Natural product; anti-osteoporotic, cytoprotective, and hepatoprotective effects
- Sweroside modulates melanogenesis by activating Akt and extracellular signal-regulated protein kinase (ERK) signaling pathways, leading to decreased MITF (microphthalmia-associated transcription factor) expression, which in turn downregulates tyrosinase, TRP-1 and TRP-2 [2].

In the course of screening for the melanogenesis inhibitors, sweroside was isolated from Lonicera japonica. Its chemical structure was determined on the basis of spectroscopic analysis, including mass spectroscopy and nuclear magnetic resonance analysis. Sweroside inhibited potent melanogenesis in melan-a cells at 300μM without cytotoxicity. Also, sweroside decreased tyrosinase, tyrosinase-related protein-1 (TRP-1) and TRP-2 protein production in melan a cells. To identify the signaling pathway of sweroside, the ability of sweroside to influence Akt and extracellular signal-regulated protein kinase (ERK) activation was investigated. Sweroside induced Akt and ERK in a dose-dependent manner. In addition, the specific inhibition of the Akt and ERK signaling pathways were studied by specific inhibitor LY294002 and U0126, respectively and it was causing the increased melanin synthesis.[2]

---

Wound healing properties of Gentian (Gentiana lutea ssp. symphyandra) extract and its main constituents, gentiopicroside, sweroside and swertiamarine (compounds 1-3, respectively) were evaluated by comparison with dexpanthenol on cultured chicken embryonic fibroblasts. The extract was also analyzed by HPLC to quantify its constituents. Chicken embryonic fibroblasts from fertilized eggs were incubated with the plant extract and its constituents, compounds 1-3. Using microscopy, mitotic ability, morphological changes and collagen production in the cultured fibroblasts were evaluated as parameters. Wound healing activity of Gentian seems to be mainly due to the increase in the stimulation of collagen production and the mitotic activity by compounds 2 and 3, respectively (p < 0.005 in all cases). All three compounds also exhibited cytoprotective effects, which may cause a synergism in terms of wound healing activity of Gentian. The findings demonstrated the wound healing activity of Gentian, which has previously been based only on ethnomedical data[1].

---

- In cultured chicken embryonic fibroblasts, Sweroside (compound 2) at 0.4 μg/mL caused a slight increase in the total number of fibroblasts (p<0.10 compared to DMSO control), while at 2 μg/mL it was ineffective (p>0.10). It did not significantly affect mitotic activity (p>0.10 for both concentrations). At 0.4 μg/mL, Sweroside significantly increased the percentage of polygonal fibroblasts (p<0.005) and at 2 μg/mL also increased polygonal cells (p<0.05). It slightly decreased the percentage of fusiform cells only at 2 μg/mL (p<0.10). Sweroside exhibited the most potent effect on collagen production among the tested compounds, increasing the number of collagen granules in fibroblasts more than 8-fold (p<0.005 at 0.4 μg/mL and p<0.01 at 2 μg/mL). It also significantly decreased the percentages of vacuole-containing cells and round cells (p<0.005 and p<0.01, respectively) [1].
- In melan‑a murine melanocytes, Sweroside at concentrations up to 300 μM showed no significant cytotoxicity. It significantly reduced melanin content in a dose-dependent manner (0–300 μM, p<0.01 and p<0.001 vs. control). Western blot analysis revealed that Sweroside decreased the protein expression of tyrosinase, TRP-1 and TRP-2 in a dose-dependent manner (p<0.001 at 300 μM vs. untreated). Sweroside also increased phosphorylation of Akt (p-Akt) dose-dependently (p<0.001 at 300 μM) and slightly increased p-ERK levels, while it significantly reduced MITF protein expression in a dose-dependent manner (p<0.01 at 100 μM, p<0.001 at 300 μM vs. untreated). The specific Akt inhibitor LY294002 and ERK inhibitor U0126 reversed the inhibitory effects of Sweroside on melanin synthesis and MITF expression, confirming that Sweroside acts via Akt and ERK pathways [2].

Sweroside presented inhibition of the body pigmentation and tyrosinase activity in zebrafish in vivo model. These results suggest that sweroside isolated from L. japonica may be an effective skin-whitening agent through the regulates the expression of MAP kinase and melanogenic enzymes[2].

