Reducing effect of intragastrically administered saikosaponin A on alcohol and sucrose self-administration in rats

Abstract Saikosaponin A (SSA) is an active ingredient of the Asian medicinal herb, Bupleurum falcatum L. When administered via the intraperitoneal (i.p.) route, SSA suppressed multiple addictive-like behaviours, including operant alcohol self-administration, in rodents. It is unknown whether these effects are retained after intragastric (i.g.) administration, a desirable prerequisite for a compound with therapeutic potential. To fill this gap, i.g. SSA (0, 50, and 100 mg/kg) was tested in Sardinian alcohol-preferring (sP) rats trained to lever-respond for oral alcohol. SSA reduced lever-responding and amount of self-administered alcohol. However, when compared to i.p. SSA, i.g. SSA resulted to be markedly less potent and effective, suggestive of reduced bioavailability after i.g. treatment. Finally, and in agreement with previous data on the suppressing effect of i.p. SSA on behaviours motivated by highly palatable foods, i.g. SSA (0, 50, and 100 mg/kg) reduced oral sucrose self-administration in a separate set of sP rats. Graphical Abstract


Introduction
Roots of the medicinal plant, Bupleurum falcatum L. (Apiaceae), are largely used in traditional Chinese, Korean, and Japanese medicine for the treatment of various inflammation-related diseases as well as psychiatric and neurological disorders (see Abbas et al. 2015;Jiang et al. 2020).The triterpenoid saponin, saikosaponin A (SSA; Figure S1), is a major component (Figure S2) and active ingredient of B. falcatum roots (see Yuan et al. 2017;Li et al. 2018).Multiple lines of experimental evidence have indicated that treatment with SSA potently and effectively suppressed several rodent behaviours motivated by drugs of abuse, including intravenous self-administration of morphine (Yoon et al. 2012) and cocaine (Yoon et al. 2013) and oral self-administration of alcohol (Maccioni et al. 2016(Maccioni et al. , 2020(Maccioni et al. , 2022)).Notably, the anti-addictive properties of SSA extended to rodent behaviours sustained by highly palatable foods, including oral chocolate self-administration (Lorrai et al. 2017) and binge-like overeating of chocolate and butter cookies (Maccioni et al. 2018).
All these sets of highly consistent data were collected after intraperitoneal (i.p.) injection of SSA.This route of drug administration is surely appropriate when investigating a drug at preclinical level, but it of course has limited translational value.When testing a drug with therapeutic potential it is imperative to assess if its 'desired' pharmacological effects are maintained after per os administration.
With the aim of contributing to filling this gap, the present study investigated the effect of intragastric (i.g.) administration of SSA on operant oral self-administration of alcohol in selectively bred Sardinian alcohol-preferring (sP) rats.To facilitate comparison with data generated by the studies with parenterally administered SSA (Maccioni et al. 2016(Maccioni et al. , 2020(Maccioni et al. , 2022)), the present study employed several identical methodological aspects, including rat line and alcohol self-administration procedure.The present study also included an experiment testing intragastrically administered SSA on operant oral sucrose self-administration; to our knowledge, this was the first 'i.g.' investigation of the effect of SSA on a food-related behaviour.

Results and discussion
The results of the present study indicate that acute i.g.treatment with SSA reduced both number of lever-responses for alcohol [F(2,33)=3.64,p < 0.05] (Figure S3A) and amount of self-administered alcohol [F(2,33)=5.62,p < 0.01] (Figure S3B).Compared to vehicle treatment, magnitude of the reducing effect averaged approximately 30% at both 50 and 100 mg/kg SSA dose.The observed limited dose-response relationship was likely due to the nonlinearity of SSA absorption after i.g.administration (Fu et al. 2019).Notably, SSA-induced reduction of alcohol self-administration was not associated to any confounding sign of sedation or malaise.These data suggest that SSA ability to reduce alcohol self-administration is maintained also after i.g.treatment.Comparison of data from i.p. (Maccioni et al. 2016) and i.g.(present study) treatment suggests however that i.g.SSA was dramatically less potent and effective than i.p.SSA.With the caution needed when comparing two different studies, although conducted using an identical experimental protocol and rats of the same line, it is indeed clear how markedly the dose-response of the reducing effect of i.g.SSA on alcohol self-administration shifted to the right in comparison to that of i.p.SSA (Figure S4).
Such a large loss of potency and efficacy after intragastric administration suggests that SSA may have poor bioavailability, likely due to unfavourable physicochemical and pharmacokinetic factors, such as high molecular weight, poor dissolution in the gastrointestinal tract, and/or saturation of transporters involved in intestinal absorption (Fu et al. 2019).Regarding metabolism, SSA proved to be remarkably stable in rat gastric juice, while being enzymatically converted by mouse intestinal bacteria into the deglucosylated metabolite, prosaikogenin F, and subsequently into the aglycone, saikogenin F (Shimizu et al. 1985).Studies examining the effects of the human gut microbiota on the hydrolysis of the SSA epimer, saikosaponin d, also found the same conversion relationship (Tang et al. 2020).Loss of one or both sugar units reduces the oral bioavailability, and thus the pharmacological activity, of SSA (Zhou et al. 2021).It should also be kept in mind that the extent of metabolic conversions of SSA depends on the composition of the gut microbiota, which varies in different rat strains (Fu et al. 2019).
Any interventions to 'protect' the SSA molecule and thus increase its bioavailability after i.g.administration could theoretically involve (i) modifying the β-glycosidic bond between glucose and fucose into an α-glycosidic bond, that might be less prone to enzymatic hydrolysis, or (ii) replacing sugar units with C-glycosides.The latter modification would significantly alter the chemical nature of SSA, since the acetal bonds of the normal glycosides would be replaced by ether bonds, which are thought to be more stable to hydrolysis catalyzed by bacterial enzymes.These modified SSAs could retain considerable affinity for intestinal transporters, but their actual pharmacological activity would still need to be evaluated.
It would also be of interest to assess if roots of B. falcatum contain ingredients with protective properties on SSA metabolism.This latter hypothesis would be consistent with the notion that plant extracts, because of favourable pharmacokinetic interactions among its multiple constituents, are often more effective than the corresponding doses of single active ingredients (see Wagner and Ulrich-Merzenich 2009).
The dose-range used in the present study is consistent with that of the very few studies demonstrating an in vivo effect of intragastrically administered SSA in rodents: anti-depressant effects in rats after i.g.treatment with 50 mg/kg SSA; analgesic effects in mice after i.g.treatment with 25 mg/kg SSA (Ahmadimoghaddam et al. 2021).
The results of the present study also indicate that acute i.g.treatment with SSA reduced both number of lever-responses for sucrose [F(2,21)=8.50, p < 0.005] (Figure S3C) and amount of self-administered sucrose [F(2,21)=7.05,p < 0.005] (Figure S3d).Compared to vehicle treatment, magnitude of the reducing effect averaged approximately 40% and 50% at the 50 and 100 mg/kg SSA doses, respectively.To our knowledge, these results constitute the very first line of experimental evidence on the ability of intragastrically administered SSA to affect addictive-like behaviours motivated by highly palatable foods.The above views on the reduced bioavailability of SSA after i.g.treatment closely fit also with these 'sucrose' data.