- RNA‑dependent RNA polymerase (RdRp, nsp12) of SARS‑CoV‑2 (predicted by molecular docking; S‑score = –8.410, interacting residues: Arg349, Ser664, Asn628, Pro677, Glu350) [3].

- Prostatitis mouse model: Male KM mice (25.0 ± 1.0 g) were injected locally with 0.02 mL of 25% Xiaozhiling injection into the prostate to induce prostatitis. Verbenalin (50.13% purity) was administered orally at 200 mg/kg (high dose), 100 mg/kg (medium dose), or 50 mg/kg (low dose) once daily for 21 days. Compared with model control, all verbenalin groups showed increased 24‑h water intake (at day 14: +2.8%, +2.4%, +0.4% respectively; at day 28: +19.1%, +14.8%, +7.7% respectively), significantly decreased white blood cell count in prostatic fluid (HD: 1.47 ± 0.38 × 10⁹/L, MD: 2.05 ± 0.89, LD: 2.19 ± 0.67 vs model 6.62 ± 1.80, P<0.01), and significantly increased lecithin corpuscle density score (HD: 3.36 ± 0.58, MD: 3.20 ± 0.80, LD: 2.82 ± 0.59 vs model 1.55 ± 0.50, P<0.01). Volume density (Vv) of inflammatory cells in prostate was significantly reduced (HD: 0.3 ± 0.1%, MD: 0.5 ± 0.1%, LD: 3.4 ± 0.2% vs model 6.2 ± 0.6%, P<0.01). Histopathological examination showed that verbenalin HD and MD groups almost normalized prostate gland structure, reduced glandular expansion, flattened epithelium, and interstitial fibrosis/inflammatory infiltration. Testicular pathological changes were significantly alleviated (eosinophilic change in spermatogonia reduced), epididymal fibrous hyperplasia and inflammatory cell infiltration were reduced (HD group most effective, P<0.01), and thymic cortex thickness and lymphocyte number were significantly increased (P<0.01), indicating enhanced immune function. Kidney pathological changes showed a potential protective trend [1].
- Focal cerebral ischemia‑reperfusion rat model: Male KM rats (25–30 g) underwent middle cerebral artery occlusion (2 h) followed by reperfusion (22 h). Verbenalin (98.07% purity) was injected intravenously (tail vein) 10 min before reperfusion at 20 mg/kg (high dose), 10 mg/kg (medium dose), or 5 mg/kg (low dose). Compared with model group, verbenalin significantly reduced mortality rate (HD: 25%, MD: 31.25%, LD: 31.25% vs model 37.5%), neurological deficit score (HD: 1.9 ± 0.5, MD: 2.0 ± 0.7, LD: 2.1 ± 0.8 vs model 3.1 ± 0.6), serum S‑100β level (HD: 16.732 ± 4.007 ng/mL, MD: 17.188 ± 2.944, LD: 17.860 ± 2.997 vs model 22.636 ± 3.563), and cerebral infarct size percentage (HD: 18.825 ± 9.213%, MD: 22.417 ± 8.917%, LD: 27.507 ± 7.884% vs model 32.477 ± 7.044%). Brain tissue analysis showed that verbenalin significantly decreased pro‑apoptotic Bax (HD: 2.805 ± 0.530 ng/mL, MD: 2.826 ± 0.440, LD: 2.995 ± 0.435 vs model 4.005 ± 0.507) and Caspase‑3 (HD: 9.812 ± 1.400 pmol/L, MD: 10.609 ± 1.491, LD: 10.841 ± 1.572 vs model 13.434 ± 0.869), and increased anti‑apoptotic Bcl‑2 (HD: 16.058 ± 1.220 ng/mL, MD: 15.140 ± 1.067, LD: 14.816 ± 1.050 vs model 11.937 ± 1.402). Na⁺K⁺‑ATPase and Mg²⁺‑ATPase activities were significantly increased in HD and MD groups (Na⁺K⁺‑ATPase: HD 4.885 ± 2.000, MD 4.212 ± 1.136 vs model 2.818 ± 0.565; Mg²⁺‑ATPase: HD 5.256 ± 1.638, MD 4.313 ± 1.853 vs model 2.551 ± 0.558). Histopathological examination of cerebral cortex and hippocampus showed that verbenalin reduced neuronal edema, necrosis, and infarct size in a dose‑dependent manner [2].

