IC50: OVA-specific IgG2a and IgG2b antibody

- Platycoside E exhibited haemolytic activity on rabbit red blood cells. The HD₅₀ value was >250 μg/ml (using 0.5% rabbit red blood cell suspension). The haemolytic activity ranking among the three saponins was PD > PD3 > Platycoside E (P < 0.001). [1]
- In splenocyte proliferation assays using splenocytes from OVA-immunized mice, Platycoside E (75 μg) significantly increased Con A‑stimulated splenocyte proliferation compared to the OVA control group (P < 0.05, P < 0.01 or P < 0.001). However, Platycoside E did not significantly enhance LPS‑ or OVA‑induced splenocyte proliferation. The order of increasing OVA‑stimulated splenocyte proliferation was PD > PD3 > Platycoside E (P < 0.05, P < 0.01 or P < 0.001). [1]

- In OVA‑immunized mice, Platycoside E at doses of 75 and 100 μg significantly increased the serum levels of OVA‑specific IgG2a and IgG2b antibodies compared to the OVA alone group and the OVA/Alum group (P < 0.05, P < 0.01 or P < 0.001). No significant differences in serum IgG and IgG1 levels were observed between mice immunized with OVA/Platycoside E and OVA alone. Among the three saponins, PD (100 μg) stimulated total‑IgG2a and IgG2b antibody responses more than Platycoside E (P < 0.05 or P < 0.01), while no significant differences were found between PD3 and Platycoside E (P > 0.05). [1]

- Haemolytic assay: Rabbit red blood cells were washed three times with sterile saline solution (0.89% w/v NaCl) by centrifugation at 180 × g for 5 min. The cell pellet was diluted to 0.5% in saline solution. Then 0.5 ml of the cell suspension was mixed with 0.5 ml of diluents containing Platycoside E at concentrations of 7.813, 15.625, 31.25, 62.5, 125, 250, and 500 μg/ml in saline. The mixtures were incubated for 30 min at 37°C and centrifuged at 70 × g for 10 min. Free haemoglobin in the supernatants was measured spectrophotometrically at 412 nm. Saline and distilled water were used as minimal and maximal haemolytic controls. The haemolytic percentage developed by the saline control was subtracted. The concentration inducing 50% of maximum haemolysis (HD₅₀) was calculated. Each experiment included triplicates at each concentration, and four independent experiments were performed. [1]
- Splenocyte proliferation assay: Spleens from OVA‑immunized mice were collected aseptically, minced, and passed through a fine steel mesh to obtain a homogeneous cell suspension. Erythrocytes were lysed with 0.8% ammonium chloride. After centrifugation (380 × g at 4°C for 10 min), pelleted cells were washed three times in PBS and resuspended in complete medium (RPMI 1640 supplemented with 12 mM HEPES pH 7.1, 0.05 mM 2‑mercaptoethanol, 100 IU/ml penicillin, 100 μg/ml streptomycin, and 10% FCS). Cell viability exceeded 95% by trypan blue exclusion. Splenocytes were seeded into 96‑well flat‑bottom microtiter plates at 5 × 10⁶ cells/ml in 100 μl complete medium. Then Con A (final concentration 5 μg/ml), LPS (final concentration 10 μg/ml), OVA (final concentration 10 μg/ml), or medium alone was added to a final volume of 200 μl. Plates were incubated at 37°C in 5% CO₂ for 44 h. Then 50 μl of MTT solution (2 mg/ml) was added to each well and incubated for another 4 h. Plates were centrifuged (1400 × g for 5 min), and untransformed MTT was removed. 150 μl of DMSO working solution (192 μl DMSO with 8 μl 1N HCl) was added to each well. Absorbance was measured at 570 nm with a 630 nm reference after 15 min. The stimulation index (SI) was calculated as the absorbance value for mitogen‑ or antigen‑cultures divided by the absorbance value for non‑stimulated cultures. [1]

- Immunization protocol: Female ICR mice (6 weeks old) were divided into groups of five. Mice were immunized subcutaneously on day 1 with OVA (100 μg) alone or with OVA (100 μg) dissolved in saline containing Alum (200 μg), PD, PD3, or Platycoside E (25, 50, 75, or 100 μg). Saline‑treated animals served as controls. A boosting injection was given 2 weeks later (day 15). Sera and splenocytes were collected 2 weeks after the second immunization (day 29) for proliferation assays and measurement of OVA‑specific antibodies. [1]

- Platycoside E showed haemolytic activity with an HD₅₀ value >250 μg/ml on rabbit red blood cells. The haemolytic activity ranking was PD > PD3 > Platycoside E (P < 0.001). The haemolytic activity of Quil A (a reference saponin) was higher (HD₅₀ = 5.76 ± 0.23 μg/ml) than that of Platycoside E. [1]
- The stock solution of Platycoside E (4 mg/ml in 0.89% saline) was sterilized by filtration (0.22 μm) and analyzed for endotoxin level using a gel‑clot Limulus amebocyte lysate assay; the endotoxin level was less than 0.5 endotoxin units (EU)/ml, excluding endotoxin contamination. [1]

[1]. Contribution of the glycidic moieties to the haemolytic and adjuvant activity of platycodigenin-type saponins from the root of Platycodon grandiflorum.Vaccine. 2008 Jun 25;26(27-28):3452-60.

