Total Alkaloids from Bamboo Shoots and Bamboo Shoot Shells of Pleioblastus amarus (Keng) Keng f. and Their Anti-Inflammatory Activities

The bamboo shoot of Pleioblastus amarus (Keng) Keng f. is a medicinal and edible plant product in China. In this study, the chemical composition of the total alkaloids from bamboo shoots and bamboo shoot shells of P. amarus (Keng) Keng f. (ABSP and ABSSP, respectively) were separated and investigated by UHPLC/QTOF-MS/MS. The results showed that a total of 32 alkaloids were extracted, with 15 common to both ABSP and ABSSP and 10 and 7 alkaloids distinct to ABSP and ABSSP, respectively. ABSP and ABSSP both decreased the lipopolysaccharide (LPS, 0.5 μg/mL)-induced nitric oxide (NO) production in RAW264.7 murine macrophages with half maximal inhibitory concentration (IC50) values of 78 and 55 μg/mL, respectively. We also found that ABSP and ABSSP (100 μg/mL) could decrease the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) at both mRNA and protein levels in LPS-exposed RAW264.7 cells. Moreover, 100 μg/mL of ABSP and ABSSP also significantly inhibited LPS-induced mRNA expression of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α). Additionally, ABSP and ABSSP (100 μg/mL) decreased the phosphorylation of extracellular regulated protein kinase (ERK) in LPS-stimulated RAW264.7 cells. Collectively, the total alkaloids from the bamboo shoots and shells of P. amarus exhibit anti-inflammatory effects in LPS-activated RAW264.7 cells through the inhibition of ERK signaling. This result can provide support for the medicinal use and further study of P. amarus.

A total of 32 alkaloids in the bamboo shoots and shells were identified according to accurate mass and the characteristic fragments at low and high collision energy. Table 1 shows the retention time, name, and characteristic fragment ions of the identified compounds. The bamboo shoots contained 25 alkaloids, and the bamboo shoot shells contained 22 alkaloids. The bamboo shoots and bamboo shoot shells shared 15 alkaloids, while the bamboo shoots contained 10 distinct alkaloids and the bamboo shoot shells contained 7 distinct alkaloids.

ABSP and ABSSP Inhibit LPS-Induced Nitric Oxide Production in RAW264.7 Cells
To test the anti-inflammatory effects of total alkaloids from bamboo shoots and bamboo shoot shells, we used RAW264.7 macrophages as models. To eliminate the interference of cytotoxic effects of ABSP and ABSSP on the detection of anti-inflammatory activity, we measured the effects of different concentrations of ABSP and ABSSP on the cell viability of RAW264.7 macrophages in the presence of LPS. Results show that treatment with ABSP or ABSSP (0-100 µg/mL) did not affect the viability of RAW264.7 cells ( Figure 3A). Thus, nontoxic concentrations from 20 to 100 µg/mL were used in the next studies.
To detect whether ABSP and ABSSP exhibited anti-inflammatory activity, we measured the effects of ABSP and ABSSP on LPS-induced release of nitric oxide (NO), a crucial inflammatory biomarker. Exposure to immunologic and inflammatory stimuli results in an overproduction of NO, which can trigger tissue injury via lipid peroxidation and DNA damage and cause chronic inflammation by upregulating the release of proinflammatory cytokines [28]. Thus, NO has been implicated as a pathogenic mediator in a variety of inflammatory diseases, such as rheumatoid arthritis (RA), Alzheimer's disease, and inflammatory bowel diseases (IBD) [29,30]. As shown in Figure 3B, both ABSP and ABSSP (20-100 µg/mL) dose-dependently suppressed LPS-induced NO production in RAW264.7 cells (p < 0.05-0.001); the inhibition rates of ABSP (100 µg/mL) and ABSSP (100 µg/mL) on NO production were 57% and 69%, respectively. The positive control BAY11-7082 also showed a potent inhibition of LPS-induced NO release in RAW264.7 cells, and the inhibition rate of BAY11-7082 at 5 µM was 92%. Therefore, both ABSP and ABSSP exhibited potent anti-inflammatory activities in vitro.

