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Year : 2011  |  Volume : 2  |  Issue : 2  |  Page : 55-57  

Lack of antiglycation activity of fresh juice of whole plant of Enicostema axillare (Lam.) Raynal

1 Department of Zoology, University of Colombo, Colombo, Sri Lanka
2 Department of Nidana Chikitsa, Institute of Indigenous Medicine, University of Colombo, Rajagiriya, Sri Lanka
3 Herbal Technology Section, Industrial Technology Institute, Colombo, Sri Lanka

Date of Web Publication25-Nov-2011

Correspondence Address:
W D Ratnasooriya
Department of Zoology, University of Colombo, Colombo 3
Sri Lanka
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0976-9234.90210

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Objective: To investigate the antiglycation potential of fresh juice of whole plant of Enicostema axillare (Lam.) Raynal. in vitro. Materials and Methods: Antiglycation activity of fresh juice of whole plant was determined in vitro using five concentrations (25, 50, 100, 200, and 400 μg/ml) by determining their ability to inhibit the formation of advanced glycation endproducts in a bovine serum albumin / glucose system using fluorescence spectroscopy. Results: There is no antiglycation activity in vitro of fresh juice of E. axillare plant. Conclusion: It is unlikely that impairment of diabetic complications by E. axillare whole plant juice as claimed by ayurvedic and traditional physicians is mediated via antiglycation activity.

Keywords: Antiglycation activity, diabetic complications, Enicostema axillare, Enicostema littorale

How to cite this article:
Ratnasooriya W D, Somarathna K, Premakumara G, Ediriweera E. Lack of antiglycation activity of fresh juice of whole plant of Enicostema axillare (Lam.) Raynal. J Pharm Negative Results 2011;2:55-7

How to cite this URL:
Ratnasooriya W D, Somarathna K, Premakumara G, Ediriweera E. Lack of antiglycation activity of fresh juice of whole plant of Enicostema axillare (Lam.) Raynal. J Pharm Negative Results [serial online] 2011 [cited 2020 Jun 2];2:55-7. Available from:

   Introduction Top

Enicostema axillare (Lam.) Raynal. syn. Enicostema littorale Blume (Family Gentianaceae) [1],[2] is a perennial herb with a short erect stem branched from base with lanceolate opposite leaves. Flowers are sessile, numerous, and whitish in color. [3] This plant is distributed in India, eastern and southern Africa, and Asia including Sri Lanka. [3] In Sri Lanka, it is found in open, sandy places among sparse grass close to the beach throughout the dry zone particularly from northwestern to northeastern coastal belt. [3]

The fresh juice of the entire plant is used in fever, worm infestation, hepatic disorders, skin diseases, in regulation of blood glucose level, and in reducing the complications associated with hyperglycemia. [4],[5] Interestingly, even today, ayurvedic physicians use this plant to treat diabetic patients. Blood glucose lowering activity of E. axillare has been proven clinically [6] and experimentally using alloxan [7],[8] and streptozotocine-induced diabetic rats. [9],[10]

Now, it is well recognized that accumulation of advanced glycation end-products (AGEs), at least in part, contributes to the pathogenesis of long-term diabetic complications (such as nephropathy, neuropathy, retinopathy, cataracts, or atherosclerosis) and progression neurodegenerative diseases (including Alzheimer's disease). [11] It also plays a major role in the development and progression of microvascular complications of the aged. [11] In fact, diabetes is a major source of AGEs in vivo.[12] AGEs are formed in vivo through non-enzymatic glycation of glucose and other reducing sugars with proteins via so-called Maillard reaction. [11]

In view of the wide occurrence of diabetes, a great demand exists for the development of antiglycation agents (AGEs inhibitors) especially from natural sources as adverse side effects are associated with potential synthetic drugs. [11],[13] Because E. axillare plant is used to treat diabetes and its associated complications in ayurvedic and traditional medicine and has potent antioxidant activity. [14],[15] It is possible that this plant may possess antiglycation activity. Further, it is known that antioxidants [11] act as AGEs inhibitors, presumably through metal-iron chelation and sequestration of free radical species. [11] . However, antiglycation activity of E. axillare plant is not scientifically tested and proven. The aim of this study was therefore to investigate antiglycation potential of E. axillare using an in vitro test model.

