Study on the identification of Pinelliae rhizoma and Pinelliae pedatisectae rhizoma based on the characteristic component triglochinic acid

For the first time, a monomeric compound, triglochinic acid, has been isolated from the tubers of Pinellia pedatisecta Schott with its structure analyzed using NMR. Its molecular formula is C7H8O6, with the chemical name (2E)-but-2-ene-1,2,4-tricarboxylic acid. Through HPLC-DAD and HPLC-MS analysis studies, it has concluded that Pinelliae rhizoma does not contain triglochinic acid, while Pinelliae pedatisectae rhizoma does. This key observation was used as a characteristic component to distinguish these two herbs. We analyzed 39 batches of Pinelliae rhizoma collected in herbal medicine market, among which triglochinic acid was detected in 27 batches, resulting in a adulteration ratio of Pinelliae rhizoma reaching 69.2%. Our method demonstrates great potential for authenticating the products, thus ensuring the quality of Pinelliae rhizoma.


Introduction
Pinelliae rhizoma is the dry tubers of Pinellia ternata (Thunb.) Breit. 1 It was documented as Diwen or Shuiyu in Shennong's Materia Medica (Shen Nong Ben Cao Jing), and other medical classics aerwards. During ancient times, counterfeit or fake products of Pinelliae rhizoma existed, such as Arisaema ringens (Thunb.) Schott in Tang Ben Cao by Su Gong and Pinellia pedatisecta Schott in Tu Jing Ben Cao. In modern times, Pinelliae rhizoma in rural areas was replaced by herbs of the same genus like Typhonii agelliformis and Arisaema heterophyllum Blume according to Modern Chinese Materia Medica. 2 Recently, Pinelliae rhizoma was discovered to be mixed with a large amount of Pinelliae pedatisectae rhizoma in the market, which greatly affected the quality of the herbs. Pinelliae pedatisectae rhizoma, also known as the palm leaf Pinellia and South Star, is the dry tubers of Pinellia pedatisecta Schott, documented in the Chinese Materia Medica Standards of Shandong, Hubei and Jiangsu, [3][4][5] as well as in the Dictionary of Traditional Chinese Medicine 6 and Chinese Flora. 7 Pinelliae rhizoma and Pinelliae pedatisectae rhizoma are both of the Pinellia genus, which can easily be identied by the leaves and owers. 7 However, their medicinal materials are extremely similar. Pinelliae rhizoma is spheroidal in shape, while Pinelliae pedatisectae rhizoma is also spheroidal with several small bulbs alongside. If the small bulbs of Pinelliae pedatisectae rhizoma are not yet formed or removed during processing, the herbs will be very similar to Pinelliae rhizoma, which makes them difficult to distinguish. Pinelliae rhizoma enjoys a wide range of clinical applications, being the raw material in 489 patent traditional Chinese medicine 8 and in 3029 prescriptions. 9 The growing period of Pinellia ternata is 4-5 months with high cost and low yield, and its market price is 85-120 yuan per kilogram. On the other hand, the clinical application of Pinelliae pedatisectae rhizoma is limited, only used in a few prescriptions. Its lifespan is shorter, about 3-4 months with lower cost, higher yield and the price is 40-50 yuan per kilogram. Therefore, the interests drive people to incorporate Pinelliae pedatisectae rhizoma into Pinelliae rhizoma.
Nowadays, the identications of Pinelliae rhizoma, Typhonii agelliformis rhizoma and Arisaematis rhizoma are mostly based on morphology identication, thin layer chromatography identication, and ngerprint identication. [10][11][12] However, the identication studies on Pinelliae rhizoma and Pinelliae pedatisectae rhizoma are very limited. In particular, it is hard to identify the incorporation of Pinelliae pedatisectae rhizoma in Pinelliae rhizoma. From 2016 to 2017, we collected Pinelliae rhizoma from several herbal medicine markets: Hehuachi, Anguo, Bozhou, Qingping, Yulin, etc. However, more than 60% of the samples were counterfeit. This situation urges us to establish an effective and rapid analytical method that can detect fake herbs and improve the quality control of Pinelliae rhizoma, meanwhile laying the foundation of a standard herbal medicine market.