ABSP and ABSSP Inhibit LPS-Induced Nitric Oxide Production in RAW264.7 Cells
To test the anti-inflammatory effects of total alkaloids from bamboo shoots and bamboo shoot shells, we used RAW264.7 macrophages as models. To eliminate the interference of cytotoxic effects of ABSP and ABSSP on the detection of anti-inflammatory activity, we measured the effects of different concentrations of ABSP and ABSSP on the cell viability of RAW264.7 macrophages in the presence of LPS. Results show that treatment with ABSP or ABSSP (0-100 μg/mL) did not affect the viability of RAW264.7 cells ( Figure 3A). Thus, nontoxic concentrations from 20 to 100 μg/mL were used in the next studies.
To detect whether ABSP and ABSSP exhibited anti-inflammatory activity, we measured the effects of ABSP and ABSSP on LPS-induced release of nitric oxide (NO), a crucial inflammatory biomarker. Exposure to immunologic and inflammatory stimuli results in an overproduction of NO, which can trigger tissue injury via lipid peroxidation and DNA damage and cause chronic inflammation by upregulating the release of proinflammatory cytokines [28]. Thus, NO has been implicated as a pathogenic mediator in a variety of inflammatory diseases, such as rheumatoid arthritis (RA), Alzheimer's disease, and inflammatory bowel diseases (IBD) [29,30]. As shown in Figure 3B, both ABSP and ABSSP (20-100 μg/mL) dose-dependently suppressed LPS-induced NO production in RAW264.7 cells (p < 0.05-0.001); the inhibition rates of ABSP (100 μg/mL) and ABSSP (100 μg/mL) on NO production were 57% and 69%, respectively. The positive control BAY11-7082 also showed a potent inhibition of LPS-induced NO release in RAW264.7 cells, and the inhibition rate of BAY11-7082 at 5 μM was 92%. Therefore, both ABSP and ABSSP exhibited potent antiinflammatory activities in vitro. (B) RAW264.7 cells were pretreated with the indicated concentrations of ABSP or ABSSP and BAY11-7082 (BAY, 5 μM) for 2 h, and then exposed to LPS for an additional 24 h. Levels of NO in the culture medium were detected using Griess reagent. Data are expressed as mean ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with LPS.

ABSP and ABSSP Suppress the mRNA Expression of Proinflammatory Cytokines in LPS-Exposed RAW264.7 Cells
The inducible nitric oxide synthase (iNOS) enzyme is responsible for the overproduction of NO under immunogenic and inflammatory stimuli [31]. We thus investigated whether the inhibitory effects of ABSP and ABSSP on NO production were due to their ability to inhibit iNOS expression. Figure 4A shows that 20 and 100 μg/mL ABSP decreased the mRNA levels of iNOS in LPS-stimulated RAW264.7 cells by 14% and 42%, respectively, while treatment with 20 and 100 μg/mL ABSSP resulted in 21% and 70% reduction in iNOS mRNA levels (p < 0.05-0.001), respectively. Consistent (B) RAW264.7 cells were pretreated with the indicated concentrations of ABSP or ABSSP and BAY11-7082 (BAY, 5 µM) for 2 h, and then exposed to LPS for an additional 24 h. Levels of NO in the culture medium were detected using Griess reagent. Data are expressed as mean ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with LPS.

