shikonin and alkannin

Shikonin and its enantiomeric analogue, alkaninn, are prevailing natural lead compounds in the drug discovery and development of anticancer agents. Despite having numerous biological effects, the most important activity reported for shikonin derivatives is the antitumor effect which is exerted through various mechanisms such as induction of apoptosis and autophagy. The design, synthesis, and development of new shikonin derivatives are continuously performed with the aim of promoting therapeutic effects through increasing cytotoxicity against cancer cells and simultaneously reducing toxicity on normal cells. In spite of significant advances in the development of shikonin derivatives in recent years and the publication of some reviews in this regard, the structural classification, synthesis methods, as well as the diversity of the anti-tumor mechanism of action of these compounds have not been well considered. This review aims to provide comprehensive data in this regard by reviewing studies conducted over the last two decades (from 2000 until now).

Topics: Antineoplastic Agents; Apoptosis; Humans; Naphthoquinones; Neoplasms

Shikonin, alkannin and their derivatives, the main ingredient of Lithospermum erythrorhizon and Arnebia euchroma (Royle) Johnst native to Inner Mongolian and Northwest of China respectively, hold promising potentials for antitumor effects via multiple-target mechanisms. This review will emphasize the importance of their antitumor activity in apoptosis, necroptosis and immunogenic cell death, and expound the relationship of their antitumor activity and naphthoquinone scaffold that could generate ROS and alkylating agent. Meanwhile, the antitumor mechanisms of naturally-occurring shikonin, alkannin and their derivatives, which were divided into the direct interaction involved in alkylating agent, covalently binding the DNA and protein, as well as the indirect interaction mediated by ROS, nonspecifically influencing the mitochondria or multiple signal pathways, will be systematically summarized and discussed.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; Cell Death; Drug Screening Assays, Antitumor; Humans; Lithospermum; Naphthoquinones; Neoplasms; Reactive Oxygen Species

Alkannins and Shikonins (A/S) are chiral-pairs of naturally occurring isohexenylnaphthazarins. They are found in the external layer of the roots of at least a hundred and fifty species that belong mainly to the genera Alkanna, Lithospermum, Echium, Onosma and Arnebia of the Boraginaceae family. Their occurrence in Jatropha glandulifera, a member of the Euphorbiaceae, should be considered as an exception. Pharmaceutical formulations with wound healing properties based on A/S have been in the market for many years. Although their wound-healing, anti-inflammatory, antimicrobial, antioxidant, antithrombotic and antitumor properties have been extensively documented, significant insight into their specific molecular pathways and mechanisms was hindered until recently. With the establishment of viable synthetic and biosynthetic routes of A/S and the synthesis of specific derivatives that were discovered the last few years, the effects of those compounds in the molecular-cell biology of human tissues in health and disease have just started being explored in depth, revealing a new class of drugs that hold promise as the basis for many valuable therapeutic targets. In the recent years, a wealth of new information arising from research efforts, on the wound healing properties of A/S has been accumulated. In this paper we review the findings and advances on the molecular and biological properties of A/S that promote wound healing.

Topics: Animals; Computer Simulation; Humans; Models, Chemical; Molecular Structure; Naphthoquinones; Stereoisomerism; Wound Healing

The enantiomeric naphthoquinones alkannins and shikonins (A/S) have been established as potent wound healing agents. The purpose of this study was to evaluate the effectiveness of an A/S based ointment for humans on second intention wound healing in the dog, as compared to wound flushing with Lactated Ringer's solution (LRS). Ten mixed breed dogs, aged 2 to 5 y, were used. One 2.5 × 2.5 cm full-thickness skin defect was created on the lateral aspect of each arm for subjective evaluation, laser-Doppler flowmetry (LDF), and planimetry. Additionally, 3 matching 2 × 2 cm wounds were created on opposite sides of the dorsal midline for histologic evaluation. Wounds were treated once daily with the A/S based ointment on the right side and by flushing with LRS on the left until healed (about 20 d). During the healing process, tissue perfusion (mean LDF value) was found to be significantly higher on the side treated with the A/S based ointment compared with the LRS-treated side. Histologically, angiogenesis (on days 4 and 11), collagen production score (on days 4, 11, and 20), and epithelial thickness score (on day 11) were significantly higher in the wounds treated with the A/S based ointment. Wound size, as evaluated by planimetry, decreased significantly from day 0 to day 20 on both sides, but no significant differences were found between the A/S based ointment and LRS-treated wounds.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Dogs; Drug Administration Schedule; Drug Combinations; Female; Forelimb; Isotonic Solutions; Laser-Doppler Flowmetry; Male; Naphthoquinones; Ringer's Lactate; Skin; Soft Tissue Injuries; Therapeutic Irrigation; Treatment Outcome; Wound Healing

The differences in catalytic mechanism and domain between the soluble (S-COMT) and membrane-bound catechol-O-methyltransferase (MB-COMT) are poorly documented due to the unavailable crystal structure of MB-COMT. Considering the enzymatic nature of S-COMT and MB-COMT, the challenge could be solvable by probing the interactions between the enzymes with the ligands with minor differences in structures. Herein, isomeric shikonin and alkannin bearing a R/S -OH group in side chain at the C2 position were used for domain profiling of COMTs. Human and rat liver-derived COMTs showed the differences in inhibitory response (human's IC

Topics: Animals; Catechol O-Methyltransferase; Humans; Molecular Docking Simulation; Protein Isoforms; Rats

Naturally occurring naphthoquinones, shikonin and alkannin, are important ingredients of traditional Chinese medicine Zicao. These constituents are reported to have many therapeutic uses, such as wound healing; scar treatment; and anti-inflammation, anti-acne, anti-ulcer, anti-HIV, anticancer, and antibacterial properties. The primary objective of this investigation was to explore the effect of shikonin and alkannin on Escherichia coli ATP synthase and its cell growth. Shikonin caused complete (100 %) inhibition, and alkannin caused partial (79 %) inhibition of wild-type E. coli ATP synthase. Both caused partial (4 %-27 %) inhibition of ATP synthase with genetically modified phytochemical binding site. The growth inhibition of strains expressing normal, deficient, and mutant ATP synthase by shikonin and alkannin, corroborated the inhibition observed in isolated normal wild-type and mutant ATP synthase. Trivial inhibition of mutant enzymes indicated αR283D, αE284R, βV265Q, and γT273A are essential for formation of the phytochemical binding site where shikonin and alkannin bind. Further, shikonin was a potent inhibitor of ATP synthase than alkannin. The antimicrobial properties of shikonin and alkannin were tied to the binding at phytochemical site of microbial ATP synthase. Selective targeting of bacterial ATP synthase by shikonin and alkannin may be an advantageous alternative to address the antibiotic resistance issue.

