Rhizobium puerariae sp. nov., an endophytic bacterium from the root nodules of the medicinal plant Pueraria candollei var. candollei.

A Gram-stain-negative, rod-shaped, motile bacterium, PC004T, was isolated from root nodules of the Thai medicinal plant Pueraria candollei var. candollei. 16S rRNA gene sequence analysis indicated that the strain is phylogenetically related to species in the genus Rhizobium, showing highest similarity (96.6 %) with Rhizobium mesosinicum HAMBI 3194T. The phylogenetic tree reconstructed based on 16S rRNA gene sequences showed that strain PC004T forms a cluster with Rhizobium petrolearium KCTC 23288T. Based on atpD, gyrB and recA gene sequences, strain PC004T also showed low similarity ( < 90 %) to reference strains. These phylogenetic data indicate that PC004T may represent a novel species. Strain PC004T also exhibited low DNA-DNA relatedness with R. mesosinicum HAMBI 3194T (8.2 %) and R. petrolearium KCTC 23288T (26.3 %). The DNA G+C content of strain PC004T was 64 mol%, which is within the range reported for the genus Rhizobium. The major fatty acid of PC004T was C18 : 1ω7c with minor amounts of C16 : 0, C16 : 0 3-OH, C18 : 0 3-OH, C18 : 1 2-OH, C19 : 0 cyclo ω8c and summed feature 2. The strain was able to grow at pH 12 and with up to 2 % (w/v) NaCl. Strain PC004T did not nodulate five tested legumes and the nifH and nodC genes were not detected by PCR. Based on the physiological, chemotaxonomic and phenotypic data from this study, strain PC004T represents a novel species of the genus Rhizobium, for which the name Rhizobium puerariae sp. nov. is proposed; the type strain is PC004T ( = BCC 73740T = NBRC 110722T).

In vitro propagation of Stemona hutanguriana W.Chuakul, an endangered medicinal plant.

In vitro propagation of Stemona hutanguriana W.Chuakul, an endangered species of Stemonaceae, was established. Nodal and internodal explants were cultured on Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) alone or in combination with 1-naphthaleneacetic acid (NAA). When cultured on MS medium supplemented with 18.16 µM TDZ with 0.54 µM NAA for 8 weeks and then on MS medium without plant growth regulator (PGR) for 8 weeks, nodal explants demonstrated a responding frequency of 91.67 % and a shoot regeneration rate of 5.46 shoots/responding explant. Furthermore, internodal explants demonstrated a responding frequency of 17.17 % with 11.17 shoots/responding explant. The regenerated shoots were rooted using a two-step protocol by culturing for 4 weeks on MS medium supplemented with various concentrations of NAA and Indole-3-butyric acid (IBA) and 8 weeks on MS medium without PGR. When 5.40 µM NAA was used as a PGR, the maximum root induction rate was 69.45 %. Regenerated plantlets were successfully transferred to soil.

The effect of Cissus quadrangularis L. on delaying bone loss in postmenopausal women with osteopenia: A randomized placebo-controlled trial.

