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Anti-diabetic molecules from Cycas pectinata Griff. traditionally used by the Maiba-Maibi.

ABSTRACT

Bioactivity guided chemical investigation on active anti-diabetic constituents of the fruits of Cycas pectinata Griff. (FCP) characterized EAFr-5 as the most potent sub fraction which significantly reduced the blood glucose level to normal in STZ induced diabetic rats. It was shown to contain the biflavonoids amentoflavone (1) and 2,3-dihydroamentoflavone (2) which exhibited significantly high inhibitory potency against [alpha]-glucosidase ([IC.sub.50] 8.09 [+ or -] 0.023 and 9.77 [+ or -] 0.032 [micro]M, respectively) and [alpha]-amylase ([IC.sub.50] 73.6 [+ or -] 0.48 and 39.69 [+ or -] 0.39 [micro]M, respectively). This is the first report of bioactivity guided isolation of anti-diabetic constituents from the traditionally used fruits of Cycas pectinata Griff.

Keywords:

Diabetes

[alpha]-Glucosidase

[alpha]-Amylase

Cycas pectinata

Maiba-Maibi

Introduction

Diabetes mellitus (DM) is a group of metabolic disorders. There has been an explosion in the number of people suffering from the disease. Worldwide 371 million people are affected and by 2035 the number is predicted to go up to almost 600 million (Zimmet et al. 2014). Asia has emerged as a "diabetic-hub". In India 61.3 million people live with diabetes. More than 90% people live with Type 2 DM (Chen et al. 2012). To prevent the progression of Type 2 DM and for the treatment of prediabetic conditions, one of the most effective therapeutic approaches is the reduction of post-prandial hyperglycemia by inhibiting the carbohydrate hydrolyzing enzymes [alpha]-glucosidase and a-amylase. Inhibition of these enzymes reduces the rate of digestion of carbohydrates, resulting in less absorption of glucose (Israili 2011).

Traditional healthcare practice of Maiba-Maibi (Male and Female healers respectively) is one of the indigenous healthcare systems of India (Devi et al. 2005). Literature on traditional medicine worldwide has abundant mention of herbal extracts having positive effect on management and treatment of diabetes (Kar et al. 2003; Mata et al. 2013). Through an extensive interaction with the local healers quite a few plant species were identified and were ranked according to use in treatment of diabetes; fruits of Cycas pectinata Griff. (FCP) occupied the top rank. It is a wild ornamental plant from the family Cycadaceae. The objective of this was to isolate and characterize the constituent(s) of FCP responsible for anti-diabetic activity and having potential to function as biomarker(s).

Materials and methods

Instruments and reagents

NMR spectra were recorded on a Bruker Avance 500 MHz instrument with TMS as an internal standard. Chemical shifts are expressed in [delta] values. Agilent LCMS was used with 1260 infinity pump and 6400 MS detector. Separations were carried out with Merck silica gel (100-200 mesh size) for column chromatography and Waters 1525 HPLC with UV detector (Waters 2487) fitted with either an analytical column [Phenomenex Luna 5[micro] C18(2), 250 x 4.6 mm] or a semi-preparative column [Phenomenex Luna 5[micro] C18(2), 250 x 10.00 mm], TLC was done on Merck pre-coated plates with silica gel 60 F254. [alpha]-Glucosidase (maltase, EC 3.2.1.20) and p-nitrophenyl-[alpha]- D-glucopyranoside were purchased from Sisco Research Laboratory (India). [alpha]-Amylase (porcine pancreas, EC 3.2.1.1), starch, and DNS were purchased from Sigma. Streptozotocin (STZ) was procured from Ozone International, Mumbai, India.

Plant materials

Fruits of C. pectinata Griff. were collected from Kakching, Manipur, India in January 2013. The identification was done by Dr. B. Thongam, Scientist, IBSD, Imphal and a voucher specimen (No. IBSD/M-193) was deposited in the IBSD Herbarium.

Preparation of enzyme and sample solutions

A stock solution of 2 U/ml of [alpha]-glucosidase enzyme in 0.1 M phosphate buffer, pH 6.8 was prepared and diluted to 0.5 U/ml with the same buffer at the time of assay. A working solution of 4 U/ml of [alpha]-amylase enzyme was prepared by dissolving the enzyme in ice cold water. The extract, fractions and compounds were dissolved in DMSO and further diluted with buffer to obtain the desired test concentrations. The final DMSO concentration was maintained below 1% (v/v).

[alpha]-Glucosidase and [alpha]-amylase inhibitory assay

[alpha]-Glucosidase and [alpha]-amylase inhibitory activities were determined spectrophotometrically in 96-well microplate reader according to a pre-incubation based reported method (Kumar et al. 2013). Acarbose was used as positive control and the uninhibited enzyme was taken as negative control (DMSO control). The assay was performed in three independent experiments.

