Biological Activities of Deer Antler-Derived Peptides on Human Chondrocyte and Bone Metabolism.

Orally administered "tortoiseshell and deer antler gelatin" is a common traditional medicine for patients with osteoporosis or osteoarthritis. From the pepsin-digested gelatin, we previously isolated and identified the osteoblast-stimulating pentapeptide, TSKYR. Its trypsin digestion products include the dipeptide YR, enhancing calcium ion uptake, and tripeptide TSK, resulting in remarkable 30- and 50-fold increases in mineralized nodule area and density in human osteoblast cells. These peptides were chemically synthesized in this study. The composition of deer antler preparations comprises not only proteins and peptides but also a significant quantity of metal ion salts. By analyzing osteoblast growth in the presence of peptide YR and various metal ions, we observed a synergistic effect of calcium and strontium on the effects of YR. Those peptides could also stimulate the growth of C2C12 skeletal muscle cells and human chondrocytes, increasing collagen and glycosaminoglycan content in a three-dimensional environment. The maintenance of bone homeostasis relies on a balance between osteoclasts and osteoblasts. Deer antler peptides were observed to inhibit osteoclast differentiation, as evidenced by ROS generation, tartrate-resistant acid phosphatase (TRACP) activity assays, and gene expression in RAW264.7 cells. In summary, our findings provide a deep understanding of the efficacy of this folk medicine.

Hypoglycemic effects of dracorhodin and dragon blood crude extract from Daemonorops draco.

BACKGROUND: Dragon blood is a red fruit resin from the palm tree Daemonorops draco and is a herbal ingredient used in the traditional Chinese medicine, "Jinchuang Ointment," which is used to treat non-healing diabetic wounds. According to the Taiwan Herbal Pharmacopeia, the dracorhodin content in dragon blood should exceed 1.0%. RESULTS: Our findings indicate that dracorhodin and dragon blood crude extracts can stimulate glucose uptake in mouse muscle cells (C2C12) and primary rat aortic smooth muscle cells (RSMC). Dracorhodin is not the only active compound in dragon blood crude extracts from D. draco. Next, we orally administered crude dragon blood extracts to male B6 mice. The experimental group displayed a decreasing trend in fasting blood glucose levels from the second to tenth week. In summary, crude extracts of dragon blood from D. draco demonstrated in vivo hypoglycemic effects in B6 male mice. CONCLUSIONS: We provide a scientific basis "Jinchuang ointment" in treating non-healing wounds in patients with diabetes.

Novel hierarchical carbon microspheres@layered double hydroxides@copper lignosulfonate architecture for polypropylene with enhanced flame retardant and mechanical performances.

Due to the inherent defect of flammability of polypropylene (PP), a novel and highly efficient carbon microspheres@layered double hydroxides@copper lignosulfonate (CMSs@LDHs@CLS) flame retardant was designed and prepared, which was attributed to the strong electrostatic interaction between carbon microspheres (CMSs), layered double hydroxides (LDHs) and lignosulfonate as well as the chelation effect of lignosulfonate on copper ions, and then it was incorporated into the PP matrix. Significantly, CMSs@LDHs@CLS not only observably improved its dispersibility in PP matrix, but also simultaneously achieved excellent flame retardant properties for composites. With the addition of 20.0 % CMSs@LDHs@CLS, the limit oxygen index of CMSs@LDHs@CLS and PP composites (PP/CMSs@LDHs@CLS) reached 29.3 % and achieved the UL-94 V-0 rating. Cone calorimeter tests indicated that the peak heat release rate, total heat release and total smoke production of PP/CMSs@LDHs@CLS composites exhibited declines of 28.8 %, 29.2 % and 11.5 %, respectively, compared with those of PP/CMSs@LDHs composites. These advancements were attributed to the better dispersibility of CMSs@LDHs@CLS in PP matrix and illustrated that CMSs@LDHs@CLS observably reduced fire hazards of PP. The flame retardant property of CMSs@LDHs@CLS might relate to condensed phase flame retardant effect of char layer and catalytic charring of copper oxides.

Isolation, Identification, and Characterization of Bioactive Peptides in Human Bone Cells from Tortoiseshell and Deer Antler Gelatin.