Topics: Adenosine Triphosphate; Escherichia coli; Naphthoquinones; Phytochemicals

Alkannin is the main coloring matter of Alkanet, a natural red dye extracted from the root of Alkanna tinctoria L. Shikonin, the optical isomer of alkannin, is extracted from Lithospermum erythrorhizon. As both red dyes are only slightly soluble in water, the application of ordinary Raman spectroscopy is limited. Thus, Surface-enhanced Raman spectroscopy (SERS) can be successfully applied to the study of the red dyes solutions. Solid alkannin and shikonin were characterized by ordinary Raman spectroscopy. Density Functional Theory (DFT) methods were used to calculate the Raman spectrum of the dyes and to assign the experimental Raman bands to their vibrational normal modes. Different pH conditions were tested in order to determine the optimal conditions for the SERS detection of alkannin and shikonin. Based on the previous results, a perpendicular orientation of the red dyes on the Ag substrate was deducted. Finally, shikonin was identify by SERS spectroscopy in a dyed paper sample from an 8th century handscroll from Japan.

Topics: Coloring Agents; Japan; Naphthoquinones; Spectrum Analysis, Raman

Alkannin/shikonin and their derivatives are specialised metabolites of high pharmaceutical and ecological importance exclusively produced in the periderm of members of the plant family Boraginaceae. Previous studies have shown that their biosynthesis is induced in response to methyl jasmonate but not salicylic acid, two phytohormones that play important roles in plant defence. However, mechanistic understanding of induction and non-induction remains largely unknown. In the present study, we generated the first comprehensive transcriptomic dataset and metabolite profiles of Lithospermum officinale plants treated with methyl jasmonate and salicylic acid to shed light on the underlying mechanisms. Our results highlight the diverse biological processes activated by both phytohormones and reveal the important regulatory role of the mevalonate pathway in alkannin/shikonin biosynthesis in L. officinale. Furthermore, by modelling a coexpression network, we uncovered structural and novel regulatory candidate genes connected to alkannin/shikonin biosynthesis. Besides providing new mechanistic insights into alkannin/shikonin biosynthesis, the generated methyl jasmonate and salicylic acid elicited expression profiles together with the coexpression networks serve as important functional genomic resources for the scientific community aiming at deepening the understanding of alkannin/shikonin biosynthesis.

Topics: Acetates; Cyclopentanes; Lithospermum; Mevalonic Acid; Naphthoquinones; Oxylipins; Pharmaceutical Preparations; Plant Growth Regulators; Salicylic Acid

Increasing genome data are coming out. Genome size estimation plays an essential role in guiding genome assembly. Several months ago, other researchers were the first to publish a draft genome of the red gromwell (i.e. Lithospermum erythrorhizon). However, we considered that the genome size they estimated and assembled was incorrect. This study meticulously estimated the L. erythrorhizon genome size to should be ∼708.74 Mb and further provided a reliable genome version (size ≈ 693.34 Mb; contigN50 length ≈ 238.08 Kb) to support our objection. Furthermore, according to our genome, we identified a gene family of the alkannin/shikonin O-acyltransferases (i.e. AAT/SAT) that catalysed enantiomer-specific acylations in the alkannin/shikonin biosynthesis (a characteristic metabolic pathway in L. erythrorhizon's roots) and further explored its evolutionary process. The results indicated that the existing AAT/SAT were not generated from only one round of gene duplication but three rounds; after different rounds of gene duplication, the existing AAT/SAT and their recent ancestors were under positive selection at different amino acid sites. These suggested that a combined power from gene duplication plus positive selection plausibly propelled AAT/SAT's functional differentiation in evolution.

Topics: Acyltransferases; Lithospermum; Naphthoquinones

Boraginales (the forget-me-not order) is a core group within the lamiids clade. However, until now, no genome from Boraginales has been reported, and published transcriptomes are also rare. Here, we report the first Boraginales species de novo genome (i.e. Echium plantagineum genome) and seven other Boraginales species transcriptomes to probe three issues: (i) Boraginales' phylogenetic position within the lamiids clade; (ii) potential whole genome duplications (WGDs) in Boraginales; and (iii) candidate key enzyme genes in the alkannin/shikonin core pathway. The results showed that: (i) Boraginales was most probably closer to the Solanales/Gentianales clade than the Lamiales clade, at least based on the single-copy orthologous genes from genome/transcriptome data; (ii) after the gamma (γ) event, Boraginaceae (classified into the Boraginales I clade) probably underwent at least two rounds of WGD, whereas Heliotropiaceae and Ehretiaceae (classified into the Boraginales II clade) probably underwent only one round of WGD; and (iii) several candidate key enzyme genes in the alkannin/shikonin core pathway were inferred, e.g. genes corresponding to geranyl cyclase, naphthol hydroxylase and O-acyl transferase.

Topics: Biosynthetic Pathways; Gene Duplication; Gene Expression Profiling; Genome, Plant; Magnoliopsida; Naphthoquinones; Phylogeny; Plant Proteins; Transcriptome

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line; Cell Line, Tumor; Cell Proliferation; Cytochrome P-450 CYP1B1; Drug Screening Assays, Antitumor; HCT116 Cells; Hep G2 Cells; Humans; K562 Cells; Mice; Naphthoquinones; Prodrugs; Xenograft Model Antitumor Assays

Isohexenylnaphthazarins are commonly found in the root periderm of several Boraginaceous plants and are known for their broad range of biological activities. The work described herein concerns the biological activity of compounds from the roots of Echium plantagineum L. and Echium gaditanum Boiss (Boraginaceae) collected from field sites in southern Spain and Australia. Bioactivity was assessed using etiolated wheat coleoptile bioassay and in vitro growth inhibitory activity in HeLa and IGROV-1 cells. The quantification of four isohexenylnaphthazarins (shikonin/alkannin, deoxyshikonin/deoxyalkannin, acetylshikonin/acetylalkannin and dimethylacrylshikonin/dimethylacrylalkannin) was performed by LC-MS/MS using juglone as internal standard. Correlation coefficient values for the activities and concentrations of these four analytes were in the linear range and were greater than 0.99. Acetylshikonin/acetylalkannin and dimethylacrylshikonin/dimethylacrylalkannin were present in the highest concentrations in extracts of both species. The results reveal that greatest overall inhibition was observed in both bioassays with E. gaditanum extracts. Strong correlations between time of collection, sampling location and bioactivity were identified.

