivery of CD206-targetable chemically modified mannose-siRNA (CMM-siRNA) conjugates to macrophages and dendritic cells (DCs). CMM-siRNA exhibited a strong binding ability to CD206 and selectively delivered contents to CD206-expressing macrophages and DCs. Furthermore, the conjugates demonstrated strong gene silencing ability with long-lasting effects and protein downregulation in CD206-expressing cells in vivo. These findings could broaden the use of siRNA technology, provide additional therapeutic opportunities, and establish a basis for further innovative approaches for the targeted delivery of siRNAs to not only macrophages and DCs but also other cell types.

Enhancement of Gene Knockdown on CD22-Expressing Cells by Chemically Modified Glycan Ligand-siRNA Conjugates.

Extrahepatic targeted delivery of oligonucleotides, such as small interfering RNA (siRNA) and antisense oligonucleotides (ASOs), is an attractive technology for the development of nucleic acid-based medicines. To target CD22-expressing B cells, several drug platforms have shown promise, including antibodies, antibody-drug conjugates, and nanoparticles, but to date CD22-targeted delivery of oligonucleotide therapeutics has not been reported. Here we report the uptake and enhancement of siRNA gene expression knockdown in CD22-expressing B cells using a chemically stabilized and modified CD22 glycan ligand-conjugated siRNA. This finding has the potential to broaden the use of siRNA technology, opening up novel therapeutic opportunities, and presents an innovative approach for targeted delivery of siRNAs to B cell lymphomas.

Quality evaluation of Pinellia tuber by LC-TOF/MS targeted to ephedrine.

Pinellia tuber (PTE, , , , , , , , ) is derived from the tuber of Pinellia ternata Breitenbach (Araceae), which is a crude drug used in traditional Japanese Kampo medicine for the purpose of antiemesis and expectoration. Since the separation of ephedrine from PTE in 1978, it has been listed as a PTE component in textbooks and internet information. Therefore, there are harmful effects on appropriate use in clinical practice because PTE is dealt with as a crude drug for doping target, and traditional Japanese Kampo medicine containing PTE must be carefully administered to the elderly. However, since the 1978 published report, there has not been any report on the isolation of ephedrine from PTE and the interpretation of biosynthesis remains questionable. In the present study, we analyzed the PTE samples in market distribution products by LC-TOF/MS. From the analysis of the result of ephedrine's m/z 148.113 [M + H-H2O]+, PTE was not detected (n = 55, detection limit: 0.5 ppb). Additionally, the tuber of P. tripartite (PTR, ), the tuber of P. pedatisecta (PPE, ), Arisaema Tuber (ART, ), and the tuber of Typhonium flagelliforme (TFI, ) that have a similar description to PTE were also not detected. Moreover, the genetic analysis of experimental samples showed that PTE is derived from P. ternata. Furthermore, our attempt to isolate ephedrine from PTE based on the past literature was unsuccessful. These results suggest that PTE in market distribution products may not contain ephedrine as a component.

Characterization of metabolites in Saposhnikovia divaricata root from Mongolia.

Saposhnikoviae Radix (SR), derived from the dried root and rhizome of Saposhnikovia divaricata, is a popular crude drug used in traditional Chinese and Japanese medicine. To evaluate the metabolites of S. divaricata roots from Mongolia and to investigate their geographical variation, we developed the HPLC method, determined the contents of 9 chromones and 4 coumarins, and conducted multivariate statistical analysis. All Mongolian specimens contained prim-O-glucosylcimifugin (1) and 4'-O-ß-D-glucosyl-5-O-methylvisamminol (3), and their total amount (5.04-25.06 mg/g) exceeded the criterion assigned in the Chinese Pharmacopoeia. Moreover, the content of 1 (3.98-20.79 mg/g) was significantly higher in the Mongolian specimens than in Chinese SR samples. The specimens from Norovlin showed the highest contents of 1 and 3. The total levels of dihydropyranochromones were higher in the specimens from Bayan-Uul. The orthogonal partial least squares-discriminant analysis revealed that the Mongolian specimens tended to be separated into three groups based on growing regions, in which several chromones contributed to each distribution. Furthermore, 1H NMR analysis revealed that Mongolian specimens had less amount of sucrose and a substantial amount of polyacetylenes. Thus, in this study, the chemical characteristics of Mongolian S. divaricata specimens were clarified and it was found that the specimens from the northeast part of Mongolia, including Norovlin, had the superior properties due to higher amounts of major chromones.

