Chemical Constituent of β-Glucuronidase Inhibitors from the Root of Neolitsea acuminatissima

Neolitsea acuminatissima (Lauraceae) is an endemic plant in Taiwan. One new carboline alkaloid, demethoxydaibucarboline A (1), two new eudesmanolide-type sesquiterpenes, methyl-neolitacumone A (2), neolitacumone E (3), and twelve known compounds (4–15) were isolated from the root of Neolitsea acuminatissima. Their structures were elucidated by spectroscopic analysis. Glucuronidation represents a major metabolism process of detoxification for carcinogens in the liver. However, intestinal bacterial β-Glucuronidase (βG) has been considered pivotal to colorectal carcinogenesis. To develop specific bacterial-βG inhibitors with no effect on human βG, methanolic extract of roots of N. acuminatissima was selected to evaluate their anti-βG activity. Among the isolates, demethoxydaibucarboline A (1) and quercetin (8) showed a strong bacterial βG inhibitory effect with an inhibition ratio of about 80%. Methylneolitacumone A (2) and epicatechin (10) exhibited a moderate or weak inhibitory effect and the enzyme activity was less than 45% and 74%, respectively. These four compounds specifically inhibit bacterial βG but not human βG. Thus, they are expected to be used for the purpose of reducing chemotherapy-induced diarrhea (CID). The results suggest that the constituents of N. acuminatissima have the potential to be used as CID relief candidates. However, further investigation is required to determine their mechanisms of action.


Introduction
The Neolitsea genus (Lauraceae) is an important component with approximately 85 species and includes evergreen shrubs and small trees in the tropical and subtropical region of Asia [1]. Some plants Molecules 2020, 25, 5170; doi:10.3390/molecules25215170 www.mdpi.com/journal/molecules of the genus Neolitsea have been used as folk/herbal medicine, such as treatment of rheumatic arthralgia, furuncle and carbuncle, and edema [2]. Moreover, crude extracts and some pure chemical constituents of the Neolitsea species exhibited antioxidant, antiaging, antimicrobial [2,3], anti-inflammation [4][5][6], tyrosinase inhibition [7], and antitumor activity [8,9]. Phytochemical studies of Neolitsea have revealed the presence of alkaloids, benzenoids, flavonoids, lignans, quinone, sesquiterpenes, steroids, terpenoids, and others [2,[4][5][6][7][8][9][10]. This success has spurred the continuing search for more bioactive constituents of Formosan Neolitsea plants. Neolitsea acuminatissima (Hayata) Kanehira & Sasaki is an endemic species and evergreen small tree distributed in broad-leaved forests at high altitude throughout Taiwan [11]. An alkaloid, (+)-laurotetanine, of the stem bark of this plant was first published in 1965 by Tomita et al. [12]. Twenty compounds have further been isolated from the stem bark and among them, eudesmanolide sesquiterpenes, such as neolitacumones B and C, are known to have cytotoxicity against Hep 2,2,15 cells [8]. However, as study of the root of N. acuminatissima has not been performed, an investigation was carried out to search for additional valuable bioactivity from N. acuminatissima roots. Irinotecan (CPT-11), a first-line chemotherapeutic agent, is essential for treating malignancies, such as brain, lung, colorectal, and pancreatic cancers [13]. One of the major dose-limiting toxicities of CPT-11 regimen is unpredictable and causes severe diarrhea: more than 80% of patients suffer from delayed-onset diarrhea, and with 30% to 40% of them having severe diarrhea (grade 3 to 5) [14,15]. This toxicity significantly affects quality of life and may threaten the success of cancer chemotherapy, thus resulting in decreasing the drug dose or even discontinuation of treatment. Therefore, treating CPT-11-induced diarrhea is a significant clinical need [16,17]. Microflora in the intestine play a pivotal role in CPT-11-induced diarrhea. As a prodrug, CPT-11 is converted by carboxylesterase to SN-38, the active metabolite responsible for both toxicity and antitumor activity [18]. SN-38 is further catalyzed to inactive SN-38 glucuronide (SN-38G) by UDP-glucuronosyltransferase in the liver and excreted into the bile with other major components, CPT-11 and SN-38 by P-glycoprotein [19]. However, bacterial β-Glucuronidase (βG) enzymes in intestinal microflora, such as Escherichia coli, may reconvert nontoxic SN-38G to toxic metabolite SN-38 and lead to damage of intestine epithelia cells and cause severe diarrhea [18,19]. Therefore, inhibiting intestinal E. coli βG (eβG) activity is expected to protect the intestines from injury and thus alleviate chemotherapy-induced diarrhea (CID), even enhancing the therapeutic index. Despite some antidiarrheal agents already used to treat CID clinically [14,15], these approaches have several drawbacks. Thus, development of eβG inhibitors from natural products are valuable and expected to mitigate CID.
In a preliminary anti-eβG screening assay, sixty-five species of Formosan lauraceous plants were selected to evaluate their anti-eβG activity. Among them, the methanolic extract of the root of Neolitsea acuminatissima (NARM) showed a strong inhibitory effect on eβG with an inhibition ratio of 68%, without affecting the activity of human βG (hβG). (The details are shown in the Supporting Information, Figure S1.) According to preliminary anti-eβG data, we proposed the existence of active constituents in N. acuminatissima, since the chemical constituents and biological activity of roots of this plant have seldom been investigated previously. It is worthy to verify the phytochemistry and medicinal treatment of NARM. Thus, NARM was selected as the candidate to investigate secondary metabolites for their anti-eβG activity. Here in this article, the structure elucidation of three new compounds and results of anti-eβG activity are reported.

