Chemical and Biological Studies on Antidiabetic Single Plants and Composite Herbal Preparations

Abstract

Eleven antidiabetic herbal preparations (ADHPs) were collected from the local herbal drug stores in Dhaka city. Among these ADHPs, four were tablet, three were capsule and rest of the ADHPs were in liquid form. Eleven ADHPs were screened at BIRDEM for their efficacy in reducing blood glucose in type 2 diabetic Long Evans model rats produced at BIRDEM and BUHS. Seven drugs showed positive response in acute tests. These seven drugs were fed to diabetic model rats for 28 days and some of them showed significant hypoglycemic activity. One with the significant activity had only one plant Salvia haematodes Lin in its composition. This plant (roots) was taken for chemical studies. The flowers of the plant Phlogacanthus thyrsiflorus Nees is used for controlling blood glucose in South Eastern Indian people. The flowers were used to study the antidiabetic effect on Type 2 diabetic rats. The powdered root (5 kg) of Salvia haematodes Linn was extracted with n-hexane, DCM, EtOAc and methanol at room temperature, respectively. Dried and powdered flowers (300 g) of P thyrsiflorus was extracted with DCM, EtOAc and methanol at room temperature, respectively. This flower also was extracted with aq 80% ethanol to get 144 g extract. The ethanol extract (144 g) was suspended in water and was partitioned with DCM followed by 1-butanol. The DCM and 1-butanol soluble part was 49.87 g and 54 g, respectively. The extracts of ADHPs and Pthyrsiflorus flowers were directly tested for their antidiabetic effects, alpha-glucosidase inhibitor activity, antioxidant activity, SPF testing, microbial contamination, insulin secratory activity, isolation of active compound (s), toxic metal analysis, OCPs testing, identification & quantification of synthetic drug and antioxidant. ADHP-2, ADHP-4 and ADHP-6 showed significant results of lowering the blood glucose level and lipid profile. The body weight of the treated T2DM rats were unchanged during the experimentation. Ethanol extract of flowers of P thyrsiflorus Nees possesses hypoglycemic and hypolipidemic properties, which reduces the oxidative changes induced by STZ administration. The results of the current study revealed that roots and flower of S haematodes & P thyrsiflorus, respectively, exhibited wide range of α-glucosidase inhibitory activities. For the root of S haematodes, n-haxane part of MeOH extract showed more significant (IC50 = 0.5226 μg/mL) activity which was better than positive control quercetin (IC50 = 1.544 μg/mL). Another fraction, root’s of S haematodes, EtOAc (EA) part of MeOH extract showed significant (IC50 = 1.318 μg/mL value) activity compare to the positive control quercetin (IC50 = 1.544 μg/mL). n-Hexane part of EtOAc extract of the same plant also showed significant (IC50 = 1.184 μg/mL) activity compared to the positive control quercetin (IC50 = 1.544 μg/mL). From the flower of P thyrsiflorus, EtOAc (EA) part showed significant activity (IC50 = 1.539 μg/mL) which is very similar to the positive control quercetin (IC50 = 1.544 μg/mL). Methanol extracts of ADHP-1, ADHP-7, ADSP-8 and ADSP-9 exihibited 94.8 ± 0.0312%, 91.88 ± 0.0534%, 94.76 ± 0.0.0045% and 94.37 ± 0.0466%, respectively, at concentration 400 µg/ml when compared with standard sample (ascorbic acid, 97.15 ± 0.0502%). The IC50 value of the methanol extracts of ADHP-1 (17±0.0243 µg /ml) was significant when compared to that of the standard Ascorbic acid, 2 ± 0.0156, respectively. Methanol extracts of ADHP-1, ADHP-3, ADHP-6, ADHP-7, ADSP-8 and ADSP-9 was found to 98.38 ± 0.0156%, 95.54 ± 0.0321%, 94.34 ± 0.0371%, 95.90 ± 0.0551 % , 96.03 ± 0.0151% and 96.03 ± 0.0212% free radical scavenging activity, respectively at concentration 400 µg/ml when control sample (ascorbic acid) exhibited 99.90 ± 0.0145%. IC50 values for the methanol extract of ADHP-1 (21±0.0251 µg /ml) was significant when compared with control sample (ascorbic acid) exhibited IC50 at 10 ± 0.0156 µg/ml. Methanol extracts of different ADHP’s and ADSP’s sample exhibited good activity for reducing power assay in the present study. Where standard ascorbic acid gives absorbance 1.250 ± 0.0075 at 700 nm. The present study reveals, ADHP-6, ADSP-8 and ADSP-9 extracts exhibited 715.54±0.0485, 830.48±0.0365 & 850.74±0.0854 absorbances, respectively for total antioxidant activity. The plant extract which possess potential antioxidant activity and UV absorption capacity can prevent photo-aging and skin cancer (Ganesan et. al., 2013). The present study showed that the S haematodes Linn has significant UV absorption capacity, especially the chloroform and ethyl acetate fractions with the SPF value 10.18 and 9.63, respectively. On the other hand, another plant’s flower (P thyrsiflorus Nees) showed the SPF value 5.82 for