Key Organic Acids in Indigenous Plants in Thailand

Organic acids had various health benefits such as citric acid can inhibit stone formation and break up beginning of small kidney stone. On the other hand, some organic acid showed negative health effects such as oxalic acid acts as anti-nutrients and can cause kidney stone. Most of Thai indigenous plants had sour taste; however general people believed that sour taste of plants could contain high ascorbic acid. In addition, there is limit report of organic acids and ascorbic acid in Thai indigenous plants. This study determined organic acids, ascorbic acid, pH, and total acidity in indigenous plants. Forty samples of 29 types of indigenous plants were analyzed. Results showed that young leaves of Cratoxylum formosum found the highest succinic acid (2454 ± 91 mg/100g fresh weight, FW) and high ascorbic acid (142 ± 35 mg/100g FW). Fruits of Antidesma ghaesembilla had high citric acid levels (5161 ± 109 mg/100g FW) but contained very low ascorbic acid (2 mg/100g FW). The sum of organic acids had significant and inverse correlations with pH (r = −0.680) and positive with total acidity (r = 0.672) but was not significantly correlated for ascorbic acid (r = 0.536). The sour taste of plants could derive from the sum of organic, citric, and formic acids, but not other organic and ascorbic acids. Against traditional belief, plants having a strong sour taste may not contain significantly high amounts of ascorbic acid.

fruits and vegetables [1] [2], where they influence sensory properties, such as giving sour or tart flavors. Some organic acids can indicate ripeness [3] [4]. Most Thai indigenous plants have a sour taste but it is not clearly known which substances cause this attribute. Vitamin C or ascorbic acid is believed to play a major role in creating a sour taste. However, this may be not true; for instance, the sour taste of Ma-mao fruits (Antidesma ghaesembilla Gaertn.) does not contain vitamin C. The sour taste may come from other substances, such as oxalic and citric acids [5].
Some organic acids have beneficial health effects; for instance, citric acid can inhibit kidney stone formation [6] [7]. Seltzer et al. [8] and Kang et al. [9] reported that 5.9 mg of citric acid significantly increases secretion of citrate in urine in kidney stone patients. Furthermore, malic acid can stimulate production of saliva in patients suffering from xerostomia symptoms [10] and succinic acid can improve postischemic cardiac function [11]. Alternatively, some types of organic acid are reported to negatively affect health. For instance, oxalic acid can form with the cation mineral to bring about kidney stones [12], while formic acid can cause acidosis [13]. Organic acids can also improve mineral absorption [14]. Greenfield & Southgate [15] stated that since organic acids provide 3 kcal/g, it should be included in energy calculations, if any, apart from protein, fat, and carbohydrate (4, 9 and 4 kcal/g, respectively).
To date, limited information exists on organic acids in indigenously consumed plant foods in Thailand and Asian countries where such plants are also eaten. Hence, this study's main purpose is to determine each organic and ascorbic acid, pH, and total acidity in Thai indigenous plants, especially since several such plants are now being commercially grown and can be found in markets, such as Ma-kham-pom (Phyllanthus emblica L.), Ma-kok-pa (Spondias pinnata [L.f.] Kurz), Ma-mao (Antidesma velutinosum Blume), and Pak-wan-pa (Melientha suavis Pierre). In addition, another aim is to evaluate the correlation of selected substances which contribute to the sour taste of plants.

Samples
In total, 40 samples (fresh and/or boiled depending commonly consumed method of each plant) of 29 indigenous plants were collected three times from two conservation areas in Kanchanaburi province (representative of the western region) and Amnatchareon province (representative of the northeastern region) of Thailand. They were identified and collected with only the edible part being selected for analysis. Each edible plant part was prepared for consumption using local preparation methods, i.e., fresh, blanching, boiling. The samples were categorized into six groups based upon the plant part that is commonly consumed, namely, young leaves, flower, fruit, pod, tuber, and young stem ( Table 1) However, each sample was analyzed in triplicate.

