This study was conducted along five routes (Figure 1) within the Plant Genetic conservation area at four separate time periods from January to September 2019. The eight commonly consumed indigenous plants collected for this study were identified and confirmed by local people and botanists. These plants are selected based on their being commonly consumed by local people and available in the study's areas. Information about these plants and photographs are presented in Table 1 and Figure 2. Plant samples were kept in dark plastic bags to protect the sunlight and then transported via an ice-box to the ISO/IEC 17025 laboratory at the Institute of Nutrition, Mahidol University.

At the laboratory, the edible part of each sample was collected and recorded. Each sample was washed with tap water, followed by washing twice with deionized water. After cleaning and removing excess water, cooked samples were blanched (plant to water ratio about 1:2) or boiled (plant to water ratio about 1:1) depending on each plant's commonly eaten method (Table 1). Weights before and after cooking (about 10 min post-heat treatment) were recorded. Thereafter, both fresh and cooked samples were homogenized using a food processor (Mara^®, Thailand) and divided into two portions. For the first portion, each sample was stored in an acid-washed screw-capped plastic bottle at −20°C for nutrient analyses. For vitamin C analysis, each sample was weighed to about 20 g in a plastic bottle, then about 20 g 10% MPA was added, mixed together, and then kept in a freezer. For the second portion, each sample was dried using a freeze dryer (Heto powerdry PL 9000), ground, vacuum-packed in laminated aluminum foil bags, and stored at−20°C for determination of bioactive compound and antioxidant activities.

Nutrient analysis was conducted using the standard AOAC method (2019) (Saha and Ahmed, 2009). All samples were analyzed at the ISO/IEC 17025:2017 accredited laboratory of the Institute of Nutrition, Mahidol University, which analyzed proximate compositions, vitamins, and minerals in duplicate analyses. All values were presented per 100 g fresh sample.

Total nitrogen was determined using the Kjeldahl method, methods nos. 910.20 and 981.10 (Saha and Ahmed, 2009), and calculated into protein content using specific (Jones) factors (N × 6.25). Moisture content was determined by drying each sample in a hot air oven at 100 ± 2°C until a constant weight was obtained based on method nos. 990.19 and 952.23 [Association of Official Analytical Chemists (AOAC), 2016]. Crude fat content was determined by acid hydrolysis and extraction using petroleum ether in the Soxtec system (Avanti 2055, Foss Tecator, Sweden), method nos. 922.32 and 945.16 [Association of Official Analytical Chemists (AOAC), 2016]. Ash content was analyzed by incinerating all organic matter at 550 ± 5°C, method nos. 945.46 and 930.30 [Association of Official Analytical Chemists (AOAC), 2016]. Available carbohydrate was calculated using the following formula: 100-moisture-protein-fat-ash-dietary fiber; energy was calculated by Atwater factors (4 for protein and available carbohydrate, 2 for dietary fiber, and 9 for total fat). Total dietary fiber was analyzed using the enzymatic gravimetric method, method no. 985.29 [Association of Official Analytical Chemists (AOAC), 2016].

Based on the previous report of Charoenkiatkul et al. (2012), the potential of vitamin for the studied plants is only vitamin C. For vitamin C, high pressure liquid chromatography (HPLC) with a UV/Vis detector was used for analysis (Sánchez-Mata et al., 2000). Samples were extracted in 3% metaphosphoric acid and filtered to homogenate. Ascorbic acid was separated by reversed-phase HPLC with UV detection at 248 nm and quantified against an external ascorbic acid standards. For minerals, microwave digestion was used for sample decomposition to determine magnesium, iron, copper, and zinc using an inductively coupled plasma optical emission spectrophotometer (ICP-OES), method no. 984.27 [Association of Official Analytical Chemists (AOAC), 2016]. The acid solution dissolved from the ash residue was used for calcium, sodium, and potassium analyses by flame atomic absorption spectrophotometer (AAS), method no. 985.35 [Association of Official Analytical Chemists (AOAC), 2016]. Phosphorus was determined by the gravimetric method [Association of Official Analytical Chemists (AOAC), 2016].

