Natural product

Effect of Arnebin-1 on the proliferation of HUVECs [1]
PCNA is a nuclear cell proliferation marker. To determine whether arnebin-1 promotes the proliferation of HUVECs, the PCNA levels were measured by western blot analysis. At concentrations ranging from 1×10−3 µM to 10−1 µM, arnebin-1 alone had no significant effect on the PCNA levels (Fig. 1B). However, in the presence of VEGF (1 ng/ml), arnebin-1 significantly increased the expression of PCNA in a concentration-dependent manner (Fig. 1C). Consistent with the results of our previous study, we found that arnebin-1 had no noticeable effect on cell viability and proliferation (as no changes were observed in PCNA expression), as evaluated by MTT assay in the test range, but had a synergistic effect with VEGF in that it promoted HUVEC proliferation (Fig. 5A).

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Arnebin-1 activates the VEGFR2 signaling pathway [1]
It has been reported that VEGFR2 phosphorylation activates extensive downstream signaling substrates that are closely related to endothelial cell proliferation, migration and tube formation. To investigate whether arnebin-1 activates VEGFR2 and its downstream signaling molecules, we screened some elementary kinases related to the VEGFR2 signaling pathway. As shown in Fig. 2, arnebin-1 significantly increased the phosphorylation of VEGFR2, FAK, ERK and Src, induced by VEGF (1 ng/ml), in a concentration-dependent manner, which suggested that arnebin-1 exerted its pro-angiogenic effect by directly targeting VEGFR2 and subsequently activating the VEGFR2-induced downstream signaling cascade. These results are consistent with those of our previous study, which demonstrated that arnebin-1 promoted the proliferation, migration and tube formation of HUVECs in a concentration-dependent manner in the presence of VEGF.

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Arnebin-1 upregulates the expression levels of eNOS, VEGF and HIF-1α in HUVECs in a PI3K-dependent manner [1]
Subsequently, we investigated the effects of arnebin-1 on the expression levels of eNOS and VEGF in HUVECs. At concentrations ranging from 1×10−3 µM to 10−1 µM, arnebin-1 significantly increased the protein expression of eNOS in the HUVECs in a concentration-dependent manner compared to the vehicle-treated (control) cells (Fig. 3A). Moreover, arnebin-1 at 10−2 and 10−1 µM also markedly increased the expression and secretion of VEGF protein compared with the control group (Fig. 3B and C). Similarly, the expression of HIF-1α was also markedly upregulated by arnebin-1 (Fig. 3D). We further examined whether the upregulation of eNOS, VEGF and HIF-1α by arnebin-1 in HUVECs is mediated by its effect on the PI3K pathway. The protein expression of HIF-1α was markedly reduced by treatment with 2 µM LY294002 1 h prior to stimulation with 10−1 µM arnebin-1 (Fig. 3E). Similarly, the protein expression of eNOS, and the expression and secretion of VEGF protein, were also significantly decreased following pretreatment with 2 µM LY294002 (Fig. 3F–H).

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Arnebin-1 promotes the proliferation, migration and tube formation of HUVECs through the PI3K-dependent pathway [1]
In a previous study, we confirmed that arnebin-1 significantly promoted the proliferation, migration and tube formation of HUVECs in the presence of VEGF (1 ng/ml) in a concentration-dependent manner, with a maximal effect at 10−1 µM. In the present study, we investigated the mechanisms responsible for these effects of arnebin-1. As shown in Fig. 5, the proliferation, migration and tube formation of the HUVECs were enhanced by stimulation with a low concentration of VEGF (1 ng/ml) compared with the controls. Moreover, arnebin-1 at 10−1 µM and VEGF had a synergistic effect and markedly increased these processes compared with the cells treated with VEGF alone. However, when the HUVECs were pre-treated with LY294002, a PI3K inhibitor, the synergistic effects of arnebin-1 and VEGF on cell proliferation, migration and tube formation were abolished (Fig. 5). As was also shown, pre-treatment with LY294002 attenuated the increase in the expression levels of eNOS, VEGF and HIF-1α induced by arnebin-1 (Fig. 3). Collectively, these results suggest that arnebin-1 promotes the processes of endothelial cell proliferation, migration and tube formation which are associated with angiogenesis through the upregulation of eNOS, VEGF and HIF-1α in a PI3K-dependent manner.

