Journal of Ethnopharmacology 156 (2014) 26–32
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Plants Fagonia cretica L. and Hedera nepalensis K. Koch contain natural compounds with potent dipeptidyl peptidase-4 (DPP-4) inhibitory activity Samreen Saleem a,1, Laila Jafri a,1, Ihsan ul Haq b, Leng Chee Chang d, Danielle Calderwood c, Brian D Green c, Bushra Mirza a,n a
Department of Biochemistry, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan Advanced ASSET Centre, Institute for Global Food Security, School of Biological Sciences, Queens University Belfast, David Keir Building, Stranmillis Road, Belfast BT9 5AG, Northern Ireland, UK d Department of Pharmaceutical Sciences, Daniel K. Inouye College of Pharmacy, University of Hawaii at Hilo, Hilo, HI 96720, United States b c
art ic l e i nf o
a b s t r a c t
Article history: Received 11 April 2014 Received in revised form 17 July 2014 Accepted 15 August 2014 Available online 26 August 2014
Ethnopharmacological relevance: The two plants investigated here (Fagonia cretica L. and Hedera nepalensis K. Koch) have been previously reported as natural folk medicines for the treatment of diabetes but until now no scientiﬁc investigation of potential anti-diabetic effects has been reported. Materials and methods: In vitro inhibitory effect of the two tested plants and their ﬁve isolated compounds on the dipeptidyl peptidase 4 (DPP-4) was studied for the assessment of anti-diabetic activity. Results: A crude extract of Fagonia cretica possessed good inhibitory activity (IC50 value: 38.1 μg/ml) which was also present in its n-hexane (FCN), ethyl acetate (FCE) or aqueous (FCA) fractions. A crude extract of Hedera nepalensis (HNC) possessed even higher inhibitory activity (IC50 value: 17.2 μg/ml) and this activity was largely retained when further fractionated in either ethyl acetate (HNE; IC50: 34.4 μg/ml) or n-hexane (HNN; 34.2 μg/ml). Bioactivity guided isolation led to the identiﬁcation of four known compounds (isolated for the ﬁrst time) from Fagonia cretica: quinovic acid (1), quinovic acid-3β-O-β-D-glycopyranoside (2), quinovic acid-3β-O-β-D-glucopyranosyl-(28-1)-β-D-glucopyranosyl ester (3), and stigmasterol (4) all of which inhibited DPP-4 activity (IC50: 30.7, 57.9, 23.5 and 4100 mM, respectively). The ﬁfth DPP-4 inhibitor, the triterpenoid lupeol (5) was identiﬁed in Hedera nepalensis (IC50: 31.6 μM). Conclusion: The experimental study revealed that Fagonia cretica and Hedera nepalensis contain compounds with signiﬁcant DPP-4 inhibitory activity which should be further investigated for their anti-diabetic potential. & 2014 Published by Elsevier Ireland Ltd.
Keywords: Fagonia cretica Hedera nepalensis Dipeptidyl peptidase 4 (DPP-4) inhibition Diabetes Incretin
1. Introduction Dipeptidyl peptidase 4 (DPP-4) is a physiological enzyme involved in cleaving dipeptide fragments from various peptide hormones, neuropeptides and chemotactic agents. Since DPP-4 performs a central role in the rapid degradation and inactivation of incretin hormones it has emerged as an important drug target for treatment of type 2 diabetes mellitus (Deacon et al., 1995).
Corresponding author. E-mail addresses: [email protected]
(S. Saleem), [email protected]
(L. Jafri), [email protected]
(I.u. Haq), [email protected]
(L.C. Chang), [email protected]
(D. Calderwood), [email protected]
(B. Green), [email protected]
(B. Mirza). 1 These authors contributed equally to this manuscript. http://dx.doi.org/10.1016/j.jep.2014.08.017 0378-8741/& 2014 Published by Elsevier Ireland Ltd.
Recently, a number of DPP-4 inhibitor drugs (commonly named “gliptins”) have been clinically approved as orally administered therapies for type 2 diabetes (Green et al., 2006a, 2006b, 2007). Diabetes is now a global pandemic affecting more than 220 million people and although many therapies are available, there is a persistent unmet medical need for alternatives (Li et al., 2005). DPP-4 is a serine-protease broadly distributed throughout the tissues of body and the blood circulation (Flatt et al., 2008). Among its many roles, DPP-4 is known for inactivating two incretin hormones called glucagon-like peptide 1 (GLP-1) and glucose dependent- insulinotropic polypeptide (GIP) (Mentlein et al., 1993; Deacon et al., 1995). These hormones are released from enteroendocrine cells of the intestine and are important stimulators of insulin secretion acting to control blood glucose levels after eating (Green et al., 2004; Baggio and Drucker, 2007).