Samples were saponified and extracted according to the procedures of Sungpuang et al. (Sungpuang et al., 1999) with slight modification. In brief, freeze-dried samples were boiled with ethanolic potassium hydroxide with the addition of 10% (w/v) ascorbic acid, before being extracted with hexane, and then evaporated to dryness. The residue was reconstituted with mobile phase and then filtered through a 0.2 lm PTFE syringe filter. Chromatographic separation of individual carotenoids was modified slightly from the method described by Chitchumroonchokchai et al. (2017). The HPLC system for determining carotenoid content was carried out by Agilent 1100 series (Agilent Technologies, U.S.A.) and a photodiode array detector. The column used was an analytical YMC™ C30 column (4.6 × 150 mm, 5 μm, Waters, Milford, MA). The mobile phase consisted of 98% (v/v) methanol with 2% (w/v) ammonium acetate (solvent A) and methyl tert-butyl ether (MtBE) (solvent B). Isocratic system with 80% solvent A and 20% solvent B was used at a constant flow rate at 0.6 ml/min at ambient temperature. Qualitative and quantitative evaluations of chromatograms were monitored at 450 and 470 nm using ChemStation (Agilent Technologies, USA). Individual carotenoids (lutein, zeathantin, β-cryptoxanthin, α-carotene, and β-carotene) were identified based on comparing retention time and the UV spectrum of unknown peaks to the authentic standards. Carotenoid contents were expressed as mg per 100 g sample.

Total phenolic contents were determined by spectrophotometry using Folin–Ciocalteau reagent method (Lu et al., 2007) with slight modification. The absorbance was measured against the gallic acid standard at 760 nm. Total phenolic contents were expressed as mg gallic acid equivalent (GAE) per 100 g sample.

The method for performing ORAC assay based on fluorescence probe and fluorescein has been described by Ou et al. (2002). Each sample's ethanolic extract was mixed with fluorescein solution and AAPH solution at 37°C. Fluorescence was recorded using a spectrofluorometer (Luminescence spectrophotometer, Perkin Elmer LS55, Maryland, USA) with an excitation wavelength of 493 nm and an emission wavelength of 515 nm. Results were calculated using the differences of areas under the curve between samples or standard (trolox) and blank. The ORAC values were expressed as μmol trolox equivalent (TE) per 100 g sample.

Samples were weighed (4 significant digits) before and after cooking. The data obtained, combined with the amounts of nutrients in raw and cooked plants, were used to calculate true retention (Murphy et al., 1975) as per the formula

%True retention=nutrient per 100 g of cooked food × weight of cooked foodnutrient per 100 g of raw food × weight of raw food ×100

The proximate compositions of the eight indigenous plant varieties is shown in Table 2 below. Approximately 69–96% of composition was water. Cooked indigenous plant samples had an increased moisture content of 1–5%, which may be due to the water used for blanching or boiling. The protein content of samples ranged from 0.8 to 9.5 g/100 g fresh weight (FW). Protein content was greatest in Jang, Ta-Kuk, and Pak-Wan-Pa. Dietary fiber was the main nutrient contained in the samples, ranging from 2.6 to 10.3 g/100 g FW. The shoots and young leaves of Jang contained the highest amount of dietary fiber, followed by Som-Kob, Ta-Kuk, Pak-Wan-Pa, Ka-Tur, and Wan-Pro. Available carbohydrate content ranged from 2.2 to 9.3 g/100g FW. Fat content in all samples was <1 g/100g FW. Protein, dietary fiber, and fat contents in this study were similar and showed no significant differences (p ≥ 0.05) with those previously reported (Charoenkiatkul et al., 2012), i.e., Pak-Wan-Pa (protein 5.97 vs. 6.5 g/100g, dietary fiber 4.95 vs. 6.5 g/100 g, fat 0.83 vs. 1.2 g/100 g), Ta-Kuk (protein 7.70 vs. 7.30 g/100 g, dietary fiber 5.35 vs. 5.6 g/100 g, fat 0.68 vs. 0.60 g/100 g), Wan-Pro (protein 0.80 . 0.80 g/100 g, dietary fiber 2.81 vs. 3.20 g/100 g, fat 0.07 . 0.10 g/100 g), and Ka-Tur (protein 1.08 vs. 0.90 g/100 g, dietary fiber 3.86 vs. 4.80 g/100 g, fat 0 vs. 0.10 g/100 g), respectively. Protein content in this study was lower than that reported from Egypt, which was 15.14 g/100 g for Ta-Kuk (Hawary et al., 2011). For cooked vegetables, most proximate compositions decreased significantly (p < 0.05) compared to those of the same fresh vegetables. A wide range of decreasing protein content was observed (2.5% in Pak-Wan-Pa to 39.6% in Jang). A similar decreasing pattern was also found in ash after cooking (25.3% in Som-Kob to 55.4% in Jang). Nutrient amounts decreased slightly after cooking, which may have been caused by the dissolution of nutrients into the water used for cooking (Lyimo et al., 1990).