Tortoiseshell and deer antler gelatin has been used to treat bone diseases in Chinese society. A pepsin-digested gelatin peptide with osteoblast-proliferation-stimulating properties was identified via LC-MS/MS. The resulting pentapeptide, TSKYR, was presumably subjected to further degradation into TSKY, TSK, and YR fragments in the small intestine. The above four peptides were chemically synthesized. Treatment of tripeptide TSK can lead to a significant 30- and 50-fold increase in the mineralized nodule area and density in osteoblast cells and a 47.5% increase in the number of chondrocyte cells. The calcium content in tortoiseshell was relatively higher than in human soft tissue. The synergistic effects of calcium ions and the peptides were observed for changes in osteoblast proliferation and differentiation. Moreover, these peptides can enhance the expression of RUNX2, OCN, FGFR2, and FRFR3 genes in osteoblasts, and aggrecan and collagen type II in chondrocyte (patent pending).

Using radial pulse wave as hemodynamic measurements to quantify effects of acupuncture therapy for patients with traumatic brain injury and ischemia stroke.

Background and aim: Traumatic Brain Injury (TBI) and stroke are major sources of death and disability worldwide. Acupuncture has been used as a supplemental therapy for patients with TBI and stroke. This study was aimed to evaluate the effects of acupuncture therapy for patients with TBI and stroke by radial pulse spectrum. Experimental procedure: 22 patients (6 TBI and 16 stroke) were enrolled and underwent radial pressure wave measurement before and after acupuncture treatment at Dubi (ST-35), Zusanli (ST-36) and Jiexi (ST-41). The harmonic analysis of the radial pressure wave was calculated and transformed into Fourier series coefficients Cn, Pn and the variation coefficient CnCV. Results: After acupuncture, systolic blood pressure, heart rate, and Glasgow Coma Scale changed very slightly. The harmonic index C4, C7, C9, C10, C3CV and C5CV had significant increases. (P < 0.05) After 3-week course acupuncture treatment, systolic blood pressure, C7, C8, C9, C10 and P10 had significant increases. (P < 0.05). Conclusion: Harmonic analysis of radial pulse waves may detect earlier circulatory system changes of acupuncture treatment before they were evident with other hemodynamic readings or scale.

Rapid Identification of Commercial Frankincense Products by MALDITOF Mass Spectrometry.

BACKGROUND: Frankincense is a resin secreted by the Boswellia tree. It is used in perfumery, aromatherapy, skincare, and traditional Chinese medicine. However, all Boswellia species are under threat owing to habitat loss and overexploitation. As a result, the market is getting flooded with counterfeit frankincense products. OBJECTIVE: This study aims to establish a high-throughput method to screen and identify the authenticity of commercial frankincense products. We report, for the first time, a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based method for rapid and high-throughput screening of frankincense samples. METHODS: MALDI-TOF MS, HPLC, thin-layer chromatography (TLC), and in vitro antiinflammatory activity assay were used to examine the frankincense samples. RESULTS: Well-resolved peaks of frankincense triterpenoids in the spectra were observed in the crude extract of commercial samples, including α-boswellic acids (αBAs), ß-boswellic acids (ßBAs), 11-keto-ß-boswellic acids (KBAs), acetyl-11-keto-ß-boswellic acids (AKBAs), and their esters. These compounds can be used as indicators for determining the authenticity of frankincense. CONCLUSION: Unlike LC-MS, which is a time-consuming and expensive method, and TLC, which requires a reference sample, our inexpensive, rapid high-throughput identification method based on MALDI-TOF MS is ideal for large-scale screening of frankincense samples sold in the market.

Role of Herbal Extracts of Catechu from Uncaria gambir in the Treatment of Chronic Diabetic Wounds.

Catechu is a dried decoction from twigs with the leaves of Uncaria gambir. Its antioxidant, anti-inflammatory, and antimicrobial activities have been previously reported because of its high catechin and epicatechin content (>21%). It is also one of the components used in traditional Chinese herbal medicine, "Jinchuang Ointment," which has excellent efficacy in treating chronic diabetic wounds. An in vivo zebrafish embryo platform and an in vitro cell-based tube formation assay were used to measure the angiogenic activity of catechu extracts. Interestingly, for the first time, catechu extracts stimulated angiogenic activity on both platforms. The expression of the IL-8 gene was induced in HMEC1 cells after treatment with catechu extracts for 1 h only. In contrast, the upregulation of FGFR2, FGFR3, NF-κB, STAT3, and vimentin persisted for 24 h. A summary of the possible mechanisms underlying the angiogenic activity of catechu extracts in HMEC1 cells is shown. Unexpectedly, catechu extracts inhibited the migration of HaCaT cells. These results can account for the intense blood flow flux in porcine excisional wound sites in our previous studies, which provides insights into the therapeutic activity of catechu extract in chronic diabetic wounds.