S. Saleem et al. / Journal of Ethnopharmacology 156 (2014) 26–32
The insulin-releasing effect of GLP-1 and GIP is eliminated by removal of His-Ala and Tyr-Ala dipeptides from the N-terminal end by DPP-4 (Deacon et al., 1995). Reﬂecting their regulatory roles, both incretins have a short plasma half-life (only 1–2 min) because of rapid degradation by DPP-4 (Mentlein et al., 1993; Deacon et al., 1995). DPP-4 inhibitors serve as enhancers of endogenous incretin hormone activity effectively acting to block DPP-4 mediated degradation of GLP-1 and GIP and improving their half-lives. This in turn improves glucose homeostasis while at the same time minimizing the risk of hypoglycemia (Lambeir et al., 2003). Examples of clinically used DPP-4 inhibitors include vildagliptin (Villhauer et al., 2003), sitagliptin (Kim et al., 2005) and saxagliptin (Augeri et al., 2005). It has been suggested that more than 1200 plants have been used to treat diabetes in folk medicine (Marles and Farnsworth, 1995), and at least 136 plants have been indicated to have anti-diabetic effects (Kavishankar et al., 2011). However, relatively few studies have been reported that investigate the DPP-4 inhibitor roles of these medicinal plants (Lendeckel et al., 2002; Bansal et al., 2012; Parmar et al., 2012). The purpose of this investigation was to identify plant and plant-derived compounds that might have potent inhibitory activity. The two plants investigated here (Fagonia cretica L. and Hedera nepalensis K. Koch) were identiﬁed by systematic screening of plant materials, and it is noteworthy that they have been previously reported as natural folk medicines for the treatment of diabetes (Ahmad et al., 2004; Gilani et al., 2007) but until now no scientiﬁc investigation of potential anti-diabetic effects has been reported. Described here is the isolation and identiﬁcation of four DPP-4 inhibitors from Fagonia cretica and one from Hedera nepalensis.
2. Materials and methods 2.1. Plant material The fresh aerial parts of plants were collected in Pakistan in September 2010. Fagonia cretica L. (Synonym Fagonia indica var. schweinfurthii Hadidi) which is the member of family Zygophyllaceae was collected from Mianwali, and Hedera nepalensis K. Koch which is the member of family Araliaceae was collected from Nathia galli, Muree (both are from Punjab Province of Pakistan). Albumbar is the local name of Hedera nepalensis and Dhamasa is local name of Fagonia cretica. Plant species were identiﬁed by Professor Dr. Rizwana Aleem Qureshi, Department of Plant Sciences, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan. Voucher specimens (HMP-461) were deposited in the “Herbarium of Medicinal Plants of Pakistan”, Quaid-iAzam University, Islamabad, Pakistan. 2.2. Extraction and isolation The fresh aerial parts of plants were rinsed with water, shade dried and crushed to dry weight to yield FC (22 kg) and HN (10 kg). Crude plant extracts (FCC (1400 g) and HNC (600 g)) were prepared by maceration in methanol–chloroform (1:1) for 7 days at room temperature. Extracts were ﬁltered, concentrated with a rotary evaporator (45 1C) under vacuum and the resulting crude extract of respective plant was then suspended in water. The water suspension was partitioned three times with n-hexane to obtain n-hexane fraction (FCN (295 g) and HNN (184 g)). The residual aqueous suspension was then partitioned with ethyl acetate (EtOAc) to obtain ethyl acetate (FCE (278 g) and HNE (170 g)) and aqueous fractions (FCA (827 g) and HNA (230 g)). Each of the above fractions were concentrated with a rotary evaporator, applied to silica gel columns and fractionated using elution with n-hexane, EtOAc and methanol (MeOH) in gradients of increasing polarity.