Topics: Australia; Cell Line, Tumor; Cell Survival; Echium; Humans; Naphthoquinones; Plant Extracts; Plant Roots; Spain; Triticum

Root bark of Onosma echioides belonging to the family Boraginaceae is reported to be rich in naphthaquinones such as alkannins and shikonins. In this study, a dimer of alkannin/shikonin was isolated from the petroleum ether (60-80 C) extract of the bark, and the structure of the same was elucidated through spectral studies (UV, IR, NMR, MS and DEPT). The petroleum ether extract was found to contain 62.4% (w/w) of the dimer of alkannin/shikonin, and the compound is found to promote wound-healing process, when studied in the excision and incision wound models in albino rats.

Topics: Animals; Boraginaceae; Molecular Structure; Naphthoquinones; Plant Extracts; Plant Roots; Rats; Wound Healing

A set of forty alkannin and shikonin oxime derivatives were firstly designed and synthesized. Their cytotoxicities against three kinds of tumor cells and a normal cell line were tested and compared with alkannin and shikonin. The cell-based investigation demonstrated that some oxime derivatives were more or comparatively effective to the lead compounds, especially their selective and excellent antitumor activities towards K562 cells with no toxicity in normal cells. We may conclude that oximate modification to the mother nucleus of alkannin and shikonin is an available approach to acquire potent antitumor agents.

Topics: Antineoplastic Agents; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; K562 Cells; MCF-7 Cells; Molecular Structure; Naphthoquinones; Oximes; Structure-Activity Relationship

The optical antipodes alkannin/shikonin (A/S) and their esters are potent pharmaceutical substances found in the roots of 150 Boraginaceous species. This study estimated and compared total and free A/S content and A/S enantiomeric ratio in roots of 11 Alkanna species (A. corcyrensis, A. tinctoria, A. pindicola, A. orientalis, A. methanaea, A. calliensis, A. graeca, A. primuliflora, A. stribrnyi, A. sieberi and A. noneiformis) growing wild in various Greek regions, to compare with cultivated species. It also re-characterized the chirality of A/S commercial samples, since most of them were misnamed by the providers. Several Alkanna species were collected (groups 1 and 3) and botanically identified, whereas some Alkanna species were cultivated from collected seeds (group 2). Free A/S and derivatives were extracted from the dried roots of Alkanna species and analyzed by high performance liquid chromatography-diode array detection (HPLC-DAD). For total A/S content the hexane extracts of Alkanna roots were hydrolyzed and analyzed by HPLC-DAD. Chirality determination and A/S enantiomeric ratio estimation was performed for several commercial samples by polarimetry,chiral LC-DAD and circular dichroism studies. Quantitative analysis revealed that A/S content varied from one region to another even within the same species. Most of the cultivated samples contained greater amounts of free and total A/S compared with the wild ones, wheras no difference was observed in A/S enantiomeric ratio. All the Alkanna samples tested contain mainly alkannin derivatives. Some of the examined Alkanna species of the Greek flora that are endemic to the Mediterranean area could serve as alternative sources for medicinally valuable A/S derivatives. Most of the commercial A/S samples tested were misnamed in terms of chirality and re-characterized.

Topics: Boraginaceae; Chromatography, High Pressure Liquid; Naphthoquinones; Plant Extracts; Plant Roots

Suspension cultures of Arnebia euchroma supported with liquid perfluorodecalin (PFD) degassed, aerated, or ethylene-saturated were investigated as a novel in situ extraction system for enhanced alkannin/shikonin production. Simultaneously, the effect of PFD applied as the liquid gas carrier on the growth of A. euchroma biomass was studied. The similar dry (4-fold) and fresh (7-fold) biomass increase was observed in the control (without PFD addition) and supplemented with PFD-degassed or PFD-aerated cultures while PFD-ethylene application impeded cell growth. The highest total of alkannin/shikonin production (23.23 mg flask(-1)) was observed when PFD-aerated has been used and it resulted in about 50% higher yield of alkannin/shikonin compared with the control culture. Chiral HPLC analysis revealed that in cultures supported with PFD, both alkannin and shikonin were produced. Their mutual ratio varied depending on culture conditions, and the accumulation of alkannin prevailed under almost all culture conditions. PFD has proved to be exceptionally efficient and cell-safe solvent for the in situ extraction of naphthoquinone red pigments without exerting any detrimental effects on cell growth. Extracellularly secreted red naphthoquinones were easily dissolved and extracted from the PFD phase, which can be regenerated and reused (e.g., in continuous culture system).

Topics: Biomass; Boraginaceae; Chromatography, High Pressure Liquid; Culture Media; Drugs, Chinese Herbal; Ethylenes; Fluorocarbons; Naphthoquinones; Plant Growth Regulators; Plant Somatic Embryogenesis Techniques

To minimize the cytotoxicity of shikonin and alkannin that arises through the generation of reactive oxygen species (ROS) and alkylation of the naphthazarin ring, two series of novel core-scaffold-modified shikonin and alkannin derivatives were designed. These derivatives, which differ in their configurational and positional isomerism (R-, S-, and 2- and 6-isomers) were synthesized in high enantiomeric excess (>99 % ee). The selectivity of the dimethylated derivatives was significantly higher than the parent shikonin in vitro, but some side effects were still observed in vivo. Surprisingly, the dimethylated diacetyl derivatives with poor anticancer activity in vitro showed tumor-inhibiting effects similar to paclitaxel without any toxicity in vivo. The anticancer activity of these derivatives is in agreement with their low ROS generation and alkylating capacity, emphasizing their potential as prodrugs. This strategy provides means to address the nonspecific cytotoxicity of naphthazarin analogues toward normal cells.