Antimicrobial and Immunomodulatory Activity of Herb Extracts Used in Burn Wound Healing: "San Huang Powder".

"San Huang Powder," a nonsterile milled herb powder, is frequently used to treat burn wounds in traditional Chinese herbal medicine. However, treating a wound with a nonsterile dressing or reagent is not compatible with the current guidelines in modern medicine. Therefore, we investigated the bactericidal and anti-inflammatory activities of four herb extracts used in "San Huang Powder" in vitro. Meanwhile, an in vivo porcine model with superficial second-degree burns was used for the experiments since the size and skin composition of pigs are the closest to that of the human body. The minimal bactericidal concentration (MBC) of the herb extracts was determined. The in vitro assay indicated that Rhubarb and Phellodendron bark extracts decreased the levels of inflammatory cytokines, IL-8, and GM-CSF on LPS-induced HMEC-1 cells. In accordance with this result, the histopathological evaluation results showed that the efficacy of "San Huang Powder" containing both herb materials was much better than the group without Rhubarb. Our results not only provide a basis to understand why "San Huang Powder" has been used to clinically treat wounds without sterilization directly since ancient times but also show the advantages of using multiple herb materials simultaneously on wound sites to prevent infection during treatment. Rhubarb is the recommended ingredient involved in the preparation of "San Huang Powder" to ensure the healing efficacy of burn wounds.

Dracorhodin perchlorate enhances wound healing via ß-catenin, ERK/p38, and AKT signaling in human HaCaT keratinocytes.

Dracorhodin can be isolated from the exudates of the fruit of Daemonorops draco. Previous studies suggested that dracorhodin perchlorate can promote fibroblast proliferation and enhance angiogenesis during wound healing. In the present study, the potential bioactivity of dracorhodin perchlorate in human HaCaT keratinocytes, were investigated in vitro, with specific focus on HaCaT wound healing. The results of in vitro scratch assay demonstrated the progressive closure of the wound after treatment with dracorhodin perchlorate in a time-dependent manner. An MTT assay and propidium iodide exclusion detected using flow cytometry were used to detect cell viability of HaCaT cells. Potential signaling pathways underlying the effects mediated by dracorhodin perchlorate in HaCaT cells were clarified by western blot analysis and kinase activity assays. Dracorhodin perchlorate significantly increased the protein expression levels of ß-catenin and activation of AKT, ERK and p38 in HaCaT cells. In addition, dracorhodin perchlorate did not induce HaCaT cell proliferation but promoted cell migration. Other mechanisms may yet be involved in the dracorhodin perchlorate-induced wound healing process of human keratinocytes. In summary, dracorhodin perchlorate may serve to be a potential molecularly-targeted phytochemical that can improve skin wound healing.

Efficient Photodynamic Killing of Gram-Positive Bacteria by Synthetic Curcuminoids.

In our previous study, we have demonstrated that curcumin can efficiently kill the anaerobic bacterium Propionibacterium acnes by irradiation with low-dose blue light. The curcuminoids present in natural plant turmeric mainly include curcumin, demethoxycurcumin, and bisdemethoxycurcumin. However, only curcumin is commercially available. Eighteen different curcumin analogs, including demethoxycurcumin and bisdemethoxycurcumin, were synthesized in this study. Their antibacterial activity against Gram-positive aerobic bacteria Staphylococcus aureus and Staphylococcus epidermidis was investigated using the photodynamic inactivation method. Among the three compounds in turmeric, curcumin activity is the weakest, and bisdemethoxycurcumin possesses the strongest activity. However, two synthetic compounds, (1E,6E)-1,7-bis(5-methylthiophen-2-yl)hepta-1,6-diene-3,5-dione and (1E,6E)-1,7-di(thiophen-2-yl)hepta-1,6-diene-3,5-dione, possess the best antibacterial activity among all compounds examined in this study. Their chemical stability is also better than that of bisdemethoxycurcumin, and thus has potential for future clinical applications.

Cloning, expression, identification and characterization of borneol dehydrogenase isozymes in Pseudomonas sp. TCU-HL1.