The n-hexane layers of both plants FCN (295 g) and HNN (184 g) were concentrated, further fractionated by silica gel chromatography using an n-hexane, EtOAc and MeOH gradient system with gradually increasing polarity. A total of 72 subfractions of Fagonia cretica (FCN) and 75 subfractions of Hedera nepalensis (HNN) were obtained. Similarly, EtOAc layers underwent similar chromatography to obtain 143 subfractions of Fagonia cretica (FCE) and 21 subfractions of Hedera nepalensis (HNE). Flash column chromatography (Merck silica gel 60, 70-230 mesh) was performed to further fractionate subfractions. From 143 subfractions, one (referred to as FCEE) was loaded (7.78 g) as an adsorbed slurry onto silica at the top of the packed column. Stepwise elution was carried out using a CHCl3:MeOH/100:11:100 as mobile phase. Fractions of 25 mL were collected and concentrated. All eluted fractions were monitored by thin layer chromatography (TLC), visualized under UV light (254 nm) and those possessing similar Rf values were combined. A total of 67 fractions were obtained. Fractions 23–35 were combined and subjected to low pressure liquid chromatography (LPLC, silica gel 60, 230-400 mesh). The column was eluted using CHCl3:MeOH/ 30:1-1:1 as the mobile phase and 17 fractions (25 mL each) were collected. Out of these 17 fractions, fraction numbers 5–12 were combined and chromatographed on reverse-phase low pressure liquid chromatography silica gel column (RP-LPLC, Bondesil-C18, 40 mm) using 100% MeOH as mobile phase. A total of 20 fractions (10 mL each) were collected. Fractions 3–17 were dried to yield compound 1 (13 mg; 0.000059% DW). One subfraction (referred to as FCEK) was loaded on 20 g of silica gel 60 (230-400 mesh) manually packed in glass column. Normal phase LPLC was carried out with gradient elution using a mixture of chloroform and MeOH in the order of increased polarity (CHCl3:MeOH/30:1-1:1). A total of 48 fractions (25 ml each) were collected. Fractions 7–14 were further puriﬁed by reverse phase low pressure liquid chromatography (RP-LPLC, Bondesil-C18, 40 mm) using 100% MeOH as the mobile phase. A total of 18 fractions were collected. Fractions 4–13 were dried to obtain compound 2 (15 mg; 0.000068% DW). Furthermore, FCEN5 (12.17 g) was subjected to normal phase low pressure liquid column chromatography (silica gel 60, 230-400 mesh) using the mobile phase CHCl3:MeOH/50:1-1:1. A total of 73 fractions (25 mL each) were collected. Fractions 16–24 were crystallized and then re-crystallized in MeOH and crystals were washed for three days with MeOH to yield compound 3 (7.5 mg; 0.000034% DW). FCNC6 (9.03 g) was subjected to normal phase low pressure liquid column chromatography (silica gel 60, 230400 mesh) using the mobile phase n-Hex:EA/20:1-1:20. A total of 60 fractions (25 mL each) were collected. Fractions 21–28 were mixed and further puriﬁed by reverse-phase low pressure liquid chromatography (RP-LPLC, Bondesil-C18, 40 mm) using 100% MeOH as the mobile phase. A total of 15 fractions were collected of which fractions 4–12 were dried to obtain compound 4 (15 mg; 0.000068% DW). Out of 75 subfractions of HNN, 7–11 (20 g) were combined on the basis of TLC and subjected to normal phase column chromatography (silica gel 60, 230-400 mesh) using a n-hexane, chloroform and methanol stepwise gradient as the mobile phase and a total of 21 fractions (100 ml each) were obtained. Fractions 12 and 13 (6 g) were combined and submitted to normal phase column chromatography (silica gel 60, 230-400 mesh) and eluted with a gradient system mobile phase of the solvents: n-hexane, chloroform and ethyl acetate. The total number of fractions collected was 42 of 25 ml each. Fractions 3–5 (800 mg) were combined on the basis of TLC and subjected to RP-LPLC (C18) using a mobile phase of MeOH:CHCl3 5:1 and a total of 40 fractions (5 ml each) were obtained. Fractions 16–36 were dried to obtain compound 5 (135 mg; 0.00135% DW). The purity of each compound was estimated by TLC using different solvent systems.