ABSP and ABSSP Suppress the mRNA Expression of Proinflammatory Cytokines in LPS-Exposed RAW264.7 Cells
The inducible nitric oxide synthase (iNOS) enzyme is responsible for the overproduction of NO under immunogenic and inflammatory stimuli [31]. We thus investigated whether the inhibitory effects of ABSP and ABSSP on NO production were due to their ability to inhibit iNOS expression. Figure 4A shows that 20 and 100 µg/mL ABSP decreased the mRNA levels of iNOS in LPS-stimulated RAW264.7 cells by 14% and 42%, respectively, while treatment with 20 and 100 µg/mL ABSSP resulted in 21% and 70% reduction in iNOS mRNA levels (p < 0.05-0.001), respectively. Consistent with their effects on iNOS transcription, repression of iNOS protein expression was observed in RAW264.7 cells treated with 100 µg/mL of ABSP and ABSSP by 34% and 64% (p < 0.01-0.001; Figure 4C,D), respectively. Cyclooxygenase 2 (COX2) is another key inducible enzyme that catalyzes the first and rate-limiting step in the formation of prostaglandin E2 (PGE2), which is an important proinflammatory biomarker [32]. Hence, COX2 is an attractive target for the development of anti-inflammatory agents [33]. As shown in Figure 4B, 20 and 100 µg/mL of ABSP or ABSSP diminished the transcription of COX2 into mRNA in LPS-exposed RAW264.7 macrophages by 17% and 40% or 22% and 67% (p < 0.05-0.001), respectively. Furthermore, treatment with 100 µg/mL ABSP or ABSSP reduced the LPS-induced protein expression of COX2 in RAW264.7 cells by 33% and 54% (p < 0.01-0.001), respectively. The resulting efficacy of BAY11-7082 (5 µM) in reducing iNOS and COX2 expression was more potent than that of ABSP or ABSSP. Taken together, ABSP and ABSSP exhibited anti-inflammatory activities by suppressing the expression of iNOS and COX2 at both transcription and translation levels.
Molecules 2019, 24 FOR PEER REVIEW 2 with their effects on iNOS transcription, repression of iNOS protein expression was observed in RAW264.7 cells treated with 100 μg/mL of ABSP and ABSSP by 34% and 64% (p < 0.01-0.001; Figure  4C, 4D), respectively. Cyclooxygenase 2 (COX2) is another key inducible enzyme that catalyzes the first and rate-limiting step in the formation of prostaglandin E2 (PGE2), which is an important proinflammatory biomarker [32]. Hence, COX2 is an attractive target for the development of antiinflammatory agents [33]. As shown in Figure 4B, 20 and 100 μg/mL of ABSP or ABSSP diminished the transcription of COX2 into mRNA in LPS-exposed RAW264.7 macrophages by 17% and 40% or 22% and 67% (p < 0.05-0.001), respectively. Furthermore, treatment with 100 μg/mL ABSP or ABSSP reduced the LPS-induced protein expression of COX2 in RAW264.7 cells by 33% and 54% (p < 0.01-0.001), respectively. The resulting efficacy of BAY11-7082 (5 μM) in reducing iNOS and COX2 expression was more potent than that of ABSP or ABSSP. Taken together, ABSP and ABSSP exhibited anti-inflammatory activities by suppressing the expression of iNOS and COX2 at both transcription and translation levels.  Various inflammatory stimuli, including LPS, cause the transcription and translation of proinflammatory cytokines, leading to the initiation and amplification of the inflammatory response [34].
We therefore assessed whether ABSP and ABSSP can alter the mRNA levels of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) in LPS-exposed RAW264.7 cells. At doses of 20 and 100 µg/mL, both ABSP and ABSSP obviously downregulated the mRNA expressions of IL-1β and TNF-α, as did BAY11-7082 ( Figure 5A,B). The inhibition rates of 20 and 100 µg/mL ABSP on IL-1β mRNA expression were 17% and 44% and were 18% and 52% on TNF-α mRNA levels (p < 0.05-0.001), respectively. Treatment with 20 and 100 µg/mL ABSP decreased the mRNA levels of IL-1β by 23% and 72%, respectively, and of TNF-α by 21% and 71%, respectively, compared with the LPS control (p < 0.05-0.001). IL-1β and TNF-α, two major proinflammatory cytokines, promote the inflammatory response both locally and systemically in many kinds of acute and chronic inflammatory diseases [35]. Therefore, the suppression of IL-1β and TNF-α is beneficial for the treatment of inflammation-related diseases [36]. The present data suggest that the anti-inflammatory effects of ABSP and ABSSP are due, at least in part, to their suppression of the expression of IL-1β and TNF-α at the transcription level.
Molecules 2019, 24 FOR PEER REVIEW 3 Various inflammatory stimuli, including LPS, cause the transcription and translation of proinflammatory cytokines, leading to the initiation and amplification of the inflammatory response [34]. We therefore assessed whether ABSP and ABSSP can alter the mRNA levels of interleukin 1β (IL-1β) and tumor necrosis factor α (TNF-α) in LPS-exposed RAW264.7 cells. At doses of 20 and 100 μg/mL, both ABSP and ABSSP obviously downregulated the mRNA expressions of IL-1β and TNFα, as did BAY11-7082 ( Figure 5A,B). The inhibition rates of 20 and 100 μg/mL ABSP on IL-1β mRNA expression were 17% and 44% and were 18% and 52% on TNF-α mRNA levels (p < 0.05-0.001), respectively. Treatment with 20 and 100 μg/mL ABSP decreased the mRNA levels of IL-1β by 23% and 72%, respectively, and of TNF-α by 21% and 71%, respectively, compared with the LPS control (p < 0.05-0.001). IL-1β and TNF-α, two major proinflammatory cytokines, promote the inflammatory response both locally and systemically in many kinds of acute and chronic inflammatory diseases [35]. Therefore, the suppression of IL-1β and TNF-α is beneficial for the treatment of inflammationrelated diseases [36]. The present data suggest that the anti-inflammatory effects of ABSP and ABSSP are due, at least in part, to their suppression of the expression of IL-1β and TNF-α at the transcription level. Figure 5. Effects of ABSP and ABSSP on the mRNA expression of IL-1β and TNF-α in LPS-exposed RAW264.7 cells. Cells were pretreated with different concentrations of ABSP, ABSSP (20 and 100 μg/mL), and BAY11-7082 (BAY, 5 μM) for 2 h, and then stimulated with LPS (0.5 μg/mL) for an additional 24 h. The mRNA levels of IL-1β (A) and TNF-α (B) were tested by a qRT-PCR method. GAPDH was used as an internal control. Data are expressed as mean ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with LPS.

