Alleviation of liver cirrhosis and associated portal-hypertension by Astragalus species in relation to their UPLC-MS/MS metabolic profiles: a mechanistic study

Liver cirrhosis is a late-stage liver disease characterized by excessive fibrous deposition triggering portal-hypertension (PH); the prime restrainer for cirrhosis-related complications. Remedies that can dually oppose hepatic fibrosis and lower PH, may prevent progression into decompensated-cirrhosis. Different Astragalus-species members have shown antifibrotic and diuretic actions with possible subsequent PH reduction. However, A.spinosus and A.trigonus were poorly tested for eliciting these actions. Herein, A.spinosus and A.trigonus roots and aerial parts extracts were subjected to comprehensive metabolic-fingerprinting using UHPLC-MS/MS resulting in 56 identified phytoconstituents, followed by chemometric untargeted analysis that revealed variable metabolic profiles exemplified by different species and organ types. Consequently, tested extracts were in-vivo evaluated for potential antifibrotic/anticirrhotic activity by assessing specific markers. The mechanistic prospective to induce diuresis was investigated by analyzing plasma aldosterone and renal-transporters gene-expression. Serum apelin and dimethylarginine-dimethylaminohydrolase-1 were measured to indicate the overall effect on PH. All extracts amended cirrhosis and PH to varying extents and induced diuresis via different mechanisms. Further, An OPLS model was built to generate a comprehensive metabolic-profiling of A.spinosus and A.trigonus secondary-metabolites providing a chemical-based evidence for their efficacious consistency. In conclusion, A.spinosus and A.trigonus organs comprised myriad pharmacologically-active constituents that act synergistically to ameliorate cirrhosis and associated PH.


Effect of Different A. spinosus and A. trigonus Extracts on Liver Cirrhosis Markers. In-vivo influence and OPLS analysis of different extracts on TGF-β.
In the present work, significant elevation in TGF-β levels were observed in untreated rats' livers compared to normal negative control rats (p < 0.001). All tested doses of A. spinosus and A. trigonus aerial and root samples extracts significantly reduced hepatic TGF-β levels (p < 0.001). The most potent reduction in hepatic TGF-β levels occurred upon the administration of A. spinosus aerial part (As-ar) extract even upon administration of a low dose; as the low dose showed the least TGF-β levels as compared to the low dose of all other tested extracts (p < 0.001). The least TGF-β levels were observed in Asar and A. trigonus root extract (At-rt). However, hepatic TGF-β levels in rats treated with high dose As-ar were not statistically significant compared to those in rats treated with the high doses At-rt or A. trigonus aerial parts (At-ar) extracts (p values are 1.00 and 0.992, respectively), Fig. 4A.
Conducting OPLS model revealed that the top efficient compounds modulating hepatic TGF-β and reducing its level were linoleic acid methyl ester, ferulic acid and naringin (naringenin 7-O-neohesperidoside), Figs. 4b1 and S1A. The former compound existed in the four tested extracts, while ferulic acid was detected only in the extracts of the At-ar and At-rt, and naringin was found in As-rt and At-ar, supplementary Tables S1 and S3.

In-vivo influence and OPLS analysis of different extracts on MMP-2.
In the present investigation, there was 4.6 fold increase in hepatic MMP-2 concentration in untreated cirrhotic livers compared to normal livers (p < 0.001). Effect of different extracts on hepatic MMP-2 was very similar to their effect on TGF-β; all treatments resulted in significant reduction in hepatic MMP-2 levels compared to the untreated positive control group (p < 0.001). Also, As-ar revealed the greatest potency in hepatic MMP-2 reduction; MMP-2 level in As-ar-low dose-treated group was comparable to its level in As-rt and At-ar high dose-treated groups (p = 0.828 and 0.319, respectively) and At-rt medium dose-treated group (p = 0.672), Fig. 4A. Moreover, similar to TGF-β results, MMP-2 levels in As-ar and At-rt high dose-treated groups were the least inspite of being not statistically significantly different from other groups treated with high doses As-rt or At-ar.
According to the OPLS model, the phytoconstituents that were found to be highly correlated to hepatic MMP-2 levels modulation were linolenic acid, tr-caffeic acid and isorhamnetin-3-O-rutinoside, Figs. 4B2 and S1A. In the tested Astragalus species, linolenic acid was detected in both As-ar and At-rt. Tr-Caffeic acid was identified in all tested extracts except for As-rt, whereas, isorhamnetin-3-O-rutinoside was found in the four tested extracts, supplementary Tables S1 and S3.

In-vivo influence and OPLS analysis of different extracts on RXR.
In the present study, RXR-α mean level in normal livers was about 2.5 folds greater than that in cirrhotic livers. Administration of different extracts significantly restored RXR-α levels compared to untreated rats with liver cirrhosis. Upon comparing extracts potency in restoring RXR-α, that is the ability to exert the most significant restoration with the administration of the low dose of the extract, extracts of the aerial parts of A. spinosus and A. trigonus showed the greatest potency with no statistically significant difference between them (p = 0.981). The maximum restoration capacity of RXR-α level was observed with the high dose As-ar. The order of extracts efficacy in restoring RXR-α level was As-ar, At-ar, As-rt then At-rt in descending order, Fig. 4A.
As revealed by OPLS model, activity on RXR-α is mainly attributed by isorhamnetin-O-rutinoside-O-arabinoside, soyasaponin I and formononetin-7-O-glucoside-6``-O-malonate, Figs. 4b3 and S1A. www.nature.com/scientificreports/ Isorhamnetin-O-rutinoside-O-arabinoside was only detected in the aerial parts of both A. spinosus and A. trigonus, and was not detected in their roots. Soyasaponin I was found in all tested extracts except for At-rt, while formononetin-7-O-glucoside-6``-O-malonate was detected in aerial parts and roots of both tested species, supplementary Tables S1 and S3.

In-vivo influence and OPLS analysis of different extracts on hepatocytes integrity and liver synthetic function.
Induction of cirrhosis resulted in 3.3 and 2.8 folds increase in Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum levels, respectively and 2.4 fold decrease in serum albumin level, Fig. 5A. The high and/or intermediate doses of three of the tested Astragalus extracts (As-rt D3, As-ar D2, As-ar D3, At-ar D2 and At-ar D3) were able to return ALT to its basal levels. P-values for comparing these extracts to normal were 0.109, 0.694, 1.00, 1.00 and 1.00, respectively. On the other hand, though no extract was able to return AST or albumin to normal base value, they all succeeded to significantly reduce AST and increase albumin level compared to positive control untreated rats (p values for comparing to normal rats or positive controls were < 0.001).
