A validated HPLC method for standardization of the most active fraction of the antihyperglycemic drug Cleome droserifolia using bioactive markers

The aqueous and ethanolic extracts of the aerial parts of Cleome droserifolia (Forssk.) Del. were assessed for their antihyperglycemic effects in male albino rats at the same dose level of the biguanide metformin (150mg/kg body weight). Diabetes was induced intraperitoneally with a single dose of alloxan (150mg/kg body weight) [1].The blood glucose level was monitored after 2 and 4 weeks from zero time (Table 1). The four sub-fractions (n-hexane, chloroform, ethyl acetate and n-butanol) of the more active aqueous extract were tested at the same dose level. A validated RP-HPLC method for standardization of the most active ethyl acetate fraction (70% as potent as metformin after 4 weeks of oral administration) was developed. Three flavonoid glycosides; Isorhamnetin-3-O-β-D-glucoside (F1), quercetin-3'-methoxy-3-O-(4''-acetylrhamnoside)-7-O-α-rhamnoside (F2) and kaempferol-4'-methoxy-3,7-dirhamnoside (F3) (were isolated from the ethyl acetate fraction and proved to increase basal glucose uptake, 2-folds as insulin, in C2C12 skeletal muscle cells [2]) were used for the standardization (Fig.1). The parameters of validation of the method (linearity, repeatability, reproducibility, ruggedness, robustness and accuracy) were evaluated. A standard calibration curve, established for the major compound F3at a concentration range of 44–174µg/ml, showed good linearity with a correlation co-efficient (R2) of 0.998. The recovery of the method was 100.5%. A high degree of repeatability and reproducibility (relative standard deviation values less than 5%) were also achieved.

Fig.1: HPLC chromatogram of the ethyl acetate fractionHPLC chromatogram of the ethyl acetate fraction showing the flavonoid glycosides F1, F2 and F3. The method involved the use of a Lichrosphere 100 RP-18 column with a guard column. Gradient elution was from 10 to 75% v/v acetonitrile/0.3% orthophosphoric acid in water, in 25min, at a flow rate of 1.0ml/min and UV detection at 325nm.

Table 1. Antihyperglycemic effect of the different extracts of Cleome droserifolia

Time	Zero	2 weeks	2 weeks	4 weeks	4 weeks
Group	M±S.E.	M±S.E.	% of change	M±S.E.	% of change
Diabetic rats (Db) non treated	243.7±8.2	256.8±9.6	-	256.8±9.6	-
Db + metformin	257.3±11.4	129.8±4.3*	49.5	81.9±3.2*	68.2
Db + ethanolic extract	249.2±8.2	216.2±7.6*	13.2	153.2±4.6*	38.5
Db + aqueous extract	256.8±10.1	173.2±6.2*	32.5	141.9±5.5*	44.7
Db + n-hexane fraction	246.9±7.8	214.3±8.6*	13.2	198.6±7.1*	19.5
Db + chloroform fraction	251.9±8.6	186.8±7.4*	25.8	138.9±5.8*	44.8
Db + ethyl acetate fraction	258.4±7.1	187.4±6.3*	27.5	135.3±4.1*	47.6
Db + n-butanol fraction	258.3±10.2	224.9±8.4	13.3	203.7±6.5*	21.1

Extracts, fractions and the standard metformin were given at a dose of 150mg/kg body weight. * Statistically significant difference from zero time at P<0.01. M, mean; S.E., standard error (n=6).

Keywords: validation, bioactive markers, RP-HPLC, Cleome droserifolia, hypoglycemia

References: 1. Eliasson SG, Samet JM (1969). Life Sci 8:493–498.

2. Abdel Motaal A, Ezzat SM, Haddad PS (2011) Determination of bioactive markers in Cleome droserifolia using cell-based bioassays for antidiabetic activity and isolation of two novel active compounds. PHYMED-D-11–00029R1 (under publication).