   Materials and Methods Top

Collection of plant

Fresh plants were uprooted from Puttalam area (N 8° 2' 5.1432", E 79° 50' 6.9961") which is situated in the dry zone of Sri Lanka, in September 2010. The plant was identified and authenticated by Dr. S. Somarathne, Department of Botany, The Open University, Nawala, Sri Lanka. A voucher specimen (KIWK Somarathna/1/2010) was deposited at the Department of Nidana Chikitsa, Institute of Indigenous Medicine, University of Colombo, Rajagiriya, Sri Lanka.

Preparation of whole plant juice

Fresh plants were cleaned and washed with tap water. These were spread on sheets of paper and shade dried for 12 h. Plants were then cut into small pieces and 470 g of this was blended for 10 min to obtain the juice using an electrical blender (Sumeet Domestic Blender, Sumeet Research and Holdings Private Limited, India). The dark green juice was squeezed through a muslin cloth and was freeze-dried. The freeze-dried product was stored air tight in 4°C.

Investigation of antiglycation activity

The antiglycation activity of fresh juice of whole plant of E. axillare against Maillard reaction on AGEs formation was determined according to Matsuura et al.[16] with some modifications. Freeze-dried juice of E. axillare whole plant was dissolved in known amount of dimethyl sulfoxide (DMSO). Reaction volume of 1 ml containing 800 μg bovine serum albumin (BSA), 400 mM glucose, and different concentrations of fresh whole plant juice of E. axillare (25, 50, 100, 200, and 400 μg/ml) in 50 mM phosphate buffer (pH 7.4) containing 0.02% sodium azide (w/v) were incubated at 60°C for 40 h. After cooling, aliquots of 600 μl were transferred to 1.5 ml Eppendorf tubes and 60 μl of 100% (w/v) trichloroacetic acid was added and stirred. Supernatant was removed after centrifugation at 15,000 rpm at 4°C for 4 min and the AGEs-BSA precipitate was dissolved in 3 ml of phosphate buffer saline (pH 10). Fluorescence intensity of the samples was measured at an excitation wave length of 370 nm and emission wave length of 440 nm using a spectrofluorometer AMINCO-Bowman, Thermo Spectronic, East Lyme, Connecticut, USA. Rutin was used as the standard antiglycation agent. Antiglycation activity (inhibition %) was calculated using the following equation:

Inhibition (%) = [(Fc - Fb ) - (Fs - Fsb )/(Fc - Fb )]΄ 100

where F c is the fluorescence of incubated BSA, glucose, and DMSO (control); Fb is the fluorescence of incubated BSA alone (blank); Fs is the fluorescence of the incubated BSA, glucose, and juice of E. axillare or rutin; and Fsb is the fluorescence of incubated BSA with juice of E. axillare or rutin. The results are expressed as mean ± SD. IC 50 values were calculated using probit analysis.

   Results and Discussion Top

This study examined the antiglycation potential of fresh juice of E. axillare whole plant using an in vitro technique. This technique is validated, reliable, sensitive, and widely used to investigate potential antiglycation agents. [16] The results obtained are depicted in [Table 1] and [Table 2]. As shown, E. axillare juice did not exhibit marked antiglycation activity compared with the reference drug, rutin [Table 2]. IC 50 value for rutin was 21.88 ± 2.82 μg/ml, whereas IC 50 value of fresh juice was 540.43 ± 10.94 μg/ml. This indicates that fresh juice was 25 times less potent than rutin. This is a novel finding. Thus, it is unlikely that claimed impairments of diabetic complications by E. axillare juice are mediated via antiglycation activity. However, it is not possible to rule out AGE cross-linked breaking activity of E. axillare juice. This is worth examining because synthetic agents are being tested as potential AGE cross-linked breakers to be used as therapeutics for diabetes and related complications. [11],[13]
Table 1: Antiglycation effect of different concentrations of fresh juice of whole plant of E. axillare in vitro (mean ± SD), (n = 3)

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Table 2: Antiglycation effect of different concentrations of rutin in vitro (mean ± SD), (n = 3)

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   Acknowledgment Top

Thanks are due to Dr. (Mrs.) A.M.P. Attanayake for collection of plant and preparation of fresh juice.

