Research Update:Gromwell root.

  seminal trace...Gromwell Root extract.10:1.Lithospermum erythrorhizon Sieb.et Zucc.Shikonin.Arnebia Root,Gromwell Root,Radix Arnebiae,Radix Lithospermi,Radix Lithospermi extract,Arnebia euchroma (Royle) Johnst., or Lithospermum erythrorhizon Sieb.et Zucc.,or Arnebia guttata Bunge,Onosma paniculatum Bur. Et Franch,Radix Arnebiae Radix Lithospermi...

 


   Phytochemical info of Gromwell root.

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 Synonym:
 Definition:Gromwell root. are majorly composed of
 Chemical information disclosed as following table:


   Research Update:Gromwell root.

  Lithospermi radix extract inhibits histamine release and production of inflammatory cytokine in mast cells.:Biosci Biotechnol Biochem. 2007 Dec;71(12):2886-92. Epub 2007 Dec 7.

 Lithospermi radix (LR, Borraginaceae, the root of Lithospermum erythrorhizon Siebold. et Zuccarinii) is used in herbal medicine to treat such conditions as eczema, skin burns and frostbite. This study investigates the effects of LR on the anti-allergy mechanism. LR inhibited the release of histamine from rat peritoneal mast cells by compound 48/80 in a dose-dependent manner. LR orally administered at 6.59 mg/100 g also inhibited the anti-DNP IgE-induced passive cutaneous anaphylaxis reaction. LR inhibited the PMA plus A23187-induced increase in IL-6, IL-8, and TNF-alpha expression in HMC-1 cells. In addition, LR also inhibited nuclear factor-kappa B (NF-kappaB) activation and I kappaB-alpha degradation. These results show that LR had an inhibitory effect on the atopic allergic reaction. Furthermore, the in vivo and in vitro anti-allergic effect of LR suggests possible therapeutic applications of this agent for inflammatory allergic diseases.

  Comparison of the cytotoxic activities of naturally occurring hydroxyanthraquinones and hydroxynaphthoquinones.:Eur J Med Chem. 2007 Sep 14;

 Seven hydroxyanthraquinone derivatives, 1-7, were isolated from the root of Rheum palmatum (Polygonaceae). Two propionated anthraquinone derivatives, 8 and 9, were synthesized. Four hydroxynaphthoquinone derivatives, 13, 14, 16 and 21, were isolated from the root of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae) and also three naphthoquinone derivatives, 19, 22 and 23, were isolated from the root of Macrotomia euchroma (Royle) Pauls. (Boraginaceae). The cytotoxicity of the anthraquinone and naphthoquinone derivatives on P-gp-underexpressing HCT 116 cells and P-gp-overexpressing Hep G2 cells was examined by MTT assay. Among the anthraquinone derivatives, compounds 3-5 which had OH, CH(2)OH and COOH substituent groups on the anthraquinone skeletons, respectively, showed potent growth inhibitory activities against both types of cancer cells (IC(50) values: 5.7+/-0.9 to 13.0+/-0.7muM in the case of HCT 116 cells and 5.2+/-0.7 to 12.3+/-0.9muM in the case of Hep G2 cells). All hydroxynaphthoquinone derivatives isolated in this study exhibited extremely potent growth inhibitory activities against both types of cancer cells (IC(50) values: 0.3+/-0.09 to 0.46+/-1.0muM in the case of HCT 116 cells and 0.22+/-0.03 to 0.59+/-0.06muM in the case of Hep G2 cells) as well as shikonin 10 (IC(50) values: 0.32+/-0.02muM in the case of HCT 116 cells and 0.24+/-0.03muM in the case of Hep G2 cells).

  Protection of human keratinocytes from UVB-induced inflammation using root extract of Lithospermum erythrorhizon.:Biol Pharm Bull. 2007 May;30(5):928-34.