Of notice, At-ar D3 showed almost similar action on ALT and albumin as As-ar D3 with no statistically significant difference between them. Nevertheless, no other group showed similar action on AST as As-ar D3 that revealed significantly less AST levels compared to all other treated groups (p < 0.001), Fig. 5A.
Top detected compounds using OPLS model that scored activities against AST and ALT were isorhamnetin-O-rutinoside-O-arabinoside, soyasaponin I and formononetin-7-O-glucoside-6``-O-malonate in descending order. Whilst the top scoring compounds against albumin was ononin (Formononetin-7-O-glucoside), followed by isorhamnetin-O-rutinoside-O-arabinoside and soyasaponin I, Figs. 5B and S1B. Noticeably, all these compounds were detected in the arial parts of the tested Astragalus species, supplementary Tables S1 and S3.

Prediction of the Diuresis Mechanism of Different Astragalus
Extracts. Different Astragalus species had shown powerful diuretic effect including Astragalus membranaceus, Astragalus glycyphyllos and Radix Astragali 23 . Limited data pointed to the diuretic effect of the species that we are concerned about in the present study. Thus, a screening of the potential diuretic effect of the four examined extracts and thorough investigation of the exact mechanism implying their diuretic action were currently performed.
Extracts acting at the proximal convoluted tubule:. Rats with induced cirrhosis in the current investigation showed significant increase in the renal gene expression of sodium-hydrogen antiporter 1 (NHE-1) and aquaporin-1 compared to normal rats (p < 0.001), Fig. 6A. All tested doses of At-rt failed to significantly decrease renal expression of either NHE-1 or aquaporin-1 compared to untreated positive control rats (p > 0.05), indicating that this is not the mechanism that accounts for its diuretic effect. On the other hand, all tested doses of As-rt, At-ar and the highest tested dose of As-ar significantly decrease the renal expression of both NHE-1 and aquaporin-1. The extract that typically affected both transporters was As-rt and this effect operated at all dose levels, Fig. 6A.
According to the data obtained from the OPLS model, most biologically active compounds towards NHE-1 were astragaloside VIII, 16-octadecenoic acid methyl ester and orientin and towards aquaporin-1 were hyperoside, 16-octadecenoic acid methyl ester and astragaloside VIII in descending order, Figs. 6B1,2 and S1C. Astragaloside VIII and orientin was detected in all extracts except for At-rt whereas 16-octadecenoic acid methyl ester existed only in As-rt and At-ar. Hyperoside was present only in As-rt, supplementary Tables S1 and S3.
Extracts acting at the thick ascending limb of the loop of Henle and the distal convoluted tubule:. Rats with CCl 4 -induced cirrhosis showed significantly higher renal expression of both Na-K-Cl co-transporter 2 (NKCC2) and sodium-chloride symporter (NCC) transporters compared to normal negative control rats (p < 0.001), Figs. 6A, 7A. Regarding the effect of different extracts on NKCC2, all extracts, except for At-rt, significantly decreased renal expression of NKCC2 with the most prominent effect observed with the highest tested dose of At-ar. Then again, two extracts (As-ar and At-ar) significantly decreased gene expression of NCC at all tested doses, while the two other extracts (As-rt and At-rt) failed to show any significant difference in NCC expression in any tested dose compared to positive control rats. The least decrease in NCC renal expression was observed in As-ar-D3 treated rats.
The top active compounds in reducing the expression of NKCC2 in reference to the OPLS model results were astragaloside VIII, 16-octadecenoic acid methyl ester and hyperoside, Figs. 6B3 and S1C. For NCC, the compounds that decreased its expression to the greatest extent were isorhamnetin-O-rutinoside-O-arabinoside, chrysin and ononin, Figs. 7b1 and S1C.
Extracts acting at the collecting ducts:. Our results that revealed significant hyperaldosteronism in cirrhotic rats with plasma aldosterone levels 1.9 folds higher than those observed with normal rats (p < 0.001). Epithelial sodium channel (ENaC) renal expression was significantly elevated in untreated cirrhotic rats compared to normal rats (p < 0.001), Fig. 7A. The only extract that significantly reduces ENaC renal expression compared to the positive control rats was the highest tested dose of At-rt.
OPLS model analysis revealed that ENaC expression was mostly decreased by the influence of eriodyctiol-7-O-glucoside (detected in As-rt and At-rt), vitexin (detected in all extracts except for As-ar) and malic acid, Figs. 7B2 and S1C. All of the three top scoring compounds are present in At-rt, supplementary Tables S1 and S3. Plasma aldosterone reduction was influenced by the action of linolenic acid (present in As-ar and At-rt), rhamnocitrin-O-glucoside (present only in At-ar) and calycosin (present in all extracts except for As-rt). As-ar and At-rt contain two of the top active compounds against aldosterone and caused lower levels of plasma www.nature.com/scientificreports/ aldosterone, while As-rt extract lack any of these top active compounds and showed little influence on plasma aldosterone, Figs. 7B3, S1C and supplementary Tables S1 and S3.
The overall effect of different extracts on diuresis and serum electrolytes. The overall diuretic effect was evaluated by comparing the volume of 24 h collected urine after placing rats of each group in metabolic cages. The rats with untreated cirrhosis showed significantly less collected urine volume compared to normal rats and all treatments succeeded to significantly increase urinary output compared to positive control rats (p < 0.001) (data not shown). Upon comparing the mean urinary output of the groups receiving the highest tested dose of each extract, the order was as follows: As-rt D3 ≥ At-ar D3 > As-ar D3 > At-rt D3 with a statistically significant difference between each group and the preceding group except for As-rt and At-ar that showed no statistically significant difference between them (data not shown). This differential effect can be clarified by identifying the dominant diuretic mechanisms for each extract (explained in the next section). Induction of cirrhosis in the current work caused significant hyponatremia (p < 0.001), supplementary Tables S4 As-rt-D3 and At-ar-D3, showed significant reduction in serum sodium level compared to untreated rats (p < 0.001), supplementary Tables S4. As-rt-D3 was the only extract that was capable to return serum sodium to its normal levels, as it shows no significant difference compared to normal rats (p = 1.00).
The present model of cirrhosis induction was associated with significant decrease in serum potassium level compared to normal rats (p < 0.001), supplementary Table S5. At-rt and As-ar showed the greatest potassium levels. The higher doses At-rt showed serum potassium level exceeding the average values in normal rats (p < 0.001 for At-rt-D3). On the other hand, rats treated with the higher doses of At-ar, As-ar and all tested doses of As-rt showed no significant difference in serum potassium levels compared to normal rats.
