Phytochemical isolation and biological screening of Cotoneaster microphyllus

ABSTRACT The root and aerial parts of Cotoneaster microphyllus were subjected to extraction and isolation of phytochemicals. The extracts were evaluated for their antioxidant anthelmintic, antimicrobial, and anticholinesterase potentials using standard protocols. Crude extract of aerial parts and roots, more potently scavenged DPPH free radicals with IC50 values of 83 and 66 μg/mL while ABTS with 92 and 90 μg/mL respectively. Chloroform fraction exhibited highest anthelmintic activity followed by ethyl acetate fraction. Ethyl acetate fraction produced high zone of inhibition against selected bacterial and fungal strains. Maximum phenolic contents and vitamin C were found in the ethyl-acetate and chloroform fractions and were therefore, biologically the most potent fractions. Ethyl acetate fraction exhibited highest anticholinesterase potential and was therefore subjected to silica gel column chromatography which resulted in the isolation of one new (1) and four known (2–5) compounds. The isolated compounds were also screened for anticholinesterase potentials. Compound 3, most potently inhibited acetyl cholinesterase and butyryl cholinesterase with IC50 value of 66 and 114 μg/mL, respectively. The extracts exhibited antioxidant, anticholinesterase, anthelmintic and antimicrobial potentials that should be further subjected for the isolation of other responsible compounds in pure state. As anticholinesterase compound 3 is a good candidate to be tested in animal models.


INTRODUCTION
The therapeutic potentials of plants have been utilized by human since the beginning of his life on the earth. [1] Plants being the complex chemical industries, prepare large variety of natural products. [2] Plants medication is preferred due to low side effects and inexpensive as well as abundantly available.
There are approximately 308,312 plant species on planet earth out of which 450,000 are vascular plants. [3][4][5][6][7] According to World Health Organization report, 80% of population in the third world countries still relies on plant medication. [8] According to a recent survey about 5700 species of medicinal plants are growing in Pakistan and 82% of Pakistanis are frequently using traditional plant based medicines to cure different diseases. [9,10] The Cotoneaster genus (family Rosacea) is comprised of 84 species found mostly in Turkey, Syria, Iran, Pakistan and Europe. . [11] In Himalayas from Kuman to Kashmir, Pakistan, Cotoneaster microphyllus species is abundantly found. A number of species of the genus Cotoneaster are used as laxative, aperient, styptic, and

Extraction and fractionation
The dried powdered samples were dipped into 85% methanol at room temperature and kept for two week with constant shaking. After filtration through cloth the extract was condensed into a semi solid mass through a rotary evaporator at 40°C which was then fractionated into ethyl-acetate, n-hexane and chloroform sub-fractions. The fractions were also condensed into semi solid mass through a rotary evaporator at 40°C and after complete drying in open air they were weighed. The resulting mass (aerial parts) of n-hexane, chloroform, and ethyl acetate fractions were 35, 22.1, and 21.1 g respectively. The crude extract of roots was also fractionated into ethyl acetate, chloroform, and n-hexane fractions.

Total phenolic contents
Antioxidant activities of plant samples are mostly due to phenolic compounds present in them. [20] Working dilutions of all extracts were prepared in range 62.5-1000 µg/mL. From each dilutions, 1 mL was further diluted into 10 mL by the addition of water. Then 1 mL of Folin-Ciocalteu reagent was added to the diluted reaction mixtures and incubated for 6 min. After incubation, 10 mL of 7% Na 2 CO 3 was added and final volume was made 25 mL. After 1.5 h the absorbance of the mixture was recorded at 760 nm. Phenolic contents were estimated as mg of GAE/g of dry sample.

Total flavonoid contents
To estimate the flavonoid contents, the method devised by Park et al (2008) was followed. [21] From working dilutions (as mentioned in above step), 1 mL was further diluted to 10 mL through the addition of water. Then 1 mL of 5% NaNO 3 was also added and allowed to stand for 6 min. The mixture was incubated for 5 min after the addition of 2 mL of 10% AlCl 3 and 2 mL of 1 M NaOH was sequentially added. The absorbance of the reaction mixtures were noted at 510 nm using UV-visible spectrophotometer. Total flavonoid contents were estimated as quercetin equivalent (mg QE/g) of dry sample from a standard quercetin curve (0 to 100 mg/mL).