Borneol is a bicyclic plant monoterpene. It can be degraded by soil microorganisms through the conversion of borneol dehydrogenase (BDH) and a known camphor degradation pathway. Recombinant BDH from Pseudomonas sp. TCU-HL1 was produced in the form of inclusion body. The refolded BDH1 tends to precipitate. Insoluble recombinant BDH1 was converted into a soluble form by adding glycerol in LB medium. The kcat and kcat/Km values of soluble form BDH1 for (+)-borneol turned out to be about 34-fold and 45-fold higher, respectively, than those of the refolded enzyme. On the other hand, a gene knockout mutant, TCU-HL1Δbdh, was constructed to investigate the possible presence of a second copy of the bdh gene in TCU-HL1 genome. A new gene, bdh2, encoding a BDH isozyme, was identified, and the recombinant BDH2 protein was produced in a soluble form. Both bdh1 and bdh2 genes are expressed in the crude extract of wild type TCU-HL1, as shown by RT-qPCR results. Both BDH isozymes prefer to degrade (+)-borneol, rather than (-)-borneol, probably because (+)-camphor is the main form present in nature.

Production of optically pure (-)-borneol by Pseudomonas monteilii TCU-CK1 and characterization of borneol dehydrogenase involved.

(-)-Borneol is a bicyclic plant secondary metabolite. Optically pure (-)-borneol can only be obtained from plants, and demand exceeds supply in China. In contrast, chemically synthesized borneol contains four different stereoisomers. A strain of Pseudomonas monteilii TCU-CK1, isolated in Hualien, Taiwan, can accumulate (-)-borneol in growth culture and selectively degrades the other three isomers when chemically synthesized borneol is used as sole carbon source. This (-)-borneol production method can be scaled-up for production of large quantities in the future. More importantly, laborious plant cultivation and harvest is no longer required. The main enzyme that appears in this degradation pathway, borneol dehydrogenase (BDH), and the genome sequence of TCU-CK1 are reported. The kcat/Km values of TCU-CK1 BDH on (+)- and (-)-borneol are 538.4 ± 38.4 and 17.7 ± 1.1 (s-1 mM-1), respectively. About ∼30 fold difference in the kcat/Km value between (+)-borneol and (-)-borneol was observed, in good agreement with the fact that TCU-CK1 prefers to degrade (+)-borneol, rather than (-)-borneol. A BDH isozyme was identified in a strain in which the primary BDH gene had been knocked out. (-)-Camphor can work as an inhibitor of BDH with a Ki of 1.03 ± 0.11 mM at pH 7.0, leading to the accumulation of (-)-borneol in culture. (Patent pending).

Structural characterization of borneol dehydrogenase from Pseudomonas sp. TCU-HL1.

During the microbial degradation of borneol, a bicyclic plant monoterpene, it is first converted into camphor by borneol dehydrogenase (BDH) and then enters a known camphor-degradation pathway. Previously, a recombinant Pseudomonas BDH was found in inclusion bodies when expressed in Escherichia coli. After refolding, it was still unstable and was difficult to concentrate. Here, the protein-expression conditions were improved by changing the medium from lysogeny broth to Terrific Broth, yielding a soluble form of the enzyme with higher activity. The protein was crystallized and its 3D structure was determined by X-ray diffraction. Like other known homologues such as quinuclidinone reductase, the protein forms a tetramer with subunits containing Rossmann folds. Structural comparison revealed major differences in the C-terminal helices and the associated loops. It is likely that these regions contain the determinants for substrate recognition.

The curative effects of the traditional Chinese herbal medicine "Jinchuang ointment" on excisional wounds.

BACKGROUND: "Jinchuang ointment" is a traditional Chinese herbal medicine for external incised wounds. This herbal medicine has been successfully used to treat patients with diabetic foot ulcers and pressure sores in Taiwan for several decades. We previously examined its biological activities on cell-based in vitro assay platforms. Because some patients refused to use animal-derived ingredients ointment during our clinical practice, the efficacy of plant oil-based reconstituted "Jinchuang ointment" was also investigated. METHODS: A porcine excisional wound model was established and used to evaluate its efficacy in vivo in this study. Besides, an unusual clinical case is also present. RESULTS: As judged from the wound appearance of animal studies on day 14 and the results of blood flow flux at the wound sites on day 28, "Jinchuang ointment" accelerated wound closure significantly better than the control group. CONCLUSIONS: The results from clinical treatment, histopathological evaluation, and the animal study showed that "Jinchung ointment" promotes wound healing significantly better than the control group. Also, sesame oil-reconstituted ointment can be a choice for patients who refuse to use lard-containing ointment.

Rapid identification of dragon blood samples from Daemonorops draco, Dracaena cinnabari and Dracaena cochinchinensis by MALDI-TOF mass spectrometry.