BACKGROUND: Osteopenia refers to bone density that is not normal but also not as low as that noted in osteoporosis. Osteopenia leads to osteoporosis and increases the risk of fractures. Current research is focused on agents that will prevent or slow the progression of bone loss. On the basis of published evidence, Cissus quadrangularis (CQ) might potentially provide a novel natural treatment for osteopenia. PURPOSE: To determine the effect of 24 weeks of consecutive treatment with CQ on delaying bone loss and safety in postmenopausal women (PMW) with osteopenia. METHODS: This study is a randomized, placebo-controlled trial. Here, 134 enrolled PMW with osteopenia (> 40 years and having no period for 1-10 years) received CQ at 1.2 (CQ1.2) or 1.6 g/day (CQ1.6) or placebo. The %change in bone mineral density (BMD) at the lumbar spine (L1-L4), femoral neck, and total hip served as the primary outcome. The %change in bone turnover markers (BTMs), including C-terminal telopeptide of type 1 collagen (CTX) and procollagen type 1 amino-terminal propeptide (P1NP), was the secondary outcome. These outcomes were compared between the CQ vs. placebo group at weeks 12 and 24. The least significant change (LSC) was used to monitor clinical changes. The adverse events (AE) were monitored. RESULTS: A total of 108 participants completed this study. The %BMD changes in the CQ-treated groups did not differ at any site after 24 weeks compared to the placebo. Statistically significant differences were detected in CQ1.6 at the lumbar spine (0.011 ± 0.025 g/cm2, p = 0.008) and CQ1.2 at the femoral neck (-0.015 ± 0.036 g/cm2, p = 0.024) compared to baseline, but these changes did not exceed the LSC. Reduced bone remodeling activity was detected in both CQ-treated groups. Compared to the placebo, the %P1NP change was significantly reduced in CQ1.6 (-2.46 ± 26.05%; p < 0.01) at week 12 and in CQ1.2 (-3.36 ± 29.47%; p < 0.01) and CQ1.6 (-9.95 ± 22.22%; p < 0.01) at week 24. These results correlated with the within-group comparison, which showed a continuously significant increase in both BTMs in the placebo group. However, a stable CTX and a significant reduction in P1NP (p < 0.05) were detected in both CQ-treated groups. This reduction exceeded the LSC of P1NP. The incidence of adverse events did not differ among the three groups. CONCLUSION: This is the first clinical report that showed a promising effect on delaying bone loss of orally administration of CQ for 24 weeks, as indicated by a slower bone remodeling process via a reduction in BTMs. However, no change in BMD was observed.

Green extraction optimization of triterpenoid glycoside-enriched extract from Centella asiatica (L.) Urban using response surface methodology (RSM).

Centella asiatica (L.) Urban extracts are widely used as food, drugs and cosmetics, and the main active compounds are glycosides (madecassoside and asiaticoside) and aglycones (madecassic acid and asiatic acid). Green extraction is an interesting concept that can produce safe and high-quality extracts that use less solvent, time and energy with the environmental friendly. This study investigated the optimum conditions for extracting a triterpenoid glycoside-enriched C. asiatica extract using microwave-assisted extraction (MAE) and ultrasound-assisted extraction (UAE). Central composite design and response surface methodology (RSM) were used for the experimental design and data analysis. Four-month-old C. asiatica tetraploid plants were selected as the elite raw material containing high amount of triterpenoid glycosides for the extraction experiments, and the triterpenoid content was determined by a validated HPLC method. The results demonstrated that the RSM models and equations were reliable and could predict the optimal conditions to enhance C. asiatica extract yield, glycoside and aglycone amounts. The percent of ethanol was the major factor that had a significant effect on C. asiatica yield and glycoside and aglycone content during MAE and UAE. The maximum triterpenoids content in extract; 7.332 ± 0.386% w/w madecassoside and 4.560 ± 0.153% w/w asiaticoside 0.357 ± 0.013% w/w madecassic acid and 0.209 ± 0.025% w/w asiatic acid were obtained by MAE with 80% ethanol at 100 watts for 7.5 min, whereas the optimal conditions for highest total triterpenoids extraction from dry plant was UAE with 80% ethanol, temperature 48 °C, 50 min enhanced 2.262 ± 0.046% w/w madecassoside, 1.325 ± 0.062% w/w asiaticoside, 0.082 ± 0.009% w/w madecassic acid and 0.052 ± 0.007% w/w asiatic acid as secondary outcome. Moreover, it was found that MAE and UAE consumed energy 59 and 54%, respectively, lower than that of the conventional method, maceration, in term of kilowatt-hour per gram of total triterpenoids. These optimized green conditions could be recommended for C. asiatica extraction for triterpenoid glycoside-enriched extracts production for the pharmaceutical or cosmeceutical industries and triterpenoids quantitative analysis in raw materials.

The efficacy of honey and a Thai Herbal Oil preparation in the treatment of pressure ulcers based on Thai traditional medicine wound diagnosis versus standard practice: An open-label randomized controlled trial.