DPPH free radical scavenging assay
DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging activities of the extracts were determined using previously reported method. [22] DPPH stock solution (20 mg/100 mL methanol) was prepared and kept for 24 h in dark in order to produce free radical in them. About 2 mL from the working dilutions were mixed with 2 mL of DPPH solution and after incubation for 15 min in dark the absorbance was noted at 515 nm using UV/visible spectrophotometer. The percent radical scavenging activity (%RSA) was calculated using the following formula:

ABTS radical scavenging activities
The antioxidant potentials of the extracts were also determined against ABTS (2,2-azinobis [3-ethylbenzthiazoline]-6-sulfonic acid) free radical using Re et al method. [23] About 100 mL each ABTS (7 mM) and K 2 SO 4 (2.45 mM) were prepared and after mixing were kept in dark for 24 h in order to develop free radical in it. From the working dilutions 2 mL were mixed with 3 mL of ABTS solution and incubated for 25 min at room temperature. The absorbance of the mixture was measured at 745 nm using a double beam spectrophotometer. The same procedure described above was used for ascorbic acid that was used as a positive control. Equation 1 was used to estimate the %RSA.

Anthelmintic activities
The anthelmintic activity of the extracts were determined against adult earthworms (Pheretimaposthuma) using a previously reported method. [24] Earthworms were collected from marshy muddy area. Different concentrations i.e. 10, 20, and 40 mg/mL of Cotoneaster microphyllus extract solution were prepared. The same size Pheretima posthuma worms were kept in Petri dishes having 35 mL fraction solution of the desired concentration. Albendazole was used as standard drug.

Antimicrobial activities
The antibacterial activity of C. microphyllus (roots and aerial parts), extracts were evaluated against two bacterial strains; Pseudomonas aeruginosa and Streptococcus pyogenes while antifungal potentials were evaluated against two fungal strains; Candida albicans and Fusarium solani using agar well diffusion method. [25] Anticholinesterase assays Acetyl cholinesterase (AChE) from electric eel and butyrylcholinesterase (BChE) from equine serum was used to evaluate the enzymes inhibitory potential of the crude extract, sub-fractions and isolated compounds using Elman's assay. [26] AChE and BChE solutions were prepared in phosphate buffer of pH 8. AChE (518 U/mg solid) and BChE (7-16 U/mg) were diluted in freshly prepared buffer (pH 8) until a final concentration of 0.03 U/mL and 0.01 U/mL respectively. Solutions of DTNB (0.0002273 M), acetyl and butyryl choline iodide (0.0005 M) were prepared in distilled water and were kept in Eppendorf caps in the refrigerator. For each analysis, an enzyme solution of 100 μL was added to the cuvette, followed by addition of plant extract dilutions and isolated compounds solution (1 mL). Finally DTNB reagent (100 μL) was added to mixture, incubated at 25°C for 15 min in an incubator, and subsequently the substrate solution (100 μL) was added to them. A double beam spectrophotometer was used to measure the absorbance of reaction mixture at 412 nm. A negative control contained all components apart from the extract/compounds, whereas the positive control contained galantamine (5 mg/5 mL) along with all the mentioned components. Percent enzyme activity and percent inhibition were calculated as follows: Where: V indicates the rate of reaction in the presence of inhibitor and V max is the rate of reaction in absence of inhibitor.

Isolation of phytochemicals
Column chromatography was used for the isolation of different phytochemicals from ethyl acetate fraction (root). Silica gel was used as an adsorbent. The desired extract was mixed in silica gel slurry which was then loaded to column with care. The developed column was eluted with n-hexanechloroform mixtures, mixed in different proportions in order of increasing polarities (1 to 100%). Several fractions were obtained which were then mixed together on the basis of TLC profiling and as result 11 sub-fractions designated as D-1 to D-11 were obtained.

