Inhibitory action on the production of advanced glycation end products (AGEs) and suppression of free radicals in vitro by a Sri Lankan polyherbal formulation Nawarathne Kalka
© The Author(s). 2016
Received: 10 December 2015
Accepted: 14 June 2016
Published: 8 July 2016
Advanced glycation end products (AGEs) and free radicals are inflammatory mediators and are implicated in many diseases such as diabetes, cancer, rheumatoid arthritis etc. Multi targeted poly herbal drug systems like Nawarathne Kalka (NK) are able to quench the overall effect of these mediators as they contain good combinations of phytochemicals that have least side effects in contrast to modern medicinal drugs. The objectives of this study were to evaluate phytochemical composition, free radical scavenging activity, cytotoxicity and the inhibitory action on the formation of AGEs by aqueous extract of NK.
Total phenolic content (TPC) and total flavonoid content (TFC) were determined using Folin ciocalteu method and aluminium chloride assay respectively. Free radical scavenging activity was assessed by DPPH radical scavenging assay (DRSA), phosphomolybdenum reduction antioxidant assay (PRAA) and nitric oxide (NO) scavenging assay. Brine Shrimp Lethality (BSL) bioassay was performed as preliminary screening for cytotoxic activity. Inhibitory action on AGE formation was evaluated using fructose mediated glycation of bovine serum albumin using fluorescence spectroscopic method.
The TPC and TFC were 75.1 ± 3.0 mg/g gallic acid equivalents and 68.7 ± 7.8 mg/g epigallocatechin gallate equivalents. The DRSA yielded EC50 of 19.15 ± 2.24 μg mL−1 for NK. DRSA of NK extract was greater than butylated hydroxy toluene (EC50 = 96.50 ± 4.51 μg mL−1) but lesser than L-ascorbic acid (EC50 = 5.60 ± 0.51 μg mL−1). The total antioxidant capacity of NK as evidenced by PRAA was 106.4 ± 8.2 mg/g L-ascorbic acid equivalents. NK showed EC50 value of 99.3 ± 8.4 μg mL−1 in the NO scavenging assay compared to the standard ascorbic acid (EC50 = 7.3 ± 0.3 μg mL−1). The extract indicated moderate cytotoxic activity in the BSL bioassay. The extract showed effective inhibitory action on the formation of AGEs with EC50 values of 116 ± 19 μg mL−1, 125 ± 35 μg mL−1 and 84 ± 28 μg mL−1 in data obtained over three consecutive weeks respectively. Comparatively the reference standard, aminoguanidine at a concentration of 500 μg mL−1 demonstrated 65 % inhibition on the formation of AGE after one week of sample incubation.
The results proved the potential of NK as a free radical scavenger, moderate cytotoxic agent and an inhibitor on the formation of advanced glycation end-products.
KeywordsAdvanced glycation end products Nawarathne Kalka Free radicals
Traditional Sri Lankan System of Medicine (TSM) was established more than 3,000 years ago and it has been useful ever since for the treatment of various ailments . In contrast to modern medicinal systems, polyherbal preparations have gained more attention for their multi-targeting ability via pathways that give fewer side effects . These TSM drug systems consist of poly herbal formulations that can suppress painful symptoms associated with various ailments such as rheumatoid arthritis, diabetes and cancer .