Structure Elucidation of Compounds 1-3
Fifteen compounds (1-15), including three new compounds, were isolated from dichloromethane (NARD) and ethyl acetate (NARE) soluble layers of NARM. Their structures ( Figure 1) were spectroscopically determined by FTIR and 1D and 2D NMR, and through comparison with those of reported analogs. The structure determination of three new compounds was described in the present paper.

Anti-E. Coli β-Glucuronidase Activity of Compounds Isolated from N. acuminatissima
Bioassay-guided isolation of NARD and NARE led to purification of 15 compounds. Among them, compounds 1, 2, 4-6, 8-10, and 12 were examined for their specific inhibition for eβG versus hβG by in vitro βG-based activity assays. The result showed that compounds 1 (1 mM) and 8 (0.3 mM) exhibited strong anti-eβG activity comparable to the positive control (1-((6,8-dimethyl-2-oxo-1,2dihydroquinolin-3-yl)methyl)-3-(4-ethoxyphenyl)-1-(2-hydroxyethyl)thiourea, 1mM) [29], and led eβG activity below 20%, respectively ( Figure 3). Compounds 2 and 10 had moderate or weak anti-eβG activity. Compounds 1 and 8 showed significant anti-eβG activity without affecting the enzyme activity of normal human intestinal (hβG > 90%); therefore, they do not influence normal function of human intestines. They may provide as a specific inhibitor to reduce eβG-induced intestinal injury and CID. Comparing the anti-eβG effect of flavone-quercetin (8), flavanone-dihydroquercetin (9), and flavan-epicatechin (10), these similar flavonoids with different saturations in the C-ring showed different behavior; compounds 9 and 10 had slight or weak anti-eβG effect, whereas 8 with a double bond between C-2/C-3 in conjugation with a 4-carbonyl group in ring C significantly decreased eβG activity. The result suggests that the flavone co-planar skeleton of 8 is better stabilized in the enzymes' binding pocket than 9 and 10 [30]. Since the inhibitory effect of β-Glucuronidase of eudesmanolide-type sesquiterpenes has never been reported, in the present paper, the anti-eβG result of methylneolitacumone A (2), neolitacumone A (4), neolitacumone B (5), and neolitacumone C (6) with the same eudesmanolide skeleton were discussed. Compound 2 exhibited moderate anti-eβG activity, while compounds 4-6 did not affect the enzyme effect. Comparison of these compounds showed that compound 2 has a methoxy group instead of a hydroxy group at C-8 and showed a better inhibitory effect than 4. Thus, it appears that presence of the lipophilic 8-methoxy group decreases eβG activity.
Molecules 2020, 25, x 6 of 11 eβG activity. Compounds 1 and 8 showed significant anti-eβG activity without affecting the enzyme activity of normal human intestinal (hβG > 90%); therefore, they do not influence normal function of human intestines. They may provide as a specific inhibitor to reduce eβG-induced intestinal injury and CID. Comparing the anti-eβG effect of flavone-quercetin (8), flavanone-dihydroquercetin (9), and flavan-epicatechin (10), these similar flavonoids with different saturations in the C-ring showed different behavior; compounds 9 and 10 had slight or weak anti-eβG effect, whereas 8 with a double bond between C-2/C-3 in conjugation with a 4-carbonyl group in ring C significantly decreased eβG activity. The result suggests that the flavone co-planar skeleton of 8 is better stabilized in the enzymes' binding pocket than 9 and 10 [30]. Since the inhibitory effect of β-Glucuronidase of eudesmanolidetype sesquiterpenes has never been reported, in the present paper, the anti-eβG result of methylneolitacumone A (2), neolitacumone A (4), neolitacumone B (5), and neolitacumone C (6) with the same eudesmanolide skeleton were discussed. Compound 2 exhibited moderate anti-eβG activity, while compounds 4-6 did not affect the enzyme effect. Comparison of these compounds showed that compound 2 has a methoxy group instead of a hydroxy group at C-8 and showed a better inhibitory effect than 4. Thus, it appears that presence of the lipophilic 8-methoxy group decreases eβG activity. β-Glucuronidase is a lysosomal enzyme and has been found in animals, plants, and bacteria [31]. This enzyme is responsible for hydrolysis of β-glucuronide conjugates of endogenous and exogenous