Effect of Arnebin-1 on the expression of HIF-1α, eNOS and VEGF in diabetic wounds [1]
To investigate the mechanisms through which neovascularization is promoted, following treatment with arnebin-1, we measured the in vivo expression level of HIF-1α and its target genes, VEGF and eNOS. Western blot analysis revealed that the protein expression levels of HIF-1α, eNOS and VEGF were markedly decreased in the diabetic wounds compared with the non-diabetic wounds (Fig. 6). No significant difference was observed in the levels of HIF-1α, eNOS and VEGF between the diabetic and vehicle-treated groups. However, the expression of HIF-1α was markedly increased in the diabetic wounds following treatment with arnebin-1 (Fig. 6A). Compared with the diabetic and vehicle-treated groups, a higher level of eNOS expression was observed in the arnebin-1-treated group (Fig. 6B). Similarly, arnebin-1 significantly increased the protein expression of VEGF on the 7th day (Fig. 6C). Taken together, these results indicate that arnebin-1 promotes neovascularization in the wounds of diabetic rats by upregulating the expression levels of HIF-1α, eNOS and VEGF.

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Effect of Arnebin-1 on neovascularization and diabetic wound healing [1]
In in vitro experiments, we demonstrated that arnebin-1 and a low concentration of VEGF significantly increased the expression of PCNA, and that the administration of arnebin-1 without VEGF did not achieve the same result. In vivo, there was still a low level of VEGF in the diabetic wound tissues, and the localized application of arnebin-1 ointment to the wounds upregulated the expression of PCNA compared with the diabetic group the and vehicle-treated group (Fig. 7A), which was in accordance with our in vitro results. To determine the role of arnebin-1 in neovascularization in diabetic wounds, the expression of CD31, a biochemical marker of angiogenesis, was examined to analyze the effects of arnebin-1. In our previous study, using histological analysis, we demonstrated that diabetic wounds treated with arnebin-1 exhibited an increased capillary density on days 4 and 7 post-wounding. In the present study, following immunofluorescence staining with an anti-CD31 antibody for endothelial cells, positive staining was present in the wounds of the non-diabetic rats (Fig. 7B). This staining appeared to be markedly reduced in the wounds of the diabetic control animals and the vehicle-treated diabetic animals. We found that the number of CD31-positive blood vessels around the granulation-formation region was increased on the 7th day following treatment with arnebin-1. The results from quantitative analysis revealed that capillary density was significantly greater in the arnebin-1-treated group than the diabetic group (Fig. 7C). Moreover, the results of western blot analysis indicated that the protein level of CD31 was significantly increased following treatment with arnebin-1 compared with the other diabetic groups not treated with arnebin-1 (Fig. 7D).

Cell proliferation assay [1]
Cell proliferation was examined by mitochondrial MTT tetrazolium assay. The HUVECs were plated at 3×103 cells/well in 96-well plates. Overnight, the HUVECs were pre-treated with or without LY294002 (2 µM), and the medium was then replaced with the test medium supplemented with the vehicle [dimethyl sulfoxide (DMSO)] and Arnebin-1 (10−1 µM) with or without 1 ng/ml VEGF. After 24 h of incubation, the number of viable cells was detected using MTT reagent according to the manufacturer's instructions. In brief, 10 µl MTT (5 mg/ml) was added to 100 µl medium, and cultivated at 37°C for 4 h. After removing the supernatant, the formazan crystals were solubilized by the addition of DMSO. The absorbance (570 nm) of the medium was determined using a Biotek Elx-800 plate reader.

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Cell migration assay [1]
Cell migration assay was performed using Transwell chambers as previously described. The bottom chamber of the device contained 600 µl of the test medium. The HUVECs (5×104 cells/well) were added to the upper chamber and cultured in M199 medium with 2% FBS. After 24 h of incubation, the non-migrated cells that were above the faces of the membranes were removed. The migrating cells were fixed with methanol for 15 min, and then stained with 0.1% crystal violet for 20 min. The membranes were then rinsed with 30% glacial acetic acid. Finally, the washing solution was examined at 540 nm for the counting of the number of HUVECs.

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Tube formation assay [1]
To examine the pro-angiogenic effect Arnebin-1, we used the experimental in vitro Matrigel system, as previously described. Growth factor-reduced Matrigel basement membrane matrix was thawed on ice at 4°C overnight, and all pipettes and 96-well flat bottom plates were pre-cooled before use. The 96-well plates were coated with 50 µl Matrigel per well for 30 min at 37°C. The HUVECs were seeded at 4×104 cells per well in 100 µl assay medium. After 16 h of incubation, tube-like structures were photographed using an inverted microscope. The total tube length was quantified using ImageJ software.