Topics: Alkylation; Animals; Antineoplastic Agents; Cell Line, Tumor; Cell Survival; Drug Design; Female; Male; Mice; Microsomes, Liver; Naphthoquinones; Neoplasms; Prodrugs; Rats; Reactive Oxygen Species; Stereoisomerism; Structure-Activity Relationship; Transplantation, Heterologous; Transplantation, Homologous

The N-acetyl glucosaminosides of shikonin/alkannin were synthesized by chemical glycosylation, which provided an ideal approach to resolve the mixture of enantiomeric shikonin and alkannin co-existed in the Chinese herbs. The glycosylated shikonin and alkannin exhibited stronger binding activity to human telomeric G-quadruplex DNA than their parent structures. This research indicated that glycosylation of natural product with amino sugars is an effective strategy to improve their DNA-binding affinity.

Topics: Aminoglycosides; DNA; G-Quadruplexes; Glycosylation; Humans; Molecular Structure; Naphthoquinones; Spectrometry, Mass, Electrospray Ionization

This work systematically investigated the enantiomeric excess (e.e.) of main components isolated from the roots of three endemic Boraginaceae plants distributed extensively in China, named Arnebia euchroma (Royle) Johnst (A.e.), Lithospermum erythrorhizon Sieb. et Zucc. (L.e.) and Onosma confertum W. W. Smith (O.c.), and the optical purity of their hydrolysis products separately, by means of three different approaches. The influence of HCl on the e.e. values of the major constituents was also studied. Analysis of the absolute configurations and e.e. values of all the derivatives acquired was performed by CD and chiral-HPLC respectively. The results of the main constituents demonstrated that A.e. mainly yields S-form naphthoquinone derivatives, while the R-form is predominant in the derivatives of L.e. and O.c. The optical purity of alkannin and shikonin and their derivatives was not influenced by acid treatment in the course of separation and hydrolysis. Additionally, it was found that 100% e.e. of shikinon could be acquired from a specific shikinon ester derivative, β,β-dimethylacrylshikonin occurring in the roots of O.c., as did 100% e.e. of alkannin from β,β-dimethylacrylalkannin contained in the roots of A.e.

Topics: Boraginaceae; China; Chromatography, High Pressure Liquid; Circular Dichroism; Naphthoquinones; Plant Extracts; Plant Roots; Stereoisomerism

Alkannin, shikonin (A/S) and their derivatives are naturally occurring hydroxynaphthoquinones with a well-established spectrum of wound healing, antimicrobial, anti-inflammatory, antioxidant and antitumor activity. Clinical studies over the years revealed that A/S derivatives-based wound healing preparations (such as HELIXDERM(®)) are among a very small group of therapeutics that modulate both the inflammatory and proliferative phases of wound healing and present significant tissue regenerative activity. The purpose of the present work was to combine the biological properties of A/S and the advantages of electrospun meshes to prepare a potent topical/transdermal biomaterial for A/S. Four biocompatible polymers (cellulose acetate, poly(L-lactide), poly(lactide-co-glycolide) LA/GA:50/50 and 75/25) were used for the first time, to produce electrospun fiber mats containing either shikonin or A/S mixture in various amounts. Both drugs were effectively loaded into the above biomaterials. The incorporation of drugs did not considerably affect fibers morphology and their mean diameter size varied from 315 to 670 nm. High drug entrapment efficiencies (ranged from 74% to 95%) and appropriate release profiles were achieved, that render these fibers as potential A/S topical/transdermal wound healing dressings. Given the multifunctional activity of the natural products alkannins and shikonins, their consideration as bioactive constituents for tissue engineering scaffolds seems a promising strategy for repairing and regenerating tissues and mainly skin.

Topics: Administration, Cutaneous; Bandages; Cellulose; Drug Combinations; Drugs, Chinese Herbal; Electrochemical Techniques; Excipients; Humans; Lactic Acid; Naphthoquinones; Polyesters; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Wound Healing

We recently reported that shikonin and its analogs were a class of necroptotic inducers that could bypass cancer drug resistance. However, the molecular targets of shikonin are not known. Here, we showed that shikonin and its analogs are inhibitors of tumor-specific pyruvate kinase-M2 (PKM2), among which shikonin and its enantiomeric isomer alkannin were the most potent and showed promising selectivity, that is, shikonin and alkannin at concentrations that resulted in over 50% inhibition of PKM2 activity did not inhibit PKM1 and pyruvate kinase-L (PKL). Shikonin and alkannin significantly inhibited the glycolytic rate, as manifested by cellular lactate production and glucose consumption in drug-sensitive and resistant cancer cell lines (MCF-7, MCF-7/Adr, MCF-7/Bcl-2, MCF-7/Bcl-x(L) and A549) that primarily express PKM2. HeLa cells transfected with PKM1 showed reduced sensitivity to shikonin- or alkannin-induced cell death. To the best of our knowledge, shikonin and alkannin are the most potent and specific inhibitors to PKM2 reported so far. As PKM2 universally expresses in cancer cells and dictates the last rate-limiting step of glycolysis vital for cancer cell proliferation and survival, enantiomeric shikonin and alkannin may have potential in future clinical application.

Topics: Antineoplastic Agents; Cell Line, Tumor; Enzyme Inhibitors; Glucose; Glycolysis; Humans; Lactic Acid; Naphthoquinones; Neoplasms; Pyruvate Kinase

In pursuit of strong shikalkin-producing cell lines, seeds of the Iranian Arnebia euchroma were collected from Dena altitudes in the central Zagross. Chemical analysis showed that the dried root of the plant contained about 8.5% (w/w) shikalkin pigment. The root explants of the young plantlets, obtained from the germinated seeds, were used for establishing callus. Then, parameters effective on proliferation and pigment production of the resulting calli were studied in detail. Accordingly, two modified media called mLS and mM9 were optimized for propagation and pigment production, respectively. Using these media, the biomass of the A. euchroma calli was increased to 600%, and the pigment production reached to a maximum of 16.3  mg per gram of the wet biomass in a period of a subculture (21 days). Parallel to these experiments, the antimicrobial activity of shikalkin pigment was examined on some fungi and gram-positive and gram-negative bacteria. Results indicated that the pigment was almost ineffective on fungi and gram-negative bacteria, but it was meaningfully effective against Micrococcus luteus.