INTRODUCTION: Dragon blood is a deep-red plant resin which has been used as folk medicine for more than a thousand years. It can be produced from at least four entirely different plant families: Asparagaceae, Arecaceae, Chamaesyce, and Fabaceae. Current pharmacopeia states that the only "authentic" source of dragon blood is the palm tree, Daemonorops draco. OBJECTIVE: The present study aims to find a high-throughput method to screen and identify the plant sources of commercial dragon blood products. METHODOLOGY: A matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) based method for rapid screening of dracorhodin in commercial dragon blood samples was established in this study. RESULTS: Well-resolved peaks of dracorhodin in spectra were observed in the crude extracts of samples. Dragon blood samples from two other plant species, Dracaena cinnabari and Dracaena cochinchinensis, were also examined. Their indicator compounds, loureirin A and B, were detected in these plants. CONCLUSION: A MALDI-TOF based method for preliminarily examination of commercial dragon blood samples is reported here. In contrast to MALDI-TOF, liquid chromatography mass spectrometry (LC-MS) is a time-consuming and costly method, not ideal for routine and large-scale screening of commercial samples.

In vivo pro-angiogenic effects of dracorhodin perchlorate in zebrafish embryos: A novel bioactivity evaluation platform for commercial dragon blood samples.

Dragon blood has been used in wound treatment for many years and can be obtained from several distinct plant species. Dracorhodin, the active substituent of dragon blood, is a characteristic compound of the palm tree, Daemonorops draco. At present, the only method to evaluate the quality of commercial dragon blood samples is a HPLC method which determines the amount of dracorhodin in a dragon blood sample. In this study, we used zebrafish embryos as a platform to demonstrate the in vivo pro-angiogenic activity of dracorhodin perchlorate, the chemically synthesized analog of dracorhodin. By using this platform, three different commercial dragon blood samples were also examined. Our results clearly show that even though the commercial dragon blood samples had similar amounts of dracorhodin, they showed highly variable biological activity, such as pro-angiogenic effects and toxicity. In short, an in vivo activity assay platform for rapidly examining the biological activity of commercial dragon blood samples was successfully established here, which complements the current HPLC-based assay method.

Resolution of isoborneol and its isomers by GC/MS to identify "synthetic" and "semi-synthetic" borneol products.

Borneol is a plant terpene commonly used in traditional Chinese medicine. Optically pure (+)-borneol and (-)-borneol can be obtained by extraction from the plants Dipterocarpaceae and Blumea balsamifera, respectively. "Synthetic borneol" is obtained from the reduction of (±)-camphor to lead to four different stereoisomers: (+)-isoborneol, (-)-isoborneol, (+)-borneol, and (-)-borneol. In contrast, "semi-synthetic borneol" is produced from the reduction of natural camphor, (+)-camphor, to afford two isomers: (-)-isoborneol and (+)-borneol. We established a convenient method to identify them by treating the four stereoisomers with two chiral reagents, (R)-(+)-α-methoxy-α-trifluoromethylphenylacetyl chloride ((R)-(+)-MTPA-Cl) and (1S)-(-)- camphanic chloride. The resulting derivatives from the above mentioned method were analyzed by gas chromatography. The enantiomers of (+)- and (-)-isoborneol were successfully separated from (+)- and (-)-borneol isomers in this study to make this a useful method in the identification of "synthetic" and "semi-synthetic" borneols. Furthermore, we also examined five different commercial borneols. During this course, a novel and unprecedented partial epimerization from isoborneol-camphanic ester to borneol-camphanic ester was observed. However, this phenomenon did not occur in isoborneol-MTPA esters epimerization to borneol-MTPA case under the same conditions. The DFT calculation of activation energies for both reactions was in a good agreement with the results obtained from GC analysis.

Investigation of borneols sold in Taiwan by chiral gas chromatography.

Borneol is a monoterpene that is widely used in traditional Chinese medicine. There are two different products sold in Taipei's traditional Chinese medicine market, natural and chemically synthesized borneol. Chemically synthesized borneol contains four stereoisomers, (+)-isoborneol, (-)-isoborneol, (-)-borneol, and (+)-borneol. The ratio of these four isomers in chemically synthesized and natural borneol products was determined by gas chromatography mass spectrometry. A huge variation between these products is highlighted in this survey. The results suggest that the Food and Drug Administrations in Asian countries should establish a regulatory standard regarding the ratio of the four different borneol isomers in both natural and chemically synthesized borneol.