Background: The herbal antidiabetic drugs are safe but effective alternate to conventional oral antidiabetic therapy. The single phytoconstituents of single plants are inadequate to produce the required efficacy therefore polyherbal can be tested for the desired results. The objective of the present study was the assessment of total phenolic, flavonoids, tannins content, in vitro antioxidant and antidiabetic activity of Azadirachta indica leaves, Picrorhiza kurroa rhizomes, Pterocarpus marsupium heartwood, and Withania coagulans berries and fruit coat. Materials and Methods: The ethanolic extract of all four drugs were prepared and mixed in a ratio of 1:1:1:1 to make the polyherbal extract. It was checked for the presence of phenols, flavonoids, and tannins. The High-Performance Liquid Chromatographic (HPLC) analysis for phenolic compounds as well as the beta carotenoids was done. The in vitro antioxidant activity as well as in vitro antidiabetic activity were calculated. Results: Quantitative estimations showed that maximum phenolic content was in Azadirachta indica and Pterocarpus marsupium, the flavonoid in Withania coagulans, and tannins content in Pterocarpus marsupium. The HPLC analysis showed the marked presence of beta carotenoids along with other phenolic compounds. The radical scavenging activity of Pterocarpus marsupium and the polyherbal extract was comparable to the standard Gallic acid. The total antioxidant capacity of polyherbal extract was found to be 15.22±.051 mg Quercetin equivalent (QE)/g dry plant extract (DPE). The polyherbal extract showed an IC50 value of 58.81 µg/ml in case of α- amylase inhibition and 64.81 µg/ml in case of α- glucosidase inhibition. Conclusion: The antioxidant activity, as well as antidiabetic activity, may be attributed to the phenolic, tannins, flavonoid content as well as beta carotene found in the polyherbal extract.
Diabetes is a serious public health concern; its global incidence has risen quickly during the last four decades. However, the World Health Organization (WHO) has programmed to inhibit the rise in diabetes incidence and premature mortality from 1/3rd by the year 2030 but the results are not promising. It has been estimated recently that the cases of diabetes from 20-79 years age will rise from 41 million in 2010 to 51 million in 2030. (1 in 10 adults).1,2
The oral antidiabetic have the undesirable side effects and toxicity along with nausea, vomiting, obstructive jaundice, hyponatremia, haematological- dermatological reactions and intolerance of alcohol and weight gain.3 The oral antidiabetic drugs have certain types of adverse effects on the body like Sulphonylureas leads to hypoglycemia and weight gain. Biguanides cause lactic acidosis, abdominal discomfort, bloating. The drug is also contraindicated in patients with liver, kidney, heart or lungs diseases. Alpha-glucosidase inhibitors cause gastrointestinal intolerance due to fermentation of undigested carbohydrates. It is also contraindicated in a patient with Irritable Bowel Syndrome (IBS) liver or kidney disorders. Thiazolidinedione use may lead to anemia, congestive heart failure, edema, pulmonary edema.4–6
The herbal medicines are considered safe as they have fewer side effects than an allopathic system of medicines. Compared to single herbal products polyherbal formulations show high performance, a broad therapeutic window, cost efficiency, easy availability and minimal side effects.7,8 Therefore,in the present study, we are going to work on polyherbal extract consisting of an equal ratio of Azadirachta indica leaves, Pterocarpus marsupium heartwood, Picrorhiza kurroa rhizomes, and Withania coagulans berries and fruit coat that could work on different targets and at the same time show improved patient adherence and therapeutic behavior.
Azadirachta indica (Family: Meliaceae) contains various phytoconstituents like Nimbin, nimbidin, azadirachtin, epicatechin, catechin, gallic acid, limonoid, flavonoid and margolone.9,10 The drug works as an antinociceptive, immunomodulatory, anti-inflammatory, antiviral, neuroprotective, antimicrobial, hepatoprotective, antidiabetic, and antioxidant.11,12 It delays diabetes onset as well as increases the glucose uptake and glycogen deposition.13
Pterocarpus marsupium Roxb. (Red Kino Tree) is used as an anti-inflammatory, anthelminthic, aphrodisiac, and antipyretic, in the management of mental disorders and ulcers.14,15 The phenolic components like marsupsin, pterosupin, and pterostilbene which reduce blood glucose levels and diabetic wounds.14,16 The drug contains alkaloids, flavonoids, saponins, phenols which may be responsible for the α- amylase and α- glucosidase inhibition activity.17,18
Picrorhiza kurroa Royle ex Benth. (Scrophulariaceae), has antiallergic, anticancer, cardiovascular, cholerectic, hypoglycemic, hypolipidemic, molluscicidal, and leishmanicidal properties.19 It contains Kutkin , kutkoside as well as picrosides I, II, III and V.20,21 It shows antioxidant activity along with α- amylase and α- glucosidase inhibition activity.22 Withania coagulans also known as ‘tukhm-e-hayat’ berry has hypoglycemic, hypolipidemic, immunosuppressive, hepatoprotective, anti-flatulent, anti-inflammatory, wound healer, anticancer, and cardiovascular effects. It contain Withanolide A, Withaferin A, Withaferin and Withanone.23 Withacoagin, withasomninne, might be the agents responsible for the free radical scavenging activity.24
MATERIALS AND METHODS
The Collection as well as Authentication of the Plant Material
Herbal drugs were gathered from the local areas of Ropar and validated at NIPER’s Natural Product Field Laboratory and Nursery, SAS Nagar, Pb. Azadirachta indica (Neem) leaves, Withania coagulans (paneer dodi) fruit, Picrorhiza kurroa (Kutki) rhizomes, and Pterocarpus marsupium (Vijay Sar) wood have all been verified. The authenticated plants’ voucher specimens were subsequently sent to the A.S.B.A.S.J.S. College of Pharmacy in Bela, Ropar, and Pb. for future reference.