- Measurement of Bax, Bcl‑2, and Caspase‑3 levels in rat brain tissue: After 22 h of reperfusion, rat brain tissue (left hemisphere) was homogenized in cold saline (9:1 v/w) and centrifuged at 3000 rpm for 10 min at 4°C. The supernatant was collected and levels of Bax (pro‑apoptotic), Bcl‑2 (anti‑apoptotic), and Caspase‑3 (executioner caspase) were determined using commercial ELISA kits according to the manufacturer’s protocols. The results were expressed as ng/mL for Bax and Bcl‑2, and pmol/L for Caspase‑3 [2].
- Measurement of ATPase activity in rat brain tissue: Brain homogenate prepared as above was used to measure Na⁺K⁺‑ATPase and Mg²⁺‑ATPase activities using a commercial ATPase test kit (based on the release of inorganic phosphorus). Activities were expressed as μmol Pi/mg protein/hour [2].
- Measurement of serum S‑100β protein level: Blood was collected from rats after reperfusion, serum was separated, and S‑100β protein level was determined using an ELISA kit. Results were expressed as ng/mL [2].
- Histopathological scoring of prostate, testis, epididymis, kidney, and thymus: Tissue sections (paraffin‑embedded, HE stained) were examined under light microscope. Pathological changes were graded semiquantitatively (‑, +, ++, +++) based on criteria defined in the literature. For prostate, volume density (Vv) of inflammatory area was calculated using a test lattice formula: Vv = P∑n / reference system ∑n × 100% [1].

- Prostatitis mouse model: SPF grade male KM mice (25.0 ± 1.0 g) were randomly divided into blank control, model control, positive control (Qianliekang Pian, 1.5 g/kg), and verbenalin high (200 mg/kg), medium (100 mg/kg), and low (50 mg/kg) dose groups (n=10 per group). Prostatitis was induced by injecting 0.02 mL of 25% Xiaozhiling injection into the dorsal prostate under sodium pentobarbital anesthesia (35 mg/kg, i.p.). Sham operation was performed on blank control. After surgery, penicillin (200,000 U/kg) was injected intramuscularly for 3 days to prevent infection. Starting from day 8 post‑surgery, verbenalin (dissolved in distilled water) was administered orally by gavage at 0.2 mL/10 g body weight once daily for 21 days. Blank and model controls received distilled water. On days 14 and 28 after modeling, 24‑h water intake was measured. On day 29, mice were sacrificed by cervical dislocation, and prostate, testis, epididymis, kidney, and thymus were collected for histopathology and white blood cell/lecithin corpuscle analysis [1].
- Focal cerebral ischemia‑reperfusion rat model: Male KM rats (25–30 g) were randomly divided into control (sham), model, positive control (nimodipine 30 mg/kg), and verbenalin high (20 mg/kg), medium (10 mg/kg), and low (5 mg/kg) dose groups (n=16 per group). All groups received oral gavage of 0.1 mL/10 g of the respective drug (or 0.5% CMC for control and model) once daily for 7 days. On day 7, after final gavage, rats were anesthetized with 10% chloral hydrate (0.03 mL/10 g, i.p.). Focal cerebral ischemia was induced by inserting a filament into the left middle cerebral artery via the internal carotid artery, occluding for 2 h. Verbenalin (dissolved in saline) was injected intravenously (tail vein) 10 min before reperfusion at the indicated doses. Reperfusion lasted 22 h. Neurological deficit scoring (Longa scale, 0–5 points) was performed after reperfusion. Mortality was recorded. Rats were then sacrificed, brains removed, and left hemispheres processed for TTC staining (infarct size), histopathology (HE staining), and homogenate for biochemical assays [2].