Animals and induction of diabetes [1]
All animal procedures were approved by the Laboratory Animal Center of Sun Yat-sen University. As previously described, male Sprague-Dawley (SD) rats (weighing 250–300 g) were kept in stainless steel cages under pathogen-free conditions. The rats were housed in a controlled environment with a constant temperature of 18–22°C and a 12-h light-dark cycle; the rats were allowed access to food and water ad libitum. The rats were allowed to acclimatize for 4 weeks before the experimental procedures commenced. The rats were fasted for 12 h and were injected intraperitoneally with alloxan monohydrate dissolved in normal saline at a double dose of 100 mg/kg every other day to induce diabetes. Following the administration of alloxan for 3 days, the fasting blood glucose (FBG) levels of the rats were measured using a glucometer. The rats exhibiting FBG levels >16.7 mmol/l were confirmed as diabetic rats for the purposes of our research. The FBG levels were monitored before and after the experiments. The animals were randomly divided into 4 groups (1 non-diabetic and 3 diabetic groups; n=6) as follows: i) the non-diabetic group: rats were administered distilled water for 7 days (non-diabetic group); ii) the first diabetic group: the diabetic animals received distilled water (diabetic group); iii) the second diabetic group: the diabetic animals received the vehicle (ointment without Arnebin-1; DM-vehicle; D + V group); and iv) the third diabetic group: the diabetic animals were treated with arnebin-1 ointment (DM-arnebin-1; D + A group) for 7 days.

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Preparation of the ointment [1]
As described in a previous study of ours, ointment containing siritch (1.5 g), beeswax (5 g) and lard oil (0.15 g) was heated at 70–75°C to become solubilized, and 6.65 mg Arnebin-1 (0.1%) was then added and mixed in. Finally, the mixture was stirred until it cooled to room temperature. This ointment was used as the test compound.

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Drug administration [1]
As stated above, each diabetic rat had 3 wounds on the dorsal surface and the non-diabetic rats had 1 wound. In the D + V group, only wounds on the top of the dorsal side were treated with only the vehicle base (without the test compound). In the diabetic group, only wounds near the tail were treated with distilled water. In the D + A group, only wounds in the middle were treated with Arnebin-1 (0.1% ointment). Thus, in each group of rats, a different wound area was treated. The wounds at the top served as the vehicle controls for the treated wounds. The test compound ointment and the vehicle were applied every other day, in quantities sufficient to cover the wounds with a thin layer. All the treatments were continued until the day of sacrifice. The rats were sacrificed with the use of an intraperitoneal injection of an overdose of barbiturate.

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Tissue collection [1]
The rats were anesthetized with an overdose of pentobarbital (200 mg/kg, injected intraperitoneally) on day 7 post-wounding. The wound and a margin of approximately 5 mm of unwounded skin was excised. These wound tissues were snap-frozen in liquid nitrogen until they were processed for protein isolation.

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Western blot analysis [1]
In order to measure the levels of PCNA, CD31, HIF-1α, VEGF and eNOS in the tissue, wounds treated with Arnebin-1 or the vehicle were harvested on day 7 post-wounding. Following excision, the tissues were homogenized in lysis buffer. The VEGF, eNOS and HIF-1α expression levels were determined by western blot analysis as described above.

[1]. Arnebin-1 promotes angiogenesis by inducing eNOS, VEGF and HIF-1α expression through the PI3K-dependent pathway. Int J Mol Med. 2015 Sep;36(3):685-97.

[2]. Anti-skin ageing activity of napthoquinones from Arnebia nobilis Reichb.f. Nat Prod Res. 2016;30(5):574-7.

Alkannin beta,beta-dimethylacrylate is a hydroxy-1,4-naphthoquinone.
Alkannin beta,beta-dimethylacrylate has been reported in Arnebia euchroma, Alkanna cappadocica, and Alkanna tinctoria with data available.