Compound 1
Sub-sub fractions C-4 and C-5 were obtained from D-2 subfraction after elution with ethyl acetatechloroform. Based on TLC analysis the fractions C-4 and C-5 were combined and further subjected to fresh silica gel column. In the obtained fraction impure crystals were obtained. The crystals were washed with n-hexane successively and 18 mg of compound 1 was obtained.

Compound 2
Using an n-hexane-chloroform solvent system sub-sub fraction C-6 was obtained from subfraction D-4. Compound 2 weighing about 23 mg was obtained from this fraction. Melting point; 144°C

Compound 5
The sub-sub fraction C-7 was obtained from a subfraction D-9 using n-hexane-chloroform (7:3) gradient which was further re-chromatogrammed over fresh silica gel column and upon elution with n-hexane-chloroform (4:6) solvent system in rising order of polarity produced compound 5 weighing about 14 mg.

Statistical analysis
All the experiments have been performed in triplicates. The results are presented as Mean ± SEM. P values were determined using Two-way ANOVA followed by Bonferroni posttest to establish the statistical differences between standard drug and test samples using Graph Pad Prism software. The value of P < .05 was considered as significant. The medium inhibitory concentration (IC 50 ) of DPPH, ABTS, AChE, and BChE has been calculated using linear regression (MS Excel program 2007).

Total phenolic and flavonoid contents
The Phenolic contents in various extracts of Cotoneaster microphyllus were investigated. The phenolic contents were found in range from 33.00 to 83.96 mg GAE/g of dried extract (Gallic acid equivalent) in aerial parts of the plants. Highest contents (83.96 mg/g) were present in ethyl acetate fraction, whereas n-hexane fraction exhibited the lowest contents (33.00 mg/g) amongst the fractions. The total flavonoid contents were expressed as quercetin equivalent (mg of QE/g of dry sample). Ethyl-acetate fraction was the richest fraction with 78.40 mg QE/g of dry sample followed by crude, chloroform, and n-hexane fractions with values of 68.70, 60.10, and 22.94 mg QE/g of dry sample respectively (Table 1). Phenolics contents were also determined in different fractions of roots (Table 1) which were ranged from 24.00 to 85.96 mg GAE/g of dry extract (Gallic acid equivalent). Ethyl acetate fraction of roots parts showed the highest phenolic contents (85.96 mg GAE/g), whereas n-hexane showed the lowest (24.00 mg GAE/g). Similarly, the total flavonoid contents were determined as quercetin equivalent (mg of QE/g of dry sample). Ethyl acetate fraction with flavonoid contents value of 80.40 mg QE/g was the richest fraction followed by crude, chloroform, and n-hexane fractions (62.70, 50.10, and 16.94 mg QE/g of the dry sample respectively).

Antioxidant potential of the extracts
Crude extract and their sub-fractions of both aerial and root parts were screened out for free radical scavenging potentials against DPPH and ABTS radicals (Tables 2 & 3). The crude extract and ethylacetate fractions showed the highest free radical inhibitions which were 89.87, and 84.17% for aerial part while 88.62, and 81.60% for root part respectively against DPPH. The n-hexane fractions exhibited a minimum inhibition of 58.27 and 50.77% for aerial and root parts. The activity of the chloroform fraction was found lower than the activity of ethyl acetate fraction. As ethyl-acetate fractions and crude extract of both parts showed the highest phenolics compounds, therefore its ABTS and DPPH radical scavenging potential are also high. While n-hexane fraction contained lower phenolic contents and hence showed the lowest %RSA activity.
The crude extract and ethyl-acetate fraction of both parts (aerial and root) of the mentioned plant showed promising antioxidant activity against ABTS free radical as well with IC 50 values of 92/90 and 178/ 240 µg/mL respectively. The rest of the fractions like chloroform and n-hexane exhibited good scavenging activities with IC 50 values of 220/320, and 880/1060 µg/mL respectively as shown in Tables 2 & 3.