[1]. Effects of verbenalin on prostatitis mouse model. Saudi J Biol Sci. 2016 Jan;23(1):S148-57.

[2]. The protective role of verbenalin in rat model of focal cerebral ischemia reperfusion. Saudi J Biol Sci. 2018 Sep;25(6):1170-1177.

[3]. An in silico approach to target RNA-dependent RNA polymerase of COVID-19 with naturally occurring phytochemicals. Environ Dev Sustain. 2021 Apr 3;1-14.

Verbenin is a terpene glycoside. It has been reported to be found in Aster tataricus, Pyrrosia lingua, and other organisms with relevant data.

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- Verbenalin is the main active glycoside from Verbena officinalis L. (Verbenaceae). In traditional Chinese medicine, verbena is used to activate blood circulation, remove blood stasis, induce diuresis, and reduce edema – actions that align with the pathogenesis of prostatitis (damp‑heat, blood stasis, deficiency). Verbenalin has shown therapeutic effects on chronic prostatitis and hematuria in clinical practice [1].
- In the prostatitis model, verbenalin not only improved prostate pathology but also protected secondary organs (testis, epididymis, kidney) and enhanced immune function (increased thymic cortex thickness and lymphocyte count), suggesting its potential to reverse the pathological process of chronic prostatitis [1].
- In cerebral ischemia‑reperfusion, verbenalin acts by inhibiting pro‑apoptotic gene expression (Bax, Caspase‑3), promoting anti‑apoptotic gene expression (Bcl‑2), improving brain microcirculation and energy metabolism (increasing Na⁺K⁺‑ATPase and Mg²⁺‑ATPase activities), and reducing serum S‑100β protein (a marker of glial cell damage). The high dose (20 mg/kg, i.v.) showed the best protective effects [2].
- In silico molecular docking predicted that verbenalin binds to the RNA‑dependent RNA polymerase (nsp12) of SARS‑CoV‑2 at the active site, interacting with residues Arg349, Ser664, Asn628, Pro677, and Glu350. It fulfills Lipinski’s rule of five (MW 388.44, HBD 2, HBA 8, LogP 2.28, molar refractivity 102) and ADMET profiling indicated non‑carcinogenic and low toxicity (though swertisin and islandicin showed minor violations). Verbenalin is considered a potential lead candidate against COVID‑19 [3].

C17H24O10

388.36646

388.136

C, 52.58; H, 6.23; O, 41.20

548-37-8

73467

White to off-white solid

1.5±0.1 g/cm3

610.1±55.0 °C at 760 mmHg

180-182ºC

219.4±25.0 °C

0.0±4.0 mmHg at 25°C

1.591

-2.39

4

10

5

27

619

9

C[C@H]1CC(=O)[C@@H]2C(=CO[C@H]([C@H]12)O[C@H]3[C@@H]([C@H]([C@@H]([C@@H](CO)O3)O)O)O)C(=O)OC

HLXRWTJXGMHOFN-XJSNKYLASA-N

InChI=1S/C17H24O10/c1-6-3-8(19)11-7(15(23)24-2)5-25-16(10(6)11)27-17-14(22)13(21)12(20)9(4-18)26-17/h5-6,9-14,16-18,20-22H,3-4H2,1-2H3/t6-,9+,10+,11-,12+,13-,14+,16-,17-/m0/s1

methyl (1S,4aS,7S,7aR)-7-methyl-5-oxo-1-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4a,6,7,7a-tetrahydro-1H-cyclopenta[c]pyran-4-carboxylate

NSC-118055; NSC 118055; NSC118055; Cornin; Verbenalin

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 : ~100 mg/mL (~257.49 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.44 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (6.44 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.44 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.)