Extraction and isolation

The dried powder of FCP (1.7 kg) was extracted at room temperature with MeOH thrice at 24 h intervals. The extract was concentrated under reduced pressure to yield 85 g of crude product. The product was suspended in water (200 ml) and fractionated successively with ethyl acetate (400 ml) and n-butanol (400 ml) to yield 7.3 g of ethyl acetate (EAFr), 5.7 g of butanol and 60 g of water fractions. The ethyl acetate fraction, the most active one, was chromatographed over a silica gel column and categorized into six sub-fractions (EAFr-1, EAFr-2, EAFr-3, EAFr-4, EAFr-5 and EAFr-6). The most active sub-fraction was EAFr-5, which was eluted with 10% methanol in chloroform and was further purified by semi-preparative HPLC to yield two amorphous solids. These were characterized as compounds 1 and 2 (Fig. 1).

Induction of experimental diabetes and hyperglycemic assay

Diabetes mellitus was induced in Wistar strain albino rats (150-250 g) by injecting streptozotocin (STZ) at the dosage of 65 mg/kg body weight (BW) in saline (2% solution, I.P.) after fasting the animals overnight. Diabetic condition was confirmed 48 h after STZ injection. The sample at the dose of 150 mg/kg BW/day was administered p.o. as suspension in distilled water. Ethical clearance was obtained from the Institutional Animals Ethical Committee (Approval No. IBSD/IAEC/lnst./Ph.cology/1) prior to the experiments.

Rats

Animals were randomly divided into three groups of six rats each. Group I, normal control rats, were administered purified water (p.o.); Group II, diabetic control rats, received purified water (p.o.); and Group III, diabetic rats, were administered the test sample (150 mg/kg, p.o.). Blood samples were collected at different time intervals on 1st (0 h), 7th and 14th day from tail vein for estimation of blood glucose level.

Statistical analysis

Statistical analyses were performed by one-way ANOVA supplemented with Tukey-Kramer multiple comparisons test. A P-value < 0.05 was considered to indicate statistical significance.

Results and discussion

Bioactivity guided extraction and isolation of compounds

The search for anti-diabetic small molecules from FCP was guided through [alpha]-glucosidase inhibition assay. The crude methanolic extract of Cycas pectinata exhibited significant inhibitory potency against [alpha]-glucosidase with [IC.sub.50] 65.88 [+ or -] 0.079 [micro]g/ml (Table 1). Successive fractionation with ethyl acetate and n-butanol followed by activity tests against [alpha]-glucosidase showed the ethyl acetate fraction (EAFr) to be the most active ([IC.sub.50] 42.82 [+ or -] 0.039 [micro]g/ml) one; the n-butanol fraction was only weakly active. The ethyl acetate sub-fraction 5 proved to be the most active one with [IC.sub.50] 15.16 [+ or -] 0.001 [micro]g/ml. From this fraction compounds 1 and 2 were isolated. These exhibited significant inhibitory potency against a-glucosidase with [IC.sub.50] 8.09 [+ or -] 0.023 and 9.77 [+ or -] 0.032 [micro]M, respectively (Table 1) and against [alpha]-amylase with [IC.sub.50] 73.60 [+ or -] 0.48 and 39.69 [+ or -] 0.39 [micro]M, respectively (supplementary Table 1). This suggests that they are most likely the active constituents responsible for the effects of the FCP for management and treatment of Type 2 diabetes and can serve as biomarkers.

Characterization of biomarkers

The LCMS of compound 1 gave a pseudo-molecular ion peak at m/z = 539.46 [[M+H].sup.+] corresponding to the molecular formula [C.sub.30][H.sub.18][O.sub.10] which was also in agreement with [sup.1]H, [sup.13]C NMR and DEPT data. It was finally characterized by spectroscopic analysis as amentoflavone having the biflavonoid linkage between C3' of one unit and C8" of the other (He et al. 1996; Chien et al. 2004).

Compound 2 was also obtained from the same sub-fraction (EAFr-5). Although it was eluted as a single product even in HPLC, its identification as a diastereomeric mixture (epimeric at C2) of 2,3-dihydroamentoflavone (2) became apparent from spectroscopic analysis (Ohmoto et al. 1983; Markham et al. 1987). The spectral patterns of 2 were very close to those of 1, the major difference being in the observation of closely lying peaks for several of the nuclei suggesting the presence of an intimate mixture of isomers. The molecular formula was established by HRMS with protonated molecular ion peak at m/z 541.1141 (Calc. 541.1135). The [sup.1]H, [sup.13]C, [sup.1]H-[sup.1]H COSY and HMBC values are in good agreement with those reported in the literature (Ohmoto et al. 1983).

