This study was authorized by the Research and Community Service of Pancasila University (LPPM – UP), Depok, Indonesia, with contract number 7915/LPPM/UP/XII/2021. The present study was conducted from January to February 2021. The sample size was determined using Slovin’s formula with a 95% confidence level according to the following equation (Gudata et al. 2019).

$\%\mathit{\text{ inhibition}}=\frac{\text{ (Slope of enzyme control }-\text{ Slope of sample) }}{\text{ Slope of enzyme control }}\times 100$

where N = total population; e = tolerance level.

According to Slovin’s formula, 387 respondents (312 females and 75 males) were selected as respondents. The gender gap among respondents between females and males at about 5:1 ratio was chosen based on the information from the community health center (Pusat Kesehatan Masyarakat – PusKesMas) as the lowest government healthcare system at the sub-district level. According to the provided information, the ratio of diabetic patients was in the ratio 3:1 (females to males). Prior to the interview process, verbal informed consent was obtained from each respondent. Data collection was conducted according to Jadid et al (2020), including the interview technique and selection of the informant. In addition, the informant’s ages ranged from 20–65 years old. Where seven were between 20–25, sixty-eight ranging from 26–35, two hundred and three between 36–45, eighty-eight ranging from 46–55 and twenty-one between 56–65 years old. The criteria for the plant’s selection were native to the region and traditionally used to treat antidiabetics as a complementary medicine.

The chemicals used in this experiment were purchased from Merck (Darmstadt, Germany) and Sigma-Aldrich (St. Louis, Missouri, USA). The in-vitro DPP-IV activity screening kit consisted of a DPP-IV assay buffer, substrate, enzyme and standard inhibitor (sitagliptin) purchased from BioVision (Waltham, Massachusetts, USA). The DPP-IV inhibitory assay was performed in Nunc F96 MicroWell Black and measured using Varioskan Flash (Thermo Scientific, Waltham, Massachusetts, USA). LC-HRMS was performed on Waters Xevo-G2 XS QTof (Waters, Milford, Massachusetts, USA).

During the ethnopharmacology study, various parts of plants were collected according to the survey results and the specimens were sent to Herbarium Bogoriense for determination. In addition, crude extracts were prepared by macerating the materials in 96% ethanol with 1:10 ratio (w/v) at room temperature for 16 hours. After sixteen hours, the mixture was separated using filter paper and the organic solvent was evaporated under reduced pressure to obtain thick crude extracts.

The presence of alkaloids, flavonoids, triterpenes/steroids, tannins, quinones and saponin in the crude extracts were identified using colorimetric methods following to the previous study (Dewi et al. 2022). The total phenolics content (TPC) was estimated using Folin-Ciocalteu method and total alkaloids (TAC) were determined according to the aluminum trichloride method with gallic acid and quercetin as the reference compounds. The value was expressed as gallic acid equivalent (mg GAE/g sample) and quercetin equivalent (mg QE/g sample) for total phenolics and alkaloids, respectively.

The elimination of tannins from the crude extracts was conducted using the gelatin precipitation method developed by Prommajak et al. (Prommajak et al. 2018) and Setyaningsih et al. (Setyaningsih et al. 2019). In detail, one percent (1%) of gelatin solution was mixed with the test solution. The mixtures were shaken at 100 rpm for 10 min at 25 °C. The supernatant was dried and dissolved with dimethyl sulfoxide (DMSO) to exclude the false positive results.