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A combination of mass spectrometry and NMR spectroscopy (Bruker AVANCE 400 MHz NMR) was used to identify candidate DPP-4 inhibitor compounds. The structures of the compounds were conﬁrmed by comparison of their chemical and spectroscopic properties reported in the literature and which are detailed below. 2.3.1. Quinovic acid (1) White amorphous powder; C30H46O5 (m/z 486.68). The 1H NMR spectrum displayed characteristic signals for six methyl groups as singlets at δH 0.69, 0.89, 0.91, 0.82, and as doublets at δH 0.85 and 0.90, respectively. In 1H NMR, characteristic ursane-type methyl signals were observed at δH 0.85 (d, J¼ 6 Hz, H-29) and 0.90 (d, J¼ 6 Hz, H-30). 1H NMR spectrum indicates an oxymethine proton at δH 2.97 (m) and an oleﬁnic proton at δH 5.53 (d, J ¼3.6 Hz), which are also characteristic of an urs-12-en-28oic acid. The 13C NMR spectrum of compound 1 showed signals for 30 carbons: six methyl groups (δC 16.4, 16.5, 17.9, 18.5, 21.5, and 28.7), nine methylenes (δC 18.4, 22.8, 25.4, 27.4, 27.3, 36.3, 36.9, 38.8 and 38.9), seven methines (δC 36.7, 36.9, 46.6, 54.1, 55.7, 77.1 and 128.4) and eight quaternary carbons (δC 38.9, 39.4, 39.5, 47.7, 55.4, 133.0, 176.5 and 178.7). In the 13C NMR spectrum, two peaks at δC 176.3 (C-27) and 178.5 (C-28) indicated that this compound has two carboxylic acids. Compound 1 was characterized as (3β)3-hydroxy-urs-12-ene-27, 28-dioic acid or quinovic acid. This is a known ursane-type triterpene previously reported by Tapondjou et al. (2002) and Fatima et al. (2002). 2.3.2. Quinovic acid-3β-O-β-D-glycopyranoside (2) White crystalline powder; C36H56O10 (m/z 648.82). The 1H NMR spectrum displayed characteristic signals for six methyl groups as singlets at δH 0.86, 1.05, 1.00, 0.92, and as doublets at δH 0.93 and 0.95. The 1H NMR spectrum indicated a methine bearing a hydroxyl group (δH 3.18, m); an oleﬁnic proton (δH 5.64, m). The 13 C NMR spectrum showed signals for 36 carbons: six methyl groups (δC 17.9, 29.3, 17.8, 19.9, 18.9 and 22.3), nine methylenes (δC 20.1, 24.7, 26.5, 27.3, 27.5, 32.0, 38.4, 38.6 and 40.8), seven methines (δC 39.1, 40.9, 48.8, 56.3, 58.1, 91.5 and 131.1), eight quaternary carbons (δC 41.2, 41.5, 38.8, 135.1, 57.7, 50.3, 179.5 and 182.0), and a glucose unit (δC 63.6, 72.4, 76.5, 78.5, 79.1 and 107.4). In the 13C NMR spectra, the two peaks at (δC 179.5, C-27 and 182.0, C-28) indicated that this compound has two carboxylic acids. The presence of a β-D-pyranosyl form of glucose unit linked at the aglycone was suggested from the 1H NMR (δH 4.33, d, J ¼8.6 Hz), and J value of the anomeric proton, and by 13C NMR (δC 107.4, C-10 ) anomeric signals. The ether glycosidation site was shown to be at C-3 of quinovic acid from HSQC experiment, at C-3 (δC 91.5) which was in agreement with an oleanolic acid substituted at C-3 (Pizza and De-Tommasi, 1987). C-12 and C-13 resonated at δC 131.1 and 135.1, respectively. These signals were diagnostic for the presence of an unsubstituted carboxyl group at C-14 in an urs-12-en-27, 28 dioic acid derivative. Hence compound 2 was named as quinovic acid-3β–O-β-D-glycopyranoside. This is a known ursane-type triterpene already reported by Aquino et al. (1989). 2.3.3. Quinovic acid-3β-O-β-D-glucopyranosyl-(28-1)-β-Dglucopyranosyl ester (3) White crystalline powder; C42H66O15 (m/z 810.96). 1H and 13C NMR analyses indicated that it is a triterpenoid-type compound. 1 H NMR spectrum displayed characteristic signals for six methyl groups (δH 0.86, 1.05, 1.00, 0.92, 0.93 and 0.95). Comparison of the 1 H and 13C NMR data of 3 and 2 indicated that they were very