Topics: Anti-Bacterial Agents; Bacteria; Boraginaceae; Fungi; Naphthoquinones; Plant Extracts; Plant Roots; Seeds; Tissue Culture Techniques

A series of naphthoquinones was tested for activity against both extracellular promastigote and intracellular amastigote Leishmania major GFP in vitro. In parallel, the compounds were evaluated for cytotoxic effects against bone marrow-derived macrophages (BMM Φ) as a mammalian host cell control. Most of the compounds noticeably inhibited the growth of extracellular parasites (IC (50) 0.5 to 6 µM) and the intracellular survival of L. major GFP amastigotes (IC (50) 1 to 7 µM) when compared with the antileishmanial drug amphotericin B (IC (50) of 2.5 and 0.2 µM, respectively). In general, antiprotozoal activity and host cell cytotoxicity seemed to increase in parallel. Conspicuously, the cytotoxic effect was less pronounced on infected host cells when compared with that on noninfected cells. Concerning structure/activity relationships for the tested naphthoquinones, some interesting structural features emerged from this study. Introduction of a methyl or methoxyl group at C-2 of the parent 1,4-naphthoquinone slightly increased the antileishmanial activity against clinically relevant amastigotes, while the presence of a hydroxyl function in this position dramatically reduced the effectiveness. In contrast, hydroxylation at C-5 and dihydroxy substitution at C-5 and C-8 significantly enhanced the antiprotozoal activity. Similarly, the presence of a side chain hydroxyl group PERI to a carbonyl function as represented in the series of shikonin/alkannin derivatives increased the activity when compared with substituted analogs. Within the series of naphthoquinones tested, the dimeric mixture of vaforhizin and isovaforhizin showed the highest activity IN VITRO against the clinically relevant intracellular amastigote with an IC (50) of 1.1 µM. With IC (50) values mostly in the range of 1-3 µM, the shikonin/alkannin derivatives proved to be similarly considerably leishmanicidal. None of the compounds tested was capable to induce NO production known to play a crucial role in the host resistance against intracellular pathogens, excluding activation of microbicidal mechanisms in macrophages. The mode of action apparently depended on the substitution pattern, associated with the electrophilicity of the naphthoquinone or the efficiency of redox cycling. Conspicuously, members oxygenated in the quinone ring proved to be leishmanicidal when coincubated with glutathione, while the majority of the remaining compounds lost activity.

Topics: Amphotericin B; Animals; Antiprotozoal Agents; Bignoniaceae; Boraginaceae; Culture Media; Drosera; Flow Cytometry; Glutathione; Green Fluorescent Proteins; Hydroxylation; Inhibitory Concentration 50; Leishmania; Macrophages; Mammals; Naphthoquinones; Organisms, Genetically Modified; Parasitic Sensitivity Tests; Structure-Activity Relationship

Isohexenylnaphthazarins (IHN), commonly known as alkannins and shikonins (A/S), are potent pharmaceutical substances with a wide spectrum of wound healing, antimicrobial, anti-inflammatory, and antitumor activity. Purification of A/S is crucial for their use in pharmaceuticals and for biological experimentation. Dimeric and oligomeric A/S derivatives co-exist with the active monomeric ones in most of the samples produced either by (semi)-synthesis or biotechnologically or isolated from natural products. Oligomeric A/S derivatives have not been studied for biological activity hitherto and a method to isolate them is essential.In the present study, solid-phase extraction (SPE) was applied for purification of commercial samples and isolation of monomeric and oligomeric A/S fractions, testing several stationary phases. Sephadex LH-20 cartridges achieved efficient purification for commercial samples containing both monomeric and dimeric A/S derivatives and also separation and isolation of both pure monomeric and dimeric A/S fractions for biological experiments. A high-performance liquid chromatography-diode array detection method was applied for detection, identification and quantification of monomeric and oligomeric shikonin fractions.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Chromatography, Liquid; Dextrans; Naphthoquinones; Solid Phase Extraction

Alkannin and shikonin (A/S) and their derivatives have been found in the roots of several Boraginaceous species and are also produced through plant tissue cultures. The chiral compounds A/S are potent pharmaceutical substances with a wide spectrum of biological and pharmacological activities like wound healing, antimicrobial, anti-inflammatory, anticancer and antioxidant activity. High-speed counter-current chromatography (HSCCC) was applied for the first time to the separation, preparative isolation and purification of A/S and their esters from extracts of Alkanna tinctoria roots, as well as commercial samples. The constituents of HSCCC fractions and their purity were determined by high-performance liquid chromatography-diode array detection-mass spectrometry (HPLC-DAD-MS), since DAD cannot detect oligomeric A/S derivatives that are present in most of the samples containing the respective monomeric derivatives. The purity of HSCCC fractions was compared with the one of fractions isolated by column chromatography (CC) using as stationary phases silica gel and Sephadex LH-20. As shown, the purity of monomeric alkannin/shikonin was greater by HSCCC than CC separation of commercial A/S samples.

Topics: Boraginaceae; Chromatography; Chromatography, High Pressure Liquid; Mass Spectrometry; Naphthoquinones; Plant Extracts; Plant Roots

Alkannin and shikonin (A/S) derivatives have been found in the roots of several Boraginaceous species and are produced through plant tissue cultures. The chiral compounds alkannins and shikonins are potent pharmaceutical substances with a wide spectrum of pharmacological activities such as wound healing, antimicrobial, anti-inflammatory, anticancer and antioxidant. Although oligomeric A/S derivatives have been detected in root extracts and commercial samples their detection and determination through high-performance liquid chromatography has not been reported. Therefore, in the present study a rapid, simple high-performance liquid chromatography-diode array detection-mass spectrometry (HPLC-DAD-MS) method was developed to detect, separate and determine monomeric and oligomeric/polymeric derivatives of alkannin/shikonin simultaneously for the first time. An optimization of HPLC-DAD parameters was performed. Both atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI) modes were applied, in order to compare detection of monomeric and oligomeric A/S. Additionally, oligomeric A/S constituents in several samples were identified and the mode of A/S polymerization was proposed.

Topics: Boraginaceae; Chromatography, High Pressure Liquid; Naphthoquinones; Plant Extracts; Plant Roots; Spectrometry, Mass, Electrospray Ionization

This work reports the extraction procedures of alkannin/shikonin mixture from roots of six populations of Onosma echioides, by means of three extraction techniques: Soxhlet extraction, maceration and rapid solid-liquid dynamic extraction (RSLDE). Five solvents with different polarity (hexane, petroleum ether, chloroform, ethyl acetate, methanol) were also studied. Analysis of the extracts was performed by an HPLC-DAD (diode array detector) system. The most efficient extraction technique was Soxhlet procedure using ethyl acetate for 6 h. Studied samples of O. echioides showed an alkannin/shikonin content in the range of 0.02-0.24 mg/kg. Other naphthoquinone derivatives (deoxyalkannin/deoxyshikonin and 5,8-dihydroxy-2-(4-methyl-6-oxo-5,6-dihydro-2H-pyran-2-yl)-[1,4]naphthoquinone and arnebin-6) were found for the first time in O. echioides and characterized in the extracts using HPLC-MS apparatus equipped with an ESI ionization source.