Medicinal materials
From Sichuan, Gansu, Guizhou, Hebei, Shanxi, Chongqing and other provinces and cities, we collected 28 batches of Pinelliae rhizoma. Also from Hebei, Heilongjiang, Gansu, Sichuan and other places, we collected 16 batches of Pinelliae pedatisectae rhizoma. All the materials were determined by Professor Min Li from College of Pharmacy, Chengdu University of Traditional Chinese Medicine. Information of these samples is shown in Table 1. Photos of Pinelliae rhizoma and Pinelliae pedatisectae are shown in Fig. 1.
In addition, 39 batches of commercial Pinelliae rhizoma were collected from the herbal medicine markets of Hehuachi Chengdu, Anguo Hebei, Yinzhou Anhui, Qingping Guangdong, and Yulin Guangxi. Information of the commercial Pinelliae rhizoma samples is shown in Table 2.

Methods and results
Aer a systematic study on the chemical components of Pinelliae rhizoma and Pinelliae pedatisectae rhizoma, we discovered a characteristic peak of Pinelliae pedatisectae rhizoma in the HPLC chromatogram of organic acid part, distinguishable from that of Pinelliae rhizoma.

Extraction, separation and purication of the characteristic component
Take the powder of Pinelliae pedatisectae rhizoma (aer passing the no. 4 sieve), and add 10 times the amount of water. Sonicate the sample for 3 times, 1 hour each time. Then, combine the extracts and add phosphoric acid, 1% of the extract volume. Shake and add an equal volume of ethyl acetate for extracting 3 times. Recover the ethyl acetate to obtain the concentrated liquid before dilute it with water. Pass through the XB-C 18 (80 Â 250 mm, 10 mm) preparative column at ow rate of 140 mL per minute, using 5% acetonitrile (containing 0.2% phosphoric acid solution) as the mobile phase, collect the eluent from 19.5 to 22.6 minutes. Then, recover the solvent and change the mobile phase to 5% acetonitrile (containing 0.1% formic acid solution). Use the column again and collect the eluate from 17.4 to 20.3 minutes. Finally, lyophilize the eluate under reduced pressure to obtain the white compound in solid form.

Structural identication of the characteristic component
We used NMR to analyze the structure of the white compound. The data was listed in   The nuclear magnetic data of this compound is generally consistent with that of triglochinic acid as reported in the literature. 13 Therefore, this compound was identied as triglochinic acid, a white powdery solid, soluble in water and methanol, with the molecular formula C 7 H 8 O 6 . The chemical name is (2E)-but-2-ene-1,2,4-tricarboxylic acid, CAS number: 31795-12-7. Its relative molecular mass is 188.13, boiling point (557.3 AE 50) C. The structure of triglochinic acid is depicted in Fig. 2.

Selection of the detection wavelength
With full-wavelength ultraviolet scanning, the maximum absorption wavelength of triglochinic acid is 210 nm. Therefore, 210 nm was selected as the detection wavelength. The spectrum is shown in Fig. 3.

Solution preparation
Preparation of the reference solution: accurately weigh an appropriate amount of triglochinic acid. Add water to make the reference solution with a concentration of 0.25 mg mL À1 . Preparation of the test solution: accurately weigh about 1 g of the sample powder (aer passing through the no. 4 sieve) and place it in an Erlenmeyer ask with 20 mL of water. Weigh before and aer 45 minutes of sonication (250 W, 40 kHz), add the lost weight with water. Filter the solution and transfer 10 mL of the ltrate to a 50 mL centrifuge tube. Add 0.1 mL phosphoric acid and shake, then add 20 mL ethyl acetate and shake. Centrifuge (5000 rpm) the solution, and aspirate the upper liquid ethyl acetate. Extract acid solution with ethyl acetate for three more times, 20 mL each time. Then combine the ethyl acetate solution and evaporate the solvent to dry under reduced pressure. Add 2 mL acetonitrile-0.1% phosphoric acid (1 : 99) into the residue to dissolve. Finally, lter it through a microporous membrane (0.45 mm) and obtain the ltrate.