closely related analogs, differing only in the presence of an additional glucose unit. That the β-glucose unit linked at the aglycone with an ether bond in this compound was suggested by detection of 1H NMR (δH 4.33, H-10 , J ¼8.9 Hz) and 13C NMR (δC 107.4, C-10 ) anomeric signals. Furthermore the β-glucose unit being linked to a carboxyl group (C-28) of the aglycone was consistent with the 1H NMR anomeric signal at δH 5.42 (d, J ¼8.9 Hz) and in full agreement with the observed carbon resonance of C-1″ at δC 96.5. The ether glycosidation site was shown to be the C-3 of quinovic acid by the 13 C NMR absorptions of C-3 (δC 91.5), C-2 (27.3) and C-4 (41.2) which were in agreement with a structure of oleanolic acid substituted at C-3 (Pizza and De-Tommasi, 1987). C-12 and C-13 signals resonated at δC 131.7 and 134.1, respectively. The C-28 esteriﬁed carboxyl group appeared at δC 178.8 and the C-27 free carboxylic acid resonated at δC 179.9. The later downﬁeld signal at δH 5.42 (J ¼8.9 Hz) was consistent with the 13C NMR upﬁeld signal of the anomeric ester-linked anomeric carbon at δC 96.5, thus locating one sugar unit at C-28. This compound was characterized as quinovic acid-3β-O-β-D-glucopyranosyl-(28-1)-β-D-glucopyranosyl ester. This is a known ursane-type triterpene already reported by Tapondjou et al. (2002) and Fatima et al. (2002). 2.3.4. Stigmasterol (4) White crystalline (needle like) powder; C29H48O (m/z 412.69). 1 H NMR spectrum showed characteristic signals of six methyl groups i.e. two methyl singlets at δH 0.69 and 1.02; three methyl doublets that appeared at δH 0.93, 0.85 and 0.83 and a methyl
90 80 IC50 µg/ml HNC = 17.2 HNN = 34.4 HNE = 34.2 HNA = 196.46
70 60 Percentage inhibition
2.3. Structure elucidation of the compounds 1–5
20 10 0
IC50 µg/ml FCC = 38.08 FCN = 48.7 FCE = 100.5 FCA = 120.39
60 Percentage inhibition
Fig. 1. Percentage DPP-4 inhibitory activity possessed by crude extracts of (a) Hedera nepalensis (HNC) and (b) Fagonia cretica (FCC) and their fractions n-hexane (N), ethyl acetate (E) or aqueous (A). Data are expressed as mean 7 SD (P o 0.05).
S. Saleem et al. / Journal of Ethnopharmacology 156 (2014) 26–32
triplet at δH 0.87. The 1H NMR has revealed the existence of signals for oleﬁnic proton at δH 5.35 which corresponds to C-6. Angular methyl protons at δH 0.69 and 1.02 corresponds to the C-18 and C-19 methyl protons, respectively. The 13C NMR spectrum indicated the presence of 29 carbon signals: six methyl groups (δC 11.8, 19.8, 21.1, 21.4, 20.1 and 12.0), nine methylenes, eleven methines (one oleﬁnic methine (δC 121.7, C-6) and one oxygenated methine (δC 71.8, C-3)) and three quaternary carbons (δC 140.7, 36.5 and 42.3 at C-5, C-10 and C-13, respectively). The 13C NMR spectrum has shown recognizable signals at δC 141.1 and 121.8 which are assigned to the C-5 and C-6 double bonds respectively. Other oleﬁnic carbons appeared at δC 138.5 and 129.5 (C-22 and C-23, respectively). The signals at δC 11.8 and 19.8 correspond to angular methyl carbon atoms C-18 and C-19, respectively. These spectral data are consistent with the reported literature values of stigmasterol (Habib et al., 2007; Jamal et al., 2009).
2.3.5. Lupeol (5) Lupeol is isolated as a white powder and has the molecular formula of C30H50O (m/z 426.72) as suggested by mass spectral data. The melting point of lupeol is 215–216 1C and the structural analysis showed that it possessed the exact mass of 426.386166. The 1H NMR spectrum showed seven singlets (tertiary methyl) at δ 0.74, 0.78, 0.84, 0.90, 0.93, 1.06, 1.72; and one secondary hydroxyl group as a doublet of doublets at 3.20 ppm. It also showed two oleﬁnic protons at 4.56 and 4.70 ppm representing the exocyclic double bond. The 13C NMR spectrum of the compound showed 30 signals for the terpenoid lupane skeleton which includes a carbon bonded to the hydroxyl group at C-3 position that appeared at 78.4 ppm, while the oleﬁnic carbons of the exocyclic double bond appeared at 151.6 and 108.6 ppm. The lupeol data was consistent
to the reported literature values as reported by Baek et al. (2010) and Chaturvedula and Prakash (2012).