methanol extract. Out of 07 antidiabetic solid and liquid samples, except ADHP-3, Bacillus subtilis (3.5 – 4.0 x 104 cfu/g) was identified from solid ADHPs and Enterococcus spp. (1.0 x104 cfu/ml) was identified from liquid ADHP, but all samples were free from fungi (yeasts and moulds). Bacillus subtillis has been identified 3.5 x 104 cfu/g, 3.7 x 104 cfu/g, 3.2 x 104 cfu/g, 3.8 x 104 cfu/g, 4.0 x 104 cfu/g in the ADHP-1, ADHP-4, ADHP-5, ADHP-6 and ADHP-7, respectively. Enterococcus spp. (1.0 x104 cfu/ml) was identified from liquid ADHP-2. ADHP-3 was free from all kinds of microbial contaminations. Compounds 1 and 4 of P thyrsiflorus showed significant effect on glucose-stimulated insulin secretion from MIN6 cells. Using repeated column and high performance liquid chromatography (HPLC) eight compounds were isolated from the dichloromethane, chloroform and 1-butanol soluble part of methanol extracts of flower and root of the plants. The structures of the isolated compounds were elucidated by extensive spectroscopic studies. Three compounds - sitosterol (1), stigmasterol (2) and 7-hydroxy coumarin (3) were isolated from the DCM and Chloroform part of roots of S haematodes. Morever, five compounds - sitosterol (4), stigmasterol (5), 8(17),13-labdadien-15,16-olide-19-oic acid (6), 19-hydroxy-8(17),13-labdadien-15,16-olide (7) and 2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-chromenone (luteolin) (8) were isolated from the dichloromethane extract and 1-butanol part of methanol extract of flowers, respectively. Among these eight compounds, compound (4) and (7) showed significant effect on glucose-stimulated insulin secretion from MIN6 cells. Toxic metal content particularly, Nickel (Ni), Copper (Cu), Manganese (Mn), Chromium (Cr), Cadmium (Cd), Lead (Pb), and Arsenic (As) were tested in the thirteen ADHPs. The amount of nickel was below detection level in all the preparations when the detection limit was 0.02 ppm. The copper concentrations varied in a wide range in all ADHPs between 0.48 to 39.58 ppm except ADHP 5 & 6. Cadmium content was detected some of the ADHP samples except ADHP-2, 4, 5, 6 & 7. Unfortunately, ADHP-3, 8, 9 & 10 samples the cadmium level were more than the level of all permissible limit (WHO, US FDA, HAS Singapore and Chinese Pharmacopoeia, Tables 3.3.1 and 3.3.2). Even if we consider a more relax permissible limit for cadmium (WHO and US FDA guidelines; Table 3.3.2), one-third of the total ADHPs (ADHP-3, 8, 9 & 10) failed to comply with the safety limit. The content of lead (Pb) in the analyzed samples ranged from less than 1.0 to 18.44 ppm. All the ADHP’s samples showed the value of Pb metal below detection level (DL=0.00005 ppm) except ADHP-3 and 8. The maximum concentrations of lead in ADHP- 3 and ADHP-8 were 0.08 ppm and 18.44 ppm, respectively. Arsenic (As) content in the analyzed samples ranged from 0.05 to 0.75 ppm. All the ADHP’s samples showed the value of As metal with the highest value in ADHP-7 (0.75 ppm) & ADHP-8 (0.74 ppm) and lowest value in ADHP-6 (0.05 ppm) as shown in Table 3.8.1. The US FDA, WHO, HSA Singapore & Chinese Pharmacopiea maximum permissible limit of As in consumed medicinal herbs is 10 ppm, 10 ppm, 5 ppm and 2 ppm respectively (Table: 3.8.2). The obtained results showed that all the analyzed ADHP samples the As contents were below the permissible limits. Four organochlorine pesticide’s residue analysis of ADHPs standards namely 4,4-DDE; 4,4-DDD; 2,4-DDT & 4,4-DDT were used to quantify the pesticides present in 07 Antidiabetic Herbal Preparations (ADHPs). 4,4-DDE was found in ADHP-1, ADHP-2, ADHP-6 & ADHP-7 amounting to 0.203 ng/g, 0.588 ng/g, 1.413 ng/g & 1.838 ng/g, respectively. 4,4-DDD amounting to 0.373 ng/g was found only in ADHP-2. 2,4-DDT was found in ADHP-2 and ADHP- 6 amounting to 1.937 ng/g and 0.756 ng/g, respectively. And 4,4-DDT was found to be present in ADHP-1, ADHP-2, ADHP-6 and ADHP-7 amounting to 0.971 ng/g, 8.608 ng/g, 3.377 ng/g and 2.917 ng/g, respectively. ADHP-03, 04, 05 were free from organochlorine pesticide residues. Small amount of metformin HCl was found in all samples of ADHP. Metformin HCl was quantified 2.25%, 2.62%, 1.28%, 1.58%, 1.47%, 1.35% & 2.47% in ADHP-1, ADHP-2, ADHP-3, ADHP-4, ADHP-5, ADHP-6 and ADHP-7, respectively. Large amounts of glaclazide (5.74%) and glibenclamide (3.82%) were quantified and isolated from ADHP-6 and ADHP-7, respectively, indicating deliberate addition. A new HPLC method to analyze seventeen antioxidant compounds simultaneously was developed and validated for, linearity, accuracy, stability and precision. All of the ADHPs and ADSPs cotainined good amount of different antioxidants. Especially ADHP-1, ADHP-2, ADHP-4, ADHP-5, ADHP-6, ADHP-7, and ADSP-8, 9 showed significat amount of antioxidants which is known to play benifical role for humans (Table-3.11.11).