 UVB irradiation is an important inducer of biological changes in skin and can activate inflammatory reactions and apoptotic pathways, leading to skin damage. A root extract of Lithospermum erythrorhizon (SK), which has naphthoquinone pigments containing shikonin and shikonin derivatives, is known for its anti-inflammatory, anti-bacterial, and anti-tumor activity, and for its scavenging of reactive oxygen species. However, the effect of SK against UV damage is not clear. The aim of this study was to evaluate the efficacy of SK against UVB induced damage in normal human epidermal keratinocytes (NHEK). UVB-irradiated NHEK showed decreased cell viability, increased production of interleukin (IL)-1alpha, IL-6, IL-8, and tumor necrosis factor-alpha, and induced apoptosis. In an apoptosis pathway assay, UVB-irradiated NHEK showed increased caspase-3 activity, p53 and its phosphorylation at serine 15 compared with non-irradiated cells. All these effects induced by UVB irradiation were clearly inhibited by treatment with SK before and after UVB irradiation for 24 h. It is suggested that SK can protect epidermal cells against harmful effects of UVB irradiation and that SK treatment is probably beneficial for photoprotection of the skin.

  Human ACAT inhibitory effects of shikonin derivatives from Lithospermum erythrorhizon.:Bioorg Med Chem Lett. 2007 Feb 15;17(4):1112-6. Epub 2006 Nov 10.

 Three naphthoquinones were isolated by bioassay-guided fractionation from the CHCl(3) extracts of roots of Lithospermum erythrorhizon. They were identified as acetylshikonin (1), isobutyrylshikonin (2), and beta-hydroxyisovalerylshikonin (3) on the basis of their spectroscopic analyses. The compounds 1-3 were tested for their inhibitory activities against human ACAT-1 (hACAT-1) or human ACAT-2 (hACAT-2). Compound 2 preferentially inhibited hACAT-2 (IC(50)=57.5microM) than hACAT-1 (32% at 120microM), whereas compounds 1 and 3 showed weak inhibitory activities in both hACAT-1 and -2. To develop more potent hACAT inhibitor, shikonin derivatives (5-11) were synthesized by semi-synthesis of shikonin (4), which was prepared by hydrolysis of 1-3. Among them, compounds 5 and 7 exhibited the strong inhibitory activities against hACAT-1 and -2. Furthermore, we demonstrated that compound 7 behaved as a potent ACAT inhibitor in not only in vitro assay system but also cell-based assay system.

  Non-radioactive and colorimetric quantification of monocyte adhesion to endothelial cells in early atherogenesis.:Biotechnol Lett. 2006 Nov;28(22):1805-10. Epub 2006 Sep 26.

 Monocyte adhesion to vascular endothelium is an initial step in atherogenesis. To quantify this, we incubated monocytes with cultured endothelial cells, and quantified the adhered live monocytes using a colorimetric assay. Endothelium activated with lipopolysaccharide attracted monocytes in a dose-dependent manner and the adhesion was attenuated with post-treatments with L-ascorbic acid (53%), alpha- (40%) and gamma-tocopherol (39%), resveratrol (39%), and Lithospermum erythrorhizon root extract (45%). This non-radioactive, colorimetric assay may be useful for screening anti-atherogenic compounds in early atherogenesis.


  The shikonin derivatives and pyrrolizidine alkaloids in hairy root cultures of Lithospermum canescens (Michx.) Lehm.:Plant Cell Rep. 2006 Oct;25(10):1052-8. Epub 2006 May 3.Pietrosiuk A, Syk?owska-Baranek K, Wiedenfeld H, Wolinowska R, Furmanowa M, Jaroszyk E.Department of Biology and Pharmaceutical Botany, Medical University of Warsaw, ul. Banacha 1, 02-097 Warsaw, Poland. ap@farm.amwaw.edu.pl

 Hairy root cultures of Lithospermum canescens were established using three strains of Agrobacterium rhizogenes: ATCC 15834, LBA 9402 and NCIB 8196. Eight lines resulting from infection with A. rhizogenes ATCC 15834 demonstrated sufficient biomass increase and were submitted to further investigations. The contents of acetylshikonin (ACS) and isobutyrylshikonin (IBS) in transformed hairy roots made up ca. 10% of those observed in natural roots of L. canescens (24.35 and 14.48 mg g(-1) DW, respectively). One line, Lc1-D, produced the largest amounts of ACS (2.72 mg g(-1) DW) and IBS (0.307 mg g(-1) DW). Traces of pyrrolizidine alkaloids (PA), canescine and canescenine, were found in all lines of transformed hairy roots.