Ingredients and proportions of Nawarathne Kalka 
Ingredients of Nawarathne Kalka
Part of the plant used
Proportions (weight basis)
1. Cedrus deodara (Vernacular name (VN): Devadara)
2. Cuminum cyminum (VN: Suduru)
3. Eugenia caryophylla (VN: Karabu)
4. Ferula asafetida (VN: Perunkayam)
5. Glycyrrhiza glabra (VN: Valmi)
6. Myristica fragrans (VN: Sadikka)
Dried kernel of the seed
7. Nigella sativa (VN: Kaluduru)
8. Picrorhiza kurroa (VN: Katukarosana)
9. Piper longum (VN: Thippili)
10. Trachyspermum roxburghianum (VN:Asamodagum)
11. Vernonia anthelmintica (VN: Sanninayam)
12. Zingiber officinale (VN:Inguru)
13. Terminalia bellirica (VN: Bulu)
Fruit (outer cover)
14. Terminalia chebula (VN: Aralu)
Fruit (outer cover)
Diseases such as RA and diabetes are inflammatory mediated, and hence require anti-inflammatory medicines to suppress the overall effects associated with inflammation. Inflammation causes pro inflammatory cytokines to be elevated such as interleukine-17 (IL-17) and tumor necrosis factor alpha (TNF-α) , which would subsequently initiate the secretion of more inflammatory mediators such as cytokines like IL-6 and IL-8  and colony stimulating factors like granulocyte macrophage colony stimulating factor (GM-CSF) . This means that propagation of inflammation activates osteoclasts in RA-cartilages to initiate osteoclastogenesis which is common in pathophysiology of RA [7, 8]. The diabetes related complications such as retinopathy , nephropathy  are also driven by similar inflammatory pathways. Accumulation of advanced glycation end-products (AGEs) resulting from protein glycation are considered to be the initiators of these complications . Advanced glycation end-products are formed due to the non-enzymatic reactions between sugars and proteins or nucleicacids [11, 12] and are associated with vascular related complications .
Oxidative stress is another factor that drives inflammation which can exert cytotoxic effects on tissues in the human body and hence there is a close association between oxidative stress and inflammation. Most common contributors of oxidative stress are hydroxyl radicals (.OH), nitric oxide (NO), superoxide anions (O2 .-) and peroxynitrites (OONO−) and these contributors are known as reactive oxygen species (ROS) . The ability to scavenge ROS is a useful quality that every anti-oxidant/anti-inflammatory drug must possess.
Suppression of the formation of ROS, AGEs and the secretion of cytokines altogether is the task of a multi targeted drug system rather than of a single targeted drug system. Hence the complex and complicated pathways by which the most dangerous diseases are associated with can be ameliorated by using the multi component formulations such as NK.
Due to lack of evidence on the pharmacologically important actions of the poly herbal formulation of NK towards suppression of various ailments, this study was focused towards investigation of NK for its phytochemical composition, antioxidant capacity and inhibitory action on formation of AGEs. Additionally, the cytotoxic effect of this herbal medicament was investigated.
2,2-diphenyl-1-picrylhydrazyl (DPPH), Glacial acetic acid, sulfanilamide, N-(1-napthyl)-ethylene diaminedihydrochloride (NEDD), sodium nitroprusside (SNP), L-ascorbic acid, potassium dihydrogen phosphate, disodium hydrogen phosphate, fructose, bovine serum albumin (BSA) and sodium azide were purchased from Sigma Aldrich USA.
Preparation of aqueous extract of NK
NK was purchased from an Ayurvedic drug store. An amount of 15 g from 3 sachet packets of NK were pooled together and the contents were then dissolved in 400 mL of deionized water. This mixture was refluxed in dark for 3 h. The refluxed solution was filtered using Whatman no.1 filter paper and the filtrate was stored under 4 °C until further use. Prepared NK specimen (voucher number NK 102) was deposited in Department of Ayurveda Pharmacology and Pharmaceutics, Institute of Indigenous Medicine, University of Colombo, Rajagiriya, Sri Lanka.
Determination of total phenolic content
The total phenolic content of the extracts were determined by Folin ciocalteu method . The extract was diluted 50, 100 and 500 times using deionized water. Folin ciocalteu’s phenol reagent (1 N, 250 μL) was added to the sample (500 μL) and the mixture was allowed to stand at room temperature for 2 min. Sodium carbonate solution (10 %, 1.25 mL) was added and samples were incubated for 45 min in the dark at room temperature. The absorbances of the resulting solutions were measured at 760 nm against a blank prepared in same manner but replacing the extract with deionized water. The calibration curve was constructed using gallic acid standards (6 – 30 μg mL−1) and the total phenolic content of the extract was expressed as mg/g gallic acid equivalents (GAE).