Topics: Boraginaceae; Chromatography, High Pressure Liquid; Molecular Structure; Naphthoquinones; Plant Extracts; Plant Roots; Solvents; Spectrometry, Mass, Electrospray Ionization

Monomeric alkannin and shikonin (A/S) are potent pharmaceutical substances with a wide spectrum of biological activity and comprise the active ingredients for several pharmaceutical preparations. Therefore, the determination of the impurities, degradation products or byproducts in alkannin and shikonin samples is of great importance. Oligomeric alkannin and shikonin are formed during biosynthesis of these bioactive secondary metabolites in Boraginaceaous root plants, during tissue culture production of A/S, during alkaline hydrolysis of A/S esters and also thermal treatment of A/S. In the present study, a dimeric alkannin/shikonin compound was isolated by size exclusion chromatography from alkannin and shikonin commercial samples and its structure was determined by one- and two-dimensional NMR spectroscopy. The structure of the most abundant oligomeric species in these samples, a dimeric naphthoquinone, was established for the fi rst time, indicating that coupling of the side chain of one naphthoquinone unit with the aromatic ring of a second naphthoquinone leads to dimer formation. This type of coupling allows further oligomerization by leaving one isohexenyl side chain available at the second monomer unit.

Topics: Boraginaceae; Chromatography, Gel; Dimerization; Naphthoquinones; Nuclear Magnetic Resonance, Biomolecular; Polymers

The effects of a naphthoquinone analogue, shikonin/alkannin (SA) and derivatives (acetylshikonin and beta,beta-dimethylacrylshikonin), on vascular reactivity were studied with isolated rat aortic rings. At lower concentrations, SA and its derivatives concentration-dependently inhibit the agonist-induced (acetylcholine and histamine) relaxation in PE precontracted aorta in an endothelium-dependent manner with IC (50) values ranging from 0.2 to 1.5 microM. In addition to the effect on agonist-induced vasorelaxation, the Ca (2+) ionophore A23187-induced vasorelaxation was also inhibited or reversed by SA. However, SA had no effect on sodium nitroprusside-induced (guanylate cyclase activator) vasorelaxation. These data suggested that SA and its derivatives might be acting as inhibitors of nitric oxide synthesis in endothelium. At a concentration greater than 10 microM, SA induced contraction of intact but not denuded aorta which could be inhibited by prior treatment with indomethacin, a cyclooxygenase inhibitor. In summary, the results from this study showed that SA and its derivatives inhibited agonist-induced relaxation at lower concentrations and induced vasocontraction at higher concentrations. All the effects seen with SA were endothelium-dependent, however, through different mechanisms. Abbreviations. SA:shikonin/alkannin PE:phenylephrine Ach:acetylcholine SNP:sodium nitroprusside eNOS:endothelial nitric oxide synthase L-NAME: Nw-nitro- L-arginine methyl ester

Topics: Acetylcholine; Animals; Aorta, Thoracic; Boraginaceae; Dose-Response Relationship, Drug; Endothelium, Vascular; Histamine; Inhibitory Concentration 50; Muscle Contraction; Naphthoquinones; Phytotherapy; Plant Extracts; Plant Roots; Rats; Rats, Wistar

Naturally occurring isohexenylnaphthazarins (IHN), such as Alkannin, Shikonin (A/S) and their derivatives, are potent pharmaceutical substances with a wide spectrum of biological activity. In the present study, inclusion complexes of alkannin and shikonin commercial samples and IHN derivatives in the form of an oily extract of Alkanna tinctoria roots were formed with beta-cyclodextrin (CD) and beta-HPCD. These complexes were investigated to evaluate the effect of complexation on their aqueous solubility, decoloration, and also the percentage of polymeric A/S and IHN derivatives enclosed in the CDs cavity, since these decrease the active monomeric IHN. Both beta-CD and beta-HPCD increased the aqueous solubility of A/S and IHN derivatives and thus inclusion complexes can be used as drug delivery systems for A/S in both internal (capsules, tablets) and external hydrophilic pharmaceutical and cosmetic preparations (creams, gels, sprays) with enhanced bioavailability. The inclusion complexes formed had a pale purple colour, contributing to the partial decoloration of the A/S and thus of the fi nal pharmaceutical preparations. Finally, CDs selectively included more monomeric and less polymeric IHN, compared with the initial each time sample that is encapsulated; thus inclusion complexes may present enhanced biological activity.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; beta-Cyclodextrins; Biological Availability; Capsules; Chemistry, Pharmaceutical; Cyclodextrins; Dosage Forms; Drug Carriers; Microscopy, Electron; Naphthoquinones; Plant Extracts; Solubility

The chiral pair alkannin and shikonin (A/S) and their isohexenylnaphthazarin (IHN) esters, which are naturally occurring hydroxynaphthoquinones (HNQ), are potent pharmaceutical substances with a wide spectrum of biological activity. The stability of A/S and their derivatives during process and storage is crucial to their use as drugs, cosmetics and food additives. The influence of alkaline media and of IHN esters hydrolysis was experimentally investigated on IHN polymerization by size exclusion chromatography (SEC). It was proved that during IHN esters hydrolysis, polymeric A/S and IHN are formed. An optimization of the hydrolysis conditions of IHN esters was also approached in terms of polymerization. Hydrolysis of IHN from a pure mixture of pigments proved preferable to that of preliminary root extracts by means of IHN polymerization, even for analytical determination; non-polar solvents are proposed for the extraction of IHN from roots, followed by hydrolysis, aiming to minimize the polymeric IHN and A/S formed. It was also proved that polymerization of IHN in alkaline media and during hydrolysis of IHN esters proceeds through the intermediate formation of semiquinones; after acidification, coupling of semiquinones with phenoxyl radicals results in polymeric IHN structures.

