Network Pharmacology-Based Prediction and Verification of Shikonin for the mechanism treating colorectal cancer

Background: Shikonin (SKN), a naturally occurring naphthoquinone, is a major active chemical component isolated from Lithospermum erythrorhizon Sieb Zucc, Arnebia euchroma (Royle) Johnst, or Arnebia guttata Bunge, and commonly used to treat viral infection, inflammation, and cancer. However, the underlying mechanism has not been elucidated Objective: This study aims to explore the antitumor mechanism of SKN in colorectal cancer (CRC) through network pharmacology and cell experiments. Methods: Using SymMap database and Genecards to predict the potential targets of SKN and CRC, while the cotargets were obtained by Venn diagram. The cotargets were imported into website of String and DA DAVID, constructing the protein-protein interaction (PPI) network, performing Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, the Compound-Target-Pathway (C-T-P) network was generated by connecting potential pathways with the corresponding targets. Results: According to the results of network pharmacological analysis, the cell experiments were used to verify the key signal pathway. The most relevant target of SKN for the treatment of CRC was PI3K/Akt signaling pathway. SKN inhibited CRC cells (HT29 and HCT116) proliferation, migration, and invasion, and promoted cell apoptosis by targeting IL6 and inhibiting the IL6R/PI3K/Akt signaling pathway. SKN promotes apoptosis and suppresses CRC cells (HT29 and HCT116) activity through the PI3K-Akt signaling pathway. Conclusion: This research not only provides a theoretical and experimental basis for more in-depth studies but also offers an efficient method for the rational utilization of a series of Traditional Chinese medicines as anti-CRC drugs.

Simultaneous determination of three naphthoquinones from Arnebia euchroma (Royle) Johnst in beagle plasma by UPLC-MS/MS and application for pharmacokinetics study

A rapid, simple and efficient ultra-performance liquid chromatography-electrospray ionization-tandem mass spectrometry (UPLC-ESI-MS/MS) method was established to simultaneous determination of shikonin, isobutyryl shikonin, β, βʹ-dimethylacryl alkanin in beagle plasma and evaluated by using esculetin as internal standard. The electrospray ionization (ESI) source was operated in negative ionization mode. Multi-reaction monitoring (MRM) was used to quantitatively analyzed shikonin m/z 287.0 → 217.9, isobutyryl shikonin m/z 357.0 → 268.9, β, βʹ-dimethylacryl alkanin m/z 370.0 → 270.1 and esculetin m/z 177.0 → 89.0, respectively. The method was validated for selectivity, linearity, lower limit of quantification, precision, accuracy, recovery, matrix effect and stability. All validation parameters met the acceptance criteria according to regulatory guidelines. This method was successfully applied for the pharmacokinetic study of shikonin, isobutyryl shikonin, β, βʹ-dimethylacryl alkanin in beagle dogs plasma after oral administration of A. euchroma extract.

De novo transcriptome analysis provides insights into formation of in vitro adventitious root from leaf explants of Arnebia euchroma

Background Adventitious root formation is considered a major developmental step during the propagation of difficult to root plants, especially in horticultural crops. Recently, adventitious roots induced through plant tissue culture methods have also been used for production of phytochemicals such as flavonoids, anthocyanins and anthraquinones. It is rather well understood which horticultural species will easily form adventitious roots, but the factors affecting this process at molecular level or regulating the induction process in in vitro conditions are far less known. The present study was conducted to identify transcripts involved in in vitro induction and formation of adventitious roots using Arnebia euchroma leaves at different time points (intact leaf (control), 3 h, 12 h, 24 h, 3 d, 7 d, 10 d and 15 d). A. euchroma is an endangered medicinal Himalayan herb whose root contains red naphthoquinone pigments. These phytoconstituents are widely used as an herbal ingredient in Asian traditional medicine as well as natural colouring agent in food and cosmetics. Results A total of 137.93 to 293.76 million raw reads were generated and assembled to 54,587 transcripts with average length of 1512.27 bps and N50 of 2193 bps, respectively. In addition, 50,107 differentially expressed genes were identified and found to be involved in plant hormone signal transduction, cell wall modification and wound induced mitogen activated protein kinase signalling. The data exhibited dominance of auxin responsive (AUXIN RESPONSE FACTOR8, IAA13, GRETCHEN HAGEN3.1) and sucrose translocation (BETA-31 FRUCTOFURANOSIDASE and MONOSACCHARIDE-SENSING protein1) genes during induction phase. In the initiation phase, the expression of LATERAL ORGAN BOUNDARIES DOMAIN16, EXPANSIN-B15, ENDOGLUCANASE25 and LEUCINE-rich repeat EXTENSION-like proteins was increased. During the expression phase, the same transcripts, with exception of LATERAL ORGAN BOUNDARIES DOMAIN16 were identified. Overall, the transcriptomic analysis revealed a similar patterns of genes, however, their expression level varied in subsequent phases of in vitro adventitious root formation in A. euchroma. Conclusion The results presented here will be helpful in understanding key regulators of in vitro adventitious root development in Arnebia species, which may be deployed in the future for phytochemical production at a commercial scale.

