Anti-oxidative , acetylcholinesterase and pancreatic lipase inhibitory activities of compounds from Dasiphora fruticosa , Myricaria alopecuroides and Sedum hybridum

Total of 35 phenolics and flavonols were isolated from flowers and leaves of Dasiphora fruticosa Rydb., from branches of Myricaria alopecuroides Schrenk. and from the herb of Sedum hybridum L. The isolated compounds were identified on the basis of spectral data and tested for their anti-oxidative, acetylcholinesterase and pancreatic lipase inhibitory activities.Quercetin glycosides, gallic acid, (-)EGCG and gossypetin-8-O-xylopyranoside tellimagrandin II exhibited strong anti-oxidative activity by the DPPH scavenging method. The acetylcholinesterase inhibitory activity of quercetin glycosides and (-)EGCG was higher than those of other compounds. Whereas, tellimagrandin II, (-)EGCG and gallic acid derivatives exhibited the most potent inhibitory activity against the pancreatic lipase enzyme among the isolated compounds. Only (-)EGCG showed a prominent activity against all assayed experiments. It was concluded that these plants could be studied further for their potential as anti-oxidative, anti-aging and lipid lowering active products.


INTRODUCTION
Traditionally, flowers, leaves and scions of Dasiphora fruticosa Rydb.(Rosaceae) were used to cure diarrhea, dysentery and other digestive organs disorders, as well as for relieving the neurological disorders [1,2].Scions and branch of Myricaria alopecuroides Schrenk.(Tamaricaceae) have generally been used to neutralize the incidents of poisoning from different kinds of origins, to alleviate the diffused and concealed chronic fever and to treat several of diseases including rash, boils, chronic ulcers, phthisis, spasms and atrophy [1,3,4].The herb of Sedum hybridum L. (Crassulaceae) has been used for the treatment of diarrhea, dysentery, sepsis, thyroid, blood vessel disease and seizures related with nervous system [1,4,5].Previous studies revealed the presence of quercetin derivatives in the leaves of D.fruticosa [6,7], gallic acid and its esterified derivatives in M.alopecuroides [8,9] and some flavonols and coumarins in S.hybridum [10].The anti-oxidative, antibacterial activities of crude extracts, fractions and some compounds from the investigated plant samples have been partly reported [11][12][13].As a part of continuing work on these plants, we report herein the content of bio-active compounds in the crude drugs, the characteristic phytochemicals and their anti-oxidative, acetylcholinesterase, and pancreatic lipase inhibitory activities.
Each crude drug (50 g) was extracted twice with distilled water at room temperature and evaporated to dryness in vacuo.Then they were tested for their biological activities.

Quantitative analysis of total phenolics, flavonoids and tannins:
The content of total phenolics in all crude drugs was measured by means of the Folin Ciocalteu reagent in pH<10, at 760 nm on a spectrophotometer.Results were expressed as gallic acid (Sigma Inc.) equivalent (GAE) from the calibration curve [14,15].The amount of total flavonoids was determined by spectrophotometry using a formation of acid stable complex with 2% AlCl 3 at 401.5 and 430.0 nm.Results were expressed as rutin (Sigma Inc.) and quercetin (TCI Co., Ltd) equivalents (RE and QE) from the calibration curve [16,17].The content of total tannins was determined by spectrophotometry using a hide powder and the Folin Ciocalteu reagent in pH<10, at 760 nm.Results were expressed as pyrogallol (Sigma Inc.) equivalent (PE) from the calibration curve [18].DPPH scavenging activity: DPPH radical (2,2-diphenyl-2-picrylhydrazyl hydrate, TCI, Co., Ltd) scavenging activity of the crude extracts, fractions and pure compounds was determined according to the known spectrophotometric assay [19].The absorbance was measured at 517 nm and the anti-oxidative activity (AA) was expressed in percentage: Methanol (1.5 ml) added to plant extract solution (1.5 ml) was used as a blank.DPPH solution (1.5 ml, 6х10 -5 М) plus methanol (1.5 ml) was used as a control and rutin was used as a positive control.