2.4. Assay for DPP-4 inhibition DPP-4 activity was determined ﬂuorometrically using the method of Fujiwara and Tsuru (1978) which measures the amount free AMC (7-amino-4-methyl-coumarin) liberated from the DPP-4 substrate, Gly-Pro-AMC. Assays were conducted in triplicate in 96-well microtiter plates with ﬂuorescence measured at Em 430 nm following excitation at Ex 351 nm using a Tecan Saﬁre desktop ﬂuorometer (Reading, England, UK). Samples were prepared in 50 mM HEPES buffer at pH 7.4. Each well contained 20 ml of porcine DPP-4 enzyme (EMD Millipore, UK) (1 U/ml), 20 ml of test sample and 30 ml of 1 mM AMC substrate. Plates were incubated at 37 1C with gentle agitation for 1 h and then 100 ml of 3 mM acetic acid was added to stop reactions. Final concentrations for plant extracts were 200, 66.6, 22.2 and 7.4 mg/ml. Final concentrations of pure compounds were 100, 33.3, 11.1, 3.7 and 1.2 mM. Berberine (IC50 ¼ 13.3 mM), a previously reported plant compound with DPP-4 inhibitory activity (Al-masri et al., 2009) was used in each experiment as a positive control. IC50 value was calculated, that was deﬁned as the concentration of plant extracts or compounds required to inhibit 50% of DPP-4 activity.
2.5. Statistics Values obtained from experiments were expressed as mean S.E.M. and further analyzed using one-way ANOVA with Tukey's test for comparison and statistical signiﬁcance was accepted for p values r0.05. Analyses were performed using GraphPad Prism software version 5.01.
Fig. 2. Reported structures of isolated plant compounds with DPP-4 inhibitory activity. Compounds (1–4) were isolated from Fagonia cretica and compound (5) was isolated from Hedera nepalensis.
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The practice of traditional medicine has described a wide range of plants and herbs which may be used in the treatment of diabetes mellitus. Such plants can play important roles in providing alternative medicines, adjuncts to existing therapies, preventive or prophylactic agents, or in the discovery of new pharmacological compounds. Indeed, one of the most widely used anti-diabetic drugs, metformin, is based on biguanide compounds isolated from the plant Galega ofﬁcinalis (Goat's rue) (Bailey and Day, 2004). Some bioactive drugs isolated from plants appear to have anti-diabetic activity more efﬁcacious than some hypoglycemic agents used in clinical therapy (Bnouham et al., 2006). Therefore, there is a need to scientiﬁcally investigate and characterize the potential anti-diabetic activities of plant and herbs used in traditional medicine. In recent years, the enzyme DPP-4 has become an important new drug target for diabetes therapy and efforts by the pharmaceutical industry have led to the development of DPP-4 inhibitors with good safety and efﬁcacy proﬁles. During the process of screening plant and herbal extracts for DPP-4 inhibitory activity, we came across two plants (Fagonia cretica and Hedera nepalensis) with potent activity. A highly speciﬁc ﬂuorimetric assay as described above was used to determine DPP-4 activity. This is based on the cleavage of the 7-amino-4-methylcoumarin (AMC) moiety from the C-terminus of the peptide substrate H-Gly-ProAMC which results in increased ﬂuorescence intensity. The DPP-4 inhibitory activity present in crude extracts of Fagonia cretica and Hedera nepalensis appears to be chemically diverse and to be result of multiple compounds within each plant. This is evidenced by the fact that crude extracts from both plants with the highest activity (IC50 values for Hedera nepalensis and Fagonia cretica were 17.20 and 38.08 mg/ml, respectively), and also by the fact that activity was spread across fractions of differing polarity (Fig. 1). For Fagonia cretica, activity was present in hexane, ethyl acetate and aqueous fractions with IC50 values of 48.7, 100.5 and 120.4 mg/ml, respectively. In the case of Hedera nepalensis, activity was also present in hexane, ethyl acetate and aqueous fractions with IC50 values of 34.4, 34.2 and 196.46 mg/ml, respectively. The DPP-4 inhibitory activity observed here in the crude and ethyl acetate extracts can be potentially explained by the ﬂavonoid-rich nature of these extracts. In our previous work we reported the highest ﬂavonoid and phenolic