Prominent diuretic mechanism of each extract. In order to identify the most prominent mechanism affected by each extract to exert its diuretic effect, the % decrease in mRNA expression of renal transporters and plasma aldosterone upon treatment with the highest tested dose of each extract were compared to the positive control group, Table 1. As-rt-D3 showed the greatest % reduction in proximal tubule transporters; NHE1 and aquaporin-1 compared to other transporters and compared to other extracts. It also showed high percentage reduction in NKCC2 of the thick ascending limb of the loop of Henle. Though As-rt D3 showed significant reduction in plasma aldosterone, this reduction was not reflected on NCC and it slightly affected ENaC. As-ar-D3 caused reduction in aldosterone level by 42.3% that more prominently affected NCC rather than ENaC. It also significantly affected NHE1, aquaporin-1 and NKCC2 but to a much lesser extent than As-rt-D3 or At-ar-D3. Compared to As-rt-D3, At-ar-D3 showed slightly less effect on NHE-1, aquaporin-1 and slightly more effect on NKCC2 renal expression with no statistically significant difference between them (p = 0.961, 0.139 and 0.524 respectively). Contrariwise, At-ar-D3 showed much greater effect on the aldosterone sensitive NCC compared to As-rt-D3 (p < 0.001). Here the reduction of plasma aldosterone more significantly affected NCC rather than ENaC.
At-rt-D3 failed to significantly decrease the expression of most renal transporters, namely NHE-1, aquaporin-1, NKCC2 or NCC (p = 0.443, 0.946, 1.000 and 0.846, respectively) compared to untreated rats with induced cirrhosis. This extract also showed the greatest reduction of plasma aldosterone that more significantly affected ENaC rather than NCC and was accompanied with hyperkalemia, supplementary Table S5. www.nature.com/scientificreports/

Effect of Different Astragalus Extracts on Portal Hypertension Markers.
In the present study, the circulating level of apelin was significantly increased in rats with induced cirrhosis compared with healthy rats (p < 0.001). With exception for At-ar-D1, all tested doses of all extracts significantly decreased serum apelin compared to rats with untreated induced cirrhosis. The least mean apelin value was observed in sera of rats treated with As-ar-D3, however, this mean value was not statistically different from those of the highest tested dose of other extracts. Hepatic DDAH-1 was significantly decreased in rats with cirrhosis compared to normal rats (p < 0.001). High doses and some medium doses of different extracts were able to significantly increase hepatic DDAH-1, Fig. 8A. The highest mean hepatic DDAH-1 was observed in At-ar-D3 treated rats which was significantly higher than As-rt-D3 and At-rt-D3 (p = 0.007 and 0.042, respectively), but showed no statistically significant difference with As-ar-D3 (p = 0.897).
According to the OPLS model, apelin is more significantly reduced in response to quinic acid (detected in all extracts), isorhamnetin-3-O-B-D-glucoside (present in all extracts except for As-ar) and caffeic acid-O-hexoside (present only in At-ar), Figs. 8B1, S1D and supplementary Tables S1 and S3. The compounds that revealed the   www.nature.com/scientificreports/ with mineral oil) as follows: The first 10 doses were received every 5 days, the subsequent 10 doses were administered every 4 days, and the last 7 doses were given every 3 days www.nature.com/scientificreports/ greatest ability to increase the level of hepatic DDAH-1 were naringenin, isorhamnetin and quercetin-3-Orhamnoside (quercitrin), Figs. 8B2, S1D, that were all detected in At-ar (supplementary Tables S1 and S3).

Discussion
Though widespread study on the chemical content of Astragalus extracts and their therapeutic effectiveness, no systematic chemical profiling of A. spinosus and A. trigonus roots and aerial parts bioactive phytoconstituents has been published to the authors' knowledge. The roots and aerial extracts of the two species were first chemically profiled using the ultra-high performance liquid chromatography/triple quadrupole mass spectrometry method (UHPLC-QqQ-MS), with the aim of establishing a correlation between the bioactive elements of the extracts and their in vivo alleviation of liver cirrhosis and associated PH. Metabolites annotation was achieved via comparison of retention times to references and examination of MS data (quasi-molecular ions and fragmentation patterns) of the detected peaks.
The MS spectrum of compound 1 showed a molecular ion peak at m/z 341.29 which was tentatively characterized as caffeic acid-O-hexoside, based on the loss of hexoside (−162.14 Da) and CO 2  Within the same context, silymarin isomers (compound 33) were identified by the precursor ion at m/z 481 with distinct fragment at (m/z 301.23) formed by the loss of a coniferyl alcohol unit as illustrated in Table 2 45,46 . 14-162.14-194.14-18.02] − and literature data 43 . The neutral loss of 146,0.14 162.14 and 194.14 Da allowed the identification of rhamnopyranosyl, galactopyranosyl and glucouronic acid moieties, respectively. Table 2. % decrease in mRNA expression of renal transporters and plasma aldosterone upon treatment with the highest tested dose of each extract compared to positive control group. As: Astragalus spinosus, At: Astragalus trigonus, ar: aerial organs, rt: root organs, D3: high dose, NKCC 2: Na-K-Cl co-transporter 2, NCC: sodium-chloride symporter, NHE1: sodium-hydrogen antiporter 1 and ENaC: epithelial sodium channel.  42 and a fragment ion at m/z 259.4, which are 2 Da lower than those of linoleic acid, was proposed to be linolenic acid 34,52 .
Compounds 50 and 51 having their molecular ion peaks at m/z 269.44 were identified as palmitic acid methyl ester and 14-methyl pentadecanoic acid methyl ester. Both compounds exhibited similar fragment ions in their MS 2 spectra, where fragment ion at m/z 238.41 [M-31.03] − represented loss of a methoxy group confirming a methyl ester compounds. Ion at m/z 74.08 is the McLafferty rearrangement ion, also a specific ion confirming that the spectra are that of methyl esters 34 . 14-methyl pentadecanoic acid methyl ester had an additional ion at m/z 43.09 due to loss of isopropyl group 34 .
Within the same context, compounds 54 and 55 giving rise to deprotonated ions at m/z 295.48 and 369.56 were proposed to be 16-octadecenoic acid methyl ester and hexanedioic acid dioctyl ester, respectively. Upon fragmentation, both compounds generated a base peak at m/z 55. Very few studies explored the hepatoprotective potential or antifibrotic activities of A. spinosus and A. trigonus. Two studies supported A. spinosus hepatoprotective potential; the first revealed that saponin glycosides in A. spinosus roots could modulate CCl 4 -induced toxicity and lethality in liver 53 . The other was conducted on rabbits where the daily administration of A. spinosus root extract to rabbits with hydrogen peroxide-induced oxidative stress showed significant improvement in liver sections upon histopathological examination 54 . To our knowledge, this is the first study to examine the effect of A. spinosus aerial parts and A. trigonus root and aerial parts extracts on cirrhosis and its complications with mechanistic insight of the role of identified phytoconstituents.