Hypoglycaemic activity of EAFr-5

The effects of EAFr-5 on STZ induced diabetes in rats at different time intervals are shown in Table 2. At the dose of 150 mg/kg, it caused significant reduction in the blood glucose levels when compared with the STZ treated group. When administered 48 h after STZ, EAFr-5 reduced blood glucose concentration of diabetic rats to normal within 14 days.

Reports on the phytochemical investigation of different species of Cycas are scarce in the literature. Nguyen et al. (2012) reported the screening of methanol extract of 38 Vietnamese medicinal plants including Cycas pectinata Griff, and mentioned the [IC.sub.50] value of 138.2 [micro]g/ml for its [alpha]-glucosidase inhibitory activity. But the isolation of active constituents was not reported. The present communication is the first report of bioactivity guided isolation of anti-diabetic constituents from C. pectinata. It identifies the biflavonoids amentoflavone (1) and 2,3-dihydroamentoflavone (2) as the active constituents. These can therefore be regarded as potential biomarkers to develop promising therapeutic agents against diabetes with higher affinity and better selectivity. Biflavonoids have already been identified as potential therapeutic agents for treatment and management of diabetes and the hypoglycaemic effect of a number of them has already been confirmed (Qi et al. 2010). Besides, amentofiavone induced cell death in HER2-positive breast cancers (Lee et al. 2013) and in MCF-7 human breast cancer cells (Pei et al. 2012). Zheng et al. (2011) showed the protective effect of 2,3-dihydroamentoflavone against anoxia in the anoxic PC12 cells assay.

Conclusion

Bioactivity guided isolation and characterization of traditionally used FCP led to the identification of amentoflavone and 2,3-dihydroamentoflavone based on [sup.1]H and [sup.13]C NMR and LCMS spectral data. The ethyl acetate sub-fraction 5 and isolated compounds 1 and 2 were most effective in inhibiting both the enzymes [alpha]-glucosidase and [alpha]-amylase. Thus it can be concluded that amentoflavone and 2,3-dihydroamentoflavone are potential anti-diabetic constituents of CPC for management of post-prandial hyperglycemia and can serve as biomarkers.

Conflict of interest

The authors declare that they have no competing interests.

Abbreviations: DM, diabetes mellitus: DMSO, dimethyl sulfoxide; DNS, 3,5-dinitrosalicylic acid; PNPG, p-nitrophenyl-[alpha]-D-glucopyranoside; STZ, streptozotocin; TLC, thin layer chromatography; HPLC, high performance liquid chromatography; LCMS, liquid chromatography mass spectrometry; NMR, nuclear magnetic resonance.

ARTICLE INFO

Article history: Received 29 April 2014

Revised 17 September 2014

Accepted 26 October 2014

Acknowledgements

The authors are thankful to IBSD Imphal and DBT, Govt. of India for financial assistance. The authors are also thankful to Dr. Basudeb Achari and Dr. Suresh Singh for their valuable suggestions.

Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.phymed.2014.10.007.

References

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Chien, S.C., Liu, H.K., Kou, Y.H., 2004. Two new compounds from the leaves of Calocedrus microtepic var. formosana. Chem. Pharm. Bull. 52,762-763.

Devi, A.K., Khan, M.L., Tripathi, R.S., 2005. Ethnomedicinal plants in the sacred groves of Manipur. Indian J. Tradit. Knowl. 4,21-32.

He, K., Timmermann, B.N., Aladesanmi, A.J., Zeng, L, 1996. A biflavonoid from Dysoxylum lenticellare Gillespie. Phytochemistry 42, 1199-1201.

Israili, Z.H., 2011. Advances in the treatment of Type 2 diabetes mellitus. Am. J. Ther. 18, 117-152.

Kar, A., Choudhary, B.K., Bandyopadhyay, N.G., 2003. Comparative evaluation of hypoglycaemic activity of some Indian medicinal plants in alloxan diabetic rats. J. Ethnopharmacol. 84, 105-108.

Kumar, D., Gupta, N., Ghosh, R., Gaonkar, R.H., Pal, B.C., 2013. [alpha]-Glucosidase and [alpha]-amylase inhibitory constituent of Carex baccans: bio-assay guided isolation and quantification by validated RP-HPLC-DAD. J. Fund. Foods 5, 211-218.