  Inhibition of lipopolysaccharide and interferon-gamma-induced expression of inducible nitric oxide synthase and tumor necrosis factor-alpha by Lithospermi radix in mouse peritoneal macrophages.:J Ethnopharmacol. 2005 Dec 1;102(3):412-7. Epub 2005 Jul 28.

 Lithospermi radix (LR, root of Lithospermum erythrorhizon Siebold. et Zuccarinii) has been used to treat various conditions, such as septic shock, eczema and burns. In this study, the effect of LR on lipopolysaccharide (LPS) and recombinant interferon-gamma (rIFN-gamma)-induced production of nitric oxide (NO) and tumor necrosis factor (TNF)-alpha were examined using mouse peritoneal macrophages. At 0.01-1 mg/ml, LR inhibited the LPS/rIFN-gamma-induced expression of inducible nitric oxide synthase (iNOS) and TNF-alpha release. To clarify the mechanism involved, the effect of LR on the activation of nuclear factor (NF)-kappaB was examined. The LPS/rIFN-gamma-induced activation of NF-kappaB was almost completely blocked by LR at 1mg/ml without cytotoxicity. These findings demonstrate that the inhibition of the LPS/rIFN-gamma-induced production of NO and TNF-alpha by LR involves the inhibition of NF-kappaB activation.

  Monoamine oxidase inhibitory naphthoquinones from the roots of Lithospermum erythrorhizon.:Arch Pharm Res. 2005 Apr;28(4):400-4.

 Activity-guided fractionation of a hexane-soluble extract of the roots of Lithospermum erythrorhizon, using a mouse brain monoamine oxidase (MAO) inhibition assay, led to the isolation of two known naphthoquinones, acetylshikonin and shikonin, and a furylhydroquinone, shikonofuran E. These compounds were shown to inhibit MAO with IC50 values of 10.0, 13.3, and 59.1 microM, respectively. Although no specificity for MAO-A and MAO-B was shown by acetylshikonin and shikonin, a Lineweaver-Burk plot analysis indicated that the inhibition was competitive for both MAO-A and MAO-B activity.

  Enhancement of natural pigment extraction using Bacillus species xylanase.:J Agric Food Chem. 2005 Apr 6;53(7):2541-5.

 Pigment extracts from the root of Lithospermum erythrorhizon are used as natural red dyes, as well as basic drugs due to their numerous pharmacological activities. In recent years, the demand for such natural pigment materials has increased; however, in natural dye production, the pigment yield is strongly affected by the source of cultivation, extracting conditions, and solvents. Accordingly, this study proposes a method of enzymatic pigment production based on the introduction of hydrolytic enzymes prior to the usual extraction to avoid repeated pigment extraction. The matrix destruction in the epidermal layer of the root by the enzymes was found to improve the pigment extractability, that is, the increment of K(L), the mass transfer coefficient, representing the pigment mobility in the epidermal layer. The root tissue maceration by the hydrolytic enzymes was also measured to evaluate the pigment extractability, and a linear relationship was observed between the K(L) values and the tissue maceration up to the addition of 3000 units/g of xylanase, indicating that the enzymatic maceration proportionally increases the interfacial area between the pigment and the solvent. Bacillus sp. DX107 xylanase only served to increase the extractability of the pigment by loosening the root shell matrix, without affecting the contents and color properties of the pigment, as almost no difference was found in the color between the pigments extracted using xylanase and those extracted according to the traditional method.

  The effect of acetylshikonin isolated from Lithospermum canescens roots on tumor-induced cutaneous angiogenesis.:Acta Pol Pharm. 2004 Sep-Oct;61(5):379-82. Pietrosiuk A, Furmanowa M, Skopi¨½iska-R¨®zewska E, Sommer E, Skurzak H, Bany J.Department of Biology and Pharmaceutical Botany, Medical University of Warsaw, 1 Banacha Str., 02-097 Warsaw, Poland

 This study has demonstrated that acetylshikonin (ACS), the isolated ingredient from Lithospermum canescens Lehm. roots, in a daily dose of 200 microg for 3 days, inhibited cutaneous angiogenesis induced by L-1 sarcoma cells in Balb/c mice.


