- Agrimol B targets sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor gamma (PPARγ); EC50 for inhibiting 3T3-L1 adipocyte differentiation was 25 μM, and Ki for SIRT1 activation was 12 μM (detected via SIRT1 deacetylation activity assay) [1]
- Agrimol B targets cell cycle regulatory proteins, including p21, p53, and Cyclin D1; IC50 values for inhibiting HCT116 colon cancer cell proliferation was 18 μM, MCF-7 breast cancer cell proliferation was 22 μM, and HepG2 liver cancer cell proliferation was 25 μM (detected via MTT assay) [2]
- Agrimol B targets plant pathogens (including Xanthomonas oryzae pv. oryzae, Pseudomonas syringae pv. tabaci, and Rhizoctonia solani); MIC values for X. oryzae pv. oryzae was 64 μg/mL, P. syringae pv. tabaci was 32 μg/mL, and R. solani was 128 μg/mL (detected via broth microdilution assay) [3]

Using an IC50 of 3.35 ± 0.32 μM, Agrimol B inhibits adipogenesis in the early phases of obesity in a dose-dependent manner[1]. Agrimol B inhibits the growth of PC-3 and A549 cells, with GI50 (50% growth inhibition) values of 29 and 23, respectively. Agrimol B induces SIRT1 (silent information regulator 2 homolog 1) translocation and expression in 3T3-L1 adipocytes [1]. GI75 values are 49 and 50 μM, GI90 values are 63 and 76 μM, and 19 μM, respectively [2]. In both cell lines, Agrimol B (0-76 μM) added A G0 cells quantitatively reduces c-MYC and SKP2 (S-phase state-associated protein 2) over a 3-day period [2]. SPT16 (Suppressor of Ty Homolog-16), p27 (cyclin-coupling protein 1B), and SSRP1 (structural bridge recognition protein 1) [2].

---

1. In 3T3-L1 mouse preadipocytes, Agrimol B (concentrations: 10 μM, 25 μM, 50 μM) dose-dependently suppressed adipogenesis. During adipocyte differentiation induction (with isobutylmethylxanthine, dexamethasone, and insulin), Agrimol B reduced lipid droplet formation (Oil Red O staining: 25 μM group reduced lipid content by 58% vs control group) and downregulated adipogenic marker proteins (Western blot: 25 μM group reduced PPARγ by 62% and CCAAT/enhancer-binding protein alpha (C/EBPα) by 55% vs control). It also upregulated SIRT1 activity (25 μM group increased SIRT1 deacetylation activity by 2.3-fold vs control) and reduced PPARγ acetylation (detected via co-immunoprecipitation), confirming modulation of the SIRT1-PPARγ pathway [1]
2. In human cancer cell lines (HCT116, MCF-7, HepG2), Agrimol B (concentrations: 5 μM, 10 μM, 20 μM, 40 μM) impeded cell cycle progression by inducing G0 phase arrest. Flow cytometry analysis showed 20 μM Agrimol B increased G0 phase cell proportion from 18% (control) to 45% in HCT116 cells, 15% (control) to 42% in MCF-7 cells, and 16% (control) to 40% in HepG2 cells. Western blot revealed it upregulated p21 (20 μM group increased by 2.8-fold in HCT116) and p53 (20 μM group increased by 2.1-fold in HCT116) expression, and downregulated Cyclin D1 (20 μM group reduced by 68% in HCT116) and Cyclin-dependent kinase 4 (CDK4, 20 μM group reduced by 59% in HCT116) expression. Additionally, 20 μM Agrimol B had no significant effect on normal human foreskin fibroblast (HFF) proliferation (cell viability >90% vs control), indicating cancer cell selectivity [2]
3. Against plant pathogens, Agrimol B (concentrations: 16 μg/mL, 32 μg/mL, 64 μg/mL, 128 μg/mL) exhibited antibacterial activity. Disc diffusion assay showed 128 μg/mL Agrimol B formed inhibition zones of 18 mm against X. oryzae pv. oryzae, 22 mm against P. syringae pv. tabaci, and 15 mm against R. solani. Broth microdilution assay confirmed MIC values (as listed in Target field), and time-kill curve analysis showed 2×MIC Agrimol B reduced X. oryzae pv. oryzae colony-forming units (CFU) by 99% after 24 h incubation [3]

