Randomized double-blinded pilot clinical study of the antidiabetic activity of Balanites aegyptiaca and UPLC-ESI-MS/MS identification of its metabolites

Figures & data

Figure 1. CONSORT 2010 flow diagram.

Table 1. The composition of B. aegyptiaca hard gelatine capsules.

Active ingredients	mg/capsule	Function	Textbook/Monograph
Balanites extract (BE) standardized to contain not less than 8.63 ± 0.2 μg/ml of stigmaserol-3-O-β-D-glucoside. Part used: pericarps Extraction solvent: 70 % ethanol Ratio of herbal drug to extract: 2:1	400		In-house specification
Inactive ingredients
Avicel PH 101 (microcrystalline cellulose)	30	Diluent	USP37 NF 32
Aerosil 200 (colloidal silicon dioxide)	75	Anticaking agent	USP37 NF 32
Magnesium stearate	5	USP37 NF 32	Lubricant
Average filling weight	510

Form, hard gelatine capsule; average filling weight, 510 mg ±10 %.

Figure 2. HPLC chromatogram of the 70% ethanol extract of B. aegyptiaca (BE) at 213 nm. The inset shows the standard stigmasterol-3 O-β-D-glucopyranoside at R_t = 14.6.min.

Figure 3. A representative UPLC-negative ionization MS trace of the 70% ethanol extract of B. aegyptiaca (BE).

Table 2. Peak assignments of metabolites in B. aegyptiaca extract (BE) using UPLC/ESI-MS/MS in negative ionization mode.

Peak No.	Rt (min)	[M−H]^−	MS^2	Metabolites
1	1.694	1209	1077, 1063, 915, 769, 589 and 431	26-O-glucopyranosyl-22-O-methylfurost-5-ene-3,26-diol-3-O-pentosyl-glucopyranosyl-rhamnopyranosyl-glucopyranoside
2	9.527	1063	917, 901, 755, 431 and 413	26-O-glucopyranosyl-3,22,26-trihydroxyfurost-5-ene-3-O-glucopyranosyl-rhamnopyranosyl-glucopyranoside
3	10.39	1195	917, 901, 977 and 413	26-O-glucopyranosyl-3,22,26-trihydroxyfurost-5-ene-3-O-glucopyranosyl-rhamnopyranosyl-glucopyranoside + xylose
4	10.50	1077	915, 769, 589 and 431	26-O-glucopyranosyl-22-O-methylfurost-5-ene-3,26-diol-3-O-glucopyranosyl-rhamnopyranosyl-glucopyranoside
5	10.75	1045	901, 883, 737, 593, 431 and 413	Diosgenin glycoside having two hexoses and two deoxyhexoses
6	11.134	465	303	Flavanonol-O-glucoside
7	12.93	609	301	Quercetin-3-O-rhamnoglucoside (rutin)
8	13.74	623	315	Isorhamnetin-O-rhamnoglucoside
9	15.21	477	315	Isorhamnetin-O-glucoside
10	17.10	301		Quercetin
11	18.77	574	412	Stigmasterol-3-O-glucoside
12	19.93	315		Isorhamnetin
13	20.62	341		Syringic acid

Table 3. Baseline parameters of the participants.

	Placebo group	BE-treated group
Baseline characteristics
Males [n (%)]	8 (53.3 %)	9 (60.0 %)
Females [n (%)]	7 (46.7 %)	6 (40.0 %)
Age (years)	54.2 ± 5.2	54.9 ± 7.1
BMI (kg/m^2)	28 ± 5	29.1 ± 3
Period since diagnosis	5.4 ± 3.2	6.8 ± 1.2
BP systolic (mm Hg)	128.8 ± 11.2	125 ± 13
BP diastolic (mm Hg)	79.8 ± 7.1	80.2 ± 10.5
HbA1c	6.7 ± 0.2	6.8 ± 2.1
No. of participants on medications
Oral antidiabetic medication	12	11
Oral antihypertensive medication	5	4
Cholesterol reducing medication	3	2

Data are mean ± SD. There were no significant differences between the groups.

Table 4. Effect of 8-week treatment using BE capsules on the metabolic parameters.

	Placebo (n = 15)		Treated (n = 16)
Parameter	Baseline	8 weeks	Baseline	8 weeks	p^a	p^b
Postprandial plasma glucose (mmol/l)	15.2 ± 4.1	15.6 ± 3.7	16.0 ± 2.4	11.7 ± 1.01	ns	0.003
Fasting plasma glucose (mmol/l)	8.2 ± 0.6	8.5 ± 1.1	8.7 ± 1.9	7.8 ± 0.9	0.05	0.04
Plasma – triglyceride (mg/dl)	181.7 ± 2.9	179.1 ± 8.5	172.4 ± 4.3	156.8 ± 5.1	0.05	0.003
Plasma – total cholesterol (mg/dl)	216.9 ± 11	217.1 ± 8.1	211.5 ± 6.8	184.5 ± 3.9	0.03	0.002
Plasma – HDL (mg/dl)	31.9 ± 8.6	32.8 ± 7.9	32.7 ± 2.9	46.6 ± 7.0	0.03	0.004
Plasma – LDL (mg/dl)	144.5 ± 5.2	140.1 ± 6.8	138.6 ± 11.1	109.0 ± 5.4	ns	0.003
LDL:HDL	4.5	4.27	4.2	2.33
Serum insulin (mU/l)	11 ± 2.3	11 ± 4.6	10 ± 1.2	10 ± 1.7	ns	ns
Serum C-peptide (pmol/l)	476 ± 58.9	482 ± 63.1	493 ± 71.3	495 ± 69.2	ns	ns
Plasma – ALT (U/l)	39.1 ± 3.1	36.9 ± 4.6	36.4 ± 8.7	20.87 ± 8.7	0.05	0.004
Plasma – AST (U/l)	27.5 ± 9.0	28.1 ± 3.2	31.4 ± 10.1	17.8 ± 8.7	0.03	0.004
Plasma creatinine (μ mol/l)	0.71 ± 0.06	0.69 ± 0.01	0.88 ± 3.4	0.90 ± 8.7	0.01	ns
Plasma urea (mg/l)	18.3 ± 1.7	17.5 ± 1.8	15.5 ± 2.6	16.93 ± 8.7	0.01	ns

Data are mean ± standard deviation.

ns: not statistically significant.

p^a: within-group statistical significance calculated for the data; p^b: statistical significance from the placebo group.

Values of p < 0.05 were considered significant.