BACKGROUND: Scientific support for Thai traditional medicine (TTM) practice is warranted for reintroduction into modern healthcare systems. A promising TTM practice for treatment of pressure ulcers was selected to conduct a clinical trial. This study aimed to evaluate the efficacy of the TTM practice for the treatment of pressure ulcers using honey or a Thai Herbal Oil preparation (THO) based on the TTM wound diagnosis comparing with the standard practice. METHODS: The study design was an open-label randomized controlled trial. Sixty-six participants, with pressure ulcers at least stage II-IV or unstageable, were allocated to two groups via minimization. A TTM practice group received honey or THO depending on the TTM diagnosis via the Thai Traditional Medicine Pressure Ulcer Assessment Tool (TTM-PUAT). A standard practice group received advanced dressings, including hydrogel, alginate, silver-impregnated, or hydrocolloid dressings. The primary outcome was the Pressure Ulcer Scale for Healing (PUSH). RESULTS: Both TTM practice and standard practice showed a significant reduction in PUSH scores after treatments. However, there was no significant difference in PUSH score reduction between the groups. The mean PUSH score reduction over the 6-week period was 2.58 ± 3.38 (95% CI 1.34-3.82) in the TTM practice group and 3.24 ± 3.49 (95% CI 1.91-4.57) in the standard practice group (p = 0.284). The TTM practice and standard practice accelerated pressure ulcer healing without statistically significant difference between the practices, during 6 weeks in a home-based care setting. This finding supported the TTM practice as an alternative treatment for pressure ulcer.

Speed of action and stage specificity of Bencha-loga-wichian, a Thai traditional antipyretic formulation, against Plasmodium falciparum and the chloroquine-potentiating activity of its active compounds, tiliacorinine and yanangcorinine.

ETHNOPHARMACOLOGICAL RELEVANCE: Bencha-loga-wichian (BLW), a Thai traditional antipyretic formulation, has been reported to have promising antiplasmodial activity, and it was previously revealed that tiliacorinine and yanangcorinine, isolated from Tiliacora triandra, were the active compounds. However, the mechanisms of action of BLW have not been investigated. In addition, these active compounds are bisbenzylisoquinoline alkaloids, many compounds of which have been reported to potentiate the efficacy of chloroquine. AIMS OF THE STUDY: To investigate the antiplasmodial mechanisms of action of BLW and evaluate the effects of chloroquine combined with tiliacorinine or yanangcorinine. MATERIALS AND METHODS: Chloroquine-resistant Plasmodium falciparum (PfW2) strains at the ring, trophozoite, and schizont stages were exposed to the extracts or compounds for 2, 4, 6, 8, 10, 12, 24 or 48 h. The percentages of parasitemia were determined by flow cytometry, and their morphologies were examined by Giemsa-stained smear to evaluate the speed of action and stage specificity. For the drug combination assay, a modified fixed-ratio isobologram method was used. RESULTS: The antiplasmodial activity of BLW possessed a slow onset of action and was the most effective against ring-stage parasites. After 48 h of extracts or compounds exposure, most of the treated parasites, at all stages, turned to the pyknotic form and could not recover even after extracts or compounds removal. The results suggested that these extracts and compounds could kill the parasites or possess parasiticidal effects. In addition, the combination of chloroquine with tiliacorinine or yanangcorinine demonstrated a synergistic effect, indicating that these compounds could potentiate chloroquine efficacy against chloroquine-resistant parasites. CONCLUSION: The antiplasmodial mechanisms of action of BLW appeared to differ from that of chloroquine and other current antimalarial drugs. In addition, tiliacorinine and yanangcorinine, the active compounds of BLW, could potentiate the efficacy of chloroquine. Accordingly, BLW was shown to be a good candidate for development as a new antimalarial and useful for drug combination therapy.

Effects of methyl jasmonate on the growth and triterpenoid production of diploid and tetraploid Centella asiatica (L.) Urb. hairy root cultures.

