This study aims to evaluate the in vitro angiotensin-converting enzyme (ACE) inhibition activity of different extracts of Orthosiphon stamineus (OS) leaves and their main flavonoids, namely rosmarinic acid (RA), sinensetin (SIN), eupatorin (EUP) and 3′-hydroxy-5,6,7,4′-tetramethoxyflavone (TMF). Furthermore, to identify possible mechanisms of action based on structure–activity relationships and molecular docking. The in vitro ACE inhibition activity relied on determining hippuric acid (HA) formation from ACE-specific substrate (hippuryl-histidyl-leucine (HHL)) by the action of ACE enzyme. A High Performance Liquid Chromatography method combined with UV detection was developed and validated for measurement the concentration of produced HA. The chelation ability of OS extract and its reference compounds was evaluated by tetramethylmurexide reagent. Furthermore, molecular docking study was performed by LeadIT-FlexX: BioSolveIT’s LeadIT program. OS ethanolic extract (OS-E) exhibited highest inhibition and lowest IC₅₀ value (45.77 ± 1.17 µg/mL) against ACE compared to the other extracts. Among the tested reference compounds, EUP with IC₅₀ 15.35 ± 4.49 µg/mL had highest inhibition against ACE and binding ability with Zn (II) (56.03% ± 1.26%) compared to RA, TMF and SIN. Molecular docking studies also confirmed that flavonoids inhibit ACE via interaction with the zinc ion and this interaction is stabilized by other interactions with amino acids in the active site. In this study, we have demonstrated that changes in flavonoids active core affect their capacity to inhibit ACE. Moreover, we showed that ACE inhibition activity of flavonoids compounds is directly related to their ability to bind with zinc ion in the active site of ACE enzyme. It was also revealed that OS extract contained high amount of flavonoids other than RA, TMF, SIN and EUP. As such, application of OS extract is useful as inhibitors of ACE.

Nidrakar Bati (NKB) is an herbal remedy consisted with seven medicinal herbs widely used to cure Somnifacient (sleeping aid) in South Asia as Ayurvedic medicinal system. In the present study, pharmacological and toxicological effects of this medicine was investigated in mice to validate the safety and efficacy of the herb. Organic solvent extracts NKB were prepared using maceration method. Effect of extracts on the central nervous system was evaluated using hypnotic activity assay. Effect of the extracts on metabolic activity, assessing involvement of thyroid was conducted using hypoxia test. analgesic and anti-inflammatory activities were assessed in mice using acetic acid induced writhing, formalin induced paw edema, xylene induced ear edema assays. Anxiolytic activity was performed using plus maze, climbing out and forced swimming tests. Effect of the extracts on psychopharmacological effect was carried out using locomotor activity tests (open field, Hole-board and Hole-cross tests). Neuropharmacological effect of the extracts was performed using motor coordination (rotarod test). Toxicological potential of the extract was evaluated using gastro-intestinal activity (gastric emptying and gastrointestinal motility tests). The studied formulation reduced the CNS stimulant effects dose independently. In the hypoxia test, only a dose of 100 mg/kg of NKB decreased the survival time. Orally administration of the NKB (200 and 400 mg/kg) produced significant inhibition (P < 0.01) of the acetic acid-induced writhing in mice and suppressed xylene induced ear edema and formalin-induced licking response of animals in both phases of the test. NKB showed locomotor activity (p < 0.05) both in higher and lower doses (100 and 400 mg/kg). NKB increased the total ambulation dose dependently (p < 0.05). NKB, at all tested doses (100, 200 and 400 mg/kg) increased some locomotion activity parameters (ambulation, head dipping and emotional defecation) in hole board test. At higher doses (200 and 400 mg/kg), NKB showed a significant increase in hole cross test. NKB showed an increase in the time on the open arms of the maze at low to medium doses (100 and 200 mg/kg). When using the Rotarod method, NKB showed a considerable increase on motor coordination of the mice. NKB produced marked gastric emptying effect and decreased gastrointestinal motility in mice at low dose. NKB demonstrated various pharmacological effects and toxicological effects due to presence of several herbs in the formulation those are not closely fit for the effect of CNS depressants.

Caffeic acid (CA) is a well-known natural compound used for both health supplement and metabolic diseases. In the present study, single, easy, and quick isolation procedure of caffeic acid was described from rosmarinic acid. A quick crystal formation also was described for highly hygroscopic CA which was spontaneous and stable with lower hygroscopic nature. In addition, the produced crystal CA was highly stable and showed neuroprotective activity with 100 μg/ml in U87 MG which revealed its dual biological effect for the treatment of Alzheimer’s disease and cancer, respectively.