---

- In a zebrafish (Danio rerio) model, Sweroside was added to embryo medium from 9 to 72 hours post‑fertilization (h.p.f.) at various concentrations. Observation under a stereomicroscope showed that Sweroside dose‑dependently reduced the number of melanin spots on zebrafish embryos. Total melanin content measured from whole embryo extracts (treated from 9 to 35 h.p.f.) was potently decreased by Sweroside (p<0.01 and p<0.001 vs. control). Tyrosinase activity measured from zebrafish embryos (treated from 9 to 48 h.p.f.) was also significantly reduced by Sweroside (p<0.01 and p<0.001 vs. control). More than 95% of zebrafish survived all treatments with Sweroside at the tested concentrations [2].

Compound treatment and phenotype-based evaluation[2]
Synchronized embryos were collected and arrayed by pipette (7–9 embryos per well in 24 well plates containing 1 ml of embryo medium). Test compounds were dissolved in 0.1% DMSO, and then added to the embryo medium from 9 to 72 h post-fertilization (h.p.f) (63 h exposure). The effects on the pigmentation of zebrafish were observed under the stereomicroscope. Occasional stirring as well as replacement of the medium was performed daily to ensure the even distribution of the compounds. In all experiments, 0.1 mM PTU was used to generate transparent zebrafish without interfering in the developmental process, and considered as a standard positive control. Phenotype-based evaluations of body pigmentation were dechorionated by forceps, anesthetized in tricaine methanesulfonate solution, mounted in 3% methylcellulose on a 35 mm dish (35 × 10 mm), and photographed under the stereomicroscope MZ16.

---

- Tyrosinase activity was measured in zebrafish embryos. Approximately 100 zebrafish embryos were treated with Sweroside from 9 to 48 h.p.f., then sonicated in protein extraction solution. The lysate was clarified by centrifugation at 10,000 g for 5 min. After protein quantification, 250 μg of total protein was mixed with 100 μL of 5 mM L‑3,4‑dihydroxyphenylalanine (L‑DOPA). A control well contained 100 μL of lysis buffer and 100 μL of 5 mM L‑DOPA. After incubation for 60 min at 37 °C, absorbance was measured at 475 nm using a microplate reader [2].

Cell culture[2]
Melan-a melanocytes are a highly pigmented, immortalized normal murine melanocyte cell line derived from C57BL/6 mice. The melan-a cell used in this study was obtained from Dr. Dorothy Bennett. Cells were cultured at 37 °C in an atmosphere of 95% air, 5% CO2 in RPMI 1640 medium supplemented to a final concentration with 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin and 200 nM 12-o-tetradecanoylphorbol-13-acetate. Cell viability was determinate by CCK-8 cell counting kit-8. Monolayers of confluent melanocytes were harvested with a mixture of 0.05% trypsin and 0.53 mM EDTA.

---

Measurement of melanin content[2]
Melan-a cells were treated with compounds for 72 h, and then the cells were dissolved in 1 N NaOH at 60 °C for 30 min. Then, the lysates were measured at 450 nm using a spectrophotometer. The data were normalized to the protein content of the cell lysates. The cell lysates were subsequently processed for the determination of the protein concentration using a BCA protein assay kit.

---

Western blot analysis[2]
Melan-a cells were washed once with 10 mM phosphate buffer (pH 7.4) containing 150 mM NaCl (PBS) and lysed in PBS containing 0.1% SDS and 10 mM-mercaptoethanol. The lysate was applied to 10% SDS–polyacrylamide gels. The protein bands in the gels were blotted onto nitrocellulose membranes using a mini-Protean system according to the manufacturer’s instructions. The membranes were then washed once with 10 mM Tris-buffered saline (TBS, pH 7.2) containing 0.1% Tween-20 (TBS-T), and then blocked for 1 h in TBS-T containing 5% skim milk. The primary antibodies were diluted at 1:1000, unless otherwise noted, and were incubated at 4 °C overnight. The membranes were washed three times for 10 min each with TBS-T buffer. The membranes were incubated with HRP-coupled secondary antibodies diluted at 1:3000 in TBS-T buffer for 1 h at room temperature, washed three times for 3 min each in TBS-T buffer, and then developed using the ECL detection kit.

