Characterization of nimbidiol as a potent intestinal disaccharidase and glucoamylase inhibitor present in Azadirachta indica (neem) useful for the treatment of diabetes

Figures & data

Table 1.  Inhibitory potency of aqueous and methanolic extract of Azadirachta indica towards intestinal and fungal glucosidases.

	IC50 values (µg/mL) of various extracts of Azadirachta indica
	Methanolic extract of root	Methanolic extract of stem-bark	Aqueous root extract	Aqueous stem-bark extract
Intestinal maltase	52 ± 2	57 ± 2	70 ± 2.5	75 ± 1.5
Intestinal sucrase	28 ± 2.5	34 ± 2.2	40 ± 2.5	42 ± 1.5
Intestinal maltase-glucoamylase (maltotetraose as substrate)	7 ± 1.5	12.5 ± 1.5	14 ± 1.5	16 ± 1.5
Intestinal maltase-glucoamylase (maltotriose as substrate)	8.5 ± 1.0	14.5 ± 1.25	15.5 ± 1.25	17.5 ± 1.0
Intestinal isomaltase	6 ± 0.75	10.5 ± 1.22	11.5 ± 1.5	14.5 ± 1.25
Intestinal trehalase	83 ± 2.5	85 ± 1.5	88 ± 1.75	89 ± 1.0
Intestinal lactase	68 ± 2.5	72 ± 1.0	81 ± 1.0	85 ± 1.0
Fungal maltase	58 ± 3.5	63 ± 2	76 ± 2	80 ± 1.5
Fungal sucrase	29 ± 2.5	34 ± 1.5	55 ± 1	60 ± 1.0
Pancreatic amylase (starch as substrate)	>150	>150	ND	ND
Pancratic amylase (dextrin as substrate)	>100	>100	ND	ND

Enzyme assay methodologies are mentioned in the text. Substrate concentration (for each) used was 1 mg/mL. Mean values shown here are the averages of triplicate similar sets of data obtained separately.

Figure 1.  Structure of various α-glucosidase inhibitors.

Table 2.  Comparison of IC50 values of nimbidiol and other glucosidase inhibitors.

IC50 value (µM)
	Nimbidiol	Acarbose^Citation26,Citation30	Voglibose^Citation30	Salacinol^Citation30	Kotalanol^Citation30	Mangiferin^Citation30	1-deoxynojirimycin^Citation40
Intestinal maltase	12 ± 1.23	2.0	1.19	9.58	6.85	>710	1.287
Intestinal sucrase	6.75 ± 0.80	1.7	0.22	2.51	1.37	206	0.091
Intestinal isomaltase	0.85 ± 0.035	154.89	2.09	1.76	4.476	511.4	–
Intestinal lactase	20 ± 1.33	Negligible	–	–	–	–	–
Intestinal trehalase	30 ± 1.75	>600	711.26	>1 mM	>942.4	–
Intestinal glucoamylase (maltotriose as substrates)	1.56 ± 0.24	–	–	–	–	–	–
(maltotetraose as substrates)	1.35 ± 0.12
Fungal maltase (A. niger)	14.23 ± 1.40	–	–	–	–	–	–
Fungal sucrase (Baker’s yeast)	10.85 ± 0.75	–	–	–	–	–

Mean values presented here (for nimbidiol) were obtained from three similar sets of experiments.

Table 3.  Comparison of inhibitor constants of nimbidiol and other glucosidase inhibitors.

Ki/Ki′ values (µM)
	Nimbidiol	Acarbose^Citation13,Citation30	Voglibose^Citation30	Salacinol^Citation30	Kotalanol^Citation30	Mangiferin^Citation30	1-deoxynojirimycin^Citation13,Citation26
Intestinal maltase	1.22 ± 0.344/3.65 ± 0.48	0.18/-	0.11/-	0.95/-	0.54/-	–	–
Intestinal sucrase	0.7 ± 0.12/1.44 ± 0.65	0.57/-	0.067/-	0.95/-	0.42/-	130.2/-	8.6 × 10^−8 M/-
Intestinal isomaltase	0.071 ± 0.008/0.19 ± 0.05	116.17/-	1.533/-	1.4/-	4.24/-	165.73/-	–
Intestinal lactase	Not determined	–	–	–	–	–	67/-
Intestinal trehalase	Not determined	–	–		–	–	–
Intestinal glucoamylase (maltotriose as substrate)	0.11 ± 0.055/0.41 ± 0.13	0.4/- (malto-oligosaccharide as substrate)	–	–	–	–	0.3/- (malto-oligosaccharide as substrate)
(maltotetraose as substrate)	0.08 ± 0.01/0.25 ± 0.11
Fungal maltase (A. niger)	1.8 ± 0.20/4.25 ± 0.54	–	–	–	–	–	–
Fungal sucrase (Baker’s yeast)	1.068 ± 0.080/2.66 ± 0.78	–	–	–	–	–	–

Enzyme inhibition assay methodologies for nimbidiol are mentioned in the text. Ki and Ki′ values were determined from Dixon plots (1/V vs I) and S/V vs I plots (Figures 2–5). Mean values shown here were obtained from three similar sets of experiments.

Figure 2.  Determination of Ki of nimbidiol on maltase and sucrase. Dixon plot (1/V vs I) for inhibition of nimbidiol on activities of intestinal maltase (a), intestinal sucrase (b), fungal maltase (c) and fungal sucrase (d). Nimbidiol (0.5–8 µM) was added against different concentrations (0.5–6 mM) of maltose and sucrose [S]. Enzyme activities were determined as mentioned in the text.

Figure 3.  Determination of Ki′ of nimbidiol on maltase and sucrase. S/V vs I plot for inhibition of nimbidiol on activities of intestinal maltase (a), intestinal sucrase (b), fungal maltase (c) and fungal sucrase (d). nimbidiol (0.5–8 µM) was added against different concentrations (0.5–6 mM) of maltose and sucrose [S]. Enzyme activities were determined as mentioned in the text.

Figure 4.  Determination of Ki and Ki′ of nimbidiol on intestinal glucoamylase. Dixon plot (1/V vs I) for inhibition of nimbidiol on activities of intestinal glucoamylase using maltotetraose as substrate (a), and maltotriose as substrate (b). S/V vs I plot for inhibition of nimbidiol on intestinal glucoamylase using maltotetraose as substrate (c) and maltotriose as substrate (d). Nimbidiol (0.05–0.8 µM) was added against different concentrations (0.5–6 mM) of maltotetraose and maltotriose. [S]. Enzyme activities were determined as mentioned in the text.

Figure 5.  Determination of Ki and Ki′ of nimbidiol on intestinal isomaltase. Dixon plot (1/V vs I) for inhibition of nimbidiol on activities of intestinal isomaltase (a) and S/V vs I plot for inhibition of nimbidiol on intestinal isomaltase (b). Nimbidiol (0.05–0.8 µM) was added against different concentrations (0.5–6 mM) of isomaltose [S]. Enzyme activities were determined as mentioned in the text.

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