Anthelmintic activity
Anthelmintic activity of C. microphyllus various extracts tested against earthworm, are presented in Table 4. Chloroform fraction of both parts showed high activity at all concentrations, while the lowest activity was observed with n-hexane fraction of both parts. Paralysis and death time of the earthworm for chloroform fraction (aerial parts) were 15 and 28 min respectively, while 19 and 31 min were for root parts at 30 and 40 mg/mL concentrations. In the case of ethyl acetate fraction time of paralysis and time of death of worms were 19 and 32 min respectively for both aerial parts and roots. When n-hexane fraction (aerial parts) was used against the worm after 34 min paralysis was observed and all the worms died after 66 min at a concentration 10 mg/mL, whereas for n-hexane fraction of roots the time of paralysis and death of warms were 41 and 72 min respectively at concentration of 10 mg/mL. The positive controlalbendazole showed paralysis and death time 10 and 16 min respectively.

Antifungal activity of various fractions of aerial parts and roots of C. microphyllus
The highest antifungal activity was recorded for ethyl acetate fraction (root parts) which was 16.04 mm against Candida albicans while 15.88 mm against Fusarium solani. Chloroform and n-hexane fractions exhibited high inhibitions against the used strains as shown in Table 5.

Antibacterial activities of various extracts of aerial parts and root of C. microphyllus
Both parts were found to be active against selected bacterial strains. Ethyl acetate fraction aerial and root parts exhibited the highest zone of inhibition as 13 and 12.3 mm respectively against Pseudomonas aeruginosa while against Streptococcus pyogenes the mentioned fraction produced zone of inhibition as 11.4 and 11.8 mm (Table 6).

Anticholinesterase potential of extracts
The enzyme inhibition potential of crude extract and their different sub fractions of C. microphyllus (aerial and root part) were tested against acetyl cholinesterase (AChE) and butyrylcholinesterase (BChE) and the results are summarized in Tables 7 and 8. Against AChE the crude and ethyl acetate fractions of aerial parts were found more potent with IC 50 values of 90 and 125 µg/mL respectively while the mentioned extract of the root produced IC 50 values equal to 80 and 145 µg/ mL. The remaining fractions like chloroform and n-hexane also showed moderate inhibition and their IC 50 were 170 and 380 µg/mL respectively (aerial parts). Similarly, the extracts were also tested against BChE. The crude and ethyl acetate fractions of aerial and root parts were more potent with IC 50 values of 105/150 and 110/210 µg/mL, respectively. Galantamine was used as a positive control.

Isolation of pure compounds (Z)-2́-Ethylundecyl 6-(chloroamino) hex-3-enoate (1)
The novel compound 1 was isolated as white solid. The molecular formula was established as C 19   Ascorbic acid was used as a positive control. Data is represented as mean ± S.E.M (n = 3). Values significantly different as compared to positive control,*: P < 0.05, ***: P < 0.001, ns: P > 0.05.  13 C and DEPT NMR spectral data of compound 1 showed that it contains 19 carbons, including 2 methyl, 12 methylene, 3 CH, and one quaternary carbon. The signal at δ C 168.2 was assigned to C-6, the quaternary (carbonyl) carbon. The peaks at δ C 128.8 and 130.9 were due to the presence of olefinic methene carbons C-3 and C-4. Peaks at δ C 68.2 and 23.7 were attributed to the aliphatic carbons C-7 and C-9 respectively. The peaks at δ C 11.0 and 14.0 showed the presence of two methyl groups at position no 10 and 18 respectively. The other peaks resonating in between δ C 23.0 (C-11) to δ C 29.7 (C-18), were attributed to methylene carbon atoms. These assignments were confirmed by advanced techniques of 2D-NMR (HMBC, HMQC, and COSY).
From the HMQC and HMBC spectra data, the connectivity was determined and clear-cut assignments were made for all of the protons and carbons. The structure of the new compound was established. The proton signal at δ H 7.52(H-3) is interrelated with the carbon at δ C 130.9 7 (C-4) in the HMBC spectrum (methylene) representing a direct connection to each other. The olefinic protons (H-3, H-4) correlated with the carbon signals at δ C 128. The proton at δ H 1.42 (H-5) and δ H 4.21(H-7) are correlated to the carbonyl carbon signal at δ C 168.27(C-6) in the HMBC spectrum. Proton signals at δ H 4.21 (H-7), δ H 1.31(H-9), and δ H 0.9 (H-7) assigned to C-9 giving a signal at δ C 23.7 protons at δ H 1.71 (H-9) correlated with C-7 (δ C 68.2). The proton at δ H 1.39 (H-15), and δ H 0.7 (H-19) correlated to C-17 which signaling at δ C 29.7 and proton at δ H 1.69 (H-18) correlate to C-16 (δ C 29.3) in the HMBC spectrum. These spectroscopic data including 1D-NMR and 2D-NMR established the structure as (Z)-2-ethylundecyl 6-(chloramine) hex-3-enoate, a novel compound, isolated for the first time from the aerial parts of C. microphyllus.   On the basis of the interpretation of its 13 C and DEPT NMR spectral data compound 2 contained 29 carbons, including six methyl, 11 CH 2 , 9 CH, and three quaternary carbons. The signal at δc 140.76 was assigned to C-5, the quaternary (olifenic) carbon. The peak at δc 121.70 is assigned to the olefinic methene carbons C-6 while δc36.51 and 42.30 were attributed to the aliphatic carbons (C-10 and C-13 respectively). The peaks indicated at δc19.83(C-18), 19.40 (C-21), 19.03 (C-27), 18.79 (C-26), 11.99 (C-29), and 11.86 (C-19) were assigned to methyl of the respective carbons. These assignments were confirmed by advance 2D-NMR techniques (HMBC, HMQC, and COSY). The spectroscopic and physical data of compound 2 agreed with those reported in the literature as β-sitosterol. [24]  Galantamine was used as a positive control. Data is represented as (mean ± S.E.M) n = 3.Values significantly different as compared to positive control, *: P < 0.05, **: P < 0.01, ***: P < 0.001, ns: P > 0.05.