  Immunomodulatory effect of shikonin derivatives isolated from Lithospermum canescens on cellular and humoral immunity in Balb/c mice.:Pharmazie. 2004 Aug;59(8):640-2. Pietrosiuk A, Skopi¨½ska-R¨®zewska E, Furmanowa M, Wiedenfeld H, Sommer E, Sokolnicka I, Rogala E, Radomska-Le?niewska D, Bany J, Malinowski M.Department of Biology and Pharmaceutical Botany, Medical University of Warsaw, Poland. ap@farm.amwaw.edu.pl

 The immunomodulatory activity of acetylshikonin (ACS) and isobutyrylshikonin (IBS) was studied in female and male inbred Balb/c mice, and in F1 hybrids (Balb/c x C3H). ACS and IBS were isolated from Lithospermum canescens Lehm. (Boraginaceae) roots. Splenocytes from mice fed 40 microg of ACS had higher proliferative potential in cultures with PHA than corresponding controls and also higher migratory in vitro activity than splenocytes obtained from control animals. ACS at a 40 microg daily dose stimulated G-v-H reaction but inhibited it at a 200 microg dose. IBS at a 40 microg dose significantly increased humoral response.

  Involvement of reactive oxygen species, but not mitochondrial permeability transition in the apoptotic induction of human SK-Hep-1 hepatoma cells by shikonin.:Planta Med. 2003 Dec;69(12):1119-24.

 Shikonin has been demonstrated to exhibit anti-cancer activity, but the underlying mechanisms are poorly understood. In this report, we showed that the administration of shikonin could result in the induction of apoptotic cell death of human hepatoma cell line, SK-Hep-1. As evident by the flow-cytometric studies, shikonin has the capability of generating increased amounts of intracellular reactive oxygen species (ROS) during the early stage of this apoptotic process (ca. one-hour), and subsequently accompanied by the dissipation of mitochondrial transmembrane potential (deltapsi (m)) at 3 hours. Further studies indicated that this apoptotic process could effectively be protected by the pretreatment of shikonin-treated cells with glutathione (GSH) and N-acetylcysteine (NAC), a precursor of GSH, but not by cyclosporin A (CyA), an inhibitor of mitochondrial permeability transition (MPT) pore. These data further proved that ROS-mediated oxidative stress was the pivotal element involved in the induction of apoptosis of SK-Hep-1 cells. Taken together, we suggest that shikonin-induced apoptosis of SK-Hep-1 cells proceeds by an oxidative stress-mediated pathway.

  Shikonins, phytocompounds from Lithospermum erythrorhizon, inhibit the transcriptional activation of human tumor necrosis factor alpha promoter in vivo.:J Biol Chem. 2004 Feb 13;279(7):5877-85. Epub 2003 Nov 25.

 Tumor necrosis factor alpha (TNF-alpha) contributes to the pathogenesis of both acute and chronic inflammatory diseases and has been a target for the development of new anti-inflammatory drugs. Shikonins, the naphthoquinone pigments present in the root tissues of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), have been reported to exert anti-inflammatory effects both in vitro and in vivo. In this study, we evaluated the effects of shikonin and its derivatives on the transcriptional activation of human TNF-alpha promoter in a gene gun-transfected mouse skin system by using a luciferase reporter gene assay. The crude plant extract of L. erythrorhizon as well as derived individual compounds shikonin, isobutyryl shikonin, acetyl shikonin, dimethylacryl shikonin and isovaleryl shikonin showed significant dose-dependent inhibition of TNF-alpha promoter activation. Among the tested compounds, shikonin and isobutyryl shikonin exhibited the highest inhibition of TNF-alpha promoter activation and also showed significant suppression of transgenic human TNF-alpha mRNA expression and protein production. We demonstrated that shikonin-inhibitory response was retained in the core TNF-alpha promoter region containing the TATA box and a 48-bp downstream sequence relative to the transcription start site. Further our results indicated that shikonin suppressed the basal transcription and activator-regulated transcription of TNF-alpha by inhibiting the binding of transcription factor IID protein complex (TATA box-binding protein) to TATA box. These in vivo results suggest that shikonins inhibit the transcriptional activation of the human TNF-alpha promoter through interference with the basal transcription machinery. Thus, shikonins may have clinical potential as anti-inflammatory therapeutics.

