Chemical constituents, anti-inflammatory and analgesic activities of iridoids preparation from Phlomoides labiosa bunge

Abstract This study reports the isolation of iridoids and cycloartane glycosides from the aerial parts of Phlomoides labiosa Bunge. Six compounds were isolated and the chemical structures were identified as phlorigidoside С (1), 8-O-acetylharpagide (2), shanzhiside methyl ester (3), cyclosiversioside A (4), cyclosiversioside E (5), and cyclosiversioside C (6). Compounds 4-6 are reported for the first time in this plant. In addition, anti-inflammatory and analgesic activities of iridoid fraction were studied. The sum of iridoids (SI) with intragastric administration is 5.2 and 52.5 times less toxic, than such market drugs as analgin and diclofenac sodium, respectively. In terms of the latitude of analgesic action (LD50/ED50), the SI exceeds analgin by 19.2 times and diclofenac sodium by 16 times. The anti-inflammatory and analgesic activities of the sum of iridoids were confirmed to be effective and nontoxic, and exceed known drugs diclofenac sodium and analgin (metamizole sodium). Graphical Abstract


Introduction
Phlomoides L. is a large genus of the Lamiaceae family, which comprises 139 accepted named species and 15 synonym named species (www.theplantlist.org). These species are distributed almost in Central and South-Western Asia where the highest biodiversity of the genus is present with more than 100 different species mainly growing in Azerbaijan, Uzbekistan, Turkey and Iran (ww2.bgbm.org). In the flora of Uzbekistan, the genus Phlomoides is represented by about 36 perennial species, in which 13 species of them are endemic (Vvedenskiy 1961).
Phytochemical studies on various species of the genus Phlomoides have resulted in the isolation of triterpenes, iridoids, phenylpropanoids, flavonoids, alkaloids, steroids, saponins, fats, carboxylic acids and diterpenes (Olennikov and Chirikova, 2017;Behlil et al. 2019;Mohammadhosseini et al., 2019;Geng et al., 2020;Doustimotlagh et al., 2020). Various species of this genus are used in Tibetan medicine to treat lung and throat diseases and different chronic conditions (Batorova et al. 2013). Some tubers of these species are used to prepare decoctions and multicomponent powders (Yang et al., 2015). Moreover, some species are used in treatment of diverse diseases and symptoms, like allergies, headache, some liver diseases, asthma, cough and common cold. Additionally, they might be used as a herbal tea (from root and flower) (Said et al., 2002).
Carrying out many biological activity studies, connected with analysis of distribution area of this genus is definite value for phytochemical science because it can help to find effective therapeutic features of these species, and as a result, can have important applications in pharmaceutical industry. In this regard, a lot biological activity studies were performed on pure natural compounds and on extracts from different Phlomoides species, including local analgesic, strong anti-inflammatory, immunomodulatory and antioxidant activities (Delazar et al., 2004;Naghibi et al., 2005;Khobrakova et al., 2017;Li et al., 2021). This paper report the first investigation about the chemical composition of the butanol extract, obtained from the aerial parts (flowers, leaves, and stems) of Phlomoides labiosa Bunge, which grows on fine-earth slopes in the lower belt of the mountains of the Nurota (Samarkand region, Uzbekistan). In addition, biological activity experiments were performed and the results of the effect of the sum of iridoid-containing preparation of this plant with anti-inflammatory and analgesic activity were discussed in this work.
The isolated compounds were identified by means of spectroscopic methods as well as comparison with literature data, including mass spectroscopy and NMR spectroscopy (see supplementary information). The mass spectra were recorded by high resolution mass spectra (HR-MS), which were measured on a Bruker micrOTOF II instrument applying electrospray ionization (ESI). The 1 and 13 NMR spectra were recorded on a Bruker AVANCE-II 600 NMR spectrometer operating at a frequency of 600 Hz equipped with a BBFO Z-gradient Smart Probe. The Table S1 represents the physicochemical data for the compounds extracted from Phlomoides labiosa Bunge. plant and their yield.
The next step was the biological investigation of the obtained iridoids amount. As you can see, from the data shown in Table S2, the experiments were performed on mice, where the investigated compound with intragastric administration is 5.2 and 52.5 times less toxic, respectively, than analgin and diclofenac sodium, which are currently applied in medical practice.
As seen from the data in Table S2, the amount of iridoids obtained from Phlomoides labiosa Bunge. in terms of the dose of efficacy (ED50) exceeds analgin 3.6 times and is inferior to diclofenac sodium by 3.2 times. In terms of the latitude of analgesic action (LD50/ED50), the amount of iridoids exceeds activity of analgin by 19.2 times and diclofenac sodium by 16 times. The average effective dose of suppression of the amount of writhing (ED50) in mice caused by intraperitoneal administration of acetic acid was 45.2 mg/kg for the amount of iridoids, and 165.5 mg/kg for analgin and 13.8 mg/kg for diclofenac sodium.
In Table S3 can be seen that the activity of the preparation in doses of 25-50-100 mg/kg exhibit pronounced analgesic activity, reducing the amount of vinegar writhing and increasing the threshold for thermal (hot plate test) pain irritation. According to the hot plate test, the claimed agent also had a distinct analgesic effect, increasing the threshold of pain sensitivity depending on the administered dose during thermal pain irritation and significantly exceeded the comparison drug analgin and diclofenac sodium. A higher analgesic index of the claimed drug indicates its greater selectivity and greater safety over analgin and diclofenac sodium in the case of practical use.
Previously, anti-inflammatory activity of methanol extract from Phlomoides labiosa was studied using an in vitro method (Zamani et al 2014). In Table S4, that the preparation have a pronounced anti-inflammatory effect and surpasses diclofenac sodium in activity depending on the administered dose (25-50-100 mg/kg) by 1.4-1.6-1.3 times, respectively.

Conclusion
In this study, we reported on the isolation and structure elucidation of three iridoids and three triterpene glycosides from the buthanol fraction of Phlomoides labiosa Bunge. Compounds 1-6 were isolated from Phlomoides labiosa Bunge. for the first time. The biological activity of isolated compounds was thoroughly investigated, including such activities as anti-inflammatory and analgesic. Furthermore, these compounds considered as steroid and alkaloid-free components. Thus, the results of the studies indicate that the preparation of iridoids (1-3) isolated from Phlomoides labiosa Bunge. has a pronounced analgesic and anti-inflammatory activity and can be used in practical medicine as an analgesic agent that also has an anti-inflammatory effect.