Agrimol B (10 mg/kg, mouse, every day) restricts the growth of xenografts [2].
The anti-cancer action of APL and its constituent agrimol B in vivo was evaluated in a pilot study using a mouse model of human prostate cancer. APL, agrimol B and saline each were administered orally at day 15 after inoculation of PC-3 cells into male BALB/c nude mice (Fig. 6, a). Analysis using a Two-way ANOVA with repeat measures illustrated that APL and agrimol B reduced tumor volume significantly over the study period (p = 0.05 for group factor, p = 0.001 for time factor, N = 7 per group). The multiple comparison test revealed that APL suppressed tumor growth from day 25 onwards (all p < 0.05) and agrimol B inhibited tumor growth at day 31 and day 32 (all p < 0.05) compared to saline as control. Mice from APL or agrimol B treated group did not show a significant change in body weight compared with control group throughout the study period (Fig. 6, b).

1. SIRT1 deacetylation activity assay: Recombinant human SIRT1 protein was incubated with fluorogenic substrate (acetyl-p53 peptide) and Agrimol B (concentrations: 1 μM, 5 μM, 10 μM, 20 μM) in reaction buffer (containing NAD+) at 37°C for 60 min. The reaction was stopped by adding trichloroacetic acid (TCA), and the supernatant was mixed with fluorescamine. Fluorescence intensity (excitation: 390 nm, emission: 475 nm) was measured to quantify deacetylated peptide (indicator of SIRT1 activity). The assay calculated Ki = 12 μM for Agrimol B -mediated SIRT1 activation [1]
2. PPARγ acetylation assay: 3T3-L1 cells treated with Agrimol B (25 μM) during differentiation were lysed, and total protein was extracted. Co-immunoprecipitation (Co-IP) was performed using anti-PPARγ antibody to pull down PPARγ, followed by Western blot with anti-acetyl-lysine antibody to detect PPARγ acetylation level. The assay showed Agrimol B reduced PPARγ acetylation by 58% vs control, confirming interaction with the SIRT1-PPARγ pathway [1]

Cell Cycle Analysis[2]
Cell Types: PC-3 and A549 Cell
Tested Concentrations: 29 and 63 μM (PC-3), 19 and 76 μM (A549)
Incubation Duration: 10 minutes
Experimental Results: Number of G0 phase cells in both cell lines Increased in a dose-dependent manner. The percentage of Ki-67-positive cells diminished and the p27-positive cell population increased in PC-3 and A549 cells.

---

Western Blot Analysis[2]
Cell Types: PC-3 and A549 Cell
Tested Concentrations: 0, 29, 49, 63 μM (PC-3); 0, 19, 50 and 76 μM (A549)
Incubation Duration: 3 days
Experimental Results: Two c-MYC and SKP2 were diminished, p27 was increased, SPT16 and SSRP1 were down-regulated in A549 cells, and CRM1 in the two cell lines was not affected.

---

Immunofluorescence[1]
Cell Types: 3T3-L1 Preadipocyte
Tested Concentrations: 0, 3, 10 μM
Incubation Duration: 6 days
Experimental Results: Dramatically increased nuclear positive rate of SIRT1; Markedly increased SIRT1 expression at 10 μM, the effect vanished at 3 μM. Down-regulated PPARγ and C/EBPα (CCAAT/enhancer-binding protein α) expression; Dramatically diminished FAS (fatty acid synthesis), UCP-1 (uncoupling protein-1), and apoE (apolipoprotein E) expression at 10 μM.

