Dipeptidyl Peptidase-4 Inhibitory Activity of Indonesian Anti-Diabetic Herbs: Carica Papaya, Tithonia diversifolia, Urena lobata

 

Yudi Purnomo¹*, Djoko W Soeatmadji², Sutiman B Sumitro³, M. Aris Widodo⁴

¹Pharmacy Department, Faculty of Medicine, University of Islam Malang, Malang, Indonesia.

²Internal Department, Faculty of Medicine University of Brawijaya, Malang, Indonesia.

³Biology Department, Faculty of Life Science, University of Brawijaya, Malang, Indonesia.

⁴Pharmacology Department, Faculty of Medicine, University of Brawijaya, Malang, Indonesia.

 

ABSTRACT:

Incretin hormone is a new therapy for type 2 diabetes related to their activity for regulating blood glucose level. Even though, this hormone is destroyed by Dipeptidyl Peptidase-4 (DPP-4) become in-active form. DPP-4 inhibition will be beneficial for type 2 diabetes therapy. Empirically, some of local herbs are used by Indonesian people to cure diabetes, however, activity on DPP-4 inhibitory has not been examined. The objective of study to examine Indonesian anti-diabetic herbs Urena lobata (U. lobata), Tithonia diversifolia (T. diversifolia) and Carica papaya (C. papaya) through inhibition of DPP-4. The in vitro study use vildagliptin and Gly-pro-p-nitroanilide (GPPN) as a reference standard and substrate respectively. C. papaya leaf extract, T. diversifolia and U. lobata were prepared both of by decoction methods and digeration with ethanol. The data were expressed as mean ± SD and the Inhibitory Concentration (IC)-50 value was calculated by non-linear regression curve fit. U.lobata leaves in ethanolic extract had more inhibitory potency on DPP-4 compare to T. diversifolia and C. papaya with an IC-50 value of 1.65mg/mL, 26.12mg/mL and 34.02mg/mL respectively. Meanwhile, on aqueous extract from U.lobata, C. papaya and T. diversifolia with IC-50 value 6.49mg/mL, 14.99mg/mL and 15.39 mg/mL respectively. U.lobata leaf extract most strongly to inhibit DPP-4 activity regulated by active compounds in its extract like stigmasterol, β-sitosterol and mangiferin. Ethanolic extract of U.lobata leaves more potent than aqueous extract to inhibit DPP-4 activity and both of them showed the most inhibitory activity on DPP-4 among other.

 

 

 

INTRODUCTION: 

Indonesia is archipelago state having a highest number of traditional communities and biodiversity in the world. Each community has local knowledge and practices in utilizing plants for primary health care. It is a part of local culture that is passed down from generation to generation and known by Local Wisdom¹.

 

One of health problem with the highest prevalence in Indonesia is Diabetes Mellitus (DM)².

 

 

 

Herbs are used people to treat DM due to they have medicinal properties, easy to obtain, economical prices and simple processing³. The herbs both of in single and combination are used to cure DM by people or traditional healers and it is known by anti-diabetic Indonesian herbs^1,4.

 

Pulutan (Urena lobata), Pahitan (Tithonia diversifolia) and Pepaya (Carica papaya) are herbs which used Indonesian people to cure DM. They have a lot of active compound which is beneficial to solve the health problems including DM^5,6,7. The herbs have a bitter taste therefore it can be used to treat DM based on their intuition⁸. The study showed administration of U. lobata roots extract and C. papaya leaf had anti-hyperglycemic on rat induced streptozotocin^9,10. It related to active substances in herbs such as sterol group, alkaloid and flavonoid^10,11,12. T.diversifolia also has anti-diabetic activity based on molecular docking study¹³. Some of anti-diabetic Indonesian herbs have not been evaluated especially on the DPP-4 inhibitor activity.

 

The unique enzyme that metabolize incretin hormone into inactive forms rapidly is Dipeptidyl peptidase-4 (DPP-4). It results a short half-life of incretin hormone of just about 1-2 minutes¹⁴. Glucose Dependent Insulinotropic Polypeptide (GIP) and Glucagon Like Peptide-1 (GLP-1) are produced by intestinal cells, both of them classified into a major incretin hormon¹⁵. GLP-1 would be beneficial to regulate blood glucose level due to its bioactivity such as to increase cell mass, stimulate insulin secretion, prevent glucagon secretion and decrease the rate of gastric emptying^14,15. One of the methods to prolong the half-life of GLP-1 is the use of DPP-4 inhibitors^16,17. It induces the exploration of DPP-4 inhibitor compounds from herbs having less side effect, economic and easy to prepare.

 

Therefore, it opens the chance to expand herbs become candidate of anti-diabetic phytopharmaca through inhibition of DPP-4 activity. The aim of study to examine anti-diabetic potency of C. papaya leaves extract, T. diversifolia and U. lobata on inhibitory activity of DPP-4.