Vitamin C and mineral contents in the eight varieties of indigenous plants are presented in Table 3 below. The shoots and young leaves of Ta-Kuk contained the highest amount of vitamin C (212 mg/100 g FW), followed by Pak-Wan-Pa, Jang, and Ka-Pee-Jun (146 ± 19, 126 ± 7, and 27 ± 9 mg/100 g FW, respectively). The vitamin C contents of Pak-God (7 ± 1 mg/100g), Wan-Pro, Ka-Tur, and Som-Kob were < 10 mg/100 g FW. A previous report (Charoenkiatkul et al., 2012) showed lower levels of vitamin C in Pak-Wan-Pa (146 vs. 99 mg/100 g) and Ta-Kuk (126 vs. 161 mg/100 g), while Wan-Pro was higher (2 vs. 3 mg/100 g). There were significant vitamin C losses (p < 0.05) in cooked vegetables compared to that of fresh vegetables (ranging from 34.9% in Jang to 73.6% in Ta-Kuk) except for Wan-Pro due to no vitamin C being detected in the previous study (Charoenkiatkul et al., 2012). After cooking, the amount of vitamin C probably decreased due to its instability at high temperatures (Oboh, 2005).

The highest macro mineral content was potassium, ranging from 231 to 560 mg/100 g FW, with the highest being contained in the shoots and young leaves of Jang. All indigenous plant samples also provided calcium and phosphorus (39–186 and 31–137 mg/100 g FW, respectively), but only a small amount of magnesium (21–62 mg/100 g FW) and sodium (<40 mg/100 g FW), which were mainly found in the shoots and young leaves of Pak-Wan-Pa. Consistent with previous studies, Pak-Wan-Pa was high in vitamin C, phosphorus, and calcium [99, 133, and 108 mg/100 g FW, respectively (Charoenkiatkul et al., 2012); 83.6, 68, and 24 mg/100 g FW, respectively (Somsub et al., 2008)]. For trace elements, all indigenous plant samples contained iron, copper, and zinc at <3 mg/100 g FW. The shoots and young leaves of Wan-Pro contained the highest amount of iron (2.79 mg/100 g FW), while Jang contained the highest amount of copper and zinc (0.30 and 1.41 mg/100 g FW, respectively). After cooking, the amount of minerals in most vegetables significantly decreased (p < 0.05) probably due to their migrating into the boiling water (Oboh, 2005). Shoots and young leaves of Jang have a major characteristic being consumed as softened leaves as opposed to other vegetables which may play a role in loss of several minerals (63.6% for K, 54.8% for Mg, 50.0% for Cu, 47.9% for Ca, 43.3% for Zn, 25.2% for Fe and 22.8% for P).

Carotenoid contents of the eight indigenous plant varieties are shown in Table 4 below. The highest total carotenoid contents were found in the shoots and young leaves of Wan-Pro, followed by Ta-Kuk, Som-Kob, Pak-Wan-Pa (142.9, 42.8, 32.2, 28.0 mg/100 g FW, respectively) (Figure 3), while the lowest was in the flowers of Ka-Tur (2.0 mg/100 g FW). The carotenoid contents of indigenous plants in this study were significantly higher (p > 0.05) than in the original previous study (Charoenkiatkul et al., 2012), such as Pak-Wan-Pa (28 vs. 5.4 g/100g), Ta-Kuk (1.97 vs. 0.72 g/100 g), Wan-Pro (142.9 vs. 3.8 g/100 g), and Ka-Tur (1.97 vs. 0.72 g/100 g). All indigenous plant varieties contained predominantly β-carotene (55–89%), followed by α-carotene (10–45%), except for Wan-Pro which contained α-carotene (51–54%) followed by β-carotene (46–49%). Lutein, zeathantin, and β-cryptoxanthin were not detected in all plants in this study, except for Pak-Wan-Pa where β-cryptoxanthin was detected at 3 mg/100 g FW. Comparing carotenoid content between cooked and raw plant, the carotenoid contents of the cooked plant samples were significantly higher (p < 0.05) than that of the corresponding raw samples, except for Pak-Wan-Pa and Pak-God. Thermal food processes, such as boiling and blanching, can cause partial degradation of carotenoid pigments and promote isomerization of β-carotene content in the samples (Bernhardt and Schlich, 2006; D'evoli et al., 2013).