Physical and chemical characteristics of soils in Ephedra gerardiana and E. pachyclada habitats of Kali Gandaki Valley in Central Nepal.

In the Kali Gandaki Valley in Central Nepal, Ephedra gerardiana and E. pachyclada show species specificity for physical and chemical characteristics of soils. Here, the relationship between soil characteristics and ephedrine and pseudoephedrine contents was examined. E. gerardiana grew in moist alpine scrub and upper alpine meadow from 3735 to 4156 m a.s.l., while E. pachyclada grew in the lower Caragana steppe and dry alpine scrub from 2629 to 3671 m a.s.l. The soil texture of E. gerardiana and E. pachyclada collection sites were classified as loam or sandy loam mainly composed of sand and silt. Loss on ignition (%) of soil in E. gerardiana habitats (28.4-35.0%) was markedly higher than for that in E. pachyclada habitats (14.2-17.2%). E. pachyclada soil (pH 8.4-9.2) was more alkaline than that for E. gerardiana (pH 8.5). The five ions (Cl-, SO42-, Ca2+, Mg2+, and Na+) in soil of E. pachyclada (Cl-, 0.01-18.97 mmol/100 g dry soil weight; SO42-, 1.95-83.33; Ca2+, 3.79-77.91; Mg2+, 1.28-27.9; Na+, 0.94-34.49) were markedly higher than those of E. gerardiana (Cl-, 0.18-0.29; SO42-, 0.07-0.08; Ca2+, 4.19-4.59; Mg2+, 0.22-0.58; Na+, 0.93-1.40). The main factor contributing to strongly alkali soils for each species was different between E. gerardiana and E. pachyclada: CaCO3 for E. gerardiana and CaSO4, MgSO4, NaCl, or a combination of these for E. pachyclada. The total ephedrine and pseudoephedrine content in E. gerardiana and E. pachyclada ranged from 1.67-1.88%DW and 1.95-4.80%DW, respectively. Both E. gerardiana and E. pachyclada were amenable for use a raw material source for extraction of ephedrine and pseudoephedrine, and the ephedrine content of both species showed a statistically significantly positive correlation with Mg2+ and Na+ contents of the soil.

Ecological, phylogenetical, and pharmacognostical characteristics of Aconitum kiyomiense endemic to Hida highlands, Takayama city, Gifu Prefecture, Japan.

Aconitum kiyomiense Kadota (Ranunculaceae) is endemic to Takayama city, Gifu Prefecture, central Japan. We collected specimens from marshes and flood plains at altitudes ranging from 852 to 1085 m and from a new habitat consisting of a mesic meadow in the subalpine belt (1681 m). Glabrous pedicels and flowering sequence of inflorescence were used for identification, but intra-species variations in the pilus of pedicels (glabrous, pilose, and chimeric types) were observed. Although the flowering sequence has been reported as both indeterminate and determinate, all specimens in the present study were determinate. No intra-species variation was detected via partial nuclear internal transcribed space, and sequences did not match another 17 East Eurasian continent subgenus Aconitum species. The chloroplast trnL-trnF intergenic spacer region (trnL-trnF) showed three different haplotypes. The trnL-trnF dominant haplotype sequence was identical to that of A. kusnezoffii growing on the Eurasian continent, suggesting that A. kiyomiense is more primitive than other Japanese aconitum and a relic species of the Eurasian continent. We report the first detection of aconitine alkaloids in the tuberous roots, which exhibited aconitine alkaloid contents varying from 0.32 to 4.05 mg/g dry weight (mg/g) for aconitine, 0.02 to 4.12 mg/g for hypaconitine, undetectable to 0.05 mg/g for jesaconitine, and 0.42 to 3.76 mg/g for mesaconitine. The variation of aconitine alkaloid components and contents appeared to be random and did not vary with inflorescence phenotype, trnL-trnF haplotype, environmental habitat conditions, or the geographic region of the collection sites. Since most populations showed no genetic intra-variation, it will be necessary to maintain the continuity of habitats and designate areas for conservation of genetic diversity at the population level.

Effects of crushed Schisandra Fruit on the content of lignans in Kampo decoction.