Acetylcholinesterase inhibiting activity:
The acetylcholinesterase (AChE) inhibiting activity of crude extracts, fractions and isolated pure substances was determined using a previously reported Ellman spectrophotometric method with DTNB (5,5'-dithiobis(2nitrobenzoisc acid), TCI, Co., Ltd) color reagent [20].The absorbance was measured at 412 nm and the AChE inhibiting activity (I) was expressed in percentage: I (%) = 100 x (∆Abs control -∆Abs sample )/∆Abs control Where, ∆A control = Abs positive control -Abs blank positive control ∆A sample = Abs sample -Abs blank sample Blank solution was prepared from 0.1 ml sample (1 mg/ml) with DTNB prepared in Tris-HCl, while the positive control was prepared from DMSO (dimethylsulfoxide) with the same amount of the sample.Eserin (physostigmine, TCI, Co., Ltd) was used as a positive control.

Pancreatic lipase inhibiting activity:
The pancreatic lipase (PL) enzyme inhibiting activity of crude extracts, fractions and isolated pure substances was determined using a spectrophotometric method with DTNB color reagent [21].The absorbance was measured at 412 nm and the PL inhibiting activity (I) was expressed in percentage:

RESULTS AND DISCUSSION
In general, the polar EA fractions of all investigated crude drugs of D.fruticosa, M.alopecroides and S.hybridum exhibited better anti-oxidative activity by the DPPH scavenging method and the inhibition activity against AChE and PL enzymes (Figure 1).
In particular, the IC 50 value (mg/ml) of the EA fraction of D.fruticosa leaves for scavenging of DPPH was determined as 24.11±0.25,which is the greatest among the all studied samples, compared to the positive control rutin Considering the results of biological activity assays and qualitative analysis, the total phenolics, flavonoids and tannins were determined by the spectrophotometric methods (Table 2).Total phenolics in flowers EtOH (17.05±0.96%)and water (14.80±1.41%)extracts of D.fruticosa were estimated in the highest amount among the ethanol and water extracts of all investigated samples.The amount of total flavonoids in the EtOH extract of D.fruticosa flowers was determined in rutin equivalent as 5.30±0.57%,which is more than twice higher than the EtOH extracts of two other plant species, while in quercetin equivalent as 5.89±0.11%,which is 3-9 times higher than the related extracts.The branch of M.alopecuroides was Fig. 1.Biological activities of crude extracts and fractions Mongolian Journal of Chemistry 17 (43), 2016, 42-49 specified by containing rather more amounts of tannins (5.33±0.11%),while its quantity of total phenolics was almost comparable to flowers and leaves of D.fruticosa.
The EtOH extract of the herb of S.hybridum contains a bit lower amount of phenolics and tannins (9.44±0.62% and 3.32±0.05%)than other two plants related samples, while the amount of flavonoids was close to M.alopecuroides.Consequently, the qualitative and quantitative analysis of these three species indicated that they could classify to the phenolics containing plants.
Another aim of this study was to evaluate the DPPH scavenging activity and the inhibitory effects against AChE and PL of isolated compounds (Table 3).Generally, the anti-oxidative activity of flavonoids and phenolic compounds was studied very well by various methods [51].Thirty four isolated compounds have been tested for their AChE inhibitory activity by the Ellman method [20].None of the compounds exhibited activity against AChE like the potential drug physostigmine, which was used as the positive control.However, quercetin and its glycosides ( -)EGCG ( 22) were found to be more active than others.On the other hand, they inhibited AChE at the concentration 0.5 mg/mL by over 50%, which was comparable with the results reported previously [55,56].Gallic acid derivatives 26 and 27 exerted a weak inhibitory activity against AChE rather than their DPPH scavenging activity.Gallic acid (23) and ethylgallate (24) were not active against AChE (see Table 3).Hence, it is noticed that both flavonols and gallic acid derivatives showed the strong anti-oxidative activity.However, flavonolos have exhibited remarkable AChE inhibitory effect, whereas effect of gallic acid derivatives on AChE was minimal.