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Arnebin-1, a naphthoquinone derivative, plays a crucial role in the wound healing properties of Zicao (a traditional wound healing herbal medicine). It has been noted that Arnebin-1, in conjunction with vascular endothelial growth factor (VEGF), exerts a synergistic pro-angiogenic effect on human umbilical vein endothelial cells (HUVECs) and accelerates the healing process of diabetic wounds. However, the mechanisms responsible for the pro-angiogenic effect of arnebin‑1 on HUVECs and its healing effect on diabetic wounds have not yet been fully elucidated. In this study, in an aim to elucidate these mechanisms of action of arnebin‑1, we investigated the effects of arnebin‑1 on the VEGF receptor 2 (VEGFR2) and the phosphoinositide 3-kinase (PI3K)‑dependent signaling pathways in HUVECs treated with VEGF by western blot analysis. The pro‑angiogenic effects of arnebin‑1 on HUVECs, including its effects on proliferation and migration, were evaluated by MTT assay, Transwell assay and tube formation assay in vitro. The expression levels of hypoxia-inducible factor (HIF)‑1α, endothelial nitric oxide synthase (eNOS) and VEGF were determined by western blot analysis in the HUVECs and wound tissues obtained from non‑diabetic and diabetic rats. CD31 expression in the rat wounds was evaluated by immunofluorescence staining. We found that the activation of the VEGFR2 signaling pathway induced by VEGF was enhanced by arnebin‑1. Arnebin‑1 promoted endothelial cell proliferation, migration and tube formation through the PI3K‑dependent pathway. Moreover, Arnebin‑1 significantly increased the eNOS, VEGF and HIF‑1α expression levels in the HUVECs and accelerated the healing of diabetic wounds through the PI3K‑dependent signaling pathway. CD31 expression was markedly enhanced in the wounds of diabetic rats treated with arnebin‑1 compared to the wounds of untreated diabetic rats. Therefore, the findings of the present study indicate that arnebin-1 promotes the wound healing process in diabetic rats by eliciting a pro-angiogenic response.[1]

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In conclusion, based on the outcomes of the present study, and in conjunction with our previous data, we confirmed that arnebin-1 markedly promotes the angiogenesis of HUVECs in vitro and that the topical application of arnebin-1 ointment accelerates the wound healing process in type I diabetic rats by inducing the expression levels of eNOS, VEGF and HIF-1α through the PI3K-dependent signaling pathway. Topical treatment with arnebin-1 ointment may thus be considered a novel therapeutic stratety for diabetic foot ulcers. Clinical tests are warranted to determine whether treatment with arnebin-1 can promote wound healing in patients with diabetes. The exact effects of arnebin-1 on fibroblasts and keratinocytes remain also to be investigated.[1]

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The present isolation and identification of napthoquinones from roots of Arnebia nobilis Reichb.f. can lead to the discovery of new anti-skin ageing ingredient in colour cosmetics. Four compounds have been isolated and purified by rigorous column chromatography. The compounds are identified as β, β-dimethylacryl alkannin (AN-I), acetoxyisovaleryl alkannin (AAN-II), acetyl alkannin (AN-III) and alkannin (AN-IV) by interpretation of spectroscopic data. This study is the first to report the isolation of Acetoxyisovaleryl alkannin (AAN-II) from A. nobilis. The IC50 values of the compounds, determined in human skin cells (human dermal fibroblasts and human keratinocytes) and mouse embryonic fibroblasts (NIH3T3) varied significantly among the four alkannins. Among the four compounds, β-acetoxyisovaleryl alkannin (AAN-II) significantly inhibited hydrogen peroxide (H2O2)-induced red blood corpuscle haemolysis and cellular senescence in human dermal fibroblasts. Collagen-I, elastin and involucrin syntheses in human dermal fibroblasts or keratinocytes were up regulated by AAN-II. These results support the potential utility of alkannins as novel anti-ageing ingredients.[2]

C21H22O6

370.3958

370.141

34539-65-6

(Rac)-Arnebin 1;5162-01-6;β,β-Dimethylacrylshikonin;24502-79-2

442720

Brown to reddish brown solid powder

1.3±0.1 g/cm3

573.9±50.0 °C at 760 mmHg

201.1±23.6 °C

0.0±1.6 mmHg at 25°C

1.589

5.98

2

6

6

27

706

1

CC(=CC[C@@H](C1=CC(=O)C2=C(C=CC(=C2C1=O)O)O)OC(=O)C=C(C)C)C

BATBOVZTQBLKIL-KRWDZBQOSA-N

InChI=1S/C21H22O6/c1-11(2)5-8-17(27-18(25)9-12(3)4)13-10-16(24)19-14(22)6-7-15(23)20(19)21(13)26/h5-7,9-10,17,22-23H,8H2,1-4H3/t17-/m0/s1

[(1S)-1-(5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl] 3-methylbut-2-enoate

34539-65-6; Alkannin beta,beta-dimethylacrylate; b,b-Dimethylacrylalkannin; NCIMech_000202; [(1S)-1-(5,8-dihydroxy-1,4-dioxonaphthalen-2-yl)-4-methylpent-3-enyl] 3-methylbut-2-enoate; CHEBI:2579; CHEMBL513640; beta,beta-dimethyl-acry-lalkannin;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~23.75 mg/mL (~64.12 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.75 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)