 

MATERIAL AND METHODS:

Chemical agent:

DPP-4 was extracted from porcine kidney, Gly-pro-p-nitroanilide (GPPN), Tris-HCl buffer. All chemicals were purchased from Sigma aldrich.

 

Plant material:

C.papaya, T. diversifolia and U. lobata leaves were obtained and identified in UPT Materia Medica, Batu, Indonesia with certificate number 074/027/101.8/2015. The leaves were dried and grinded to maintain the green color.

 

Extraction of herbs:

C.papaya, T. diversifolia and U. lobata leaves powder each were taken 50g by analytical balance. Each plant materials were extracted in 250ml ethanol for 4 hours by waterbath shacker and reiterated 2 times with fresh ethanol. In the same weight, three of herbs each were extracted in 250ml hot aqueous 90ºC for 30 minutes or called a decoction method. Both of the extracts were then evaporated until produce paste form and dilute with solvent according to concentration which have been fixed.

 

Dipeptidyl peptidase-4 (DPP-4) assays:

The assay referred to Bharti et al with slight modification¹⁸. A pre-incubation volume of 50μL solution contains of 35μL Tris-HCl buffer, 15μl DPP-4 enzyme and various concentration of test material or standard. This mixture was incubated at 37ºC for 10 minutes, followed by the adding of substrate 50μL Gly-pro-p-nitroanilide. The mixture was incubated for 30 minutes at 37ºC and the absorbance was measured at 405nm every 10 seconds. Vildagliptin was used as reference drugs of DPP-4 inhibitor.

 

Statistical Analysis:

Data are stated as the mean±SD. The Inhibitory Concentration (IC)-50 rate was calculated by non-linear regression curve fit using SPPS version 16.0.

 

RESULTS AND DISCUSSION:

Indonesian anti-diabetic herbs, including C.papaya leaf extracts, T. diversifolia and U. lobata were examined on Dipeptidyl peptidase-4 (DPP-4) inhibitory activity by in vitro method. The DPP-4 inhibitory activity of herbs is shown in Table 1 and Figure 1.

 

Table 1. DPP-4 inhibitory activity of Indonesian anti-diabetic herbs and Vildagliptin

Group	Sample	n	Ethanolic ext Dose (mg/mL)	% inhibition	IC-50 (mg/mL)	Aqueous ext Dose (mg/mL)	% inhibition	IC-50 (mg/mL)
1	C.papaya	3 3 3 3 3	2.50 5.00 10.00 20.00 40.00	7.14 ± 0.00 11.90 ± 0.00 14.29 ± 0.00 30.37 ± 2.54 58.26 ± 3.85	34.02	1.25 2.50 5.00 10.00 20.00	14.29 ± 0.00 21.43 ± 0.00 35.71 ± 0.00 45.24 ± 4.12 54.05 ± 0.00	14.99
2	T. diversifolia	3 3 3 3 3	2.50 5.00 10.00 20.00 40.00	12.20 ± 0.00 14.63 ± 0.00 21.95 ± 0.00 39.60 ± 0.00 73.59 ± 0.00	26.12	1.25 2.50 5.00 10.00 20.00	7.69 ± 0.00 15.38 ± 0.00 23.08 ± 0.00 30.77 ± 0.00 53.84 ± 0.00	15.39
3         4	U. lobata         Vildagliptin	3 3 3 3 3 3 3 3 3	0.625 1.25 2.50 5.00 10.00 0.0125 0.0250 0.0500 0.1000	36.17 ± 0.00 48.94 ± 0.00 55.32 ± 0.00 61.70 ± 0.00 74.47 ± 0.00 16.07 ± 4.12 37.50 ± 0.00 46.63 ± 3.85 60.71 ± 0.00	1.65         0.0574	0.625 1.25 2.50 5.00 10.00	0.00 ± 0.00 13.33 ± 0.00 26.67 ± 0.00 42.22 ± 3.85 62.22 ± 3.85	6.49

 

 

Figure 1. Inhibitory Concentration-50(IC-50) of Indonesian anti-diabetic herbs on DPP-4

 

Table 1. showed inhibition percentage of anti-diabetic herbs on DPP-4 both in ethanol extract and aqueous extract. Ethanolic extract of U. lobata indicate the strongest inhibition activity on DPP-4 at the same concentration, meanwhile aqueous extract from T. diversfolia and C. papaya showed a stronger activity compare to its ethanolic extract.

 

Figure 1. showed Inhibitory concentration-50 (IC-50) of Indonesia anti-diabetic herbs on DPP-4. Ethanolic extract from U.lobata have IC-50 value lowest then followed by its aqueous extract and other herbal extract. Aqueous extract of C. papaya and T. diversifolia indicate a same of IC-50 value and they were stronger than its ethanolic extracts. On the other hand, IC-50 value of three herbs on DPP-4 were lower than vildagliptin as reference drug. 