Determination of flavonoid content
The flavonoid content was measured by the aluminium chloride colorimetric assay . The extract was diluted 3, 4 and 5 times using deionized water. The diluted extract (100 μL) was mixed with deionized water (400 μL) and sodium nitrite (5 %, 30 μL). After 5 min aluminium chloride (10 %, 30 μL) was added followed by sodium hydroxide (1 M, 200 μL) at 6th minute. The total volume was adjusted to 1000 μL with deionized water and absorbance was measured at 510 nm against a blank prepared in similar manner but replacing the extract with deionized water. The calibration curve was plotted using (−)-epigallocatechingallate (EGCG) standards (300–1000 μg mL−1) and flavonoid content was expressed mg/g EGCG equivalents.
1,1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity
The effective concentration needed to scavenge 50 % of the DPPH free radical (Half maximal effective concentration, EC50) was calculated by regression analysis of the dose response curve plotted between percentage inhibition versus concentration of the test sample and the standard.
Phosphomolybdenum reduction antioxidant assay
The total antioxidant capacity of the extract was evaluated based on the method developed by Prieto et al. . The reduction of Mo (VI) to Mo (V) by the antioxidants present in the extract will subsequently form a green phosphate-Mo (V) complex at an acidic pH.
The extract was diluted 50, 100 and 500 times. Diluted extract (0.5 mL) was combined with 2.5 mL of reagent solution (0.6 M sulfuric Acid, 28 mM trisodium phosphate and 4 mM ammonium molybdate). The reaction mixture was then incubated at 95 °C for 90 min. Finally, after cooling the reaction mixture to room temperature, the absorbance was measured at 695 nm against a blank prepared in the same manner but using deionized water instead of extract. The calibration curve was constructed using L-ascorbic acid standards (25 – 100 μg mL−1) and the total antioxidant capacity of the above extracts was expressed as mg/g L-ascorbic acid equivalents.
NO scavenging activity
The NO scavenging activity of NK extract was determined according to a method published previously . Sodium nitroprusside (10 mM) solution was mixed with phosphate buffer (pH 7.4) in the ratio of 1:3 and kept for 20 min until the required aerobic conditions were obtained. Auto oxidation products (nitrites/nitrates) of NO generated by SNP were produced under these conditions. The SNP and buffer mixture (2.0 mL) was added to 1.0 mL of NK (0.1-19.8 mg mL−1) and the samples were incubated for 150 min at 25 °C.
Sulfanilamide (0.33 % in 20 % glacial acetic acid, 1.0 mL) was added to 0.5 mL of the previously incubated solution and allowed to stand for 5 min. Then 1.0 mL of NEDD (0.1 % w/v) was added to the mixture and further incubated for 30 min at 25 °C. The pink chromophore generated during diazotization of nitrite ions with sulphanilamide and NEDD was measured spectrophotometrically at 540 nm against a blank sample which consisted of NEDD, SNP and buffer only. The control was prepared by replacing NK with phosphate buffer which lacks a NO scavenger. L-Ascorbic acid was used as the positive control. Each analysis was performed in triplicates. The percentage inhibition (% I) of NO radicals by NK/positive control was calculated according to equation 1.
Brine shrimp lethality bioassay
(Where, Nc = Number of living nauplii in the control sample, Ns = Number of living nauplii in the test sample)
The effective concentration required to kill 50 % of the living nauplii with respect to the control (Half maximal lethal dosage, LD50) was calculated by the dose response curves plotted between %L versus concentration of the extract.
Inhibitory action on the formation of Advanced Glycation End-products
(Where FC = Fluorescence intensity of control with fructose, FCB = Fluorescence intensity of blank of control without fructose, FS = Fluorescence intensity of sample with fructose, FSB = Fluorescence intensity of blank of sample without fructose)
Results are presented as mean ± standard deviation (Mean ± SD) of at least three independent experiments. Statistical analysis including student’s t-test was performed using Microsoft Excel. Value of p < 0.05 was considered as significant.