Topics: Boraginaceae; Chromatography, Gel; Cosmetics; Drug Stability; Esters; Food Additives; Hydrogen-Ion Concentration; Hydrolysis; Naphthoquinones; Pharmaceutical Preparations; Plant Roots; Polymers

Polymerization of naturally occurring isohexenylnaphthazarins (IHN), such as alkannin, shikonin (A/S) and their derivatives, which are potent pharmaceutical substances, significantly affects their use in pharmaceuticals, cosmetics and as food colorants, because it leads to reduction of the lustre of their red coloration, a decrease in their solubility and reduces the active monomeric IHN derivatives. In the present study, the influence of several crucial variables (processing and storage) was experimentally investigated on IHN polymerization by size exclusion chromatography (SEC). Temperature and solvent polarity increased significantly the concentration of hydroxynaphthoquinone (HNQ) polymers, while air and light exposure conditions did not significantly affect IHN polymerization. Low temperatures are proposed for all processes of industrial production of pharmaceutical preparations containing IHN and HNQ. An optimization of the industrial conditions used for the preparation of pharmaceutical and cosmetic preparations containing IHN, maximizing the active monomeric IHN fraction, was performed.

Topics: Air; Chemical Phenomena; Chemistry, Physical; Chromatography, Gel; Light; Models, Molecular; Molecular Structure; Naphthoquinones; Polymers; Solvents; Temperature

The chiral pair alkannin and shikonin (A/S) are potent pharmaceutical substances with a wide spectrum of biological activity; their enantiomeric ratio does not influence the major biological activity studied hitherto. Nevertheless, in pharmaceutical development and approval of chiral drugs from the Health and Regulatory Authorities, full documentation of methods of analysis of enantiomeric drugs, is required in order to evaluate the enantiomeric purity of starting materials and final products and to control the stability of enantiomers in pharmaceutical formulations under several experimental conditions. In the present study, the enantiomeric ratio of A/S was determined in several commercial samples of alkannin and shikonin and also the proportion of A/S derivatives in several Alkanna root samples, which are all used as active ingredients in pharmaceuticals. Light and air proved not to influence the enantiomeric ratio of A/S on a shikonin commercial sample, and temperature also did not alter the A/S ratio on shikonin and alkannin commercial samples. Microencapsulation of alkannin and shikonin commercial samples in ethylcellulose microspheres and also molecular inclusion of a shikonin commercial sample in beta-hydroxypropyl-cyclodextrin, which are used as drug delivery systems, did not alter the A/S enantiomeric ratio.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Capsules; Chromatography, High Pressure Liquid; Naphthoquinones; Pharmaceutical Preparations; Stereoisomerism; Temperature

Naturally occurring gums and resins with beneficial pharmaceutical and nutraceutical properties were tested for their possible protective effect against copper-induced LDL oxidation in vitro. Chiosmastic gum (CMG) (Pistacia lentiscus var. Chia resin) was the most effective in protecting human LDL from oxidation. The minimum and maximum doses for the saturation phenomena of inhibition of LDL oxidation were 2.5 mg and 50 mg CMG (75.3% and 99.9%, respectively). The methanol/water extract of CMG was the most effective compared with other solvent combinations. CMG when fractionated in order to determine a structure-activity relationship showed that the total mastic essential oil, collofonium-like residue and acidic fractions of CMG exhibited a high protective activity ranging from 65.0% to 77.8%. The other natural gums and resins (CMG resin 'liquid collection', P. terebinthus var. Chia resin, dammar resin, acacia gum, tragacanth gum, storax gum) also tested as above, showed 27.0%-78.8% of the maximum LDL protection. The other naturally occurring substances, i.e. triterpenes (amyrin, oleanolic acid, ursolic acid, lupeol, 18-a-glycyrrhetinic acid) and hydroxynaphthoquinones (naphthazarin, shikonin and alkannin) showed 53.5%-78.8% and 27.0%-64.1% LDL protective activity, respectively. The combination effects (68.7%-76.2% LDL protection) of ursolic-, oleanolic- and ursodeoxycholic- acids were almost equal to the effect (75.3%) of the CMG extract in comparable doses.

Topics: Cholesterol, LDL; Dose-Response Relationship, Drug; Gum Arabic; Humans; Karaya Gum; Mastic Resin; Naphthoquinones; Oils, Volatile; Oxidation-Reduction; Pigments, Biological; Pistacia; Plant Extracts; Resins, Plant; Structure-Activity Relationship; Triterpenes

Bioassay-directed fractionation of extract of Arnebia euchroma led to the isolation of alkannin (1), shikonin (2), and their derivatives (3-8) as the active principles against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). The stereochemistry of alpha-methylbutyryl alkannin (8) is revealed for the first time, and the antimicrobial activity of 8 was compared with its corresponding diastereomer (9). The derivatives 3-9 showed stronger anti-MRSA activity [minimum inhibitory concentrations (MICs) ranged from 1.56 to 3.13 microg/mL] than alkannin or shikonin (MIC = 6.25 microg/mL). Anti-MRSA activity of derivatives was bactericidal with minimum bactericidal concentration (MBC)/MIC < or = 2. In a time-kill assay, the bactericidal activity against MRSA was achieved as rapidly as 2 h. The derivatives 3-9 were also active against vancomycin-resistant Enterococcus faecium (F935) and vancomycin-resistant Enterococcus faecalis (CKU-17) with MICs similar to those with MRSA. Aromatic ester derivatives were also synthesized for antimicrobial activity comparison. None of these compounds were active against Gram-negative bacteria tested. Their cytotoxicity was also evaluated on selected cancer cell lines, and they expressed their activity in the range 0.6-5.4 microg/mL (CD(50)). Our results indicate that the ester derivatives of alkannin are potential candidates of anti-MRSA and anti-VRE agents with antitumor activity.