Arboreous fruit of Schisandra chinensis Baillon, Schisandra Fruit (SF), is a crude drug used in Japanese traditional Kampo medicine. The marker compounds of SF for quality control are lignans, such as schizandrin (Sz) and gomisin A (GmA). Kampo formulation containing SF is usually prepared as decoctions in the dosage form of whole crude drug (W), as its size is small enough to measure using a spoon. However, in some traditional books, it has been described that SF must be used in the dosage form of crushed or cut pieces (Cr). In this study, we evaluated the transferring ratio of lignans from SF to the decoction, and the stability and taste of the decoctions of shoseiryuto (SST) and ninjin'yoeito (NYT) using each dosage form, i.e., Cr and W, of SF. The transferring ratio of Sz and GmA was significantly higher in the decoction prepared with the Cr form than that prepared using the W form in both SST and NYT. The concentration of Sz and GmA in the decoctions was stable when maintained at 4 °C for 35 days. The taste of SST decoction prepared using the Cr form was more acidic, harsher, and bitterer than SST decoction prepared using the W form, and the taste of NYT decoction prepared using the Cr form was harsher than NYT decoction prepared using the W form. In conclusion, when SF is used in Kampo prescription, crushing the fruits and seeds can increase its effectiveness.

Comparison of byakujutsu (Atractylodes rhizome) and sojutsu (Atractylodes lancea rhizome) on anti-inflammatory and immunostimulative effects in vitro.

The Japanese Pharmacopoeia defines byakujutsu (Atractylodes rhizome) as the rhizome of Atractylodes japonica or A. macrocephala and sojutsu (Atractylodes lancea rhizome) as the rhizome of A. lancea, A. chinensis, or their interspecific hybrids. Because their pharmaceutical uses differ in traditional Japanese Kampo medicine and traditional Chinese medicine, with less apparent scientific evidence, we compared the pharmacological properties between byakujutsu and sojutsu. Crude drug specimens of byakujutsu (n = 40) and sojutsu (n = 49) obtained in markets were identified by their species using DNA profiling. Their pharmacological properties were evaluated by the inhibitory effect of a MeOH extract of the samples on nitric oxide (NO) production by lipopolysaccharide-stimulated murine macrophage-like RAW264.7 cells and by the inducing effect of boiling water extract of the samples on granulocyte-colony stimulating factor (G-CSF) secretion from murine normal colonic epithelial MCE301 cells. We authenticated A. macrocephala (n = 8), A. japonica (n = 35), and the hybrid between A. macrocephala and A. japonica (n = 1), and they were used as byakujutsu. We authenticated A. chinensis (n = 25), A. lancea (n = 14), and the hybrid between A. chinensis and A. lancea (n = 6), and they were used as sojutsu. The inhibitory effects of byakujutsu on NO production were significantly higher than those of sojutsu (P < 0.05). This activity of A. japonica rhizome was significantly higher than that of A. macrocephala rhizome and A. lancea rhizome (P < 0.01). The activity of A. chinensis rhizome was significantly higher than that of A. lancea rhizome (P < 0.05). The extract of A. japonica rhizome significantly induced G-CSF secretion from MCE301 cells in a concentration-dependent manner. These effects of byakujutsu samples were not significantly different from those of sojutsu samples. A. japonica rhizome had significantly higher activity than A. macrocephala rhizome; however, there were no statistically significant differences among A. japonica, A. chinensis, and A. lancea. The pharmacological differences of byakujutsu and sojutsu may not be large among highly variated crude drug samples with average values, and quality control with the identification of the original plant species of byakujutsu and sojutsu may guarantee their pharmacological properties.

Survey on the Original Plant Species of Crude Drugs Distributed as Cynanchi Wilfordii Radix and Its Related Crude Drugs in the Korean and Chinese Markets.