Structure of β-Sitosterol β-Sitosterol 3-O-β-D-glucopyranoside (3):
The IR spectrum of compound 3 displayed bands at 3452, 1648, 1379 and 1065 cm −1 indicating the presence of OH and unsaturation respectively. The molecular formula was determined as C 29 13 C NMR the peak of anomeric carbon at C-3 with additional sugar moiety C-3́, C-4́, C-2́, C-5`. The spectroscopic and physical data of compound 3 in as β-sitosterol glycoside was showed good agreement with previously compiled data. [16][17][18][19] Stigma sterol (4) Compound 4 was obtained as an amorphous powder the molecular formula C 29 H 48 O, was established from 13 C -NMR, DEPT, and EIMS. Its steroidal nature was indicated by the appearance of six methyl groups. The IR spectrum showed characteristic peaks for hydroxyl (3412 cm −1 ), CH stretching ( were assigned to H-19. All the spectral data (IR, UV, 1 H NMR 13 C NMR and mass spectrum) and other data are in agreement with the reported values for 3β-hydroxylup -20(29)-ene) (Lupeol) . [16][17][18][19] The structure was confirmed using 1D and 2D NMR techniques.

Anticholinesterase effect of the isolated compound
The enzyme inhibition potential of the isolated compounds against acetylcholinesterase and butyrylcholinesterase are summarized in Table 9. The percent AChE inhibition observed for compound 1 were 72.87 ± 1.27, 60.95 ± 2.01, 49.08 ± 1.04, and 38.41 ± 0.99 at the concentration ranging from 125-1000 µg/ml respectively with the IC 50 of 178 µg/mL while the compound 2 and 3 showed potent percent inhibitions with the IC 50 of 80 and 66 µg/ml respectively as shown in Table 9. Similarly, compounds 4 and 5 showed moderate percent inhibition with IC 50 of 268 and 242 µg/ml respectively. The BChE was inhibited by compounds; 1, 2, 3, 4 and 5 resulted with IC 50 values of 320, 132, 144, 334, and 316 µg/ml, respectively. Galantamine was used as a positive control that has produced IC 50 values of 44 and 60 µg/ml respectively against AChE and BChE.