  Shikonin, a component of chinese herbal medicine, inhibits chemokine receptor function and suppresses human immunodeficiency virus type 1.:Antimicrob Agents Chemother. 2003 Sep;47(9):2810-6.Chen X, Yang L, Zhang N, Turpin JA, Buckheit RW, Osterling C, Oppenheim JJ, Howard OM.Basic Research Program, SAIC-Frederick, Inc, National Cancer Institute-Frederick, Frederick, Maryland 21702-1201, USA.

 Shikonin is a major component of zicao (purple gromwell, the dried root of Lithospermum erythrorhizon), a Chinese herbal medicine with various biological activities, including inhibition of human immunodeficiency virus (HIV) type 1 (HIV-1). G protein-coupled chemokine receptors are used by HIV-1 as coreceptors to enter the host cells. In this study, we assessed the effects of shikonin on chemokine receptor function and HIV-1 replication. The results showed that, at nanomolar concentrations, shikonin inhibited monocyte chemotaxis and calcium flux in response to a variety of CC chemokines (CCL2 [monocyte chemoattractant protein 1], CCL3 [macrophage inflammatory protein 1alpha], and CCL5 [regulated upon activation, normal T-cell expressed and secreted protein]), the CXC chemokine (CXCL12 [stromal cell-derived factor 1alpha]), and classic chemoattractants (formylmethionyl-leucine-phenylalanine and complement fraction C5a). Shikonin down-regulated surface expression of CCR5, a primary HIV-1 coreceptor, on macrophages to a greater degree than the other receptors (CCR1, CCR2, CXCR4, and the formyl peptide receptor) did. CCR5 mRNA expression was also down-regulated by the compound. Additionally, shikonin inhibited the replication of a multidrug-resistant strain and pediatric clinical isolates of HIV in human peripheral blood mononuclear cells, with 50% inhibitory concentrations (IC(50)s) ranging from 96 to 366 nM. Shikonin also effectively inhibited the replication of the HIV Ba-L isolate in monocytes/macrophages, with an IC(50) of 470 nM. Our results suggest that the anti-HIV and anti-inflammatory activities of shikonin may be related to its interference with chemokine receptor expression and function. Therefore, shikonin, as a naturally occurring, low-molecular-weight pan-chemokine receptor inhibitor, constitutes a basis for the development of novel anti-HIV therapeutic agents.

  An extract of the root of Lithospermun erythrorhison accelerates wound healing in diabetic mice.:Biol Pharm Bull. 2003 Mar;26(3):329-35.

 Many people suffer from intractable bedsores, which sometimes develop because of chronic metabolic failure in patients. An extract of the root of Lithospermun erythrorhison (SK) has been reported to have an effect on wound healing. However, the effects of SK have not been studied in chronic wounds, such as bedsores. The healing-impaired diabetic (db/db) mouse is a good model for the investigation of clinical healing therapies. Therefore, we examined whether SK accelerates wound healing in db/db mice. Full-thickness round wounds of 6-mm diameter were created on the backs of mice. After applying SK, we covered the wound with a film dressing to keep it moist. At three weeks, wound closure was complete in SK-treated mice but not in controls. Capillary vessel number and collagen synthesis increased early in wound healing in SK-treated wounds. At this time, vascular endothelial growth factor (VEGF)-positive neutrophils had infiltrated the wound and the appearance of apoptotic fibroblasts and endothelial cells in the granulation tissue was more advanced than in the controls. Where the wound was covered with epithelium, there tended to be less infiltration of VEGF-positive cells and apoptotic cells. These results suggest that the inflammatory phase was shortened, and the proliferative and maturation phases were advanced by SK. It is known that SK also has antibacterial activity. Therefore, we conclude that SK is useful for wound healing in db/db mice, and could potentially help patients with intractable bedsores.


  High level expression of chorismate pyruvate-lyase (UbiC) and HMG-CoA reductase in hairy root cultures of Lithospermum erythrorhizon.:Plant Cell Physiol. 2002 Aug;43(8):894-902.K?hle A, Sommer S, Yazaki K, Ferrer A, Boronat A, Li SM, Heide L.Pharmazeutische Biologie, Pharmazeutisches Institut, Eberhard-Karls-Universit?t T¨¹bingen, Auf der Morgenstelle 8, D-72076 T¨¹bingen, Germany.