In this study, the effects of methyl jasmonate (MeJA) on the phytomass and triterpenoid production of diploid and tetraploid Centella asiatica hairy roots were investigated. Hairy root cultures were obtained from diploid and induced tetraploid plants of C. asiatica infected by Agrobacterium rhizogenes strain ATCC 43057. MeJA triggered triterpenoid production in both ploidy hairy roots, whereas triterpenoids were not produced in the untreated hairy roots. Among the treatments, the 50 µM MeJA treatment yielded the maximum triterpenoid production in diploid hairy roots of 27.25 ± 0.27 µg/mg Dry weight (DW) total triterpenoid at day 21. For the tetraploid hairy root cultures, the 28th-day hairy root culture produced a maximum amount of triterpenoids of 16.29 ± 6.32 µg/mg DW in response to the 50 µM MeJA treatment, whereas the 100 µM MeJA treatment produced a similar triterpenoid amount (16.31 ± 9.24 µg/mg DW) at day 14. Moreover, in response to 50 µM MeJA, we obtained different ratios of aglycone to glycoside, i.e., 1:7 and 1:2, between the diploid and tetraploid hairy root cultures. Asiaticoside was the dominant phytochemical, followed by asiatic acid and madecassic acid. This study provides valuable information for producing triterpenoids for C. asiatica commercial products and preparations by using hairy root cultures.

Antiplasmodial activities of a Thai traditional antipyretic formulation, Bencha-Loga-Wichian: A comparative study between the roots and their substitutes, the stems.

ETHNOPHARMACOLOGICAL RELEVANCE: Bencha-Loga-Wichian (BLW) is a polyherbal antipyretic formulation that is comprised of Capparis micracantha, Clerodendrum indicum, Ficus racemosa, Harrisonia perforata, and Tiliacora triandra. A traditional medical textbook has documented the use of this formulation for the treatment of many types of fever, including malaria-like fever. Traditionally, BLW is composed of the root parts of those plants. However, in current practice, the stem parts are frequently substituted. Thus, this study aimed to evaluate the antiplasmodial activities of BLW and compare the efficacy between the stem and root parts. MATERIALS AND METHODS: BLW formulations produced from either the stem or root parts of the various constituent plants as well as the stems or roots of the individual plants were separately extracted and tested against the chloroquine-sensitive (Pf3D7) and -resistant (PfW2) strains Plasmodium falciparum using flow cytometry. The cytotoxicity against peripheral blood mononuclear cells was evaluated using the WST-8 assay to determine the selectivity index (SI). The active compounds of BLW were isolated using antiplasmodial-guided isolation and quantified using Ultra-Performance Liquid Chromatography (UPLC). RESULTS: The stem and root parts of BLW and the individual plants exhibited antiplasmodial activities at the same levels with good SI values in the range of 3.55-19.74. The extracts of BLW exhibited promising antiplasmodial activity against both Pf3D7 (IC50<5µg/mL) and PfW2 (IC50=6-10µg/mL). Among the five component plants, T. triandra was the most active and exhibited an IC50<5µg/mL against both strains of parasites with SI values >10. We isolated tiliacorinine and yanangcorinine as the major active compounds (IC50<2µg/mL). However, these two compounds demonstrated cytotoxic effects (SI<1). The UPLC analysis identified these compounds in both the stem and root parts of BLW in the range of 0.57-7.66%, which correlated with the antiplasmodial activity. The concentrations of these compounds in BLW, at comparable efficacy, were much less than those at the IC50s for the single compounds alone. It suggested that synergistic interactions increased the antiplasmodial effects as well as alleviated the toxicity of the active compounds in BLW. CONCLUSION: This study described a promising antiplasmodial activity of BLW that had good selectivity and a toxicity-alleviating effect. The results provided scientific support for the use of this formulation for the treatment of malaria. In addition, the stem and root parts of the plants in BLW exhibited equivalent activities, which indicates the potential for the substitution of the stem parts in the formulation. Thus, we recommend additional study of the stem parts of these plants for further development on the basis of the availability and sustainability.

Alkaline phosphatase activity-guided isolation of active compounds and new dammarane-type triterpenes from Cissus quadrangularis hexane extract.