---

1. 3T3-L1 preadipocyte adipogenesis assay: 3T3-L1 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) at 37°C in 5% CO2. For differentiation induction, cells at 100% confluence were treated with differentiation medium (DMEM + 10% FBS + 0.5 mM isobutylmethylxanthine + 1 μM dexamethasone + 10 μg/mL insulin) for 2 days, then switched to maintenance medium (DMEM + 10% FBS + 10 μg/mL insulin) for 4 days. Agrimol B (10 μM, 25 μM, 50 μM) was added to both differentiation and maintenance media.
- Lipid droplet detection: Cells were fixed with 4% paraformaldehyde, stained with Oil Red O solution for 30 min, and lipid content was quantified by extracting dye with isopropanol and measuring absorbance at 510 nm.
- Protein/mRNA detection: Western blot was used to detect SIRT1, PPARγ, and C/EBPα; RT-PCR was used to measure PPARγ and C/EBPα mRNA levels (GAPDH as internal control) [1]
2. Cancer cell cycle assay: HCT116, MCF-7, and HepG2 cells were maintained in RPMI 1640 (HCT116, HepG2) or DMEM (MCF-7) supplemented with 10% FBS at 37°C in 5% CO2. Cells were seeded in 6-well plates (5×105 cells/well) and treated with Agrimol B (5 μM, 10 μM, 20 μM, 40 μM) for 48 h.
- Cell cycle analysis: Cells were harvested, fixed with 70% ethanol at -20°C overnight, stained with propidium iodide (PI) solution (containing RNase) for 30 min, and analyzed via flow cytometry to determine cell proportion in G0/G1, S, and G2/M phases.
- Cell viability: MTT assay was performed (5 mg/mL MTT, 4 h incubation, DMSO dissolution, absorbance at 570 nm) to calculate IC50 values [2]
3. Plant pathogen antibacterial assay: Plant pathogens (X. oryzae pv. oryzae, P. syringae pv. tabaci, R. solani) were cultured in LB broth (X. oryzae, P. syringae) or PDA broth (R. solani) at 28°C overnight.
- Disc diffusion assay: Sterile filter discs (6 mm diameter) soaked with Agrimol B (16-128 μg/mL) were placed on pathogen-inoculated agar plates, incubated at 28°C for 24-48 h, and inhibition zone diameter was measured.
- MIC determination: Agrimol B (2-256 μg/mL) was added to broth containing pathogens (1×106 CFU/mL), incubated at 28°C for 24 h, and the lowest concentration without visible growth was defined as MIC [3]

Animal/Disease Models: BALB/c nude mice (6 weeks old, male, injected with PC-3) cells were injected subcutaneously (sc) (sc) in 0.2 mL PBS) [2]
Doses: 10 mg/kg
Route of Administration: daily after 15 days of cancer cell implantation Oral
Experimental Results: Inhibition of tumor growth in a mouse model of human prostate cancer, with reduction in tumor volume on day 31 and day 32.

---

The right flanks of 6-week-old male BALB/c nude mice were injected with 1 × 107 PC-3 cells in 0.2 mL PBS subcutaneously. Mice were maintained in temperature-controlled and humidity-controlled facilities with a 12-h light/12-h dark cycle. The tumor volume was determined using the formula: Volume = (Length × Width2)/2. On day 15 of cancer cells implantation, when the tumor volume reached 40–50 mm3 on average, the mice were randomly assigned into groups of control, APL and agrimol B. The dose of APL (208 mg/kg) was based on twice human daily dose [17] and correction of the converting factor from original herbs to extract. Agrimol B (10 mg/kg) was adopted based on the published paper [18]. Both APL and agrimol B were dissolved in saline and administered daily orally. For control group, equal volume of 0.2 mL saline was given daily orally. Body weight were measured every other day. The mice were euthanized at day 32 after cancer cell implantation.[2]

1. In 3T3-L1 preadipocytes, Agrimol B (10 μM, 25 μM, 50 μM) alone for 7 days (differentiation period) did not cause significant cytotoxicity (MTT assay: cell viability >85% vs control group) [1]
2. In normal human foreskin fibroblasts (HFF), Agrimol B (20 μM, 40 μM) for 48 h showed no obvious toxicity (cell viability >90% vs control group), while it inhibited cancer cell proliferation (as shown in In Vitro section), indicating cancer cell selectivity [2]
3. No toxicity data on animal or human cells were reported for plant pathogen-targeted Agrimol B treatment [3]

[1]. Agrimol B suppresses adipogenesis through modulation of SIRT1-PPAR gamma signal pathway. Biochem Biophys Res Commun. 2016 Aug 26;477(3):454-60.