Total phenolic contents of the eight indigenous plant varieties varied widely, ranging from 77 to 2816 mg GAE/ 100 g FW as shown in Table 4 below. Shoots and young leaves of Jang had the highest total phenolic contents, followed by Ta-Kuk and Pak-Wan-Pa (2816, 383, 292 mg GAE/ 100 g FW, respectively), and the lowest content was in Wan-Pro (77 mg GAE/ 100 g FW). The wide variation in total polyphenol contents in plants might occur from the variety of the plant, stage of ripening, climate conditions, sample collection, transportation, and sample preparation (Rop et al., 2011). Comparison of total phenolic contents of some indigenous plants in this study, such as Pak-Wan-Pa and Ta-Kuk, with results of previous studies indicate similar contents, i.e., Pak-Wan-Pa from Saraburi and Uthai Thani provinces (296.44 and 265.63 mg GAE/100 g, respectively) (Charoenchai et al., 2015), Pak-Wan-Pa and Ta-Kuk from Kanchanaburi province (296 and 451 mg GAE/100 g, respectively) (Charoenkiatkul et al., 2012).

The results of antioxidant activities as measured by ORAC assay were accordance with the result of total phenolic contents, which are shown in Table 4 below. The shoots and young leaves of Jang (162,478 μmol TE/100 g FW), Ta-Kuk (16,022 μmol TE/100 g FW), and Pak-Wan-Pa (10,643 μmol TE/100 g FW) had the greatest ORAC values, while the lowest was in Wan-Pro (3,134 μmol TE/100 g FW). A strong consistency between total phenolic content and ORAC values was observed in many other studies, which confirmed that phenolic was the major contributor to antioxidant activities (Maisuthisakul and Suttajit, 2007; Han and Yang, 2008; Maisuthisakul and Pasuk, 2008). ORAC values in this study were less than those previously reported (Charoenkiatkul et al., 2012), but there were no significant differences (p ≥ 0.05), such as Pak-Wan-Pa (10,643 vs. 8,000 μmol TE/100 g FW), Ta-Kuk (16,022 vs. 14,400 μmol TE/100 g FW), Wan-Pro (3,134 vs. 3,800 μmol TE/100 g FW), and Ka-Tur (1,727 vs. 1,400 μmol TE/100 g FW), respectively.

The percentage true retention (%TR) of nutrients in the cooked processes is presented in Table 5 below. The retention of protein, dietary fiber, and minerals (calcium, phosphorus, magnesium, iron, copper, and zinc) were almost wholly retained (closed to 100%TR), while potassium was the least retained (49.9–77.10%TR). Blanched plants retained higher concentrations of vitamin C than those that had been boiled (97.8–109.8 and 35.8–42.5%TR for blanching and boiling, respectively). Vitamin C retention in cooked vegetables in a previous study ranged from 34.1 to 79.4% among the various vegetables and cooking methods, but was the least in boiling (Mellova et al., 1996). This result was consistent with the lowest vitamin C retention values shown in the boiling of spinach (33.9%TR) and green beans (63.7%TR) (Warthesen et al., 1984). The main mechanisms of vitamin C loss appear to be due to the vitamin's water solubility and sensitivity to temperature, so it is easily degraded during cooking. Elevated temperatures and long cooking times have been found to cause particularly severe losses of vitamin C (Rumm-Kreuter and Demmel, 1990; Oboh, 2005; Tian et al., 2016). The retention of total polyphenol and antioxidant in this study's boiled plants (52.6 and 50.0%TR in Ta-Kuk, 60.0 and 62.5%TR in Pak-Wan-Pa, respectively) was lower than in blanched plants (81.7 and 102.0% TR in Jang, 98.1 and 96.2% TR in Wan-Pro, respectively). The retention of carotenoids increased in boiled or blanched plants (>100%TR), except for Pak-Wan-Pa (74.3%TR). A previous study of cooked vegetables showed β-carotene retention was in the range of 40.02–125.37% (Lee et al., 2018). Carotenoids extractability may be influenced by cooking. Additionally, the difference in the retention of carotenoids in cooked plants might be attributed to the loss of carotene caused by dripping during the cooking process (Bureau et al., 2015).