ETHNOPHARMACOLOGICAL RELEVANCE: The stem of Cissus quadrangularis L. (CQ) is used in traditional medicine to treat bone fractures and swelling. Anti-osteoporotic activity of CQ hexane extract has been reported, but the active compounds in this extract remain unknown. Thus, we aimed to identify the active compounds in CQ hexane extract using bioassay-guided isolation. MATERIALS AND METHODS: The CQ hexane extract was fractionated sequentially with benzene, dichloromethane, ethyl acetate, and methanol. The examination of CQ extract and its fractions was guided by bioassays for alkaline phosphatase (ALP) activity during the differentiation of MC3T3-E1 osteoblastic cells. The cells were treated with or without the CQ extract and its fractions for a period of time, and then the stimulatory effect of the alkaline phosphatase enzyme, a bone differentiation marker, was investigated. The compounds obtained were structurally elucidated using spectroscopic techniques and re-evaluated for activity during bone differentiation. RESULTS: A total of 29 compounds were isolated, viz., triterpenes, fatty acid methyl esters, glycerolipids, steroids, phytols, and cerebrosides. Four new dammarane-type triterpenes were isolated for the first time from nature, and this report is the first to identify this group of compounds from the Vitaceae family. Seven compounds, viz., glycerolipids and squalene, stimulated ALP activity at a dose of 10µg/mL. Moreover, the synergistic effect of these compounds on bone formation was demonstrated. CONCLUSION: This report describes, for the first time, the isolation of active compounds from CQ hexane extract; these active compounds will be useful for the quality control of extracts from this plant used to treat osteoporosis.

New compounds, nanaomycin F and G, discovered by physicochemical screening from a culture broth of Streptomyces rosa subsp. notoensis OS-3966.

Two new compounds, nanaomycin F and G, were isolated by physicochemical screening method from cultured broth of Streptomyces rosa subsp. notoensis OS-3966, which is known to produce nanaomycin A, B, C, D, and E. Nanaomycin F is a new nanaomycin analog, a 4a-hydroxyl analog of nanaomycin B. Nanaomycin G has a unique skeleton with 1-indanone infused with a tetrahydropyran ring. Nanaomycin A possesses broad antimicrobial activity but nanaomycin F and G demonstrated no bioactivity against all bacteria and fungi tested in this study. In addition, in both nanaomycin F and G, the production of superoxide radicals was majorly decreased in comparison to nanaomycin A. It was considered that the antimicrobial properties were lost as a result of the decrease in production of the superoxide radicals.

Antifungal activity and local toxicity study of Alangium salviifolium subsp hexapetalum.

Alangium salviifolium subsp hexapetalum is a medicinal plant which has been traditionally used for tonic and treatment of hemorrhoid. This plant showed promising antimicrobial activity in our preliminary experiments, this study was, therefore, conducted to investigate its inhibitory effect against dermatomycotic organisms and its toxicity. The lyophilized powder extract (4.59%) of pulverized wood was tested for its inhibitory effect by agar disc diffusion test. The extract gave inhibitory zone diameters of 25.23 and 14.78 mm against 26 and 14 isolates of dermatophytes and Candida albicans, respectively. Ketoconazole, used as a reference antifungal agent, had inhibitory zone diameters of 33.15 and 27.93 mm against dermatophytes and C. albicans, respectively. There was no significant difference between the extract and ketoconazole in their inhibition against dermatophytes (p > 0.01), but their difference was significant against C. albicans (p < 0.01). Using Buehler' s method, different amounts of extract (3, 6, and 9 mg/inch2 gauze pad) were tested in five male New Zealand white rabbits. All tested amounts of extract did not induce dermatitis among those rabbits within 1 week. The results demonstrated the inhibitory effect of Alangium salviifolium subsp hexapetalum against fungi without any local toxicity; a tendency to further develop a herbal preparation for the treatment of some dermatomycotic infections.

Field evaluation of in vitro-induced tetraploid and diploid Centella asiatica (L.) Urban.

Centella asiatica-a medicinal plant that produces high-value active triterpenoids-is in increasing demand by the pharmaceutical and cosmetic industries. The aim of this study was to field-test one induced tetraploid and three diploid C. asiatica lines for the selection of high-quality plants with high phytomass and triterpenoid content and to determine their optimal harvesting time. All tested C. asiatica were micropropagated using an established protocol. One-month-old plantlets were acclimatized for the field experiment. The plants were grown in a randomized complete block design (RCBD) with three replications, ten plantlets per replication, and the experimental bed site was 0.6 × 1.0 m. Growth parameters, phytomass and the amounts of four active triterpenoids were evaluated. All lines exhibited the highest growth, yields and triterpenoids at 4 months after cultivation. The tetraploid line showed significantly better characteristics, i.e., larger leaf area, leaf width, petiole length, and greater yields, than diploid lines. Dry weight per cultivated area (77.53 ± 3.07 g/m(2)) and total triterpenoids (15.38 ± 0.76 % dry weight) were increased significantly in tetraploid plants of C. asiatica. Furthermore, the harvesting time had an effect on the yield and triterpenoid content (P < 0.001). In all tetraploid and diploid lines, the yields and triterpenoid content per cultivated area reached their maximum at 4 months after planting. Our results demonstrated that polyploidy induction is a beneficial tool that can be used to improve the medicinal value of C. asiatica.