Cynanchi Wilfordii Radix (CWR) is used in Korea as a substitute for Polygoni Multiflori Radix (PMR), which is a crude drug traditionally used in East Asian countries. Recently, the use of Cynanchi Auriculati Radix (CAR) in place of PMR and CWR has emerged a major concern in the Korean market. In Japan, PMR is permitted to be distributed as a pharmaceutical regulated by the Japanese Pharmacopoeia 17th edition (JP17). Although CWR and CAR have not traditionally been used as medicines, CWR was recently introduced as a health food. The distribution of unfamiliar CWR-containing products could lead to the misuse of original species for PMR and CWR like in Korea. To prevent this situation, the original species of plant products distributed as PMR, CWR, and CAR in the Korean and Chinese markets were surveyed and identified by their genes and components. The results revealed that all two PMR in the Korean market were misapplied as CAR, and that CAR was incorrectly used in eight of thirteen products distributed as CWR in both markets. As PMR is strictly controlled by JP17, the risk of mistaking PMR for CWR and CAR would be low in Japan. In contrast, the less stringent regulation of health food products and the present situation of misidentification of CWR in the Korean and Chinese markets could lead to unexpected health hazards. To ensure the quality and safety of crude drugs, it is important to use the information about the genes and components of these crude drugs.

A Novel Clerodane Diterpene from Vitex cofassus.

New clerodane diterpene, 16-hydroxy-pentandralactone (1) and known diterpene acuminolide (2) were isolated from the methanol extract of Vitex cofassus leaves. The chemical structure and the absolute configuration of 1 were determined by MS, NMR and electron circular dichroism (ECD) experiments. The isolated compounds were evaluated for their antiproliferative activities against a panel of human tumor cell lines, including a multidrug-resistant (MDR) cell line. Both compounds showed potent antiproliferative activities against all the tested cell lines with IC50 values of 5.4-11.4 µM. Their effects on cell viability were also tested using vascular endothelial growth factor (VEGF)-stimulated human umbilical vein endothelial cells (HUVECs). Compound 1 inhibited VEGF-stimulated HUVEC proliferation in a dose-dependent manner. Based on these results, compound 1 could be a candidate for antitumor agent and inhibitor of angiogenesis.

Naringenin interferes with the anti-diabetic actions of pioglitazone via pharmacodynamic interactions.

Pioglitazone is a peroxisome proliferator-activated receptor gamma (PPARγ) full agonist and useful for the treatment of type 2 diabetes mellitus. Naringenin is a citrus flavonoid with anti-inflammatory actions, which has been shown to prevent obesity-related diseases and to activate PPARγ. The aim of this study was to investigate whether dietary naringenin affects the actions of pioglitazone. We administered naringenin (100 mg/kg) and pioglitazone (10 mg/kg) to Tsumura Suzuki Obese Diabetes (TSOD) mice for 4 weeks and then conducted an oral glucose tolerance test. We found that oral administration of naringenin attenuated the hypoglycemic action of pioglitazone in TSOD mice. However, pioglitazone and naringenin did not affect fasting blood glucose levels, epididymal fat pad weight and body weight changes in this administration period. Pioglitazone suppressed expression of obesity-related adipokines such as tissue inhibitor of metalloproteinases-1 in adipose tissue of TSOD mice, but this effect was attenuated by naringenin. However, naringenin did not affect the pharmacokinetics of pioglitazone after single or repeated administration. Naringenin exhibited weak partial agonist activity in time-resolved fluorescence resonance energy transfer assay, but naringenin interfered with pioglitazone agonism, consistent with partial agonism. Our results suggest that it is advisable to avoid administering a combination of naringenin and pioglitazone.

Synthesis of a new glycosphingolipid, neurosporaside, from Neurospora crassa.

The glycosphingolipid neurosporaside (α-D-Glcp-(1 → 2)-ß-D-Galp-(1 → 6)-ß-D-Galp-(1 → 6)-ß-D-Galp-(1 →)-Cer) occurs in Neurospora crassa. We attempted to synthesize neurosporaside by block synthesis (route A) and linear synthesis (route B). Oligosaccharide derivatives were synthesized using trimethylsilyltrifluoromethanesulfonate and N-iodosuccinimide/trifluoromethane sulfonic acid as promoters. The target tetrasaccharide could not be attained via route A, but route B showed potential: glycosidic bonds (ß-D-Galp-(1 → 6)-ß-D-Galp-(1 → 6)-ß-D-Galp) were formed stereoselectively, leading to the synthesis of glycosphingolipid 2.

Diversity in aconitine alkaloid profile of Aconitum plants in Hokkaido contrasts with their genetic similarity.