   References Top

1.Khare CP. Indian medicinal plants: An illustrated dictionary. 1 st ed. New York: Springer Science+Business Media, LLC.; 2007.  Back to cited text no. 1
2.Gentian Research Network. USA: Gentian Research Network and Rutgers University; 2002-2008. Available from: [Last cited on 2011 Jan 24].  Back to cited text no. 2
3.Dassanayake MD, Fosberg FR. A Revised Handbook to the Flora of Ceylon. New Delhi: Oxford and IBH Publishing Co.; 1981.  Back to cited text no. 3
4.Kirtikar KR, Basu BD. Indian Medicinal Plants. Dehradun: International Book Distributors; 1935.  Back to cited text no. 4
5.Warrier PK, Ramankutty C. Indian Medicinal Plants: A compendium of 500 species. Hyderabad: Orient Longman Limited; 1994.  Back to cited text no. 5
6.Upadhyay UM, Goyal RK. Efficacy of Enicostemma littorale in type 2 diabetic patients. Phytother Res 2004;18:233-5.  Back to cited text no. 6
7.Maroo J, Vasu VT, Gupta S. Dose dependent hypoglycemic effect of aqueous extract of Enicostemma littorale Blume in alloxan induced diabetic rats. Phytomedicine. 2003;10:196-9.   Back to cited text no. 7
8.Prince PSM, Srinivasan M. Enicostemma littorale Blume acqueous extract improves antioxidant status in alloxan-induced diabetic rat tissues. Acta Pol Pharm 2005; 62:363-7.  Back to cited text no. 8
9.Murali B, Upadhyaya UM, Goyal RK. Effect of chronic treatment with Enicostemma littorale in non-insulin-dependent diabetic (NIDDM) rats. J Ethnopharmacol 2002;81:199-204.  Back to cited text no. 9
10.Vishwakarma SL, Rakesh SD, Rajani M, Goyal RK. Evaluation of effect of acqueous extract of Enicostemma littorale Blume in streptozotocine-induced type 1 diabetic rats. Indian J Exp Biol 2010;48:26-30.  Back to cited text no. 10
11.Reddy VP, Beyaz A. Inhibitors of the Maillard reaction and AGE breakers as therapeutics for multiple diseases. Drug Discov Today 2006;11:646-54.  Back to cited text no. 11
12.DeGroot J. The AGE of the matrix: chemistry, consequence and cure. Current Opin Pharmacol 2004;4:301-5.  Back to cited text no. 12
13.Bakris GL, Bank AJ, Kass DA, Neutel JM, Preston RA, Oparil S. Advanced glycation end-product cross-link breakers: A novel approach to cardiovascular pathologies related to the aging process. Am J Hypertens 2004;17:23S-30S.  Back to cited text no. 13
14.Deore SL, Khadabadi SS, Bhagure L, Ghorpade DS. In vitro antimicrobial and antioxidant studies on Enicostemma axillare (Lam.) Raynal. leaves. Natural Product Radiance (Nat Product Radiance) 2008;7:409-12.  Back to cited text no. 14
15.Vaijanathappa J, Badami S, Bhojraj S. In vitro antioxidant activity of Enicostemma axillare. J Health Sci 2008;54:524-8.  Back to cited text no. 15
16.Matsuura N, Aradate T, Sasaki C, Kojima H, Ohara M, Hasegawa J, Ubukata M. Screening system for Maillard reaction inhibitor from natural product extract. J Health Sci 2002;48:520-6.  Back to cited text no. 16


  [Table 1], [Table 2]

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