Sample Preparation
Each indigenous plant sample was cleaned twice with tap water and once with deionized water. In each source of sample, the edible part of each sample was collected, weighed, and divided into two portions. The first portion was prepared as a homogeneous sample for moisture analysis only. The other portion entailed cooked samples that were blanched or boiled depending on the commonly used preparation method. Thereafter, both raw and cooked samples were homogenized using a food processor (Mara®, Thailand) and dried using a freeze-dried system (Heto®, Power Dry PL9000 Freeze Dryer, Thermo Fisher Scientific). They were then weighed, ground, vacuum-packed in laminated aluminum foil bags, and stored at 20˚C until analysis.

Moisture Content Determination
Moisture content was analyzed by drying according to the Official Analytical Chemists, Method 927.05 [16] [17]. The homogenized sample was weighed into an aluminum container and dried in a hot air oven at 100˚C ± 1˚C until constant weight. The results were expressed as gram per 100 g of fresh weight (FW).

Organic Acid Determination (Oxalic Acid, Citric Acid, Malic Acid, Succinic Acid, Formic Acid, Acetic Acid)
Organic acids were extracted from 1 g of finely ground-dried sample with 50 mL 2M HCl at temperature 80˚C for 15 minutes. They were centrifuged at 3000 rpm for 15 minutes. Supernatant was passed through a filter of 0.45 µm cellulose acetate membrane. Organic acid contents were determined by the HPLC method. A 20 µL of filtrated sample was injected into the HPLC system (Waters chromatography system, Milford, USA) with UV detector (Waters® 486), set at 210 nm. The separation of each organic acid was carried out on a 300 × 7.8 mm Biorad Aminex ion exclusion column (HPX-87H), using an isocratic elution at 0.5 mL/min with 0.0125M sulphuric acid as a mobile phase. Figure 1 shows a chromatogram corresponding to a six-component mixture of standards and an indigenous sample. One can observe the good resolution and separation of the identified organic acids in a real sample. All samples were analyzed in triplicate. Organic acid contents were calculated against their standard curves (calibration of mix cocktail standards) [18] [19].

Ascorbic Acid Determination
Ascorbic acid or vitamin C was determined using the HPLC method [20]. In brief, each sample was extracted in 3% metaphosphoric acid and the homogenate was filtered. Ascorbic acid was separated by reversed-phase HPLC with UV detection at 248 nm (Agilent® 1100 series, USA) and quantified against external standards.

Total Acidity and pH Determination
Total acidity and pH were determined by the potentiometric method using a B. Sukontaprapun et al. glass electrode according to the AOAC Method 942.15B [16] [17]. Each sample was dissolved with deionized water, stirred, measured for pH value, and then titrated to the range of pH 8.10 ± 0.2. Total acidity was calculated, as percentage, based on each type of dominant organic acid in the sample.

Quality Control System
For precision in the organic acid analysis, Cha-moung leaves were dried at 50˚C ± 1˚C until dryness then ground into fine particles. They were then used as in-house quality control (QC) samples for representative organic acids, since these leaves are known to contain a significant amount of oxalic acid and several other organic acids. The assigned values of oxalic were developed from duplicate analysis of 10 single samples of the in-house QC samples on 10 different days. The QC sample was then analyzed along with the unknown samples in each run of the organic acid measurement over time. Due to a lack of an available certified reference material, spike standard organic acids in a QC sample were performed (n = 10) to demonstrate accuracy. Limit of detection (LOD) and limit of quantitation (LOQ) were studied using spiked lowest concentration of mixed standard into the QC sample and then calculated as 3 and 10 times of standard deviation from 10 times analysis, respectively.

Quality Control System
The Cha-moung (Garcinia cowa Roxb.) dried leaf, as the in-house QC sample, American Journal of Plant Sciences contained a high amount of oxalic acid (1120 ± 103 mg/100g of fresh weight, FW). The organic acid content in each analytical set stayed within the acceptable range (mean ± 2 standard deviation of the assigned value). Recoveries of oxalic acid added (10 mg/g of sample) prior to sample extraction were performed in both the in-house QC sample and the dried food samples. Percentages of recovery of oxalic acids ranged from 90.6% to 99.4% (Table 2) which stayed in the acceptable recovery range (90% -107%) [17]. LOD of oxalic, citric, malic, succinic, formic, and acetic acids were 0.9, 2.2, 2.0, 0.2, 0.1, and 3.0 mg/100g, respectively. LOQ of oxalic, citric, malic, succinic, formic, and acetic acids were 3.0, 7.3, 12.0, 0.5, 0.5, and 9.0 mg/100g respectively.