Aconite tuber is a representative crude drug for warming the body internally in Japanese Kampo medicine and Chinese traditional medicine. The crude drug is used in major prescriptions for the aged. Varieties of Aconitum plants are distributed throughout the Japanese Islands, especially Hokkaido. With the aim of identifying the medicinal potential of Aconitum plants from Hokkaido, 107 specimens were collected from 36 sites in the summer of 2011 and 2012. Their nuclear DNA region, internal transcribed spacer (ITS), and aconitine alkaloid contents were analyzed. Phylogenic analysis of ITS by maximum parsimony analysis showed that the majority of the specimens were grouped into one cluster (cluster I), separated from the other cluster (cluster II) consisting of alpine specimens. The aconitine alkaloid content of the tuberous roots of 76 specimens showed 2 aspects-specimens from the same collection site showed similar aconitine alkaloid profiles, and cluster I specimens from different habitats showed various alkaloid profiles. Environmental pressure of each habitat is presumed to have caused the morphology and aconitine alkaloid profile of these genetically similar specimens to diversify.

Naringenin suppresses macrophage infiltration into adipose tissue in an early phase of high-fat diet-induced obesity.

Obese adipose tissue is characterized by increased macrophage infiltration, which results in chronic inflammation in adipose tissue and leads to obesity-related diseases such as type 2 diabetes mellitus and atherosclerosis. The regulation of macrophage infiltration into adipose tissue is an important strategy for preventing and treating obesity-related diseases. In this study, we report that naringenin, a citrus flavonoid, suppressed macrophage infiltration into adipose tissue induced by short-term (14 days) feeding of a high-fat diet in mice; although naringenin did not show any differences in high-fat diet-induced changes of serum biochemical parameters in this short administration period. Naringenin suppressed monocyte chemoattractant protein-1 (MCP-1) in adipose tissue, and this effect was mediated in part through inhibition of c-Jun NH2-terminal kinase pathway. Naringenin also inhibited MCP-1 expression in adipocytes, macrophages, and a co-culture of adipocytes and macrophages. Our results suggest a mechanism by which daily consumption of naringenin may exhibit preventive effects on obesity-related diseases.

The development of new molecular tools containing a chemically synthesized carbohydrate ligand for the elucidation of carbohydrate roles via photoaffinity labeling: carbohydrate-protein interactions are affected by the structures of the glycosidic bonds and the reducing-end sugar.

Photoaffinity labeling technology is a highly efficient method for cloning carbohydrate-binding proteins. When the carbohydrate probes are synthesized according to conventional methods, however, the reducing terminus of the sugar is opened to provide an acyclic structure. Our continued efforts to solve this problem led to the development of new molecular tools with an oligosaccharide structure that contains a phenyldiazirine group for the elucidation of carbohydrate-protein interactions. We investigated whether carbohydrate-lectin interactions are affected by differences in the glycosidic formation and synthesized three types of molecular tools containing Galp-GlcpNAc disaccharide ligands and a photoreactive group (1, 2, 3). Photoaffinity labeling validated the recognition of the new ligand by different glycosidic bonds. Photoaffinity labeling also demonstrated that both the reducing end sugar and non-reducing end sugar recognized the Erythrina cristagalli agglutinin.

Hot water extract of adzuki (Vigna angularis) suppresses antigen-stimulated degranulation in rat basophilic leukemia RBL-2H3 cells and passive cutaneous anaphylaxis reaction in mice.

The hot water extract of adzuki (HWEA), which is produced as a byproduct in the adzuki bean boiling process, has anti-tumor, antioxidative, and anti-diabetic activities. In this study, we fractionated HWEA to 4 fractions using stepwise gradient column chromatography with water and ethanol, and demonstrated the effects of each fraction on antigen (Ag)-stimulated degranulation in rat basophilic leukemia RBL-2H3 cells. The 40% ethanol eluate extract (EtEx.40) showed the strongest inhibition level of these fractions. To reveal the inhibitory mechanisms underlying degranulation by EtEx.40, we investigated intracellular reactive oxygen species (ROS) production, intracellular free Ca²âº concentration ([Ca²âº]i), and early intracellular signaling pathways. Treatment with EtEx.40 markedly inactivated Lyn following Ag stimulation, resulting in the suppressions of intracellular elevation of [Ca²âº]i and production of ROS. To identify the active compound in EtEx.40, we isolated 7 flavonoids from EtEx.40 and calculated their inhibition levels on Ag-stimulated degranulation. These flavonoids inhibited degranulation by about 25-60%. We further examined the in vivo effects of HWEA or EtEx.40 using a passive cutaneous anaphylaxis (PCA) reaction. Both extracts strongly suppressed the PCA reaction. These findings suggest that HWEA and/or EtEx.40 are beneficial for alleviating type I allergic symptoms.