The potency of several Indonesian medicinal plants as DPP-IV inhibitors have been conducted previously. Ten medicinal plants were screened for DPP-IV inhibitor and the highest activity was achieved by Caesalpinia sappan (84.25%) at a concentration of 100 µg/mL (Setyaningsih et al. 2019). Twelve edible plants from Indonesia were also screened and it was found that Ipomoea batatas (L). have the highest inhibition (28.8±7.7%) at a concentration of 10 µg/mL (Amin et al. 2019). According to the previous report, the presence of tannins in the crude extract may give a positive result in DPP-IV inhibitory activity (Kim et al. 1998). Since qualitative phytochemical analysis indicated the presence of tannins in all crude extracts, tannin elimination was performed prior DPP-IV inhibitory activity screening using the gelatin precipitation method (Prommajak et al. 2018). In-vitro DPP-IV inhibitory activity on selected medicinal plants was presented in Fig. 2. According to our results, at a concentration of 250 µg/mL, crude extracts exhibited inhibition ranging from 34.30 to 78.33%, while the positive control, sitagliptin showed 100% inhibitory activity. The highest inhibition was achieved by crude extract of the leaves of Piper ornatum (78.33±1.35 %) and the lowest activity was exhibited by Syzygium polyanthum with an inhibition value of 34.30±1.57 %. In accordance, the leaves of Psidium guajava, which have previously been screened at a concentration of 100 µg/ml and exhibited 29.50% inhibition. Those results corroborated our finding that by using 2.5 fold higher concentration (250 µg/ml), the inhibition was increased to 65.38 ± 4.88%. Compared to sitagliptin as a positive control, all crude extracts were statistically significant different.

Figure 2. 

In-vitro DPP-IV inhibitory activity of crude extracts from selected Indonesian plants. Sitagliptin was used as the positive control and showed 100% inhibition at 250 µg/mL. All data presented as mean ± SD of triplicates (n=3) experiments. The different letters indicated significant differences compared to sitagliptin (p value = 0.05).

Piper ornatum N.E.Br. is widely known as Celebes pepper and native to Sulawesi. According to the literature survey, no reported compounds have been isolated from Piper ornatum. Instead, thirty-six compounds have been isolated from Piper crocatum until the year 2021 (Heliawati et al. 2022). Interestingly, a specific search using a filtered keyword biological source “Piper ornatum” or “Piper crocatum” in the Dictionary of Natural Products on USB database version 29.2 gave no results. These indicated that 36 compounds isolated from Piper ornatum or Piper crocatum have previously been isolated from another source. Only crocatin A and B (Arbain et al. 2018), pipercroside A and B (Li et al. 2019a) and 5α,6β-dihydroxy-3β-(β-D-glucopyranosyloxy)-7E-megastigmen-9-one (Li et al. 2019b) were reported for the first time isolated from Piper crocatum. Furthernore, Piper ornatum crude extract analysis using LC-HRMS revealed that at least eleven compounds were present in the crude extract. Among those compounds, pipcroside B and crocatins were tentatively identified in the crude extract, which was isolated previously from Piper crocatum. (Figs 3, 4 and Table 4).

Figure 3. 

Chromatogram of Piper ornatum crude extract showed tentatively assigned compounds.

Figure 4. 

Mass spectrum of tentatively identified compounds.

Several classes of compounds have been screened for DPP-IV inhibitor activity. Flavonoids compounds, i.e., kaempferol 7-O-α-L-rhamnoside (IC₅₀=20.81 µM), vitexin (IC₅₀=33.12 µM) and rutin (IC₅₀=32.93 µM) isolated from the leaves of Smilax china L. showed moderate DPP-IV inhibitory activities (Zhao et al. 2016). An isolated compound from Rheum palmatum L., emodin (phenolic) was also shown to have the capability to inhibit DPP-IV activity with an IC₅₀ of 5.76 µM (Wang et al. 2017). In addition, stigmasterol isolated from Fagonia cretica L. was found to have DPP-IV activity with IC₅₀ >100 µM (Saleem et al. 2014). We found that the crude extract of Piper ornatum as the most active DPP-IV inhibitors in this study has an IC₅₀ value of 192.67±1.53 µg/mL. The quantitative phytochemical screening and LC-HRMS analysis of our samples suggested that a combination of total phenolics and alkaloids content may affect the DPP-IV activity, which was indicated by the highest activity of Piper ornatum crude extract.