Antioxidant and Compositional HPLC Analysis of Three Common Bamboo Leaves

Bamboo leaves of Phyllostachys nigra (PN), Lophatherum gracile (LG), and Pleioblastus amarus (PA) are three common herbs in China. In this work, a new high performance liquid chromatography (HPLC) method for the simultaneous determination of seven compounds in bamboo leaves has been developed; and PN, LG, and PA leaves were analyzed. PN showed four times as much chlorogenic acid (CA) than the other two, and contained the most isoorientin (iso-ORI) and isovitexin (iso-VIT) as well. The PA presented the most orientin (ORI) and LG covered a majority of cynaroside (CYN). We measured the antioxidant activity by scavenging the stable 2,2-diphenyl-1-pyridinohydrazinyl (DPPH) free radicals, and found that Luteolin (inhibitory concentration (IC)50 = 0.42 µM, LUT) and CYN (IC50 = 0.43 µM) showed 2–3 times higher antioxidant activity than iso-ORI (IC50 = 0.81 µM), ORI (IC50 = 0.84 µM), and other related antioxidant standards such as trolox (IC50 = 0.97 µM) and ascorbic acid (IC50 = 0.93 µM, VC). Among extracts, PN and PA showed considerable antioxidant activity, which was related well with the contents of CA, iso-ORI, and iso-VIT (p < 0.05). This study firstly provides evidence for functional antioxidant compounds of bamboo leaves based on statistical analysis of the HPLC analysis and DPPH assay, and it lays a foundation for its further development or utilization.


Introduction
China, the world's leading bamboo producing country, is rich in bamboo resources and has more than 500 genus of more than 500 species of bamboo [1]. The "Chinese Traditional Medicine Resources Guide" lists more than 30 species of medicinal bamboo [2,3] including Lophatherum gracile (LG), Pleioblastus amarus (PA), and Phyllostachys nigra (PN), among others. The medical stems and leaves of Lophatherum gracile were usually called "Dan Zhu Ye", the leaves of Pleioblastus amarus were commonly named "Ku Zhu Ye", and "Zhu Ye" was often used to define the leaves of Pleioblastus amarus and other bamboo plants. All three types of bamboo leaves have an extremely long history of medicinal and edible use in China [3]. The LG are the only medicinal bamboo contained in the Pharmacopoeia of the People's Republic of China (hereinafter referred to as the "Pharmacopoeia"). It was first recorded in "Materia Medica of Southern Yunnan" in the Ming Dynasty. "Compendium of Materia Medica" for the first time, described in detail the morphological characteristics of LG, "Pharmacopoeia" was included in 1963 [4,5]. It has the functions of clearing heat and purging fire, removing annoyance and quenching thirst, as well as anti-inflammatory, antibacterial, antiviral, diuretic, and other functions. It is used to The stability was evaluated by measuring and comparing the peak area of one sample at 0, 1,2,4,8,12, and 24 h. The standard solutions, at three different contents (50%, 100%, and 150%), were added into known extracts in recovery test. Further, the mean recovery was calculated using the following formula: Recovery (%) = 100 × (amount found − original amount)/amount spiked. (1) The recovery test results were in the range from 92.90% to 122.32%.

HPLC Analysis of Sample
Quantitative analysis of seven components was performed on each specimen of bamboo leaves. The method specificity was assessed by comparing the consistency of the retention time between a sample and corresponding reference standard. Figure 1 showed a typical separation of Lophatherum gracile (LG), Pleioblastus amarus (PA), and Phyllostachys nigra (PN) leaf-extracts (A)-(C), and a standard mixture (D) under the optimized chromatographic conditions. All markers were well resolved from background peaks within the analysis time and the seven peaks in the chromatogram of LG, PA, and PN could be identified by the corresponding standards. As listed in Table 3 (results expressed as mean ± SD), PN indicated a four times higher content of CA than the other two, and contained the most iso-ORI and iso-VIT as well. PA presented the highest content of ORI and LG covered a majority of CYN. Figure 2 presented the average relative abundance of compounds in leaves of LG, PN, and PA.
Molecules 2020, 25, x FOR PEER REVIEW 4 of 15 mean ± SD), PN indicated a four times higher content of CA than the other two, and contained the most iso-ORI and iso-VIT as well. PA presented the highest content of ORI and LG covered a majority of CYN. Figure 2 presented the average relative abundance of compounds in leaves of LG, PN, and PA.