Liver cirrhosis can be sensitively assessed using a combination of multiple direct and indirect biomarkers. Usually, direct markers reflect fibrogenesis process and ECM turnover whereas indirect biomarkers indicate liver functionality alterations of hepatic function 55 . Herein, hepatic levels of TGF-β and RXR-α were determined as direct biomarkers reflecting HSCs activation and proliferation, while ECM turnover was evaluated by assessing hepatic MMP-2. Hepatocellular damage and alterations in liver functions were estimated by determining serum level of ALT, AST and albumin.
TGF-β is a key profibrogenic mediator involved in all fibrogenesis processes starting from initiation of inflammation-induced liver injury to development of fibrosis and cirrhosis ending to hepatocellular carcinoma. Elevated levels of TGF-β augment hepatocyte destruction and facilitate HSCs activation and ECM deposition 4 . Though effect of other Astragalus species, such as A. membranaceus, A. trojanus and A. oleifolius, on TGF-β in different conditions was formerly examined 56-58 , influence of A. spinosus and A. trigonus on TGF-β was not studied before. OPLS model revealed that the top efficient compounds modulating hepatic TGF-β and reducing its level were linoleic acid methyl ester, ferulic acid and naringin. It was proved earlier that circulating plasma linoleic acid in patients with simple hepatic steatosis, the progressive nonalcoholic steatohepatitis or any endstage liver diseases were significantly lower than in healthy subjects, hence, plasma fatty acids level may serve as a prognostic marker in cirrhosis 59,60 . Thus, administration of extracts containing this essential fatty acid may compensate for cirrhosis-associated linoleic acid and its derivatives deficiency. Furthermore, upon comparing the involvement of different dietary lipids on the modulation of TGF-β in the platelets, it was found that platelets lysates treated with corn oil, composed primarily of linoleic acid, showed the least levels of TGF-β 61 . Ferulic acid potently improved hepatic fibrosis via inhibition of the TGF-β1/Smad pathway in vitro and in-vivo 62 . Yet, naringenin was proved to inhibit TGF-β ligand-receptor interaction which is the initial step of TGF-β signaling 63 . Consequently, naringenin exerted potent antifibrotic effect in ameliorating hepatic, cardiac and lung fibrosis via its action on TGF-β pathways [64][65][66] . Since, among the top efficient compounds modulating hepatic TGF-β, As-ar contains only linoleic acid methyl ester, this means that this compound accounts for the activity of As-ar towards TGF-β to a great extent and is present in high concentration within this extract.
MMP-2 is a key fibroproliferative marker in chronic liver diseases with high diagnostic accuracy 67 . It degrades the excessively produced ECM during fibrogenesis 55 . Hepatic MMP-2 levels modulation was mostly affected by linolenic acid, tr-caffeic acid and isorhamnetin-3-O-rutinoside according to the OPLS model. Previous reports strongly agree with the correlation between these three compounds and MMP-2. For example, Hubbard et al., proved that linolenic acid administration could decrease MMP-2 levels in isolated mammary tumors from mice, .besides, it could potently inhibit MMP-2 activity 68 . Further, Eleonora et al., revealed that linolenic acid protective role in periodontal diseases could be the result of being an inhibitor to MMP-2 and MMP-9 69 . Caffeic acid was reported to down regulate MMP-2 gene expression and suppress MMP-2 enzyme activity in HepG2 cells 70 . Potent MMP-2 enzyme inhibition was also observed with caffeic acid derivatives such as esters and amides 71,72 . Furthermore, isorhamnetin-3-O-rutinoside is a polyphenolic flavone found in many herbal species that can depress MMP-2 levels in various conditions 73,74 . The combined presence of these top scored compounds affecting MMP-2 in As-ar and At-rt may explain the observed less hepatic MMP-2 levels in rats treated with these extracts than other treated groups.
Vitamin A regulates many physiological processes, including epithelial growth and immunological processes. Vitamin A pleiotropic effects are exerted either by vitamin A itself or by the main active metabolite, all-trans retinoic acid. They regulate the expression of a battery of target genes through several nuclear receptors such as www.nature.com/scientificreports/ RARs, RXRs, and PPARβ/δ 75 . Vitamin A is primarily stored in HSCs that lose vitamin A storage capacity during active liver injury and convert into activated ECM-producing myofibroblasts, which contributes to hepatic fibrosis then cirrhosis 76 . Consequently, the mRNA expression levels of RARβ and RXR-α are down-regulated in cirrhotic patients, suggesting the major role of these nuclear receptors in HSC activation and liver fibrogenesis 77 . Restoration of RAR/RXR levels suppresses liver fibrosis by blocking HSCs activation and proliferation 5 . As shown by OPLS model, activity on RXR-α is accredited mainly to isorhamnetin-O-rutinoside-O-arabinoside, soyasaponin I and formononetin-7-O-glucoside-6``-O-malonate. It was observed that extracts especially containing isorhamnetin-O-rutinoside-O-arabinoside caused significant elevation in RXR-α level which points to the key role of in restoring vitamin A storage capacity within the liver. In a number of studies, isorhamnetin suppressed HSCs activation via inhibition of TGF-β/Smad signaling pathway 78,79 . Suppression of HSCs activation is usually accompanied by restoration of their vitamin A storage capacity and subsequent upregulation of RXR 80 . The presence of glycoconjugates to isorhamnetin in the isorhamnetin-O-rutinoside-O-arabinoside compound may result in intensifying its ability to suppress HSCs activation, as in many cases it was observed that glycoconjugated derivatives are more active than the parent compound 81 .
ALT and AST are normally present in the hepatocyte cytosol. They serve as sensitive markers for evaluating the liver functional status in hepatic diseases as increased circulating ALT and AST level reflects cell damage and leakage 82 . ALT is predominantly found in hepatocyte cytosol whereas AST is found within the cytosol and mitochondria of the hepatocytes as well as many other organs 83 . Though ALT is more accurate than AST as hepatocyte injury marker, AST is more indicative for hypoxic and toxic damage, because zone 3 of the acinus with higher AST concentration is more susceptible to toxic damage 84 . On the other hand, albumin is synthesized in the liver and is an indicator of liver function 85 . Induction of cirrhosis was coupled with many folds increase in ALT and AST serum levels which indicates significant loss of hepatocytes integrity as well as liver synthetic function as a consequence of cirrhosis. Effect of different extracts was more pronounced on ALT than AST reflecting the liver-specific injury reversal by Astragalus species. Amongst all treated groups, the least ALT, AST and highest albumin levels were observed with the highest tested dose of As-ar. This result matches the observed greatest ability of this extract to regress hepatic cirrhosis, indicated by the highest significant reduction of TGF-ꞵ and MMP-2, and reversal of HSCS activation, marked by restoration of HSCs vitamin A storage capability and RXR expression level. Fibrosis reduction led to hepatocytes repair and increasing hepatic synthetic capabilities. As-ar D3 revealed significantly less AST levels compared to all other treated groups which reflects a potent ability of the arial parts of Astragalus spinosus to repair hypoxic damage and may indicate the presence of significant amount of antioxidant compounds in their extracts.