ABSP and ABSSP Block LPS-Induced ERK Activation in RAW264.7 Cells
MAPKs are evolutionarily conserved kinases that play pivotal roles in signal transduction mediated by cytokines, growth factors, and various environmental stresses [37]. In mammals, the MAPK family include three distinct members: the extracellular regulating kinase (ERK), p38 MAPK, and the c-Jun N-terminal kinase (JNK) [37]. A growing number of studies suggest that MAPKs play pivotal roles in the release of proinflammatory mediators and in downstream signaling events leading to acute or chronic inflammatory responses [38]. LPS can trigger the phosphorylation of MAPKs by binding and activating Toll-like receptor 4 (TLR4) in macrophages [39]. We therefore determined whether ABSP and ABSSP alters the LPS-induced activation of MAPK signaling, thereby inhibiting the inflammatory response. As shown in Figure 6, ERK, p38 MAPK, and JNK were phosphorylated in RAW264.7 cells after treatment with LPS. Inhibitors of ERK, p38 MAPK, and JNK, which were PD98059, SB203580, and SP600125 (10 μM each), respectively, attenuated the phosphorylation of the corresponding kinases by up to about 80% (p < 0.001). Pretreatment with 100 μg/mL ABSP or ABSSP significantly decreased the phosphorylation of ERK by 47% and 60% (p < 0.001), respectively. In contrast, LPS-induced activation of p38 MAPK and JNK1/2 in RAW264.7 cells were not affected by treatment with ABSP or ABSSP (p > 0.05). These results show that the inhibition by ABSP and ABSSP of LPS-induced activation of ERK may contribute to their anti-inflammatory potencies. Figure 5. Effects of ABSP and ABSSP on the mRNA expression of IL-1β and TNF-α in LPS-exposed RAW264.7 cells. Cells were pretreated with different concentrations of ABSP, ABSSP (20 and 100 µg/mL), and BAY11-7082 (BAY, 5 µM) for 2 h, and then stimulated with LPS (0.5 µg/mL) for an additional 24 h. The mRNA levels of IL-1β (A) and TNF-α (B) were tested by a qRT-PCR method. GAPDH was used as an internal control. Data are expressed as mean ± SD, n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001, compared with LPS.

ABSP and ABSSP Block LPS-Induced ERK Activation in RAW264.7 Cells
MAPKs are evolutionarily conserved kinases that play pivotal roles in signal transduction mediated by cytokines, growth factors, and various environmental stresses [37]. In mammals, the MAPK family include three distinct members: the extracellular regulating kinase (ERK), p38 MAPK, and the c-Jun N-terminal kinase (JNK) [37]. A growing number of studies suggest that MAPKs play pivotal roles in the release of proinflammatory mediators and in downstream signaling events leading to acute or chronic inflammatory responses [38]. LPS can trigger the phosphorylation of MAPKs by binding and activating Toll-like receptor 4 (TLR4) in macrophages [39]. We therefore determined whether ABSP and ABSSP alters the LPS-induced activation of MAPK signaling, thereby inhibiting the inflammatory response. As shown in Figure 6, ERK, p38 MAPK, and JNK were phosphorylated in RAW264.7 cells after treatment with LPS. Inhibitors of ERK, p38 MAPK, and JNK, which were PD98059, SB203580, and SP600125 (10 µM each), respectively, attenuated the phosphorylation of the corresponding kinases by up to about 80% (p < 0.001). Pretreatment with 100 µg/mL ABSP or ABSSP significantly decreased the phosphorylation of ERK by 47% and 60% (p < 0.001), respectively. In contrast, LPS-induced activation of p38 MAPK and JNK1/2 in RAW264.7 cells were not affected by treatment with ABSP or ABSSP (p > 0.05). These results show that the inhibition by ABSP and ABSSP of LPS-induced activation of ERK may contribute to their anti-inflammatory potencies.