---

- Chicken embryonic fibroblasts: Fertilized chicken eggs were incubated at 38.5 °C for 8 days. Embryos were dissociated using a cell dissociation sieve, and fibroblast cells were isolated after trypsinization. Cells were cultured in DMEM supplemented with glucose, NaHCO₃, glutamine, penicillin, streptomycin, phenol red, and fetal calf serum. Sweroside (compound 2) was dissolved in DMSO and added to culture media at concentrations of 0.4 and 2 μg/mL. After incubation, cells were stained using Masson’s trichrome technique. Morphometric analysis was performed under a light microscope at 400× and 800× magnification to evaluate total cell number, mitotic cells, percentages of polygonal/fusiform/round cells, vacuole‑containing cells, and collagen granules. Each experiment was repeated with n=5 or n=10 as indicated [1].
- Melan‑a cells (murine melanocytes): Melan‑a cells were cultured at 37 °C in 10% CO₂ atmosphere using RPMI 1640 medium supplemented with 10% heat‑inactivated fetal bovine serum, 1% penicillin/streptomycin, and 200 nM 12‑O‑tetradecanoylphorbol‑13‑acetate. Cell viability was assessed using a CCK‑8 assay after treatment with various concentrations of Sweroside (0–300 μM). For melanin content measurement, cells were treated with Sweroside for 72 h, then dissolved in 1 N NaOH at 60 °C for 30 min, and absorbance was measured at 450 nm. Protein concentration was normalized using a BCA assay. For Western blot analysis, cells were treated with Sweroside for 12 h (for Akt/ERK/MITF) or 72 h (for tyrosinase, TRP‑1, TRP‑2), lysed in PBS containing 0.1% SDS and 10 mM β‑mercaptoethanol, and proteins were separated by 10% SDS‑PAGE, transferred to nitrocellulose membranes, and probed with primary antibodies (anti‑tyrosinase, anti‑TRP‑1, anti‑TRP‑2, anti‑p‑Akt, anti‑Akt, anti‑p‑ERK, anti‑ERK, anti‑MITF) followed by HRP‑coupled secondary antibodies. Detection was performed using an ECL kit. In inhibitor studies, cells were pretreated with 20 μM LY294002 (Akt inhibitor) or 10 μM U0126 (ERK inhibitor) for 1 h before adding 300 μM Sweroside for 12 h, then Western blot was performed [2].

Tyrosinase activity and melanin contents in zebrafish[2]
Tyrosinase activity was spectrometrically determined. Briefly, about 100 zebrafish embryos were treated with sweroside from 9 to 48 h.p.f. and sonicated in Pro-Prep protein extraction solution. The lysate was clarified by centrifuging at 10,000g for 5 min. After quantification, 250 μg of total protein was added, followed by 100 μl of 5 mM L-3,4-dihydroxyphenylalanine (L-DOPA). Control well contained 100 μl of lysis buffer and 100 μl of 5 mM L-DOPA. After incubation for 60 min at 37 °C, absorbance was measured at 475 nm using the microplate leader. For the determination of melanin content, briefly, about 100 zebrafish embryos were treated with sweroside from 9 to 35 h.p.f, and sonicated in Pro-Prep protein extraction solution. After the centrifugation, the pellet was dissolved in 500 μl of 1 N NaOH at 100 °C for 10 min. The mixture was then vigorously vortexed to solubilize the melanin pigment. Optical density of the supernatant was measured at 490 nm

---

- Zebrafish pigmentation assay: Adult zebrafish were maintained at 28.5 °C with a 14/10 h light/dark cycle. Embryos were obtained from natural spawning within 30 min after lights on. Synchronized embryos (7–9 per well in 24‑well plates containing 1 mL embryo medium) were treated with Sweroside dissolved in 0.1% DMSO from 9 to 72 h post‑fertilization (h.p.f.) for pigmentation observation. The medium was replaced daily to ensure even distribution of the compound. A positive control group was treated with 0.2 mM 1‑phenyl‑2‑thiourea (PTU). At the end of treatment, embryos were anesthetized in tricaine methanesulfonate solution, mounted in 3% methylcellulose on a 35 mm dish, and photographed under a stereomicroscope (MZ16) [2].

