Mass production of the ginsenoside Rg3(S) through the combinative use of two glycoside hydrolases

The ginsenoside Rg₃(S), which is one of the exceptional components of Korean red ginseng extract, has been known to have anti-cancer, anti-metastatic, and anti-obesity effects. An enzymatic bioconversion method was developed to obtain the ginsenoside Rg₃(S) with a high specificity, yield, and purity. Two glycoside hydrolases (BglBX10 and Abf22-3) were employed to produce Rg₃(S) as a 100 g unit. The conversion reaction transformed ginsenoside Rc to Rd using Abf22-3, followed by Rb₁ and Rd to Rg₃(S), using BglBX10. It was performed in a 10 L jar fermenter at pH 6.0 and 37 °C for 24 h, with a high concentration of 50 mg/ml of purified ginsenoside mixture obtained from ginseng roots. Finally, 144 g of Rg₃(S) was produced from 250 g of root extract with 78 ± 1.2% chromatographic purity. These results suggest that this enzymatic method would be useful in the preparation of ginsenoside Rg₃(S) for the functional food and pharmaceutical industries.     

The processing of Panax notoginseng and the transformation of its saponin components

Notoginseng, the root of Panax notoginseng (Burk.) F. H. Chen (Araliaceae), a famous traditional Chinese medicine, has been used in both raw and processed forms due to their different therapeutic functions. In this study, HPLC analyses on saponin composition of processed notoginseng were conducted, which revealed that, during the steaming process, the five main saponin constituents (ginsenosides Rg₁, Rb₁, Rd, and Re, and notoginsenoside R₁) in raw notoginseng decreased gradually and some other new saponins were formed. Among these, eight newly converted ginsenosides were identified as 20(S)-Rh₁, 20(R)-Rh₁, Rk₃, Rh₄, 20(S)-Rg₃, 20(R)-Rg₃, Rk₁ and Rg₅. Different processing methods, including steaming and baking, were studied, along with the correlative dynamic curves of the transformation of saponins. The results indicated that the moisture content and temperature were the determinant effecting factors in the preparing process, and water content was necessary and high temperature was helpful for saponin transformation.     

Increase in antioxidant effect of ginsenoside Re-alanine mixture by Maillard reaction

Ginsenoside Re, one of the major triol type ginsenosides contained in Panax ginseng, has a hydrophobic four-ring steroid-like structure with hydrophilic sugar moieties at carbon-3 and -20. The aim of the present study was to identify the changes in structure and antioxidant activity of ginsenoside Re by the Maillard reaction, which has not been reported yet. The free radical-scavenging activity of ginsenoside Re-alanine mixture was increased by heat-processing. Ginsenoside Re was gradually changed into Rg₂, Rg₆ and F₄ by heat-processing, and the glucosyl moiety at carbon-20 was separated. The improved-free radical-scavenging activity by heat-processing was mediated by the generation of antioxidant Maillard reaction products (MRPs). Antioxidant MRPs were generated from the reaction of glucose and alanine. Based on the viability results of LLC-PK1 renal epithelial cells, MRPs and less-polar ginsenosides contributed to the combined renoprotective effect against oxidative renal damage. Maillard reaction is importantly involved in the increased antioxidant effect of ginsenoside by heat-processing.     

Investigation of ginsenosides in different parts and ages of Panax ginseng

Panax ginseng (P. ginseng) has been used as a traditional medicine for thousands of years. It includes P. ginseng root, leaf, root-hair, rhizome and stem. P. ginseng root is usually considered to be the main part for medicine, and other parts of P. ginseng are neglected. In this paper the content of ginsenosides in different parts and ages of P. ginseng was determined. Separation and determination of seven major ginsenosides, including Rg₁, Re, Rb₁, Rc, Rb₂, Rb₃ and Rd, has been achieved by high performance liquid chromatography (HPLC) with UV detector at 203 nm. The extraction of ginsenosides from P. ginseng material was performed by microwave-assisted extraction (MAE). The results indicate that the content of ginsenosides is higher in the leaf and root-hair, and lower in stem than that in other parts of P. ginseng. The content of ginsenosides in root and root-hair increases with increase in age of P. ginseng from one to five years. However, the total content of ginsenosides in P. ginseng leaf decreases with the increase in age.     