Top detected compounds scoring activities against AST and ALT using OPLS model were identical to each other and to those of RXR. Whilst the top scoring compounds against albumin was ononin, followed by isorhamnetin-O-rutinoside-O-arabinoside and soyasaponin I that were the top scoring compounds against ALT, AST and RXR. The coincidence of the top scoring compounds that were able to restore RXR, hepatocytes integrity and synthetic function implies a strong correlation between hepatic vitamin A content and hepatic regeneration. In support of this conclusion, a study was done to test the effect of vitamin A deficiency on hepatic regeneration in male and female rats after partial hepatectomy. This study proved that vitamin A is essential for the survival of hepatocytes 86 . Therefore, compounds capable of restoring vitamin A content with subsequent elevation of RXR, would restore hepatocytes integrity and synthetic function as indicated by the significant decrease in ALT and AST and increase in albumin synthesis.
Cirrhosis is associated with altered sodium and water metabolism regulation. During the course of cirrhosis, progressive impairment of kidney ability to maintain the volume of extracellular fluid within normal limits occurs. This is a consequence of an abnormal increase in tubular sodium and kidney disability to adjust the amount of sodium excreted. As a result, accumulation of solute-free water and an increase in total body water relative to total sodium content follows 87 . Diuretics are a heterogenous group of medications that function to control sodium and water metabolism in various sites within the renal tubules via various mechanisms of action. The use of diuretics is one of the widely used strategies to control PH and edema in cirrhotic patients 88,13 .
The proximal convoluted tubule is the site where the majority of sodium is reabsorbed. Within the proximal convoluted tubule, NHE-1 is probably the most important isoform due to its multi-functionalities unlike other isoforms such as NHE-3 that carries out the sole function of mediating the renal sodium absorption. For example, NHE-1 regulates intracellular pH, cell volume and intracellular sodium homeostasis 89,90 . NHE-1 may also modulate other transporters, such as apical membrane NHE-3, possibly through actin cytoskeleton remodeling, as well as Na + -K + ATPase through functional interaction with these transporters 90,91 . NHE-1 has been also shown to regulate cell cycle, proliferation, migration and adhesion, resistance to apoptosis and to implicate hypertension pathogenesis 91,92 . Thus NHE-1 downregulation can result in diuresis and relief of PH. Aquaporin-1 is the principal water-transporter in cell plasma membranes in both apical and basolateral membranes of kidney proximal tubule, where it facilitates > 70% of transepithelial water transport and causes urine concentration 15 . Aquaporin-1 has been shown to affect cell migration, cell proliferation, and angiogenesis. Aquaporin-1 inhibition or downregulation causes significant urinary concentrating defect with subsequent diuretic effect in many tested diuretics 93 . In the current investigation, induction of cirrhosis was associated with significant increase in both transporters. In accordance, the abundance of aquaporin-1 was formerly detected in the kidneys of rats with CCl 4 -induced cirrhosis, pointing to potentially vital effects of having cirrhosis on the proximal tubules. On the other hand, renal NHE-1 was not previously detected in rats with induced cirrhosis, yet, hepatic NHE-1 up regulation was found to contribute to the pathogenesis of liver fibrotic diseases 94 . These effects may account for the sodium retention and increased fluid absorption in this segment during cirrhosis progression 95 . According to data obtained from OPLS model, both NHE-1 and aquaporin-1 share the two active compounds astragaloside VIII and 16-octadecenoic acid methyl ester indicating that the presence of either of these compounds may be essential for selective activity at the proximal tubules. Since At-rt lack the presence of any of the top scoring www.nature.com/scientificreports/ compounds for either NHE-1 or aquaporin-1, it does not show any effect on the expression of these transporters. Though no previous study was done to show the diuretic effect of the claimed compounds in their purified form, several studies proved diuretic effect of plant extracts containing these compounds 96,97 .
The thick ascending limb of the loop of Henle accounts for a total of 20-25% of the filtered NaCl reabsorption. The major apical entry pathway for Na + is provided by Na-K-Cl co-transporter (NKCC2), which accounts for ∼80% of the total Na + reabsorption in this region of the nephron 98 . This area of the nephron is the site of action of loop diuretics where they bind to NKCC and inhibit its action. Inhibition of NKCC2 or its diuretic effect results in impairing reabsorption of Na + , K + , and Cl − and delivering an increase in Na + to the distal tubule. These changes decrease the osmotic driving force with subsequent diuretic effect 14 . At the distal convoluted tubule, reabsorption of 5-10% of glomerular filtrate occurs by the action of Na-Cl co-transporter (NCC) 99 . Blockade of NCC, which is the mechanism of action of thiazide diuretics, or its down regulation result in increased Na + delivery to collecting ducts, enhanced K + wasting and diuresis 14 . Induction of cirrhosis using CCl4 significantly increased renal expression of NKCC2 and NCC. This is in accordance with previous reports that proved significant increased renal abundance of sodium transporter proteins, including NKCC2 and NCC, and sodium retention in cirrhotic rats 95 . Top active compounds in reducing NKCC2 expression were also identical to the top scoring active compounds that decrease aquaporine-1 expression. Thus, similar activities of different extracts towards both transporters were obtained. Also, two compounds (astragaloside VIII and 16-octadecenoic acid methyl ester) were common with the top scoring compounds for reducing NHE-1 expression. For NCC, isorhamnetin-O-rutinoside-O-arabinoside, the compound that reduced the expression for the greatest extent was also the compound that mostly decreased ALT and AST, besides ononin was the top compound responsible for the restoration of albumin synthetic function which indicates a very close association between cirrhosis degree and the NCC renal expression. Thus, the extracts of the arial parts of the tested Astragalus species, especially As-ar, that showed greater effect on restoring hepatocytes integrity and synthetic function induced diuresis mainly by affecting NCC expression.