 Shikonin, a red naphthoquinone pigment, is produced by cell cultures of Lithospermum erythrorhizon (Boraginaceae). It is biosynthetically derived from two key precursors, 4-hydroxybenzoate (4HB) and geranyldiphosphate (GPP). The bacterial ubiC gene, encoding chorismate pyruvate-lyase (CPL) which converts chorismate to 4-hydroxybenzoate, was expressed in L. erythrorhizon under the control of the strong (ocs)(3)mas-promoter. This introduced an efficient biosynthetic pathway to 4HB, i.e. a one-step reaction from chorismate, in addition to the endogeneous multi-step phenylpropanoid pathway. Feeding experiments with [1,7-(13)C(2)]shikimic acid showed that in the most active transgenic line, 73% of 4HB was synthesized via the genetically introduced pathway. However, there was no correlation between CPL activity and 4HB glucoside or shikonin accumulation in the transgenic lines. HMG-CoA reductase (HMGR) is involved in the biosynthesis of GPP in L. erythrorhizon. Two forms of HMGR1 of Arabidopsis thaliana were expressed in Lithospermum under control of the (ocs)(3)mas promoter. Only moderate increases in enzyme activity were obtained with the complete enzyme, but high activity was achieved using the soluble cytosolic domain of HMGR1. Shikonin accumulation remained unchanged even upon high expression of soluble HMGR.

  Cellular pharmacology studies of shikonin derivatives.:Phytother Res. 2002 May;16(3):199-209. Review.Chen X, Yang L, Oppenheim JJ, Howard MZ.Laboratory of Molecular Immunoregulation, Division of Basic Sciences, National Cancer Institute-Frederick, MD 21702-1201, USA.

 The naphthoquinone pigment, shikonin, isolated from Lithospermum erythrorhizon Sieb. et Zucc.(Boraginaceae) and its derivatives are the active components isolated from the Chinese herbal therapeutic, Zicao. Historically, Zicao root extracts have been used to treat macular eruption, measles, sore-throat, carbuncles and burns. Multiple pharmacological actions have been attributed to shikonin, e.g. antiinflammatory, antigonadotropic and anti-HIV-1 activity. In this review, several therapeutic applications of shikonin will be summarized including its pleiotropic, antiinflammatory and antitumour effects. Widely diverse and sometimes conflicting activities have been attributed to shikonin, e.g. wound healing, enhanced granuloma formation, suppression of local acute inflammatory reactions, inhibition of angiogenesis, inhibition of select chemokine ligands, inhibition of DNA topoisomerase activity, inhibition of platelet activation and antimicrobial activity. Comparison of the various reported mechanisms of action for shikonin lead us to hypothesize that shikonin is an effective inhibitor of protein-protein interaction with multiple targets in both the intracellular and extracellular compartments. This general inhibitory effect can account for the broad spectrum of shikonin biological and pharmacological activities.

  Cell-specific production and antimicrobial activity of naphthoquinones in roots of lithospermum erythrorhizon.:Plant Physiol. 1999 Feb;119(2):417-28.Brigham LA, Michaels PJ, Flores HE.Department of Plant Pathology, The Pennsylvania State University, University Park, Pennsylvania 16802-4507, USA

 Pigmented naphthoquinone derivatives of shikonin are produced at specific times and in specific cells of Lithospermum erythrorhizon roots. Normal pigment development is limited to root hairs and root border cells in hairy roots grown on "noninducing" medium, whereas induction of additional pigment production by abiotic (CuSO4) or biotic (fungal elicitor) factors increases the amount of total pigment, changes the ratios of derivatives produced, and initiates production of pigment de novo in epidermal cells. When the biological activity of these compounds was tested against soil-borne bacteria and fungi, a wide range of sensitivity was recorded. Acetyl-shikonin and beta-hydroxyisovaleryl-shikonin, the two most abundant derivatives in both Agrobacterium rhizogenes-transformed "hairy-root" cultures and greenhouse-grown plant roots, were the most biologically active of the seven compounds tested. Hyphae of the pathogenic fungi Rhizoctonia solani, Pythium aphanidermatum, and Nectria hematococca induced localized pigment production upon contact with the roots. Challenge by R. solani crude elicitor increased shikonin derivative production 30-fold. We have studied the regulation of this suite of related, differentially produced, differentially active compounds to understand their role(s) in plant defense at the cellular level in the rhizosphere.


  Scientific References:

  1.Research Update:Gromwell root.