Preparation of Extracts
The authenticated drugs Azadirachta indica (Neem) leaf, Picrorrhiza kurroa (Kutki) rhizomes, Pterocarpus marsupium (Vijay Sar) wood, and Withania coagulans (paneer dodi) fruit, were dried, and powdered properly in a grinder and were extracted using ethanol by using cold maceration method for 18 hr individually. The extracts so obtained were concentrated to dryness on a water bath below 60oC. The dried extracts were then stored in a cool place for further usage. A polyherbal extract was prepared using the above-obtained extracts by mixing them in the ratio of 1:1:1:1.
Qualitative Phytochemical Identification Test
Quantitative Estimation of Phytoconstituents Total phenolic content
This method was taken from Stoilova et al. The UV spectrophotometer was used to check the absorbance at 750 nm using Gallic acid as standard. The result was expressed in mg of Gallic acid Equivalent (GAE)/1g of DPE (dry plant extract).28 (y = 2.51x + 0.0921R² = 0.9958).
Total Flavonoids contents
The procedure given by Ajay et al. was used. The UV spectrophotometer was used to check the absorbance at 510 nm using rutin trihydrate as standard. The results were expressed in rutin trihydrate equivalents (RE)/1 g of DPT as standard was used to check the UV spectrophotometer.28 (y = 0.6733x + 0.0214, R² = 0.9926).
Total Tannins content
The Folin—Ciocalteu technique was used for the estimation of Total tannins content. The UV spectrophotometer was used to check the absorbance at 725nm using gallic acid as standard. The results were expressed in mg of Gallic acid Equivalent to mg/g GAE.29 (y = 6.0367x +0.0555, R² = 0.995).
High-Performance Liquid Chromatographic analysis
The method used by Piyush Kashyap et al. was used. The phenolic compounds as well as beta carotene were analysed using High Performance Liquid Chromatography technique. The qualitative as well quantitative evaluation was done. The ethanolic extracts of Azadirachta indica (Neem) leaf, Withania coagulans (paneer dodi) fruit, Picrorhiza kurroa (Kutki) rhizomes, Pterocarpus marsupium (Vija Sar) wood as well as polyherbal extract samples were investigated using Water’s HPLC 2489 equipped with C18 column (Phenomix; 100 mm × 4.6 mm, 5 μm). The system was operated at a flow rate of 1 mL/min consisting of (A) Acetonitrile and formic acid (99.8:0.2 v/v) and (B) water, acetonitrile, and formic acid (96:3.8:0.2 v/v). The gradient of elution comprised of A:B (95:5) at 5min, 85:15 at 25 min, 80:20 at 30 min, 75:25 at 39 min, 55:45 at 43 min, 5:95 at 48 min to 55 min, 80:20 at 55 min and 100:0 at 60 min. The injection volume of 10 μL was used. The mobile phase was run for total of 60 min. and phenolic as well as beta carotenoids were noticed at 280 nm. The column was washed using the mobile phase concentration increased to 100% of B for 1 min and upheld for 5 min prior to coming back to the earlier situation. The peak of each phenolic compound as well beta carotene was recognized with the standards by comparing the retention time and their quantification was done using standard curves.30
Antioxidant activity α- α-Diphenyl-β-picryl-hydrazyl radical scavenging (DPPH) Assay.
The procedure mentioned by Ajay et al. where gallic acid was used as a standard was employed. The results were evaluated in mg GAE/g dried extract. The percentage inhibition (I%) was calculated using the equation given below:
Total Antioxidant capacity
In vitro Antidiabetic Activity Determinations of α-Amylase inhibition activity
The method given by Demelash Z et al. was used to estimate the in vitro antidiabetic activity using acarbose as a positive control.
Where AC- control aborbance-samples % ||