DPPH Assay and Statistical Analysis
Before the DPPH assay, dry ointment yield of extracts was obtained. The antioxidant inhibitory concentration (IC)50 values of 26 extracts, 7 compounds, and 2 positive antioxidants were evaluated by scavenging DPPH free radical. These results were listed in Tables 4 and 5. LUT (IC50 = 0.42 µM) and CYN (IC50 = 0.43 µM) showed 2-3 times higher antioxidant activity than iso-ORI (IC50 = 0.81 µM), ORI (IC50 = 0.84 µM), and other related antioxidant standards such as trolox (IC50 = 0.97 µM) and ascorbic acid (IC50 = 0.93 µM). Among extracts, LG yielded the weakest antioxidant activity, DPPH free radical scavenging IC50 values of LG varied from 2.10 to 10.17 mg·mL −1 , PA and PN showed a considerable and stable ability to scavenge the DPPH free radical (1.25 to 5.07 mg·mL −1 and 1.59 to 14.64 mg·mL −1 , respectively) as shown in Figure 3, and the activity of LG was significantly different from that of PA and PN by one-way analysis of variance (ANOVA) (p < 0.05).

DPPH Assay and Statistical Analysis
Before the DPPH assay, dry ointment yield of extracts was obtained. The antioxidant inhibitory concentration (IC) 50 values of 26 extracts, 7 compounds, and 2 positive antioxidants were evaluated by scavenging DPPH free radical. These results were listed in Tables 4 and 5 Figure 3, and the activity of LG was significantly different from that of PA and PN by one-way analysis of variance (ANOVA) (p < 0.05). As a result of the correlation analysis ( Table 5), contents of CA (r = −0.508, p < 0.01), iso-ORI (r = −0.643, p < 0.01), and LUT (r = 0.431, p < 0.05) were significantly correlated with the IC 50 of PA extracts, the activity of PN extracts was found in relation to the content of ORI (r = −0.501, p < 0.01), and LG was associated with the content of API (r = −0.491, p < 0.05). Because of a significant correlation between the content of each component (p < 0.05), the stepwise regression method was used in our analysis. We found that the content of iso-ORI and total content of seven compounds jointly explained 53.4% of the variation in the activity of PA extracts, which means iso-ORI is the main active substance in PA. The main antioxidant compounds in PN is ORI (R 2 = 21.9%), and the activity of LG extracts was influenced greatly by the contents of ORI, CYN, and API (R 2 = 95.9%).   Besides, as shown in Figure 4, as a result of correlations between observed and expected antioxidant activities based on established multiple regression equation (Table 6) As a result of the correlation analysis ( Table 5), contents of CA (r = −0.508, p < 0.01), iso-ORI (r = −0.643, p < 0.01), and LUT (r = 0.431, p < 0.05) were significantly correlated with the IC50 of PA extracts, the activity of PN extracts was found in relation to the content of ORI (r = −0.501, p < 0.01), and LG was associated with the content of API (r = −0.491, p < 0.05). Because of a significant correlation between the content of each component (p < 0.05), the stepwise regression method was used in our analysis. We found that the content of iso-ORI and total content of seven compounds jointly explained 53.4% of the variation in the activity of PA extracts, which means iso-ORI is the main active substance in PA. The main antioxidant compounds in PN is ORI (R 2 = 21.9%), and the activity of LG extracts was influenced greatly by the contents of ORI, CYN, and API (R 2 = 95.9%).
Besides, as shown in Figure 4, as a result of correlations between observed and expected antioxidant activities based on established multiple regression equation (Table 6)