Cyclooxygenase inhibitory activity of ginsenosides from heat-processed ginseng

Ginsenosides, from heat-processed ginseng, and sapogenins were evaluated for their inhibitory effects on the enzyme catalytic activities of cyclooxygenases-1 and -2 (COX-1 and -2). The ginsenosides, 20(S)-Rg₃, Rg₅, and Rk₁, inhibited COX-2 activity, but did not affect the enzyme activity of COX-1. Protopanaxatriol (PPT) moderately inhibited both COX-1 and -2. The ginsenosides, 20(R)-Rg₃, Re, and protopanaxadiol (PPD) showed a minimal effect on COX-1 and -2 activities. Taken together, ginsenosides Rg₃ (20S-form), Rg₅ and Rk₁ showed selective inhibitory activity on COX-2 by behaving as an inhibitor of the enzyme–substrate reaction.     

Analysis of ginsenosides in Panax ginseng in high pressure microwave-assisted extraction

High pressure microwave assisted extraction (HPMAE) was applied to extract the ginsenosides from Panax ginseng root. The influences of extraction solvent, extraction pressure and extraction time were individually investigated. HPMAE has been compared with other extraction methods, including Soxhlet extraction, ultrasound-assisted extraction and heat reflux extraction. The determination of ginsenosides was performed by HPLC–ESI-MS. The results indicated that the HPMAE not only took a shorter time but also afforded higher extraction yields of ginsenosides, especially ginsenoside Rb₁, Rc, Rb₂ and Rd. Furthermore, the neutral ginsenosides and malonyl ginsenosides in Panax ginseng root extracts by HPMAE were investigated. The malonyl ginsenoside m-Rb₁, m-Rc, m-Rb₂ and m-Rd degraded in HPMAE at 400 kPa (109–112 °C) in 70% (v/v) ethanol–water and at 600 kPa (112–115 °C) in methanol, and transformed into corresponding neutral ginsenoside Rb₁, Rc, Rb₂ and Rd. Using water as extraction solution, the neutral ginsenosides degraded under HPMAE at 400 kPa (135–140 °C), and transformed into less polarity rare ginsenosides.     

Study on ginsenosides in different parts and ages of Panax quinquefolius L.

The contents of 12 ginsenosides (Rg₁, Re, F₁₁, Rf, Rg₂, Rh₁, Rb₁, Rc, Rb₂, Rb₃, Rd, Rh₂) in different parts and ages of Panax quinquefolius L. (American ginseng) were quantified by high pressure microwave-assisted extraction (HPMAE) high-performance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD). The analytical method was established and analytical performances were evaluated. The chemical marker of American ginseng F₁₁ was detected, and Rf which is the chemical marker of Asian ginseng was not found. Rare ginsenoside Rh₁, Rg₂ and Rh₂ were also studied in this experiment. The total contents of these 12 ginsenosides in the different parts of 5-year-old American ginseng follow this order: leaf > root-hair > rhizome > root > stem. Therefore, compared with the other parts of American ginseng, the leaf is a better available source of ginsenosides. The contents of ginsenosides in root and leaf also change with age.     

Biotransformation of ginsenoside Rd in the ginseng extraction residue by fermentation with lingzhi (Ganoderma lucidum)

Ginseng and lingzhi (Ganoderma lucidum) both are valuable traditional Chinese medicines and have been extensively utilised in functional foods and traditional medicines in many Asian countries. However, massive quantity of ginseng residue is produced after extraction of ginseng which still contains a lot of bioactive compounds such as ginsenosides. The goal of this study was to reuse the American ginseng extraction residue as the fermentation medium of G. lucidum to produce bioactive ginsenoside enriched biotransformation products. The changes of ginsenosides in the fermentation products were analysed during fermentation. Our results showed that after 30 days of fermentation, ginsenoside Rg1, Rd, and compound K (CK) significantly increased, especially Rd, while other ginsenosides (Re, Rb1 and Rc) decreased during fermentation. Ginsenoside Rd is the major ginsenoside in the final fermentation product. Furthermore, the biotransformation of ginsenosides was the major reaction in this fermentation process.     