Collecting ducts possess two mechanisms that contribute to sodium reabsorption control; the epithelial Na + channel (ENaC) and aldosterone via binding to aldosterone receptor. Since only 3% of the filtered Na + is reabsorbed at the collecting duct, drugs inhibiting or down regulating ENaC or aldosterone receptor do not result in appreciable diuresis and lead to minimal antihypertensive efficacy as monotherapies. Instead, they are often used with other agents to correct K + deficiency 14 . Aldosterone can also affect aldosterone-sensitive apical renal transporters, namely NCC as well as ENaC 95 . Cirrhosis is accompanied with a hyperaldosteronism status that plays a vital role in the pathogenesis of sodium retention and ascites formation in cirrhosis 95 which is consistent with our results that revealed significant hyperaldosteronism in cirrhotic rats with plasma aldosterone levels 1.9 folds higher than those observed with normal rats. Consequently, the aldosterone-sensitive ENaC renal expression was significantly elevated in untreated cirrhotic rats. Though all tested extracts, except for the lowest tested dose of At-ar, showed significant reduction in plasma aldosterone levels, the only one that significantly reduces ENaC renal expression compared to the positive control rats was the highest tested dose of At-rt. This indicates that the significant reduction in plasma aldosterone level observed in rats treated with different extracts most probably affected the binding to aldosterone receptor or the aldosterone-sensitive NCC rather than ENaC. ENaC expression was mostly decreased by the influence of eriodyctiol-7-O-glucoside, vitexin and malic acid. All of the three top scoring compounds are present in At-rt, consequently, it was the only extract capable of significantly decreasing the expression of ENaC. Unlike vitexin and malic acid, the correlation between eriodyctiol-7-Oglucoside and diuresis was not formerly studied. Pure vitexin exerted potent hypotensive effect in volunteers and rabbits through its diuretic effect 100 . In addition, extracts containing malic acid induced significant diuresis 101 . Plasma aldosterone reduction was influenced by the action of linolenic acid, rhamnocitrin-O-glucoside and calycosin. As-ar and At-rt contain two of the top active compounds against aldosterone (linolenic acid and calycosin) and resulted in lower levels of plasma aldosterone. In support with our results, linolenic acid was found to affect aldosterone level; the oxidized derivatives of linoleic acid showed biphasic action, stimulating aldosterone production at low concentrations and inhibiting it at higher concentrations 102 . Accordingly, it may be inferred that linolenic acid is present As-ar and At-rt at high concentration to cause the observed inhibitory effect on aldosterone production. While linolenic acid seems to act via direct modulation of steroid synthesis and aldosterone production by adrenal cells, rhamnocitrin-O-glucoside and calycosin mostly act through affecting renin-angiotensin-aldosterone system. Rhamnocitrin and calycosin downregulate angiotensin (Ag) II/Ag converting enzyme (ACE)/Ag II type I receptor pathway and upregulate Ag 1-7/ACE2/Mas receptor pathway with subsequent inhibition of aldosterone release from adrenal cortex 103,104 .
Induction of cirrhosis in the current model caused significant hyponatremia. Hyponatremia is a frequent complication of advanced cirrhosis correlated to an impairment in the renal capacity to eliminate solute-free water. This causes a disproportional water to sodium retention resulting in hypoosmolality which is linked pathogenically to renal aquaporins 105 . Thus, extracts that most significantly affected renal aquaporin-1, namely As-rt-D3 and At-ar-D3, showed significant reduction in serum sodium level compared to untreated rats. As-rt-D3 was the only extract that was capable to return serum sodium to its normal levels which may be attributable to its significant downregulation of aquaporin-1.
Potassium derangement is commonly detected in cirrhotic patients with both hypokalemia and hyperkalemia occurrence is possible and are linked to unfavorable prognosis 106 . The present model of cirrhosis induction was associated with significant hypokalemia which might be linked to the significantly elevated plasma aldosterone in cirrhotic rats; circulating potassium is tightly regulated by aldosterone and in high aldosterone states, surface cells contribute to active potassium secretion following an ENaC-dependent mechanism 107,108 . Extracts that showed the greatest potassium levels (At-rt and As-ar) were those with less plasma aldosterone indicating that aldosterone is one of the dominant regulators of circulating potassium in the currently applied cirrhosis animal www.nature.com/scientificreports/ model. It is noteworthy to say that higher doses At-rt elevated serum potassium level to an extent that significantly exceeded average values in normal rats meaning the occurrence of hyperkalemia. The most prominent mechanism affected by each extract to exert its diuretic effect was identified by determining the % decrease in mRNA expression of renal transporters and plasma aldosterone upon treatment with the highest tested dose of each extract in comparison to the positive control group. As-rt-D3 showed the greatest % reduction in proximal tubule transporters and high % reduction in the thick ascending limb transporter, both nephron areas are responsible for the majority of Na + reabsorption and their downregulation would result in urine dilution and significant diuresis. This effect was confirmed by measuring the 24 h urine volume that was significantly higher than other tested extracts (p < 0.05) (data not shown). As-ar-D3 more considerably affected the distal areas of the nephron more than the proximal tubule or the thick ascending limb of the loop of Henle. Since As-ar showed relatively small effect on the proximal tubule, responsible for the greatest portion of Na + reabsorption, and aquaporin-1, key regulator of water reabsorption, it caused moderate diuresis. At-ar-D3 exhibited very similar effect on renal transporters as As-rt-D3 in showing greater effect on non-aldosterone sensitive transporters, resulting in the high diuretic effect observed from the collected 24 h urinary output. Compared to As-rt-D3, At-ar-D3 showed slightly less effect on NHE-1and aquaporin-1 which accounts for the observed lesser diuresis. At-rt-D3 failed to significantly decrease the expression of most renal transporters (NHE-1, aquaporin-1, NKCC2 or NCC) accounting for the weakest diuretic effect observed in rats treated with this extract.