Medicinal plants used with Thai Traditional Medicine in modern healthcare services: a case study in Kabchoeng Hospital, Surin Province, Thailand.

ETHNOPHARMACOLOGICAL RELEVANCE: Thai Traditional Medicine (TTM) is available in many modern hospitals in Thailand. However, there have been difficulties in integrating TTM, particularly the practices of the use of herbal medicines, into modern healthcare services. Kabchoeng Hospital is one hospital that has been able to overcome these difficulties. Thus, this study aimed to document the successful utilization of herbal medicine at Kabchoeng Hospital. The documentation focused on both the knowledge of medicinal plants and the success factors that facilitated the utilization of herbal medicine in the context of a modern hospital in Thailand. MATERIALS AND METHODS: Kabchoeng Hospital was intentionally selected for this case study. Participatory observation was used for the data collection. There were six groups of key informants: three applied Thai Traditional Medicine practitioners (ATTMPs), a pharmacist, two physicians, two folk healers, the head of an herbal cultivation and collection group, and 190 patients. The plant specimens were collected and identified based on the botanical literature and a comparison with authentic specimens; these identifications were assisted by microscopic and thin layer chromatography (TLC) techniques. RESULTS: Eighty-nine medicinal plants were used for the herbal preparations. The ATTMPs used these plants to prepare 29 standard herbal preparations and occasional extemporaneous preparations. Moreover, in this hospital, seven herbal preparations were purchased from herbal medicine manufacturers. In total, 36 preparations were used for 10 groups of symptoms, such as the treatment of respiratory system disorders, musculo-skeletal system disorders, and digestive system disorders. Four success factors that facilitated the utilization of herbal medicine at Kabchoeng Hospital were determined. These factors included a proper understanding of the uses of herbal medicines, the successful integration of the modern and TTM healthcare teams, the support of an herbal cultivation and collection group, and the acceptance of the local people. CONCLUSIONS: The practices that support the use of herbal medicine at Kabchoeng Hospital illustrated the successful application of TTM and also represented a model for the integration of TTM, and particularly the use of herbal medicine, into modern hospitals. This integration will be beneficial for sustainable healthcare systems in Thailand and in other countries where modern medicine is the mainstream medical system.

Micropropagation of a Thai medicinal plant for women's health, Curcuma comosa Roxb., via shoot and microrhizome inductions.

We studied the effects of explant types, plant growth regulators, and sucrose concentrations on shoot and microrhizome inductions of Curcuma comosa Roxb., an important Thai medicinal plant for women's health. Explant types significantly affected shoot induction of the plant. The maximum shoot multiplication rate of 11.82 ± 1.03 shoots/responding explant was obtained when culturing terminal bud explants on semi-solid Murashige and Skoog (MS) medium supplemented with 18.16 µM thidiazuron for 8 weeks. Subsequently, they were transferred to a semi-solid MS medium without plant growth regulators for 4 weeks. The regenerated shoots produced roots spontaneously. Rooted plantlets were successfully transferred to the soil. Microrhizome induction was significantly influenced by sucrose concentrations, but not by 6-benzyladenine (BA). Liquid MS medium with a combination of 17.76 µM BA and 50 g L(-1) sucrose was optimal for microrhizome induction of C. comosa. After 12 weeks of culture, the microrhizome induction rate was 3.36 ± 0.44 microrhizomes/responding explant. Starch accumulation in microrhizomes increased with higher sugar concentration and with longer duration of culture. The microrhizomes were allowed to germinate under greenhouse conditions and further developed into normal plants. The protocols established will be used for the production of uniform plantlets suitable for field plantation for the herbal industry.