Determination method
Inject 10 mL of the reference solution and test solution into HPLC, record the chromatogram. 3.7.4 Detection limit. Take the reference solution, and dilute it to 0.10 mg mL À1 with water. Accurately inject 10 mL into the HPLC, and calculate the signal-to-noise ratio (S/N ¼ 3 as the detection limit). The detection limit was 545.5 mg kg À1 (Table 4).

Sample tests
28 batches of Pinelliae rhizoma, 16 batches of Pinelliae pedatisectae rhizoma and 39 batches of commercial medicinal materials were prepared according to the method in 3.5, and determined following the chromatographic conditions in 3.4. If there is a peak consistent with the retention time of triglochinic acid, and the absorption spectrum of the corresponding chromatographic peak is the same within the wavelength range of 190-400 nm by the diode-array detector, the sample will be identied as Pinelliae pedatisectae rhizoma. The results were shown in Table 5.
The results showed that triglochinic acid was not detected in the 28 batches of Pinelliae rhizoma, while detected in all the 16 batches of Pinelliae pedatisectae rhizoma. However, triglochinic acid were detected in 27 of the 39 batches of the commercial samples. Therefore, the rate of fake products of Pinelliae rhizoma was 69.2%.
3.9 HPLC-MS verication of triglochinic acid in the samples 3.9.1 Liquid chromatographic conditions. The Agilent 1290-6460 LC/MS was used with Agilent ZORBAX Eclipse Plus C 18 column (2.1 Â 50 mm, 1.8 mm) and the mobile phase of methanol-0.02% ammonia solution (5 : 95) at 0.15 mL min À1 ow rate, 45 C column temperature. The injection volume was 2 mL.
3.9.2 Mass spectrometry conditions. Multi-reaction monitoring (MRM) was performed with a mass spectrometer detector, in electrospray negative ion mode (ESI À ) at 3500 capillary voltage. Flow rate of dryer was 9 L per minute, temperature of dryer was 280 C, 40 psi Nebulizer, and 0 V Fragmentor (secondary). The parameters of triglochinic acid were shown in Table 6.
3.9.3 Preparation of the solution. In the preparation of the test solution, the acidifying reagent phosphoric acid was replaced by formic acid, while the remaining was as same as in 3.5.
3.9.4 Results verication. The multi-reaction detection of ion ratios for triglochinic acid, Pinelliae rhizoma, and Pinelliae pedatisectae rhizoma samples were shown in Table 7. The molecular ion peaks of Pinelliae pedatisectae rhizoma and triglochinic acid were the same (187), which is consistent with the mass of triglochinic acid ion (C 7 H 7 O 6 À ). The molecular ion peaks were further conrmed by MS/MS, showing that the secondary ions were the same: 143 and 99.
According to the results of HPLC-MS, the peak retention time detected in Pinelliae pedatisectae rhizoma was consistent with that of triglochinic acid. Furthermore, the mass-to-charge ratio of the selected two pairs of daughter ions were consistent. The relative abundance of qualitative ions of Pinelliae pedatisectae rhizoma and triglochinic acid were within the range of tolerance (AE20%), 14,15 while their retention times were also consistent. Since the triglochinic acid peak was not detected in Pinelliae rhizoma, it can be veried that Pinelliae rhizoma does not contain triglochinic acid, while Pinelliae pedatisectae rhizoma does.