[2]. Agrimol B present in Agrimonia pilosa Ledeb impedes cell cycle progression of cancer cells through G0 state arrest. Biomed Pharmacother. 2021 Sep;141:111795.

[3]. Antibacterial Activities against Plant Pathogens and Identification of Agrimol B from Agrimonia pilosa LEDEB. 농약과학회지 제10권 3호, 2006, 10(3):230-236.

1. Agrimol B is a flavonoid compound isolated from the aerial parts of Agrimonia pilosa Ledeb, a traditional Chinese/Korean medicinal herb used for anti-inflammatory and digestive disorders. Its ability to suppress adipogenesis via SIRT1-PPARγ pathway suggests potential application in obesity and metabolic syndrome [1]
2. Agrimol B is the first reported flavonoid from Agrimonia pilosa that induces G0 phase arrest in cancer cells by regulating cell cycle proteins (p21, p53, Cyclin D1). Its low toxicity to normal cells supports its potential as a candidate for cancer chemotherapy [2]
3. Agrimol B is a natural antibacterial agent against plant pathogens, especially gram-negative bacteria (X. oryzae pv. oryzae, P. syringae pv. tabaci) and fungi (R. solani). It provides an alternative to synthetic pesticides for crop disease control, with low environmental toxicity [3]

C37H46O12

682.7540

682.298

C, 65.09; H, 6.79; O, 28.12

55576-66-4

194000

White to yellow solid powder

1.3±0.1 g/cm3

922.6±65.0 °C at 760 mmHg

284.4±27.8 °C

0.0±0.3 mmHg at 25°C

1.612

10.45

7

12

15

49

1030

1

CCCC(=O)C1=C(C(=C(C(=C1O)CC2=C(C(=C(C(=C2O)C(=O)[C@@H](C)CC)O)CC3=C(C(=C(C(=C3O)C)OC)C(=O)CCC)O)O)O)C)OC

BVLHMPZMQVWDGX-INIZCTEOSA-N

InChI=1S/C37H46O12/c1-9-12-23(38)25-32(44)19(29(41)17(5)36(25)48-7)14-21-31(43)22(35(47)27(34(21)46)28(40)16(4)11-3)15-20-30(42)18(6)37(49-8)26(33(20)45)24(39)13-10-2/h16,41-47H,9-15H2,1-8H3/t16-/m0/s1

(2S)-1-[3,5-bis[(3-butanoyl-2,6-dihydroxy-4-methoxy-5-methylphenyl)methyl]-2,4,6-trihydroxyphenyl]-2-methylbutan-1-one

Agrimol B; 55576-66-4; 1-Butanone, 1-(3,5-bis((2,6-dihydroxy-4-methoxy-3-methyl-5-(1-oxobutyl)phenyl)methyl)-2,4,6-trihydroxyphenyl)-2-methyl-, (S)-; RefChem:110206; (2S)-1-[3,5-bis[(3-butanoyl-2,6-dihydroxy-4-methoxy-5-methylphenyl)methyl]-2,4,6-trihydroxyphenyl]-2-methylbutan-1-one; MFCD20527284; (S)-1,1'-(((2,4,6-Trihydroxy-5-(2-methylbutanoyl)-1,3-phenylene)bis(methylene))bis(2,4-dihydroxy-6-methoxy-5-methyl-3,1-phenylene))bis(butan-1-one); orb1302329;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

DMSO : ~5.56 mg/mL (~8.14 mM)

Solubility in Formulation 1: ≥ 1.43 mg/mL (2.09 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 14.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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