Platycodon E is a triterpenoid saponin that acts as a metabolite. Platycodon E has been discovered in Platycodon grandiflorus, and relevant data have been reported.

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- Platycoside E is one of three platycodigenin‑type saponins isolated from Platycodon grandiflorum root. Its chemical structure includes a platycodigenin aglycone with sugar chains attached at C‑3 and C‑28. At C‑3, Platycoside E has a trisaccharide chain: Glc-(1→6)-glc-(1→6)-glc- (attached to Api-(1→3)-Xyl-(1→4)-Rha-(1→2)-Ara-). The same C‑28 sugar side chain is shared with PD and PD3. The study demonstrated that the haemolytic and adjuvant activities of platycodigenin‑type saponins decrease with an increased number of monosaccharide residues at the C‑3 position of the aglycone, with Platycoside E having the longest sugar chain and the lowest activity. [1]
- Platycoside E did not significantly enhance OVA‑specific IgG and IgG1 antibody responses but did enhance IgG2a and IgG2b responses, suggesting that the C‑28 attached carbohydrates are particularly relevant for inducing IgG2a and IgG2b subtypes, while the C‑3 attached carbohydrates are related to inducing IgG and IgG1 responses. [1]

C69H112O38

1549.6048

1548.683

237068-41-6

70698202

White to off-white solid

1.63±0.1 g/cm3(Predicted)

-8

23

38

21

107

3050

41

O([C@@]1([H])[C@@]([H])([C@]([H])([C@@]([H])([C@@]([H])(C([H])([H])O[C@@]2([H])[C@@]([H])([C@]([H])([C@@]([H])([C@@]([H])(C([H])([H])O[C@@]3([H])[C@@]([H])([C@]([H])([C@@]([H])([C@@]([H])(C([H])([H])O[H])O3)O[H])O[H])O[H])O2)O[H])O[H])O[H])O1)O[H])O[H])O[H])[C@@]1([H])[C@]([H])(C([H])([H])[C@@]2(C([H])([H])[H])[C@@]([H])(C([H])([H])C([H])([H])[C@@]3(C([H])([H])[H])[C@]4(C([H])([H])[H])C([H])([H])[C@]([H])([C@@]5(C(=O)O[C@@]6([H])[C@@]([H])([C@]([H])([C@]([H])(C([H])([H])O6)O[H])O[H])O[C@@]6([H])[C@@]([H])([C@@]([H])([C@]([H])([C@]([H])(C([H])([H])[H])O6)O[C@@]6([H])[C@@]([H])([C@]([H])([C@@]([H])(C([H])([H])O6)O[H])O[C@@]6([H])[C@@]([H])([C@@](C([H])([H])O[H])(C([H])([H])O6)O[H])O[H])O[H])O[H])O[H])C([H])([H])C([H])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[C@@]5([H])C4=C([H])C([H])([H])[C@@]32[H])O[H])C1(C([H])([H])O[H])C([H])([H])O[H])O[H]

TZSYJZBVJYXHEK-SNQGWRGYSA-N

InChI=1S/C69H112O38/c1-25-50(103-57-49(90)51(30(76)18-94-57)104-61-53(91)68(93,23-73)24-98-61)44(85)48(89)58(99-25)105-52-37(78)29(75)17-95-60(52)107-62(92)69-12-11-63(2,3)13-27(69)26-7-8-34-64(4)14-28(74)54(67(21-71,22-72)35(64)9-10-65(34,5)66(26,6)15-36(69)77)106-59-47(88)43(84)40(81)33(102-59)20-97-56-46(87)42(83)39(80)32(101-56)19-96-55-45(86)41(82)38(79)31(16-70)100-55/h7,25,27-61,70-91,93H,8-24H2,1-6H3/t25-,27-,28-,29-,30+,31+,32+,33+,34+,35+,36+,37-,38+,39+,40+,41-,42-,43-,44-,45+,46+,47+,48+,49+,50-,51-,52+,53-,54-,55+,56+,57-,58-,59-,60-,61-,64+,65+,66+,68+,69+/m0/s1

[(2S,3R,4S,5S)-3-[(2S,3R,4S,5R,6S)-5-[(2S,3R,4S,5R)-4-[(2S,3R,4R)-3,4-dihydroxy-4-(hydroxymethyl)oxolan-2-yl]oxy-3,5-dihydroxyoxan-2-yl]oxy-3,4-dihydroxy-6-methyloxan-2-yl]oxy-4,5-dihydroxyoxan-2-yl] (4aR,5R,6aR,6aS,6bR,8aR,10R,11S,12aR,14bS)-5,11-dihydroxy-9,9-bis(hydroxymethyl)-2,2,6a,6b,12a-pentamethyl-10-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-[[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-[[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxymethyl]oxan-2-yl]oxymethyl]oxan-2-yl]oxy-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylate

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 (~64.53 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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