Moisture and Organic Acids
The major component in the studied plants was water, ranging from 69.9 g/100g to 97.4 g/100g FW (Tables 3-6). In general, organic acids can be divided into two groups. The first group entails organic acids that have been reported to have negative health effects, such as oxalic acid. The second group comprises those that provide health benefits, such as citric, malic, succinic, and acetic acids. Results and discussion for each organic acid are presented in Tables 3-6.     [18], Judprasong et al. [19], and Virginia et al. [21], which reported that cooking in water can lead to the leaching of soluble oxalate into the water thus reducing the oxalate content in foods.  [22] presented values of oxalic acid content higher than those exhibited in this study. and 295 ± 10 mg/100g FW, respectively.

Citric Acid
Citric acid has both positive and negative health benefits. One positive benefit of citric acid is that it can prevent kidney stone formation and can break up the formation of small kidney stones [6] [7]. Moreover, Seltzer et al. [8] and Kang et al. [9] reported that 5.9 mg of citric acid significantly increases secretion of citrate in urine when it used as a medical therapy in kidney stone patients. Most  [24] that noted that unripe fruits had higher citric acid content than ripe fruits in two species of the family Averrhoa bilimbi and star fruits.

B. Sukontaprapun et al.
For parts of consumed leaves, Kra-don (Careya sphaerica Roxb) and Ma-ra-pa (Momordica charantia) are the two plants in this group that contained citric acid content higher than 100 mg/100g FW. For flowers of consumed plants, blanched and fresh flowers of the Pak-wan-pa (Melientha suavis Pierre) contained citric acid levels at 207 ± 11 and 167 ± 8 mg/100g FW, respectively. For tubers of consumed plants, fresh tubers of Ka-min-pa (Curcuma sp.) and boiled tubers of Kra-chai-pran (Zingiber citriodorum J. Mood and T. Theleide) had citric acid levels at 133 ± 24 and 159 ± 12 mg/100g FW, respectively.

Malic Acid
One benefit of malic acid is that it stimulates the production of saliva that can improve antihypertensive-induced xerostomia [10]. Kra-don (Careya sphaerica Roxb.) had the greatest amount of malic acid, especially in certain parts of leaves and flowers (1381 ± 986 and 952 ± 138 mg/100g FW, respectively). The taste of this leaf and flower is tart, similar to the taste of malic acid [25]. For parts of leaves consumed, fresh young leaves of Tew-deang (Cratoxylum formosum subsp. Pruniflorum [Kurz] Gogel.) and Tew-kaw (Cratoxylum formosum [Jack] Dyer) also contained the highest malic acid content (1361 ± 61 and 1083 ± 12 mg/100g FW, respectively) when compared to other studied plants. Fresh flowers of Pak-wan-pa (Melientha suavis Pierre) had a high amount of malic acid (777 ± 16 mg/100g FW, respectively); whereas it had a moderate content in fruits and blanched leaves (319 ± 15 and 291 ± 4 mg/100g FW). A study on the effect of minerals in sand cultures on the malic acid content of Valencia orange leaf tissue demonstrated that the high calcium level in sand culture positively influences increasing malic acid content [26].

Succinic Acid
No studies have reported on the content of succinic acid in young plant leaves.
Succinic acid is assumed not to be a main acid in plants [27]. However, this study found a very high amount of succinic acid in fresh young leaves of Tew-daeng (Cratoxylum formosum subsp. Pruniflorum [Kurz] Gogel.), Tew-kaw (Cratoxylum formosum [Jack] Dyer) and Tew-mon (Cratoxylum cochinchinense [Lour.] Blume) (2452 ± 91, 2330 ± 101, and 1689 ± 145 mg/100g FW, respectively). These three plants are in the family of Guttiferae, which generally has a sour taste. Sakamoto et al. [11] reported that one benefit of succinic acid is that its salt improved post ischemic cardiac function, which can guard against ischemia/reperfusion cardiac injury. On the other hand, other parts of plants consumed contained low succinic acid contents.