Constituents of Sideritis syriaca. ssp. syriaca (Lamiaceae) and their antioxidant activity

The aerial parts of Sideritis syriaca ssp. syriaca (Lamiaceae) were extracted, after defatting, with diethyl ether, ethyl acetate and n-butanol. The antioxidant activities of the extracts were evaluated through in vitro model systems, such as 1,1-diphenyl-2-picryl hydrazyl (DPPH) and Co(II) EDTA-induced luminol chemiluminescence. In both model systems the ethyl acetate extract was the most effective. Phytochemical analysis of ethyl acetate extract showed the presence of two new isomeric compounds (1 and 1′), identified as 1-rhamnosyl, 1-coumaroyl, dihydrocaffeoyl, protocatechuic tetraester of quinic acid, as well as chlorogenic acid (2), apigenin 7-O-glucoside (3), apigenin (4), 4′-O-methylisoscutellarein 7-O-[6′′′-O-acetyl-β-D-allopyranosyl-(1 → 2)-β-d-glucopyranoside] (5), isoscutellarein 7-O-[6′′′-O-acetyl-β-D-allopyranosyl-(1 → 2)-β-d-glucopyranoside] (6), 4′-O-methylisoscutellarein 7-O[β-d-allopyranosyl-(1 → 2)-β-d-glucopyranoside] (7) and 4′-O-methylisoscutellarein 7-O-[β-d-allopyranosyl-(1 → 2)-6′′-O-acetyl-β-d-glucopyranoside] (8). The above compounds were identified by spectroscopic methods.     

Change of ginsenoside composition in red ginseng processed with citric acid

The purpose of this research was to investigate the composition of ginsenosides and to identify the chemical structures of ginsenosides generated from red ginseng by citric acid pre-treatment (RGC). The amount and the composition of ginsenosides in red ginseng (RG) and RGC were determined by high performance liquid chromatography (HPLC) analysis. The content of Rg₃, a well-known bioactive ginsenoside in RGC increased significantly (p<0.05) over 2 fold (0.411 mg/g) when compared with RG (0.186 mg/g). Moreover, the structures of 5 novel ginsenosides in RGC were investigated by liquid chromatography/mass spectrometry (LC/MS/MS) analysis. Three peaks were completely fragmentized from their mother ions to aglycones and suggested to be less polar ginsenosides Rk₃/Rh₄, Rk₁, and Rg₅. This study suggested that by processing red ginseng with citric acid it is possible to enhance the yield of both ginsenoside Rg₃ and less polar ginsenosides.     

Studies on cytotoxic triterpene saponins from the leaves of Aralia elata

Aralia elata has long been used as a tonic, anticancer and antidiabetic agent in China and Japan, and is widely consumed as food. Phytochemical investigation of the leaves of A. elata has led to the isolation of four new compounds, 3-O-[β-d-glucopyranosyl(1 → 3)-β-d-glucopyranosyl] echinocystic acid 28-O-β-d-glucopyranosyl ester (congmuyenoside I, 1), 3-O-[β-d-glucopyranosyl(1 → 2)-β-d-glucopyranosyl] hederagenin 28-O-β-d-glucopyranosyl ester (congmuyenoside II, 2), 3-O-{[β-d-glucopyranosyl(1 → 2)]-[β-d-glucopyranosyl(1 → 3)-β-d-glucopyranosyl(1 → 3)]-β-d-glucopyranosyl} echinocystic acid 28-O-β-d-glucopyranosyl ester (congmuyenoside III, 3) and 3-O-β-d-glucopyranosyl caulophyllogenin 28-O-β-d-glucopyranosyl ester (congmuyenoside IV, 4), and eight known triterpene saponins (5–12). The structural determination was accomplished with spectroscopic analysis, in particularly ¹³C NMR, 2D NMR and HR-ESI-MS techniques. In addition, compounds 5–10 were found for the first time in the genus Aralia. Compounds 1–12 were tested for their inhibition of the growth of HL60, A549 and DU145 cancer cells. In addition, compound 8 showed significant cytotoxic activities against HL60, A549 and DU145 cancer cells with IC₅₀ values of 15.62, 11.25 and 7.59 μM, respectively.     

Isolation and structural characterisation of five new and 14 known metabolites from the commercial starfish Archaster typicus

From the commercially available starfish Archaster typicus, five new (1–5) and 14 known (6–19) metabolites were isolated and identified. Detailed 1D and 2D NMR spectroscopic data, including ¹H, ¹³C, DEPT, HSQC, HMBC, and NOESY, established the structures of the new metabolites as sodium 5α-cholesta-9(11),24-dien-3β,6α,20β-triol-23-one 3-sulphate (1), sodium 5α-cholesta-9(11)-en-3β,6α,20β-triol-23-one 3-sulphate (2), sodium (25R)-5α-cholestane-3β,4β,6α,8,14α,15β,26-heptaol-15-sulphate (3), sodium (25R)-5α-cholestane-3β,6α,8,14α,15β,26-hexaol 15-sulphate (4), and sodium cholest-25(27)-ene-3β,4β,5α,6α,7β,8β,14α,15α,24,26-decanol 6-sulphate (5). Other spectroscopic techniques, including IR, ESI–MS, and HR-ESI–MS, were also adopted to further confirm the structures of the metabolites. These five steroids (1–5) are reported in nature for the first time. All of the steroids found in A. typicus (1–12) were tested for anticancer activities against MDA-MB-435 and Colo205 tumour cells. However, only sodium 5α-cholesta-9(11)-en-3β,6α,20β-triol-23-one 3-sulphate (2) and 27-nor-5α-cholestane-3β,4β,5,6α,7β,8,14,15α,24α-nonaol (6) exhibited weak activities.     