Discussion
Pinelliae pedatisectae rhizoma and Pinelliae rhizoma belong to the same genus, and share similar characteristics. The processed or young tubers of Pinellia pedatisecta are sold as Pinelliae rhizoma in herbal medicine market. While the identi-cation has always been difficult to achieve. In addition, Pinelliae pedatisectae rhizoma in large size consists of the main tuber and a few small attached ones, resembling a tiger's claw. 16 Due to the factors like provenance, soil, pests and diseases, and cultivation, some trait variations occur to Pinelliae rhizoma as well: one or several small tubers appear around the major tuber, which is similar to the characters of Pinelliae pedatisectae rhizoma and is easily misidentied as Pinelliae pedatisectae rhizoma. Pinelliae rhizoma, with its various forms of prepared drug in pieces, is popular in clinical use. On the market, the products of Pinelliae pedatisectae rhizoma are sold as the counterfeit of Pinelliae rhizoma because aer processing, their color and surface characteristics further change. Especially aer slicing, Pinelliae rhizoma and Pinelliae pedatisectae rhizoma are almost indistinguishable from their appearances.
The chemical constituents of Pinelliae rhizoma and Pinelliae pedatisectae rhizoma are similar. Pinelliae pedatisectae rhizoma contains a variety of alkaloids, dipeptides, amino acids, organic acids, nucleosides, and polysaccharides, 17-20 the same does Pinelliae rhizoma except for dipeptides. [21][22][23][24][25][26][27] At present, morphological identication, thin layer chromatography iden-tication and ngerprint identication are the main identication methods for Pinelliae rhizoma. However, morphological identication faces great difficulty especially aer processing or slicing. Chen et al. 28 identied one more spot in Pinelliae rhizoma which was not detected in Pinelliae pedatisectae rhizoma by means of thin-layer chromatography, but this method could not determine whether Pinelliae rhizoma was incorporated or not. Lu 9 established the ngerprints of Pinelliae rhizoma and Pinelliae pedatisectae rhizoma, in which Pinelliae rhizoma had three more chromatographic peaks than Pinelliae pedatisectae rhizoma, but it was also unable to identify the incorporated Pinelliae rhizoma.
Triglochinin, which can be hydrolyzed into triglochinic acid, was found in the owers of Triglochin maritima, 29 the young leaves of Alocasia 30,31 and Ranunculaceae genus. 32 The Alocasia genus is toxic, and the whole plant contains cyanogenic glycoside. However, whether cyanogenic glycoside is the main substance causing the toxicity of Alocasia macrorrhizos is not fully understood. According to the literatures, cyanogenic glycoside itself is not toxic, but it can be degraded by b-glucosidase and a-hydroxynitrile lyase, thereby releasing the toxic hydrogen cyanide (HCN) as well as glucose and aldehydes or ketones, 33 resulting in toxic effects. The triglochinic acid was found in Pinelliae pedatisectae rhizoma, but whether it is toxic remains to be conrmed in future research. Pinelliae rhizoma and Pinelliae pedatisectae rhizoma are of the same genus Araceae, which is inherently toxic: mainly stimulating toxic effects, caused by the shared raphides and lectin proteins. 34 Furthermore, their efficacy and clinical applications are different. Therefore, the use should be strictly differentiated, and the safety of Pinelliae rhizoma mixed with Pinelliae pedatisectae rhizoma should be concerned as well.
In addition, the history of articial cultivation of Pinellia ternata is short. The seeds of Pinellia ternata are mostly wild,  sometimes being mixed with that of Pinellia pedatisecta, resulting in a small amount of Pinelliae pedatisectae rhizoma identied in Pinelliae rhizoma. Now it is highly necessary to strengthen the research on the seeds standard of Pinellia ternata, and control its quality from the source. In this study, for the rst time we isolated the triglochinic acid from Pinelliae pedatisectae rhizoma and established the HPLC identication method and LC-MS verication method. This method is stable, accurate, and widely applicable. It can be used for the identication of Pinelliae pedatisectae rhizoma in Pinelliae rhizoma materials or prepared drugs. As a supplement to the quality control of Pinelliae rhizoma in the Chinese Pharmacopoeia, this method effectively combats the situation of adulteration and counterfeiting in the market, protecting the interests of farmers, planting enterprises, merchants, and companies. It also promotes the quality control of Pinelliae rhizoma, effectively ensuring the safety of clinical use.

Conflicts of interest
There are no conicts to declare.