Topics: Anti-Bacterial Agents; Boraginaceae; Carcinoma, Hepatocellular; China; Drug Screening Assays, Antitumor; Electron Spin Resonance Spectroscopy; Enterococcus faecalis; Female; HeLa Cells; Humans; Methicillin Resistance; Microbial Sensitivity Tests; Molecular Structure; Naphthoquinones; Nuclear Magnetic Resonance, Biomolecular; Ovarian Neoplasms; Plant Bark; Plants, Medicinal; Staphylococcus aureus; Stereoisomerism; Tumor Cells, Cultured; Vancomycin

Alkannin and shikonin, two natural products from Alkanna tinctoria and Lithospermum erhythrorhizon (Boraginaceae), are used in folk medicine where they are claimed to possess, among other properties, wound healing and anti-inflammatory activity. We investigated, together with the structurally related naphthazarin, their in vitro antioxidant and hydroxyl radical scavenging activity as well as their in vivo antiinflammatory activity. I was found that all examined compounds significantly inhibited in vitro lipid peroxidation of ra hepatic microsomal membranes, competed with DMSO for free hydroxyl radicals, and reduced inflammation (mouse paw edema induced by FCA) very efficiently. The examined compounds proved equal or superior to the common reference compounds for each of these properties. I is concluded that the claimed and/or proven actions of alkannin and shikonin are attributable at least partly to their intervention in free radical processes.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Dimethyl Sulfoxide; Edema; Female; Free Radical Scavengers; Freund's Adjuvant; Lipid Peroxidation; Naphthoquinones; Rats; Rats, Inbred F344

The present study was carried out to compare the accelerating effect of shikonin and alkannin and to elucidate the expression of CD antigen and histological changes on the proliferation of granulation tissue in rats. Shikonin and alkannin produced a dose-dependent acceleration of the cotton pellet-induced granuloma formation and this accelerating potency of both compounds on the proliferation of granulation tissue was about the same 5 and 10 d after implantation of the cotton pellet. Also, both compounds increased the ratio of CD11b+ cells in the granulation tissue 5 and 10 d after implantation of the cotton pellet. Both compounds increased the expression of CD11b+ cells with granulocytes such as macrophages and histiocytes, and then accelerated the proliferation of fibroblasts and collagen fiber. On the other hand, neither compound increased the ratio of CD3+ cells in the granulation tissue after 5 and 10 d. These results suggest that shikonin and alkannin accelerate the proliferation of granulation tissue induced by the cotton pellet and this accelerating effect may be attributed to an increase in the expression of CD11b+ cells, and the acceleration of the proliferation of fibroblasts and collagen fiber in the granulation tissue.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; CD3 Complex; Flow Cytometry; Gossypium; Granulation Tissue; Granuloma, Foreign-Body; Lymphocytes; Macrophage-1 Antigen; Male; Naphthoquinones; Rats; Rats, Wistar; Skin; Stimulation, Chemical

The effects of shikonnin (SK) and its optical isomer alkannin (AK) on the hydroxyl radical (HO.) generation system including iron ions were evaluated using the spin trap method by ESR spectroscopy. 5,5-Dimethyl-1-pyrroline-1-oxide (DMPO) was used as a spin trap agent and HO. was generated by a reaction between an iron ion and hydrogen peroxide, which is called Fenton reaction system. SK inhibited the HO. spin adduct (DMPO-OH) yielded in a dose-dependent manner. In this effect no difference was observed between SK and AK. When different concentrations of DMPO were used for the confirmation of its competitive reaction, no difference was also observed in the concentration of SK required to reduce the amount of the DMPO-OH by 50% (ID50). These findings suggested that the inhibitory effect of SK against the thus yielded DMPO-OH was not generated by the scavenging for HO., but by the inhibition on the Fenton reaction system. The mechanism of the inhibition on this system may be based on the formation of a complex between SK and the iron ion. The molar ratio of SK to the iron ion in the complex was considered 2 to 1 (2:1), because the concentrations of the observed ID50 and the used iron ion exhibited the same value. In addition, the same result was also obtained from the study using spectroscopic analysis.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antineoplastic Agents, Phytogenic; Cyclic N-Oxides; Depression, Chemical; Dose-Response Relationship, Drug; Electron Spin Resonance Spectroscopy; Hydrogen Peroxide; Hydroxyl Radical; Iron; Isomerism; Naphthoquinones; Spin Trapping

The present study was carried out to compare the accelerative effect of shikonin (R-type), alkannin (S-type), and acetylshikonin on the proliferation of granulation tissue in rats, and to elucidate the correlation between the potency of the effect and their optical activity. Koushikon mainly contained the R-type of acetylshikonin, and Nanshikon mainly contained the S-type of acetylshikonin. Each compound produced a dose-dependent acceleration of the cotton pellet-induced granuloma formation. In comparing identical doses of shikonin, alkannin and acetylshikonin, the potency of their accelerative effects on the proliferation of granulation tissue was about the same. This result suggests that their absolute configurations (R-type or S-type) and their acetylation on the hydroxy group of the sidechain of shikonin or alkannin may not be important in producing the effect.

Topics: Animals; Anthraquinones; Anti-Inflammatory Agents, Non-Steroidal; Cell Division; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Gossypium; Granulation Tissue; Isomerism; Male; Naphthoquinones; Rats; Rats, Wistar

The circular dichroism (CD) and electronic absorption spectra of seven derivatives of shikonin and alkannin isolated from the roots of Onosma confertum W.W. Smith (from Sichuan) and Arnebia euchroma (Royle) Johust (from Xinjiang) were described. Each pair of CD spectra obtained was exact mirror images and the UV spectra of these compounds were very similar. In comparison with the CD spectra of shikonin given in literature, the configuration of the pigments from Sichuan was assigned R and from Xinjiang were all S.

Topics: Circular Dichroism; Drugs, Chinese Herbal; Molecular Conformation; Naphthoquinones; Species Specificity; Stereoisomerism

This study was carried out to compare the accelerative effect in ether extracts of "Koushikon" and "Nanshikon" on proliferation of granuloma tissue in rats, and to elucidate this effect on optical isomer of naphthoquinone derivatives in those extracts. The content of total naphthoquinone derivatives in the ether extracts of Koushikon and Nanshikon were found to be 56.1% and 25.4%. Among naphthoquinone derivatives, Koushikon contained mostly acetyl derivative and Nanshikon mostly teracryl derivative. The percentage of R-type (shikonin-type) in total naphthoquinone derivatives of the extracts was 85.5% and 3.8%. Each ether extract showed a dose-dependent acceleration on the cotton pellet-induced granuloma formation. Comparison with corresponding doses containing the same quantity of naphthoquinone derivatives showed the accelerative potency of ether extracts of Koushikon and Nanshikon to be about the same. The result suggests that the accelerative effect on proliferation of granuloma tissue depends primarily on the total content of naphthoquinone derivatives, and not on the ratio of the optically active isomers.

Topics: Animals; Cell Division; Drugs, Chinese Herbal; Granuloma; Male; Naphthoquinones; Rats; Rats, Wistar; Stereoisomerism