Spirostane and cholestane glycosides from the bulbs of Allium nigrum L

A phytochemical investigation of the fresh bulbs of Allium nigrum L. led to the isolation of new spirostane-type glycosides as two inseparable isomer mixtures, nigrosides A1/A2 (1a/1b) and nigrosides B1/B2 (2a/2b), two new cholestane-type glycosides, nigrosides C and D (3 and 4), together with the known compounds, 25(R,S)-5α-spirostan-2α,3β,6β-trio1-3-O-β-d-glucopyranosyl-(1 → 2)-O-[β-d-xylopyranosyl-(1 → 3)]-O-β-d-glucopyranosyl-(1 → 4)-β-d-galactopyranoside (5a/5b) and 25(R,S)-5α-spirostan-2α,3β,6β-trio1 3-O-β-d-glucopyranosyl-(1 → 2)-O-[4-O-(3S)-3-hydroxy-3-methylglutaryl-β-d-xylopyranosyl-(1 → 3)]-O-β-d-glucopyranosyl-(1 → 4)-β-d-galactopyranoside (6a/6b), isolated from this plant for the first time. All structures were elucidated mainly by spectroscopic analysis (1D and 2D NMR experiments, FABMS, HRESIMS) and by comparison with literature data. Cytotoxicity of the isolated compounds was assessed against human colon carcinoma (HT-29 and HCT 116) cell lines. Compounds 5a/5b and 6a/6b were found to be the most active with IC₅₀ values 1.09 and 2.82 μM against HT-29 and 1.59 and 3.45 μM against HCT 116, respectively.     

Terpenoids and hexenes from the leaves of Crataegus pinnatifida

Crataegus pinnatifida have long been used in traditional Chinese medicine and European herbal medicine, and are widely consumed as food, in the form of juice, drink, jam and canned fruit. Four new compounds, a sesquiterpene and its glycoside (1–2), two monoterpene glycosides (3–4), together with eight known compounds (5–12), were isolated from the leaves of C. pinnatifida. Their structures were elucidated as (5Z)-6-[5-(2-hydroxypropan-2-yl)-2-methyltetrahydrofuran-2-yl]-3-methylhexa-1,5-dien-3-ol (1), (5Z)-6-[5-(2-O-β-d-glucopyranosyl-propan-2-yl)-2-methyl tetrahydrofuran-2-yl]-3-methylhexa-1,5-dien-3-ol (2), 5-ethenyl-2-[2-O-β-d-glucopyranosyl-(1″ → 6′)-β-d-glucopyranosyl-propan-2-yl]-5-methyltetrahydrofuran-2-ol (3), 4-[4β-O-β-d-xylopyranosyl-(1″ → 6′)-β-d-glucopyranosyl-2,6,6-trimethyl-1-cyclohexen-1-yl]-butan-2-one (4), (Z)-3-hexenyl O-β-d-glucopyranosyl-(1″ → 6′)-β-d-glucopyranoside (5), (Z)-3-hexenyl O-β-d-xylopyranosyl-(1″ → 6′)-β-d-glucopyranoside (6), (Z)-3-hexenyl O-β-d-rhamnopyranosyl-(1″ → 6′)-β-d-glucopyranoside (7), (3R,5S,6S,7E,9S)-megastiman-7-ene-3,5,6,9-tetrol (8), (3R,5S,6S,7E,9S)-megastigman-7-ene-3,5,6,9-tetrol9-O-β-d-glucopyranoside (9), (6S,7E,9R)-6,9-dihydroxy-4,7-megastigmadien-3-one 9-O-[β-d-xylopyranosyl-(1″ → 6′)-β-d-glucopyranoside] (10), Linarionoside C (11), and (3S,9R)-3,9-dihydroxy-megastigman-5-ene 3-O-primeveroside (12), using a combination of mass spectroscopy, 1D and 2D NMR spectroscopy and chemical analysis. Cytotoxicity of the new compounds was assayed against selected human glioma (U87) cell lines.     