PH is primarily caused by the increase in resistance to portal outflow, as a consequence of hepatic cirrhosis, and secondly by an increase in splanchnic blood flow. Alterations in vascular tone play a central role in the pathophysiology of PH by contributing to increased intrahepatic resistance, hyperdynamic circulation, and expansion of the collateral circulation 109 . PH can be estimated by assessing serum biomarkers, such as serum apelin 110 . Apelin is an endogenous ligand for angiotensin-like receptor 1 (APJ) that is known to have diverse physiological and pathological effects, including regulation of cardiovascular function, fluid homeostasis and so on. Serum apelin levels have shown close relationships with both intrahepatic cirrhosis and splanchnic hemodynamics 111 . In cirrhosis, the hepatic nitric oxide (NO) levels are significantly reduced, with associated elevated sinusoidal vascular resistance. On the other hand, asymmetric dimethylarginine (ADMA), the competitive endogenous inhibitor of endothelial nitric oxide synthase (eNOS) that synthetizes NO, is metabolized by dimethylarginine dimethylaminohydrolase-1 (DDAH-1) within the liver. Impaired liver function is associated with increased blood levels of ADMA and correlates with the severity of PH and decompensation 10 . Induction of cirrhosis in the present work significantly increased the circulating level of apelin, which matches early reports 112 , indicating notable PH. Hepatic DDAH-1 was significantly decreased in rats with cirrhosis, reflecting increased availability of ADMA and NO synthesis inhibition. Under normal circumstances, apelin/APJ cause a decrease in blood pressure, but, under pathological conditions that cause vascular damage, such as in cirrhosis where elevated ADMA is a risk factor for endothelial dysfunction, apelin functions as a vasopressor peptide 113 . Apelin/APJ system induces hypertension via affecting sympathetic nervous system, renin-angiotensin-aldosterone system, endothelial injury, excessive endothelin, sodium retention and vascular remodeling 113 . Since serum apelin is elevated only after vascular damage induced by cirrhosis development, reversal of cirrhosis associated fibrosis and hepatic damage would be the driving factors for decreasing serum apelin and counteracting apelin-induced PH. Therefore, extracts that more profoundly reduced fibrosis markers and restored hepatic integrity were those that more significantly reduced serum apelin. According to the OPLS model, apelin is mosly reduced by effect of quinic acid, isorhamnetin-3-O-B-D-glucoside and caffeic acid-O-hexoside. Quinic acid is the only compound from these top active compounds that is present in As-ar, the extract that showed the greatest reduction in serum apelin, which means that this compound may possess a great capability to modulate apelin. In accordance, polyphenols, such as quinic acid, showed the ability to counteract the hazardous effects of elevated apelin in obese diabetic pregnant females 114 . Generally, decreased levels of DDAH-1 result in increased ADMA levels which is linked to up-regulation of circulating renin-angiotensin-aldosterone system components 115 , sodium retention and blood pressure increase 116 . In the present work, the activity of different extracts towards DDAH-1 may be linked to their combined diuretic effect and influence on circulating aldosterone. The compounds that revealed the greatest ability to increase the level of hepatic DDAH-1 were naringenin, isorhamnetin and quercetin-3-Orhamnoside. All of these compounds previously have proved efficacy in modulating DDAH-ADMA-NO pathway and were all exclusively detected in At-ar, therefore, At-ar-D3 showed the highest hepatic DDAH-1 level. For instance, Naringenin had been shown to stimulate eNOS and NO in endothelial cells exposed to high glucose 117 . Isorhamnetin also showed protective effects on endothelial cell line EA.hy926 and caused upregulation of eNOS expression 118 . Further, Quercetrin affected endothelial NO ⋅ signaling by inhibiting both arginase-1 activity and expression 119 .

Methods Preparation and analysis of plants extracts. Collection of plant materials:. Astragalus spinosus and
Astragalus trigonus whole plant materials were collected in August 2020 from Borg El Arab, Alexandria, Egypt, with permission from the Agriculture Research Center, Giza, Egypt at "9 Cairo University Road, Giza District, Giza Governorate". The plant collection was accomplished in accordance with the national guidelines. The identity of the plant materials was confirmed by Prof. Dr. Salama El Dareer, Professor of Botany, Faculty of Science, Alexandria University. A voucher specimens of A. spinosus and A. trigonus (No.: A-SP-019 and A-TR-019, respectively) were deposited at the herbarium of the Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University. The roots and the aerial parts of the two species were separated and left for complete dryness at room temperature. www.nature.com/scientificreports/ Plant extracts and samples preparation. The air-dried powdered A. spinosus and A. trigonus aerial parts (100 g from each species) and roots (50 g from each species) were separately extracted with methanol, filtered and dried under reduced pressure using rotary evaporator at 45 ˚C to afford dry solid residues (3.5, 2.8, 1.5 and 1.3 g, respectively). The four samples were prepared at (1 mg/ml) concentration using HPLC-grade methanol, filtered using membrane filters (0.2 μm). The forementioned process was repeated three times for each sample to ensure reproducibility.
Standard solutions for UPLC-MS quantification. The stock standard solutions of caffeic acid, malic acid, sucrose, quercetin, astragaloside I and linoleic acid were prepared as follow: an accurate weight of each compound (10 mg) was separately placed in 10-mL volumetric flask. HPLC-grade methanol was added, and the solution was diluted to the working concentrations ranging from 0.0125 to 0.625 mg mL −1 using the same solvent as shown in Table S2. Five μL aliquots of each standard solution were applied onto the chromatographic column and the injections were carried out in triplicates for each concentration level. The calibration curves were generated by plotting peak areas of the standards versus their concentrations.
Chromatographic parameters and conditions. The UPLC system consists of a Waters Acquity QSM pump, a LC-2040 (Waters) autosampler, degasser and Waters Acquity CM detector. 10 μl of each of the previously prepared samples (full loop injection volume) were separately injected into the chromatographic column. Chromatographic separation was conducted using a Waters Acquity UPLC BEH C18 column (50 mm × 2.1 mm ID × 1. ESI-MS parameters and conditions. Electrospray ionization (ESI) source in conjunction with triple quadrupole (TQD) mass spectrometer were utilized to analyze the samples in a negative ionization mode. ESI operating conditions briefly were: capillary voltage of 3 kV, cone voltage; 35 V, the ion source temperature was 150 • C, the nebulizer (nitrogen gas) pressure was 35 psi, drying and sheath gas (N 2 ) temperature was 440 °C and 350 °C, respectively. The drying and sheath gas flows were applied at 900 L/h and 50 L/h, respectively. The analytical run time was extended to 30 min. MS spectra were achieved by full range acquisition covering 100-1000 m/z. For automatic MS/MS fragmentation analyses of the precursor ions which were mass-selected by the first quadrupole (Q1), the collision-induced dissociation (CID) energy was ramped from 30 to 70 eV using nitrogen gas as a collision gas in the second quadrupole collisional cell (Q2). Finally, the daughter ions yielded from CID are consequently related to the molecular structure of the precursor ions and can be monitored by a third quadrupole mass analyzer (Q3).
Characterization of compounds. For annotation of secondary metabolites of A. spinosus and A. trigonus aerial parts and roots samples, comparison of the quasi-molecular ions and MS/MS data together with retention times with reference literature was implemented.
Mass spectrometry data processing for multivariate data analysis. MZmine 2.0 (http:// mzmine. sourc eforge. net/) freely downloadable data analysis software was utilized for relative quantitation of the UPLC-MS derived metabolic fingerprints of the four Astragalus extracts as well as rapid processing of the raw data according to the protocol adopted by Ghallab et al. 120 .
Chemometric analysis of metabolic fingerprints. The UPLC-MS processed data were subjected to multivariate data analyses for complex data mining and dimensionality reduction of Astragalus extracts metabolic fingerprints. The data matrix was imported to SIMCA-P software (Version 14.0, Umetrics, Umea, Sweden) for building various chemometric models.