Discussion
Bamboo leaf has been used as an antioxidant material to prevent food deterioration since ancient times. Studies indicated that flavonoids, polysaccharides, and phenolic acids might be the major active constituents [30,31]. Crude extracts and pure compounds from the leaves have been shown to possess multiple biological activities such as anti-inflammatory, antivirus, cardiovascular protection, cancer prevention, and particularly antioxidant activity [32]. However, as far as chemical constituents are concerned, pharmacological reports most often attribute some activities to various extracts rather than specific compounds. This makes it difficult to build a connection between the chemical constituents and pharmacological activities, resulting in the difficulty of identifying the major active compounds. Besides, bamboo leaves are various in varieties and confused in classification, and thus many scientific questions remain, which need to be answered through further laboratory research and clinical trials.
First, we developed a new HPLC method for the quantification of seven main active components in bamboo leaves simultaneously. Several chromatographic conditions using different elution systems were tested according to optimum resolution, peak shape, and efficiency of samples. Conditions were ensured with a Diamonsil Plus C18 column (4.6 × 250 mm, 5 µm) and a water/methanol mixture containing 0.1% (v/v) formic acid elution system for the detection of seven compounds. Formic acid (0.1%) was selected as the mobile phase additive to provide improved pH control, simplified preparation, and was found to achieve good peak intensity and resolution. Our preliminary experiments have measured individual maximum UV absorbance of CA, iso-ORI, ORI, iso-VIT, CYN, LUT, and API at 324, 350, 346, 336, 350, 350, 332, and 346 nm, respectively. To achieve optimal UV absorbance for all references, 350 nm was selected and demonstrated a high degree of sensitivity and precision. The method was applied successfully for the analysis and identification of the components of the three bamboo leaves. Then, the antioxidant activity of six flavonoids, one phenolic acid, and methanol extracts from bamboo leaves of LG, PN, and PA were detected by DPPH.
According to Chinese Pharmacopoeia published in 2015, there is no standard assay for determining the contents of LG. In fact, HPLC fingerprints could be used to improve quality control. Assays of biological activities could be applied as well. In addition, studies on this medicinal plant were presently limited to the antioxidants and preservatives, even though ancient books report that the flesh can clear heat and remove vexation. More efforts should be made to fill the blank.

Reagents and Chemicals
Chlorogenic acid, isoorientin, and apigenin were purchased from Shanghai Natural Biological Technology Co., Ltd. (Shanghai, China). Iso-vitexin, cynaroside, and luteolin were obtained from Shanghai Yuanye Biological Technology Co., Ltd. (Shanghai, China). Orientin was bought from Chengdu Push Biological Technology Co., Ltd. (Chengdu, China). The purity of all standards was more than 98%, and their chemical structures are listed in Figure 5. DPPH was gained from Sigma-Aldrich (St. Louis, MO, USA). Trolox was from Beyotime Institute of Biotechnology Co., Ltd. (Shanghai, China). Ascorbic acid (VC) was ordered from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Diamonsil Plus C18 column (4.6 × 250 mm, 5 µm) was used. All liquid chromatography solvents (methanol, acetonitrile) were of HPLC grade, obtained from Anhui Fulltime Specialized Solvent and Reagent Co., Ltd. (Anqing, China), and used after filtration by a 0.45 µm organic membrane. All other solvents were of analytical grade and used without further purification.

Plant Materials
Twenty-six commercial herbal samples (Table 7) of Phyllostachys nigra, Lophatherum gracile, and Pleioblastus amarus were collected via purchase from commercial Chinese markets and authenticated by Dr. Long Song from Shanghai University of Traditional Chinese Medicine. The air-dried samples were smashed into powder (65 mesh) and stored in a desiccator.

Plant Materials
Twenty-six commercial herbal samples (Table 7) of Phyllostachys nigra, Lophatherum gracile, and Pleioblastus amarus were collected via purchase from commercial Chinese markets and authenticated by Dr. Long Song from Shanghai University of Traditional Chinese Medicine. The air-dried samples were smashed into powder (65 mesh) and stored in a desiccator.

Sample Solutions
Each powdered material (1.0 g) was accurately weighed and extracted with 25 mL of methanol under ultrasonication for 30 min followed by centrifugation. The supernatant solutions were filtered through a 0.22 µm filter membrane prior to injection.

Preparation of Solutions for DPPH Assay
Twenty-six extracts were prepared as the sample preparation for HPLC analysis. The seven characteristic compounds, as well as positive antioxidants including VC and trolox, were configured to be 20 mM with DMSO. All samples were evaluated by DPPH radical scavenging activity, and 50% methanol was used as the blank solution. For the DPPH assay, we have some modifications on the basis of reported methods [15]. Samples of 10 µL at various concentrations were added into 100 µL of DPPH solution (0.65 mM, methanol) and 140 µL of 50% methanol. After one hour of incubation at room temperature in the dark, the absorbance of mixtures (test samples, control samples, blank samples) was recorded at 517 nm. The scavenging rates (SR) were calculated based on the following formula: where Abs blank and Abs sample or control represent the absorbance of the blank sample, test sample, and control sample, respectively.

Statistical Analysis of Content and Antioxidant Activity
The correlation between the content of components and antioxidant activity of extracts was explored by reported multivariate regression analysis [33]. All statistical analyses were performed with the SPSS Statistics (SPSS vision 21, IBM, Camp Takajo, NY, USA).