Lee, J.S., Sul, J.Y., Park, J.B., Lee, M.S., Cha, E.Y., Song, I.S., Kim, J.R., Chang, E.S., 2013. Fatty acid synthase inhibition by amentoflavone suppresses HER2/neu (erbB2) oncogene in SKBR3 human breast cancer cells. Phytother. Res. 27, 13-20.

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Nguyen, M.T.T., Nguyen, N.T., Nguyen, H.X., Thuy, N.N., Min, B.S., 2012. Screening of [alpha]-glucosidase inhibitory activity of Vietnamese medicinal plants: isolation of active principles from Oroxylum indicum. Nat. Prod. Sci. 18,47-51.

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Qi, L.-W., Liu, E.-H., Chu, C., Peng, Y.-B., Cai, H.-X., Li, P., 2010. Antidiabetic agents from natural products--an update from 2004 to 2009. Curr. Top. Med. Chem. 10, 434-457.

Zheng, J.-X., Zheng, Y., Zhi, H., Dai, H., Wang, N.-L, Fang, Y.-X., Du, Z.-Y., Zhang, K., Li, M.-M., Wu, L-Y., Fan, M., 2011. New 3',8"-Linked Biflavonoids from Selaginella uncinata Displaying Protective Effect against Anoxia. Molecules 16, 6206-6214.

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S. Laishram, Y. Sheikh, D.S. Moirangthem, L. Deb, B.C. Pal, N.C. Talukdar, J.C. Borah

Natural Product Chemistry & Pharmacology Programme, Institute of Bioresources & Sustainable Development, Takyelpat, Imphal 795001, Manipur, India

* Corresponding author. Tel.:+91 385 2051278; fax:+91 385 2051277.

E-mail address: jcborah03@yahoo.com, jagatborah.ibsd@nic.in (J.C. Borah).

http://dx.doi.org/10.1016/j.phymed.2014.10.007

Table 1
Half maximum inhibitory concentration
([IC.sub.50]) of the tested samples on the
enzyme [alpha]-glucosidase.

Treatment      [Ic.sub.50] ([micro]g/ml)

MeOH extract     65.88 [+ or -] 0.079a
EAFr             42.82 [+ or -] 0.039b
EAFr-5           15.16 [+ or -] 0.001c
Compound 1        4.37 [+ or -] 0.0Id
                 (8.09 [+ or -] 0.023)
Compound 2        5.27 [+ or -] 0.014d
                 (9.77 [+ or -] 0.032)
Acarbose        348.85 [+ or -] 16.47e
               (540.36 [+ or -] 25.51)

Each value represents the mean [+ or -] SD
(n = 3). Data in parentheses are
expressed in [micro]M. Values with different
letters are significantly different
(P < 0.05-0.001). Data were analysed by
one way ANOVA analysis supplemented
with Tukey-Kramer multiple comparisons test.

Table 2
Hyperglycemic activity of EAFr-5 on streptozotocin (STZ) induced
diabetes in rats at weekly interval (study has been done on 1st (0
h), 7th and 14th day of treatment after 48 h of STZ injection).

Group                                           Blood glucose (mg/dl)

                                                1st day (Oh)

Group I (control)                               101.33 [+ or -] 5.11a
Group II (diabetic control) administered STZ    433.50 [+ or -] 26.83b
  (65 mg/kg, i.p.)
Group III (administered EAFr-5,150 mg/kg,       448.16 [+ or -] 28.33b
  p.o., and STZ, 65 mg/kg, i.p.)

Group                                           Blood glucose (mg/dl)

                                                7th day

Group I (control)                               101.33 [+ or -] 4.9a
Group II (diabetic control) administered STZ    434.33 [+ or -] 36.12b
  (65 mg/kg, i.p.)
Group III (administered EAFr-5,150 mg/kg,       133.83 [+ or -] 10.43a
  p.o., and STZ, 65 mg/kg, i.p.)

Group                                           Blood glucose (mg/dl)

                                                14th day

Group I (control)                               101.33 [+ or -] 4.77a
Group II (diabetic control) administered STZ    454.00 [+ or -] 37.88b
  (65 mg/kg, i.p.)
Group III (administered EAFr-5,150 mg/kg,        92.33 [+ or -] 5.9a
  p.o., and STZ, 65 mg/kg, i.p.)

Each value is the mean [+ or -] SEM for six rats in each group. Means
marked with different letters, within each column, are significantly
different (P < 0.05-0.001). Analysed by one-way ANOVA supplemented
with Tukey-Kramer multiple comparisons test.
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Title Annotation:Short communication
Author:Laishram, S.; Sheikh, Y.; Moirangthem, D.S.; Deb, L.; Pal, B.C.; Talukdar, N.C.; Borah, J.C.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9INDI
Date:Jan 15, 2015
Words:2836
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