Plant Materials and Extract of Alkaloids
The bamboo shoots of P. amarus (Keng) Keng f. were collected from Leshan, Sichuan Province, China. The bamboo shoots were harvested in May. The plant sample was authenticated by Xianming Lu (College of Pharmacy, Chengdu University of Traditional Chinese Medicine, China).
The crude ethanol extracts from dried bamboo shoots and bamboo shoot shells of P. amarus (Keng) Keng f. were obtained by treatment with 95% ethanol at 25 °C. After the solvent was evaporated under reduced pressure, the crude ethanol extract was mixed with 1% HCl and then extracted with CHCl3. The total alkaloids from bamboo shoots (ABSP) and bamboo shoot shells (ABSSP) of P. amarus (Keng) Keng f. were obtained after solvent evaporation and drying.

Conditions of UHPLC/QTOF-MS/MS
Alkaloids were analyzed by a UHPLC LC-30A system (Shimadzu, Kyoto, Japan) equipped with an Xbridge BEH amide column (100 mm × 2.1 mm, 2.5 μm) (Waters, Milford, MA, USA) and coupled RAW264.7 cells were pretreated with different concentrations of ABSP and ABSSP or specific inhibitors (PD98059, SB203580, and SP600125; 10 µM) for 4 h, and then exposed to LPS for an additional 30 min. Next, cell lysates were used to detect the phosphorylation levels of ERK, JNK, and p38 MAPK using specific antibodies. The nonphosphorylated MAPK proteins were used as the loading control. Results from representative experiments are shown (A). Quantification of the ratio of phosphorylated ERK (B), p38 MAPK (C), and JNK (D) normalized to the loading control. Data are expressed as mean ± SD, n = 3. *** p < 0.001, compared with LPS.

Plant Materials and Extract of Alkaloids
The bamboo shoots of P. amarus (Keng) Keng f. were collected from Leshan, Sichuan Province, China. The bamboo shoots were harvested in May. The plant sample was authenticated by Xianming Lu (College of Pharmacy, Chengdu University of Traditional Chinese Medicine, China).
The crude ethanol extracts from dried bamboo shoots and bamboo shoot shells of P. amarus (Keng) Keng f. were obtained by treatment with 95% ethanol at 25 • C. After the solvent was evaporated under reduced pressure, the crude ethanol extract was mixed with 1% HCl and then extracted with CHCl 3 . The total alkaloids from bamboo shoots (ABSP) and bamboo shoot shells (ABSSP) of P. amarus (Keng) Keng f. were obtained after solvent evaporation and drying.

Cell Culture
RAW264.7 cells were provided by the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Hyclone, Beijing, China) in atmosphere with 5% CO 2 at 37 • C.

Determination of Cell Viability
Cell viability was measured by the CCK-8 reagent. Briefly, RAW264.7 cells (2.5 × 10 4 cells/well) were seeded into a 96-well plate and incubated overnight at 37 • C. Next, cells were treated with different concentrations of ABSP or ABSSP for 24 h with LPS (0.5 µg/mL). Subsequently, 10 µL of CCK-8 were added into each well, and then incubated for 2 h at 37 • C. Next, absorbance at 450 nm was measured using a microplate reader (Thermo Fisher, Waltham, MA, USA). Relative cell viability was defined as the ratio of the absorbance in test wells compared to control wells.

Measurement of Nitric Oxide (NO)
Briefly, about 2.5 × 10 5 of RAW264.7 cells were seeded into 24-well plates and incubated overnight. Cells were then treated with different concentrations of ABSP or ABSSP for 2 h, followed by stimulation of LPS (0.5 µg/mL) for an additional 24 h. Levels of NO in cell culture medium were evaluated using Griess reagent.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
RAW264.7 cells (2 × 10 6 cells/well) were seeded onto 6-well plates and incubated overnight at 37 • C. After treatment with different concentrations of ABSP or ABSSP and BAY 11-7082 for 2 h, cells were then exposed to LPS for an additional 24 h. Total RNA from each treatment was extracted using a UNLQ-10 Column total RNA Purification Kit (Sangon, Shanghai, China) according to the manufacturer's instructions. Total RNA from the treatments were then reverse-transcribed to complementary