Arnebia euchroma, a Plant Species of Cold Desert in the Himalayas, Harbors Beneficial Cultivable Endophytes in Roots and Leaves

The endophytic mutualism of plants with microorganisms often leads to several benefits to its host including plant health and survival under extreme environments. Arnebia euchroma is an endangered medicinal plant that grows naturally in extreme cold and arid environments in the Himalayas. The present study was conducted to decipher the cultivable endophytic diversity associated with the leaf and root tissues of A. euchroma. A total of 60 bacteria and 33 fungi including nine yeasts were isolated and characterized at the molecular level. Among these, Proteobacteria was the most abundant bacterial phylum with the abundance of Gammaproteobacteria (76.67%) and genus Pseudomonas. Ascomycota was the most abundant phylum (72.73%) dominated by class Eurotiales (42.42%) and genus Penicillium among isolated fungal endophytes. Leaf tissues showed a higher richness (Schao1) of both bacterial and fungal communities as compared to root tissues. The abilities of endophytes to display plant growth promotion (PGP) through phosphorus (P) and potassium (K) solubilization and production of ACC deaminase (ACCD), indole acetic acid (IAA), and siderophores were also investigated under in vitro conditions. Of all the endophytes, 21.51% produced ACCD, 89.25% solubilized P, 43.01% solubilized K, 68.82% produced IAA, and 76.34% produced siderophores. Six bacteria and one fungal endophyte displayed all the five PGP traits. The study demonstrated that A. euchroma is a promising source of beneficial endophytes with multiple growth-promoting traits. These endophytes can be used for improving stress tolerance in plants under nutrient-deficient and cold/arid conditions.

Diversity and plant growth-promoting potential of actinomycetes associated with the rhizosphere of Arnebia euchroma from Himachal Pradesh (India)

Aim: The present study aimed to explicate the diversity and plant growth promoting (PGP) potential of actinomycetes present in the rhizosphere of an endangered medicinal plant, Arnebia euchroma. Methodology: Rhizospheric soil samples of A. euchroma collected from trans-Himalayan region of Himachal Pradesh were processed for elucidating actinomycetes diversity and load by employing Standard Plate Count Technique. All recovered isolates were screened for their PGP potential using standard protocols. The potential strains were identified through 16SrRNA ribotyping and were deposited in the National Culture Collection Centre, NCMR, Pune, (India). Results: The rhizosphere of A. euchroma harboured a great deal of actinomycetes diversity (33 diverse morphotypes). Starch casein agar was best for isolating the actinomycetes. The same actinomycete isolate showed variations in their morphological features including pigments production on different isolation media. They exhibited multifarious plant growth-promoting activities like P- solubilization, phytase activity, N- fixation, siderophore production etc. The potential isolates were identified as Streptomyces silaceus CA7 (MK836019) and Streptomyces rectiviolaceus NA8 (MK836018). Interpretation: The actinomycetes evinced a considerable plant growth promoting potential that might be helpful in the adaptation and perpetuation of A. euchroma under prevailing harsh environmental conditions of Himachal Pradesh.

Treatment of Pressure Ulcer with Abukhalsa (Arnebia euchroma) Ointment: A Case Report

Pressure ulcers, caused by reduced tissue blood flow because of long pressure on the site, are of the problems of people with immobility. Treatment of this problem has always been one of the problems of medical community with disabled and immobile people. Thus, many studies are always conducted to seek new approaches to reduce this problem. Additionally, given the acceptance of herbal medicines in recent years, using medicinal plants is on the rise. The case was a 52-year-old woman admitted to the wound clinic with a complaint of sensory and motor weakness of the upper limb and sudden paralysis of the lower limb since one year ago, with a diagnosis of myelitis .Her complaints were two wounds on both sides of the right and left buttocks measuring 9 by 5 and another 6 by 4.5 cm from three months ago and a wound with dimensions of 5 by 4.5 in the sacral region (sacrum) from twenty days before the visit. It had necrotic tissue without muscle involvement, osteomyelitis, or tunneling, also there were not bad odor and sludge, considered in class 3 in the category of wounds. Treatment was started for her by Arnebia euchroma ointment. The treatment was very successful from the second week on, and the wounds healed completely in 8 weeks. Using Arnebia euchroma as a wound treatment and reduction of infection and inflammation could be of great help in the treatment of pressure ulcers and a good suggestion in treating these ulcers. Keywords:Pressure ulcer, Wound healing, Arnebia euchroma, Bed sore, Persian traditional medicine