Formic Acid
Formic acid is not a major organic acid in plants. It is mainly founded in coffee [28]. In this study, fresh fruit of Ma-kok-pa (Spondias pinnata (L.f.) Kurz) contained the highest amount of formic acid (1507 ± 298 mg/100g FW). Fresh young leaves of Ta-keuk (Albizia lebbeck (L.) Benth) and Song-fa (Clausena wallichii Oliv. var. guillauminii) had moderate amounts of formic acid (326 ± 57 and 198 ± 13 mg/100g FW, respectinvely). Interestingly, fresh young fruits of Ma-mao (Antidesma velutinosum Blume) had a very low amount of formic acid (Not detectable, ND), but the amount of formic acid increased during the ripening process (115 ± 15 mg/100g FW). For other parts of consumed plants, most contained low or no amounts of formic acid.

Sum of Organic Acids Contribution to Energy
Organic acids have been used as a component in calculating total energy apart from protein, fat, and carbohydrate, by providing 3 kcal/g [15]. Only some fermented foods and foods containing high amounts of total organic acids could affect the energy levels of those foods. Limited information exists in the different food composition databases around the world concerning energy from organic acids as part of total energy.

Correlation between Organic Acid and Other Parameters
According to traditional belief, sour-tasting food is probably high in vitamin C or ascorbic acid. However, this belief may not always be true, because organic acids in a plant may also play an important role in causing a sour taste. Pearson's coefficient correlations (r) were used here due to the common and complex interactions between all parameters related with the sour taste (Table 7). In general, it is well-known that lower pH and high total acidity could indicate a sour taste in food. Results from this study demonstrated that the sum of organic acids in the studied plants presented significant and inverse correlations with pH (r = −0.680) and the total acidity positive correlations (r = 0.672) but were not significantly correlated with ascorbic acid (r = 0.536). For all individual organic acids, only oxalic acid had a significantly positive correlation with ascorbic acid (r = 0.670), whereas a study by Suarez et al. [30] reported a negative correlation (r = −0.156). The pH is strongly inversely correlated with total acidity (r = −0.913) which agrees with the trend in the Suarez et al. [30] study in different cultivars of tomatoes (r = −0.384). The pH also had a notable negative correlation with the sum of organic, citric, and formic acids by order of r = −0.680, −0.660, and −0.595, respectively, but was not correlated with ascorbic acid (r = −0.494). Total acidity presented a significantly positive correlation with the sum of organic, citric, formic, and ascorbic acids (r = 0.672, 0.648, 0.539, and 0.570, respectively).
In terms of the sour taste in plants, as indicated by low pH and high total acidity, this finding suggests that this taste could come from a sum of organic, citric, and formic acids, but not from ascorbic acid.

Conclusion
Forty samples from 29 indigenous plants were collected three times from two conservation areas in Thailand. They were analyzed for organic acids (citric, malic, succinic, formic, acetic, and oxalic acids), ascorbic acid, pH, and total acidity, and evaluated in terms of the relationship of sour taste, using low pH and high total acidity, amongst other parameters. Young leaves in the family of Guttiferae, which has a strong sour taste, such as Tew-mon (Cratoxylum cochinchinense [Lour.] Blume), Tew-kaw (Cratoxylum formosum [Jack] Dyer), and Tew-deang (Cratoxylum formosum subsp. Pruniflorum [Kurz] Gogel) contained a high sum of organic acid, especially succinic, high ascorbic acid, and low pH. On the other hand, fresh fruits of Ma-kok-pa (Spondias pinnata [L.f.] Kurz), and Ma-mao (Antidesma velutinosum Blume), which has a strong sour taste, contained high amounts of sum organic acids, especially oxalic acid, total acidity, and low pH, but low ascorbic acid. This correlation of sour taste, indicated by low pH and high total acidity, indicates that the sour taste of plants could mainly come from the sum of organic, citric, and formic acids, but not only from ascorbic acid. This study is the first to provide database information for organic acids in Thai plants and especially in terms of the correlation of organic acid to the sour taste. This study also showed that the energy from organic acid of plants minimally contributes to recommended energy.