contents in HNC and HNE while HPLC proﬁling of Hedera nepalensis revealed the presence of catechin and caffeic acid in HNA and HNE respectively (Jafri et al., 2014) and rutin and chlorogenic acid in crude extracts of Hedera nepalensis (Inayatullah et al., 2012). These ﬂavonoids have previously been reported to have anti-hyperglycemic activity (Coskun et al., 2005; Jung et al., 2006; Cho et al., 2010) and other ﬂavonoid compounds from plants have been reported to be effective inhibitors of DPP-4 (Bansal et al., 2012; Parmar et al., 2012) and to improve the glycemic status in animal models. Due to their antioxidant properties, ﬂavonoids are continually indicated to possess anti-diabetic potential (Rauter et al., 2010), and growing evidence suggests that DPP-4 inhibition could be a plausible mechanism of action. Bioassay-guided separation of extracts using column chromatography led to the isolation of some of the compounds responsible for DPP-4 inhibition (Fig. 2). From Fagonia cretica we identiﬁed quinovic acid (1), quinovic acid-3β-O-β-D-glycopyranoside (2), quinovic acid-3β-O-β-D-glucopyranosyl-(28-1)-β-D-glucopyranosyl ester (3) and stigmasterol (4). From Hedera nepalensis, one compound, lupeol (5), was isolated from HNN (and was also found to be present in HNE). Lupeol (5) is a previously characterized compound but this work provides the ﬁrst conﬁrmation of its presence in Hedera nepalensis. Of these 5 compounds 1, 2 and 3 have not previously been reported to have DPP-4 inhibitory activity, whereas 4 and 5 have previously been reported to inhibit this enzyme (Micaela et al., 2010; Zhang et al., 2011). Their DPP-4
inhibitory activities were directly compared by performing sigmoidal dose-response curves at concentrations ranging from 1.2 to 100 mM (Fig. 3). This demonstrated that compounds 1, 3 and 5 were the most potent inhibitors (IC50 values: 30.7, 23.5 and 31.6 mM, respectively) followed closely by compound 2 (IC50 value 57.9 mM). Compound 4 appears to be a very weak inhibitor of DPP4 (IC50: 4100 mM) but stigmasterols from other plant have been reported with greater activity than observed here (Zhang et al., 2011). Compounds 1, 2 and 3 are structurally related chemicals. Quinovic acid (1) is a triterpenoid compound and 2 and 3 are its glycoside derivatives. There are very few reported activities of these compounds, although it is known that they are natural inhibitors of the phophosphodiesterase I enzyme in snake venom (Fatima et al., 2002; Mostafa et al., 2006) and that they possess
(1) (2) (3)
% DPP-4 inhibition
3. Results and discussion
IC50 µM 1 = 30.7 2 = 57.9 3 = 23.5
log concentration [M]
% DPP-4 inhibition
IC50 µM 5= 31.6 0 -6
log concentration [M] Fig. 3. Percentage DPP-4 inhibitory activity possessed by compounds isolated from (a) Fagonia cretica (1–3) and (b) Hedera nepalensis (5). Data are mean 7SD and curves are ﬁtted by sigmoidal dose-response analysis. Compound (4) had weak inhibitory activity and is not shown.
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anti-parasitic activity (Di Giorgio et al., 2006). This is the ﬁrst time that quinovic acid related compounds have been reported as having anti-diabetic or DPP-4 inhibiting activity.
4. Conclusion In conclusion, Fagonia cretica and Hedera nepalensis are plants that have not previously been investigated for anti-diabetic activity and we report them to be rich sources of DPP-4 inhibitors. We have identiﬁed 5 compounds that contribute to this inhibitory activity, 3 of which have not previously been reported as DPP-4 inhibitors. Quinovic acid and its derivatives are potentially important naturally occurring DPP-4 inhibitors and their efﬁcacy in animal models of diabetes should now be investigated. Finally, more studies of Fagonia cretica and Hedera nepalensis are warranted including screening for other anti-diabetic activities and the efﬁcacy of these medicinal plants for in patients with type 2 diabetes.
Acknowledgments We thank Dr. John M. Pezzuto, College of Pharmacy for start-up funding, and for the provision of the NMR spectroscopic facilities used in this study. We are also thankful to Higher Education Commission of Pakistan (17-5-7(Bm7-088)/HEC/Sch-Ind/2011) for ﬁnancial support.
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