Influence of Aging Process on the Bioactive Components and Antioxidant Activity of Ginseng (Panax ginseng L.)

The effects of aging process on the ginsenosides and antioxidant activity of ginseng was investigated. Fresh ginseng roots were aged in oven at 70 or 80 °C for 7, 14, 21, or 28 d. Their ginsenosides, phenolics, and antioxidant activity were analyzed. Ginseng aged at 80 °C for 14 d exhibited the highest amounts of total saponins and phenolics. It also showed markedly higher free radical scavenging activity, reducing power, and ferrous ion chelating ability than the other aged ginsengs. The ginsenosides Rb₁, Rb₃, Rg₃, Re, Rg₁, and Rg₂ were generated during aging. The Rg₂ was the most abundant ginsenoside in aged ginseng, with samples treated at 80 °C for 14 d having the highest amount. These findings provide the first evidence that aging, particularly at 80 °C for 14 d, could increase the bioactive compounds, indicating that this heating process may be useful in enhancing the biological activity of ginseng.     

Selective and differential enumerations of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium spp. in yoghurt--a review

Yoghurt is increasingly being used as a carrier of probiotic bacteria for their potential health benefits. To meet with a recommended level of ≥ 10⁶ viable cells/g of a product, assessment of viability of probiotic bacteria in market preparations is crucial. This requires a working method for selective enumeration of these probiotic bacteria and lactic acid bacteria in yoghurt such as Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, Lb. acidophilus, Lb. casei and Bifidobacterium. This chapter presents an overview of media that could be used for differential and selective enumerations of yoghurt bacteria. De Man Rogosa Sharpe agar containing fructose (MRSF), MRS agar pH 5.2 (MRS 5.2), reinforced clostridial prussian blue agar at pH 5.0 (RCPB 5.0) or reinforced clostridial agar at pH 5.3 (RCA 5.3) are suitable for enumeration of Lb. delbrueckii subsp. bulgaricus when the incubation is carried out at 45 °C for 72 h. S. thermophilus (ST) agar and M17 are recommended for selective enumeration of S. thermophilus. Selective enumeration of Lb. acidophilus in mixed culture could be made in Rogosa agar added with 5-bromo-4-chloro-3-indolyl-β-d-glucopyranoside (X-Glu) or MRS containing maltose (MRSM) and incubation in a 20% CO₂ atmosphere. Lb. casei could be selectively enumerated on specially formulated Lb. casei (LC) agar from products containing yoghurt starter bacteria (S. thermophilus and Lb. delbrueckii subsp. bulgaricus), Lb. acidophilus, Bifidobacterium spp. and Lb. casei. Bifidobacterium could be enumerated on MRS agar supplemented with nalidixic acid, paromomycin, neomycin sulphate and lithium chloride (MRS-NPNL) under anaerobic incubation at 37 °C for 72 h.     

Antifungal activities of triterpenoids from the roots of Astilbe myriantha Diels

During a search for plant extracts that possess activity against the fungi that cause diseases in plants, a 95% ethanol extract from the roots of Astilbe myriantha Diels showed inhibitory activity against Colletotrichum gloeosporioides. Bioassay-guided fractionation of the 95% ethanol extract led to the isolation of seven triterpenoids (1–7). Their structures were elucidated by spectroscopy and by a comparison with literature data. Among these compounds, 3β,6β,24-trihydroxyurs-12-en-27-oic acid (7) was the most active inhibitor of C. gloeosporioides, with >68.0% inhibition at 100 mg/ml after 72 h. Moreover, compound 7 displayed broad-spectrum inhibitory activity against Rhizoctonia solani, Fusarium oxysporum f. sp. Niveum, Fusarium oxysporum f. sp. Cubens, C. gloeosporioides, Penicillium citrinum, Colletotrichum lagenarium, and Penicillium digitatum, with EC50 values ranging from 13.9 to 34.0 μg/ml. The potential of compound 7 as a fungicide is therefore promising. In addition, compound 1 was found to be a new triterpenoid, 3β,6β-dihydroxyurs-12-en-7α,27α-olide, which possesses six rings.     

Toxicity of ethyl formate to adult Sitophilus oryzae (L.), Tribolium castaneum (herbst) and Rhyzopertha dominica (F.)