DISCUSSION
In the current investigations, the medicinal aspects of C. microphyllus have been evaluated to revive its medicinal importance. The quantitative analysis of secondary metabolites in C. microphyllus showed the presence of significant amounts of phenolics and flavonoids. The phenolics and flavonoids have been the focus of research for decades due to their beneficial role in the management of the various types of health anomalies. One of the best role of phenolics and flavonoids, which have been reported from time to time, is the free radical scavenging role which is very important from human physiological point of view and their accumulation inside body leads to high oxidative stress which may cause the peroxidation of lipid membranes of various cells in the body and neuronal membranes as well. They also cause damage to DNA and other biologically important molecules. As mentioned above the high oxidative stress also causes neuronal damage, which may lead to a decreased amount of various neurotransmitters within the body. The decreased amount of the neurotransmitters i.e., acetylcholine or butyrylcholine leads to the demonstration of various symptoms including the memory impairment and movement disorders collectively known as Alzheimer's disease. As far as the role of phenolics, flavonoids, and other secondary metabolites are concerned, they are capable of scavenging the free radicals in the body thereby minimizing the effects of oxidative stress and being from plant origin are considered nontoxic. [27][28][29] The scavengers of the free radicals are compounds mostly containing phenolic ring that prevent or ameliorate the symptoms of oxidative stress including Alzheimer's disease. The presence of phenolics and flavonoids in a considerable amount along with significant antioxidant activities as demonstrated by extracts of the selected plant signifies the beneficial role of C. microphyllus that would be helpful in the prevention of Alzheimer's disease and other oxidative stress related complications in human. [27][28][29] DPPH and ABTS free radicals were more potently inhibited by crude extract of aerial parts with IC 50 values of 83 and 92 μg/mL respectively followed by ethyl acetate fraction (IC 50 = 140 and 178 μg/mL respectively) while against the same free radicals crude extract exhibited IC 50 values of 66 and 90 μg/mL respectively. Against AChE and BChE good inhibitions were observed for crude and ethyl acetate extracts. As anthelminthic agent chloroform fraction was more potent than other extracts followed by ethyl acetate fraction. High zone of inhibitions were formed by ethyl acetate fraction of aerial parts against the selected fungal strains (16.04 mm against Candida albicans and 15.88 mm against Fusarium solani). Similar trend was observed for the root extracts. However, the intensity of inhibition of root extract was lower than that of the aerial parts. Against Pseudomonas aeruginosa zone of inhibitions equal to 13 and 12.3 mm respectively was produced by ethyl acetate fraction of aerial and root parts while against Streptococcus pyogenes the zone of inhibitions produced by aerial and root parts, ethyl acetate fractions were 11.4 and 11.8 mm respectively. Similar role of C. microphyllus as a significant antimicrobial agent has been reported previously. [27][28][29] Although all the extracts exhibited good antibacterial, antifungal, antioxidant, anthelmintic and anticholinesterase activities. However, among them comparatively good activities were observed for ethyl acetate fraction which was then subjected to isolation of phytochemicals. Five compounds mentioned above were isolated in pure state. In neurodegenerative diseases the role of AChE and BChE is prominent, and to cure them the general strategy used is the inhibition cholinesterases. As these enzymes are responsible for the breakdown of acetylcholine and other neurotransmitters leading to its deficit results a number of neurodegenerative diseases. So by inhibiting the AChE and BChE, the acetylcholine level can be restored. Among the isolated compounds, compound 3 exhibited high percent inhibition of both of these enzymes as depicted in Table 7 with IC 50 values of 66 and 114 µg/mL respectively against AChE and BChE followed by compound 2.

CONCLUSION
The results of the current investigational study and the previous literature reveals the importance of C. microphyllus. Being a good source of phenolics and flavonoids the extracts exhibited high antioxidant, anthelminthic, anticholinesterase, and antimicrobial potential potentials. Among the extracts, ethyl acetate fraction due to its high phenolic and flavonoid contents was subjected to silica gel column isolation that resulted in isolation of one new and four known compounds. Among the isolated compounds, compound 3 exhibited promising anticholinesterase potentials. Further investigation are needed to isolate other biologically active compounds from this plant and investigate the anticholinesterase potential of compound 3 in animal models along with its toxicological evaluations.