 

Ethanolic extract from U. lobata leaf indicated a stronger DPP-4 inhibitory activity compare to its aqueous extract. It is related to active compound which extracted in ethanolic extract of U. lobata was stronger to constrain DPP-4 activity. U. lobata both of in ethanolic extract and aqueous showed the strongest activity on DPP-4 inhibition among other extracts. U. lobata leaf extract have been identified containing some active substances like stigmasterol, β-sitosterol, mangiferin, gossypetin and chrysoeriol^12,19. Ethanolic extract of U. lobata contain more stigmasterol than in aqueous extract, meanwhile gossypetin was found more in aqueous extract^19,20. The differences of composition result DPP-4 inhibitory activity in ethanolic extract more potent than aqueous extract in U. lobata by in vitro study.

However, aqueous extract of C. papaya and T. diversifolia more active to inhibit DPP-4 activity compare to its ethanolic extract. Active compound in herbs for example phytosterol, alkaloid and flavonoid groups are predicted to inhibit DPP-4 activity and contained more in aqueous extract than in ethanolic extract^7,21. This activity maybe controlled by lead compound as mentioned above which is more found in aqueous extract than in ethanolic extract. However, some studies showed phytosterols do not inhibit DPP-4 activity and they are more found in semi polar or non-polar solvent²².  T. diversifolia in ethanolic extract more active to inhibit DPP-4 activity compare to C. papaya, meanwhile their activity was not different in aqueous extract. Hispidulin and tithonine are active compound of T. diversifolia predicted as DPP-4 inhibitor based on in silico study¹³.

 

Molecular docking study showed also that phytosterol groups such as stigmasterol, β-sitosterol and alkaloid groups like mangiferin in U. lobata have a strong binding to DPP-4 particullary on inhibition activity²³. The lowest inhibition constant was mangiferin followed β-stigmasterol and sitosterol respectively therefore it influences their activity^23,24. Based on previous study, stigmasterol have the highest value of surface interaction that followed by β–sitosterol and mangiferin respectively²³. A great result of surface interaction showed a stronger bond between ligand and protein target, it produces a huge biology activity²⁴. Based on the in silico analysis, mangiferin has the lowest rate in binding free energy while stigmasterol and β–sitosterol were in the second and third position²³. Meanwhile, T. diversifolia contain cathecin, hispidulin and tithonine having the low score of binding free energy based on bioinformatic approach. The binding are strong between DPP-4 with three substances mentioned above, however their potency are not stronger than sitagliptin as reference drug¹³. The lowest value of binding free energy produces a strong binding molecule therefore causes the increase of its biology activity²⁴. Free energy binding and surface interaction between ligand and protein target affect the inhibitory activity of the leaf extract on DPP-4²⁵.

 

This study explores the anti-diabetic potential of a natural plant extracts through the inhibition of DPP-4 activity by in vitro methods. Inhibition of DPP-4 activity will improve the half-life of incretin hormone which contribute to control glucose level in blood for T2DM by the stimulation of insulin release, inhibition of glucagon, β-cells proliferation, decreasing the rate of gastric-emptying and inducing satiety^26,27. DPP-4 is involved in the inactivation of GLP-1, a powerful insulinotropic peptide furthermore inhibitory of DPP-4 activity could be an effective method to cure T2DM by increasing insulin secretion^15,28. The previous study indicated U.lobata leaf extract has anti-diabetic effect by inhibiting DPP-4, alpha-glucosidase and alpha-amylase^29,30.

 

DPP-4 inhibitory activity of Indonesian anti-diabetic herbs are lower compared to vildagliptin as a reference drugs. U. lobata, C. papaya and T. diversifolia indicated DPP-4 inhibitory activity approximately were lower 30-100 times folds compared to vildagliptin as a DPP-4 inhibitor reference. This may be caused by different active compound both in vildagliptin and herbs^13,20,31,32. Vildagliptin is single compound or isolate, therefore, the purity is higher compared to crude extract of herbs³³. In generally, crude extract of herbs contained many active substances in low concentration, moreover, the biology activity is lower than pure compound^12,13,31. Many active compound in herbs can interact one with others and result the modulation of biology activity^4,17. The interaction produce synergistic effect and or antagonistic moreover it can decrease or increase inhibitory activity on DPP-4 as anti-diabetic^17,34. The multicomponent in herbs reduce also its side effect and toxicity by their interaction mechanism that is known by Side Effect Eliminating Substances (SEES)^11,35.

 

CONCLUSION:

Ethanolic extract from U. lobata more potent to inhibit DPP-4 activity than its aqueous extract and U. lobata leaf extract have more inhibitory activity on DPP-4 compare to C. papaya and T.diversifolia.

 

ACKNOWLEDGEMENT:

This work was funded by Research Grant of Directorate General of Higher Education Indonesia.

 

CONFLICT OF INTEREST:

The authors declare no conflicts of interest.

 

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Accepted on 19.12.2022           © RJPT All right reserved

Research J. Pharm. and Tech 2023; 16(1):273-277.