CONCLUSIONS
The flowers and leaves of Dasiphora fruticosa, the branch of Myricaria alopecuroides and the herb of Sedum hybridum were rich in phenolics, which were identified as the major bio-active characteristic compounds.Quercetin-, myricetin-, gossypetin glycosides, kaempferol-3-O-β-D-(6"-coumaroyl)glucopyranoside and (-)EGCG exhibited as active inhibitors against the AChE enzyme associated with the anti-oxidative activity.Whereas, multigalloylglucopyranose derivatives and (-)EGCG demonstrated to be as the active inhibitors against the PL enzyme associated with the anti-oxidative activity.Consequently, the flowers of Dasiphora fruticosa and the herb of Sedum hybridum could be identified as antioxidants and AChE enzyme inhibitors, the branch of Myricaria alopecuroides as the antioxidant and the PL enzyme inhibitor.Therefore, the crude drugs of these plants could be studied further in view of their potential uses as anti-oxidative, brain anti-aging and lipid lowering active product (22.66±0.29).The EA and n-BuOH fractions of the branch of M.alopecuroides (27.11±0.58 and 26.14±0.31)and the herb of S.hybridum (28.63±0.32 and 29.13±0.13)showed comparable results.The EtOH and water extracts, including the n-BuOH fraction of D.fruticosa samples gave almost twice lower activities than rutin.The ethanol extracts of M.alopecuroides and S.hybridum gave comparable results (31.93±0.48 and 35.61±0.36),which were better than the activity of related water extracts (52.35±0.85 and 48.59±0.64).The AChE inhibition percentage ofall assayed samples at the 1 mg/ml concentration was considerably less than the positive control eserin, which can inhibit by 100% at the 0.1 mg/ml.However, in this tested concentration the EtOH extract, EA and n-BuOH fractions of D.fruticosa leaves, as well as the EA fraction of the branch of M.alopecuroides could inhibit by over 60%, while activities of other samples were in the 40-59% range.The PL inhibition percentages of all assayed samples were less than the positive control orlistat (62.5±1.27%) at the same 1 mg/ml concentration.The EA fractions of flowers and leaves of D.fruticosa gave the best activity as 50.25±0.86%and 48.82±1.27%,respectively, which were the closest results to orlistat.Whereas, the EA fractions of two other plant species and all EtOH extracts exhibited ca.2-1.5 times lower activity than the positive control.Nonpolar DCM fractions of all assayed samples did not contribute any effect to scavenging of DPPH, inhibiting of AChE and PL enzymes.Results of biological activity experiments indicate that the EtOH extracts of all assayed plant samples exhibited better activity than the related water extracts and biologically active key components are presented in the polar EA and n-BuOH fractions.

Table 1 .
[42][43][44][45][46][47][48][49] gMongolian Journal of Chemistry 17(43), 2016,[42][43][44][45][46][47][48][49]DTNB prepared in Tris-HCl and porcine pancreatic lipase was prepared in water in the same amount as the sample analysis.Orlistat (TCI, Co., Ltd), a selective inhibitor for PL, was used as a positive control.Statistical analysis: All data are expressed in mean ± standard deviation (S.D).The IC 50 value, defined as the amount of the sample that could reduce the initial concentration of DPPH, PL and AChE by 50%, was calculated from the linear regression plots of test samples concentration (µg/mL and mg/mL) against the mean inhibition in percentage.IC 50 values were calculated using concentration of tested plant extracts, fractions and isolated substances and average percent of the antioxidant activity from three separate tests, PL and AChE inhibiting activities 6-8 separate tests.

Table 3 .
[62]ated compounds and their biological activities the brain cognition function and an excellent inhibitor for the PL enzyme or preventer against accumulation of lipids.Previously, Zhao et al.[60], Okello et al.[61]and Nakai et al.[62]reported about these three activities of (-)EGCG.