Results and Discussion
Phytochemical composition of NK aqueous extract
Total Phenolic Content
75.1 ± 3.0 mg/g gallic acid equivalents
Total Flavonoid Content
68.7 ± 7.8 mg/g epigallocatechin gallate equivalents
Phosphomolybdenum reduction antioxidant assay is a single electron transfer system which is useful in measuring the capacity of an antioxidant in reduction of an oxidant which changes its color when reduced. A higher degree of color formation indicates higher reducing power of the antioxidant . NK extract demonstrated an antioxidant capacity of 106.4 ± 8.2 mg/g L-ascorbic acid equivalents in this assay. This proves that the extract has a higher reducing power to almost neutralize many oxidants generated in vivo as well as arising from exogenous sources.
Several studies conducted with different types of honey have proven their antioxidant properties. This is due to the compounds present such as vitamin C, monophenolics, flavonoids and polyphenolics. Antioxidant compounds like caffeic acid, chrysin, galangin, quercetin, acacetin, kaempferol, pinocembrin, pinobanksin, apigenin and enzymes like glucose oxidase and catalase are found to predominate in most of the types of honeys . They have received special attention due to their role in preventing diseases associated with oxidative stress such as cancer, cardiovascular diseases, inflammatory diseases and infections [40, 41]. Honey being a main ingredient in the formulation of NK may be responsible for this therapeutic potential of the formulation itself and causing a synergistic effect along with phyto-constituents derived from plant materials to enhance the activity of the medicament. The next most abundant ingredients Terminalia belerica and Terminalia chebula present in NK have been scientifically proven for many biological activites including antioxidant and anti-diabetic effects [42, 43]. NK being a polyherbal formulation comprising of these two ingredients also would have added to and enhanced the overall effects of NK. Future studies will be focused at identification and quantification of individual compounds present in NK.
Our findings provide evidence of potent antioxidant activity, moderate NO scavenging activity and cytotoxic effects as well as the ability to inhibit the formation of advanced glycation end products possessed by the poly herbal formulation Nawarathne Kalka. This can be attributed to very high levels of phenolic and flavonoid compounds being present, thus justifying the use of this particular herbal remedy in the treatment of various inflammatory conditions including arthritis in the Traditional Sri Lankan System of Medicine. However further studies including identifying potent individual chemical components present in NK, their mechanistic pathways of action and clinical trials should be conducted to understand the holistic effects caused by this poly herbal medicament on human body.
% I, Percentage inhibition; .OH, Hydroxyl radicals; AGEs, Advanced glycation end products; BHT, Butylated hydroxy toluene; BSA, Bovine serum albumin; BSL, Brine Shrimp Lethality bioassay; DPPH, 2,2-diphenyl-1-picrylhydrazyl; DRSA, DPPH radical scavenging assay; EC50, Half maximal effective concentration; EGCG, (−)-epigallocatechingallate; GAE, Gallic acid equivalents; GM-CSF, Granulocyte macrophage colony stimulating factor; IL-17, Interleukine-17; LD50, Half maximal lethal dosage; NEDD, N-(1-napthyl)-ethylene diaminedihydrochloride; NK, Nawarathne kalka; NO, Nitric oxide; O2 .-, Superoxide anions; OONO−, Peroxynitrites; PRAA, Phosphomolybdenum reduction antioxidant assay; RA, Rheumatoid arthritis; ROS, Reactive oxygen species; SNP, Sodium nitroprusside; TFC, Total flavonoid content; TNF-α, Tumor necrosis factor alpha; TPC, Total phenolic content; TSM, Traditional Sri Lankan System of Medicine
The authors wish to thank College of Chemical Sciences, Institute of Chemistry Ceylon for providing financial assistance to conduct this study. Professor S A Deraniyagala, Department of Chemistry, University of Colombo, Sri Lanka is gratefully acknowledged for donating a sample of aminoguanidine.
Financial assistance to conduct this study was received from College of Chemical Sciences, Institute of Chemistry Ceylon.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article.
Laboratory work was conducted by DTK, CDF, SDG and MCDC. CU, PKP and CDF supervised the project. Conception of the project hypothesis was by CDF, CU, PK and PKP. The manuscript was written by DTK and CDF. The manuscript was revised by CDF, CU and PKP. All authors read and accepted the final draft of the manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
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