Unsupervised pattern recognition analysis. Principal Component Analysis (PCA) and Hierarchial Clustering Analysis (HCA) were used as exploratory data approaches, where PCA summarized the information in the data set through searching the direction of maximum variance through using a small number of orthogonal latent variables. PCA was performed using nine components and 0.95 confidence interval for Q and T2 hotelling limits for outliers. HCA-heat map based on average-linkage method was used for building dendrograms with the Euclidean distance and the centered correlation metric using MetaboAnalyst (https:// www. metab oanal yst. ca/).
Animals. The current study was performed on 112 healthy male Sprague Dawley rats of a local bred strain, weighing 200 ± 20 g each. The rats were purchased and housed in the animal house of Faculty of Pharmacy, Pharos University in Alexandria (PUA); (Alexandria, Egypt), under standard environmental conditions of light and temperature. Rats were allowed to acclimatize for a period of one week prior to experimentation. The use of animals and all experimental procedures were approved by the PUA Research Ethics Committee and were performed in strict accordance with the regulations of ARRIVE guidelines. All methods were performed in accordance with ARRIVE guidelines and regulations.
Experimental design. Animals were divided into 14 groups; each composed of eight rats, as follows: Plain normal control group (eight rats) that received 1 mL saline i.p. daily. Liver cirrhosis was induced in the remaining rats by intraperitoneal administration of CCl 4 after dilution with mineral oil at the ratio 1:6 as follows: The first 10 doses were received every 5 days, the subsequent 10 doses were administered every 4 days, and the latest 7 doses were given every 3 days 121 . Different doses (40, 80 and 160 mg/kg) of A. spinosus and A. trigonus aerial parts and roots extracts were orally administered starting from the 10th week to the end of the experiment (16th week). At the end of experimentation period, all rats were put in metabolic cages for 24-hr urine collection and then euthanized under desflurane anesthesia. Blood samples were collected for sera and plasma separation then killed by decapitation. Livers were quickly isolated and an appropriate part was sectioned and kept in 10% formal-saline for Masson's trichrome staining and histopathological examination for confirming cirrhosis induction defined by formation of hepatic nodules. The remaining parts of the livers and kidneys were stored in liquid nitrogen and then in −80 °C until performing quantitative RT-PCR (qRT-PCR), ELISA and biochemical testing.

Effect of Different Astragalus Extracts on Liver Cirrhosis Markers.
Determination of hepatic TGF-ꞵ, MMP-2 and RXR-α by ELISA. TGF-ꞵ, MMP-2 and RXR-α levels were assessed using ELISA kits following manufacturers' guidance. Levels were assayed in the supernatant obtained following centrifugation of the homogenized liver tissue (20% homogenates in PBS (pH7.4)) in cooling-centrifuge (Centurion-scientific-K3series, UK) at 15,000 rpm at 5 °C for 5 min.
Biochemical assessment of serum ALT, AST and albumin. ALT, AST concentrations (U/L) and albumin (g/dL) in the sera obtained from different groups were assayed colorimetrically using colorimetric assay kit according to instructions provided by suppliers.
Prediction of the diuresis mechanism of different Astragalus extracts. Assessment of mRNA expression (2^-ΔΔCt) of different renal transporters acting at different nephron parts using qRT-PCR. Using real time one step qRT-PCR, the renal expression of Aquaporin 1, Na-K-Cl co-transporter 2 (NKCC 2), sodium-chloride symporter (NCC), sodium-hydrogen antiporter 1 (NHE1) and epithelial sodium channel (ENaC) was assessed. Total RNA isolation using total RNA isolation kit and purity determination at 260 and 280 nm were performed as previously reported 122 . Reverse transcription was done using High-capacity cDNA reverse transcription kit (Applied Biosystems; California, USA). Then qRT-PCR was performed, using SYBR Green PCR master mix and SYBR green RT-PCR reagents kit according to manufacturer's instructions, to amplify the cDNA using sets of specific primer for each gene (Table 3). During the extension step, data acquisition was collected using Rotor-Gene Q-Pure detection, software version 2.1.0 (build 9); (Qiagen; Hilden, Germany). Quantification was expressed as normalized ratio of the relative expression of target genes relative to reference gene in the same sample by calculating the threshold cycles (Ct) values using ΔΔCt method_ENREF_41.
Determination of plasma Aldosterone. Plasma aldosterone (nM) obtained from different groups was assayed using ELISA kit according to instructions provided by supplier.
Determination of serum electrolytes. Serum sodium (mM) and potassium (mM) were analyzed spectrophotometrically by using colorimetric assay kits following manufacturer's instructions.

Effect of Different Astragalus Extracts on Portal Hypertension Markers (serum apelin and hepatic DDAH1 by ELISA).
Both apelin (ng/mL) and DDAH1 (pg/g protein) were assessed in supernatants, separated by centrifuging 20% liver homogenates from different groups, respectively, using ELISA kits following steps provided by their suppliers.
Statistical analysis of the data. Data were analyzed using IBM SPSS software package version 20.0. (Armonk, NY: IBM Corp). The normality of distribution was verified using Kolmogorov-Smirnov test. Quantitative data were described using mean and SD. Significance of the obtained results was judged at the 5% level. In order to compare between more than two groups, F-test (ANOVA) was used for normally distributed quantitative variables, followed by Post Hoc test. Outliers were excluded from data set. www.nature.com/scientificreports/ Supervised pattern recognition analysis. Orthogonal projection to latent structures (OPLS) was utilized to model the metabolite differences among the different Astragalus extracts and to investigate chemical variability within the X-matrix (via employing LC-MS data as independent variables) that were related to the investigated pharmacological activities (Y-dependent variables). Accordingly, the algorithm exposed the main efficacy associated markers via biplots and coefficient plots.

Conclusion
Different extracts of A. spinosus and A. trigonus roots and aerial parts significantly ameliorate cirrhosis/fibrosis and associated PH to variable extents. They also induced powerful diuresis by inconsistent mechanisms that affect different nephron portions. This study represents the first approach to reveal the metabolites variation among Astragalus species using UPLC-ESI-TQD -MS coupled with multivariate data analysis. The study showed that the differences among studied accessions mainly emanate from saponins and flavonoid content. Based on MS2 analysis, a number of phenolic acids, saponins, flavonoids and fatty acids were reported for the first time in A. spinosus and A. trigonus. OPLS models assisted in identifying the exact metabolites from each extract responsible for each pharmacological action.

Data availability
All data generated or analyzed during this study are included in this published article and its supplementary information files. The datasets (RNA sequences) analysed during the current study are available in the GenBank repository, https:// www. ncbi. nlm. nih. gov/ genba nk/, accession numbers are included in Table 3.
Received: 3 May 2022; Accepted: 1 July 2022 Table 3. Used oligonucleotide primers in the present study for RT-PCR analysis.