Fumigations were conducted using a continuous flow-through laboratory process to maintain constant concentrations of ethyl formate and low levels (<0.8%) of respiratory carbon dioxide. The procedure minimised the effects of sorption by exposing test insects without media and minimised the effect of carbon dioxide by use of continuous flow. The concentration×time (Ct) products of ethyl formate for adult Sitophilus oryzae, Tribolium castaneum and Rhyzopertha dominica at 25 °C and 70% relative humidity for the 6 h exposure were, respectively: (1) LD₅₀ 107.8, 108.8 and 72.8 mg h L⁻¹ and (2) LD_99.5 207.4, 167.1 and 122.2 mg h L⁻¹. Endpoint mortality was reached within 24 h of initial exposure.     

Spirostane,furostane and cholestane saponins from Persian leek with antifungal activity

A phytochemical investigation of the seeds of Persian leek afforded the isolation of two new spirostane glycosides, persicosides A (1) and B (2), four new furostane glycosides, isolated as a couple of inseparable mixture, persicosides C1/C2 (3a/3b) and D1/D2 (4a/4b), one cholestane glycoside, persicoside E (5), together with the furostane glycosides ceposides A1/A2 and C1/C2 (6a/6b and 7a/7b), tropeosides A1/A2 and B1/B2 (8a/8b and 9a/9b), and ascalonicoside A1/A2 (10a/10b), already described in white onion, red Tropea onion, and shallot, respectively. Structure elucidation of the compounds was carried out by comprehensive spectroscopic analyses, including 2D NMR spectroscopy and MS spectrometry, and by chemical evidences. The chemical structure of new compounds were identified as (25S)-spirostan-2α,3β,6β-triol 3-O-[β-d-glucopyranosyl-(1 → 3)] [β-d-xylopyranosyl-(1 → 2)]-β-d-glucopyranosyl-(1 → 4)-β-d-galactopyranoside (1), (25S)-spirostan-2α,3β,6β-triol 3-O-[β-d-xylopyranosyl-(1 → 3)] [α-l-rhamnopyranosyl-(1 → 2)]-β-d-glucopyranosyl-(1 → 4)-O-β-d-galactopyranoside (2), furosta-1β,3β,22ξ,26-tetraol 5-en 1-O-β-d-glucopyranosyl (1 → 3)-β-d-glucopyranosyl (1 → 2)-β-d-galactopyranosyl 26-O-α-l-rhamnopyranosyl (1 → 2)-β-d-galactopyranoside (3a,3b), furosta-2α,3β,22ξ,26-tetraol 3-O-β-d-glucopyranosyl (1 → 3)-β-d-glucopyranosyl (1 → 2)-β-d-galactopyranosyl 26-O-β-d-glucopyranoside (4a,4b), (22S)-cholesta-1β,3β,16β,22β-tetraol 5-en 1-O-α-l-rhamnopyranosyl 16-O-α-l-rhamnopyranosyl (1 → 2)-β-d-galactopyranoside (5).     

Insecticidal activity of asarones identified in Acorus gramineus rhizome against three coleopteran stored-product insects

The insecticidal activities of Acorus gramineus rhizome-derived materials against adults of Sitophilus oryzae (L.), Callosobruchus chinensis (L.) and Lasioderma serricorne (F.) were examined using direct contact application and fumigation methods. The biologically active constituents of the Acorus rhizome were characterized as the phenylpropenes (Z)- and (E)-asarones by spectroscopic analysis. Responses varied with insect species, compound and exposure time rather than dose. In a filter paper diffusion test, (Z)-asarone caused 70% and 90% mortality against S. oryzae adults at 0.064 and 0.255 mg/cm² at 4 days after treatment, respectively, with 100% mortality at 7 days after treatment. (E)-Asarone at 0.255 mg/cm² was almost ineffective against S. oryzae adults at 7 days after treatment. Against C. chinensis adults at 0.064 mg/cm², (Z)- and (E)-asarones gave 100% mortality at 3 and 7 days after treatment, respectively. Against L. serricorne adults, (Z)-asarone gave 90% and 83% mortality at 0.255 and 0.064 mg/cm² at 7 days after treatment, respectively, whereas (E)-asarone at 0.255 mg/cm² was almost ineffective at 7 days after treatment. These results indicate that the toxicity of asarones might be due to the cis configuration rather than to the position of the double bond. In a fumigation test, (Z)-asarone at 0.577 mg/cm² was much more effective against adults of all three insect species in sealed containers than in open ones, indicating that the insecticidal activity of the compound was largely attributable to fumigant action.