Difference in cultivation characteristics and genetic polymorphism between Chinese and Japanese strains of Wolfiporia cocos Ryvarden et Gilbertson (Poria cocos Wolf).

Poria, a dried sclerotium of Wolfiporia cocos Ryvarden et Gilbertson (Polyporaceae) has been used as a crude drug in both Chinese and Japanese (Kampo) traditional medicines. Recently, cultivated products of Chinese Poria strains have accounted for most of the market, while the cultivation of Japanese Poria strains has not been successful. Aiming to determine the relationship between the differences in cultivation characteristics and genetic polymorphism, we conducted a field cultivation experiment, a rot test, and rapid amplification of polymorphic DNA (RAPD) analysis of Poria strains collected from China and Japan: 3 Chinese and 7 Japanese strains. In field cultivation, although there was no marked inferiority of Japanese strains to Chinese ones in either mycelium propagation or the rate of sclerotium formation, Chinese strains formed whiter sclerotia with a mean size more than twice that of Japanese ones. Representatives of Chinese and Japanese strains, Yunnan and Kaimondake, respectively, were tested for wood-rotting ability. More wood was utilized and the wood color was darker in trials of the Yunnan strain. Amplifications of total DNA of these 10 fungal strains with 2 primers, PC-6 and PC-11, in RAPD analysis showed a difference in the amplicon profile between Japanese and Chinese strains, suggesting differences in their genetic background.

The relationship between growth of the aerial part and alkaloid content variation in cultivated Aconitum carmichaeli Debeaux.

Processed root of aconite, Aconitum carmichaeli Debeaux--known as bushi in Japan--is indispensable for treating diseases among elderly persons in Japanese and Chinese traditional medicine. Its active component is bushi diester alkaloid (BDA), which consists of aconitine (ACO), mesaconitine (MES), hypaconitine (HYP), and jesaconitine (JES). Since an overdose of BDA results in severe side effects, the BDA content should be within safe limits. However, the BDA content of raw aconite root, even that produced by standard cultivation procedures, varies greatly. In this study, to clarify the cause of BDA variation, we examined the weight and BDA content of each part of cultivated A. carmichaeli: the aerial part, the mother tuberous root (MT), the daughter tuberous root (DT), and the rootlet (RL). We found the following positive relationships: between aerial part weight and DT weight, aerial part weight and BDA content in stem of apex, and BDA content in stem of apex and total BDA of DT attached to the plant. Furthermore, DT belonging to a higher weight group showed less BDA content variation. In addition, BDA of DT and those of MT and RL differ in both content and composition. In conclusion, it was suggested that the weight or the size of the aerial part was a good marker for monitoring BDA content and its variation in the tuberous root, and it was found to be desirable to prevent mixing MT and RL at harvest.

Novel phenolic glycosides, adenophorasides A-E, from Adenophora roots.

Five novel phenolic glycosides, adenophorasides A (1), B (2), C (3), D (4), and E (5), were isolated from commercial Adenophora roots, together with vanilloloside (6), 3,4-dimethoxybenzyl alcohol 7-O-beta-D: -glucopyranoside (7), and lobetyolin (8). The structures of the new compounds (1-5) were characterized as 4-hydroxy-3-methoxyphenylacetonitrile 4-O-beta-D: -glucopyranoside (1), 4-hydroxy-3-methoxyphenylacetonitrile 4-O-beta-D: -glucopyranosyl-(1-->6)-beta-D: -glucopyranoside (2), 4-hydroxy-3-methoxyphenylacetonitrile 4-O-alpha-L: -rhamnopyranosyl-(1-->6)-beta-D: -glucopyranoside (3), 4-hydroxyphenylacetonitrile 4-O-beta-D: -glucopyranosyl-(1-->6)-beta-D: -glucopyranoside (4), and 4-hydroxy-3-methoxybenzyl alcohol 4-O-beta-D: -glucopyranosyl-(1-->6)-beta-D: -glucopyranoside (5), respectively, by means of spectroscopic and chemical analyses.