- In melan‑a cells, Sweroside at concentrations up to 300 μM showed no significant cytotoxic effect as determined by CCK‑8 assay [2].
- In zebrafish, more than 95% of embryos survived treatment with Sweroside at the concentrations used (no specific lethal dose reported) [2].
- No information on hepatotoxicity, nephrotoxicity, drug‑drug interactions, plasma protein binding, or LD50 was provided [1][2].

[1]. Effects of gentiopicroside, sweroside and swertiamarine, secoiridoids from gentian (Gentiana lutea ssp. symphyandra), on cultured chicken embryonic fibroblasts. Planta Med. 2006 Mar;72(4):289-94.

[2]. Modulation effects of sweroside isolated from the Lonicera japonica on melanin synthesis. Chem Biol Interact. 2015 Aug 5;238:33-9.

Sweroside is a glycoside. It has been reported to be found in Gentiana macrophylla, Gentiana scabra, and other organisms with relevant data. See also: honeysuckle (partial); Mallotus trifoliata (partial); Cornflower safflower (partial).

---

- Sweroside is a secoiridoid glycoside with a glucose moiety attached to C‑1 in the pyrane ring. It has been reported to exhibit hepatoprotective, antibacterial, antifungal, antiosteoporotic and antiallergic activities [2].
- In the wound healing model using chicken embryonic fibroblasts, Sweroside was the most potent among the tested secoiridoids in stimulating collagen production (more than 8‑fold increase), and it also showed cytoprotective effects by reducing vacuole‑containing and round cells. These findings suggest that Sweroside contributes to the wound healing activity of Gentiana lutea extract, likely through a synergistic interaction with other secoiridoids (gentiopicroside and swertiamarine) [1].
- In melanogenesis inhibition, Sweroside acts by activating Akt and ERK signaling pathways, leading to MITF phosphorylation and degradation, which subsequently downregulates tyrosinase, TRP‑1 and TRP‑2 expression. This mechanism was confirmed using specific inhibitors LY294002 (Akt) and U0126 (ERK) that reversed the anti‑melanogenic effect of Sweroside. The compound also reduced pigmentation in zebrafish in vivo, indicating its potential as a natural skin‑whitening agent for the treatment of hyperpigmentation disorders [2].

C16H22O9

358.3405

358.126

C, 53.63; H, 6.19; O, 40.18

14215-86-2

161036

White to off-white solid powder

1.5±0.1 g/cm3

630.3±55.0 °C at 760 mmHg

100 °C

231.8±25.0 °C

0.0±4.2 mmHg at 25°C

1.602

Gentiana macrophylla; Gentiana algida; Lonicera japonica

-2.81

4

9

4

25

548

8

O([C@@]1([H])[C@]([H])(C([H])=C([H])[H])[C@@]2([H])C(C(=O)OC([H])([H])C2([H])[H])=C([H])O1)[C@@]1([H])[C@@]([H])([C@]([H])([C@@]([H])([C@@]([H])(C([H])([H])O[H])O1)O[H])O[H])O[H]

VSJGJMKGNMDJCI-ZASXJUAOSA-N

InChI=1S/C16H22O9/c1-2-7-8-3-4-22-14(21)9(8)6-23-15(7)25-16-13(20)12(19)11(18)10(5-17)24-16/h2,6-8,10-13,15-20H,1,3-5H2/t7-,8+,10-,11-,12+,13-,15+,16+/m1/s1

(3S,4R,4aS)-4-ethenyl-3-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4,4a,5,6-tetrahydro-3H-pyrano[3,4-c]pyran-8-one

(−) Sweroside; (−)-Sweroside; 1,9-trans-9,5-cis-Sweroside

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 : ~100 mg/mL (~279.06 mM)
H2O : ~50 mg/mL (~139.53 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.98 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (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 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (6.98 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (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 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (6.98 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.

---

Solubility in Formulation 4: 100 mg/mL (279.06 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

---

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