compounds in the body, such as benzo[a]pyrene glucuronides and natural plant glucuronides. It has been found that βG released from macrophages and neutrophils is necessary for bioactivation of glucuronide conjugates into the aglycone [30,32], and that the enzyme requires acidic conditions for its catalytic activity. For hβG, it exhibits maximal catalytic activity at pH 4-4.5, while eβG shows optimal activity at neutral pH [32][33][34]. Inhibition or dysfunction of hβG may disturb glycosaminoglycan degradation and cause mucopolysaccharidosis (MPS), affecting appearance, physical abilities, organ and system functioning, and mental development [34,35]. Thus, it is worthy to figure out candidates that can specifically inhibit eβG activity but not hβG.
CID is a common side effect experienced by cancer patients; it affects quality of life and may result in early death either directly from life-threatening sequelae or indirectly from adjustments in cancer treatment that result in suboptimal therapy [14][15][16]. Many studies indicate that inhibition of β-Glucuronidase is a lysosomal enzyme and has been found in animals, plants, and bacteria [31]. This enzyme is responsible for hydrolysis of β-glucuronide conjugates of endogenous and exogenous compounds in the body, such as benzo[a]pyrene glucuronides and natural plant glucuronides. It has been found that βG released from macrophages and neutrophils is necessary for bioactivation of glucuronide conjugates into the aglycone [30,32], and that the enzyme requires acidic conditions for its catalytic activity. For hβG, it exhibits maximal catalytic activity at pH 4-4.5, while eβG shows optimal activity at neutral pH [32][33][34]. Inhibition or dysfunction of hβG may disturb glycosaminoglycan degradation and cause mucopolysaccharidosis (MPS), affecting appearance, physical abilities, organ and system functioning, and mental development [34,35]. Thus, it is worthy to figure out candidates that can specifically inhibit eβG activity but not hβG.
CID is a common side effect experienced by cancer patients; it affects quality of life and may result in early death either directly from life-threatening sequelae or indirectly from adjustments in cancer treatment that result in suboptimal therapy [14][15][16]. Many studies indicate that inhibition of βG activity can reduce CPT-11-induced intestine mucosal damage and CID [19,20,33]. Although use of antibiotics against intestinal βG could relieve CPT-11-induced diarrhea, allergies and resistance effects must be consulted or monitored for choosing an antibiotic; meanwhile, antibiotics will kill all native gut floras, including probiotics within the digestive tract, which is not recommended for long-term use in chemotherapeutic patients [14,34]. Development of eβG-specific inhibitors can be used as a chemotherapy adjuvant to reduce CID [33].
To sum up these results, demethoxydaibucarboline A (1), methylneolitacumone A (2), and quercetin (8) isolated from N. acuminatissima may be candidate-specific eβG inhibitors to reduce CID and intestinal injury. The best inhibitor among these isolates is quercetin (8). As we know, quercetin is the major flavonoid in our diet. Diets rich in quercetin and other flavonoids may relate to decreasing incidence of cardiovascular, neoplastic, and neurodegenerative diseases [30,32,36]. Thus, a specific eβG inhibitor, such as quercetin, could be used as a nutrient supplement for chemoprevention and health promotion. Further experiments are needed to pinpoint their mechanisms of action.

General
Melting points were determined on an Electrothermal MEL-TEMP 3.0 apparatus (manufacturer, city, state abbrev. if USA, country) and were uncorrected. Optical rotations were recorded on a Jasco P2000 digital polarimeter. UV spectra were obtained in methanol on a Beckman Coulter TM-DU 800 UV-visible spectrophotometer. IR spectra were measured on a Perkin Elmer system 2000 FTIR spectrophotometer. 1D ( 1 H, 13

Plant Material
Roots of N. acuminatissima were collected in July 2012 from Yilan County, Taiwan and positively identified by one of the authors, Prof. I. S. Chen. A voucher specimen (2012-07-NA) was deposited at the Herbarium of the Department of Fragrance and Cosmetic Science, Kaohsiung Medical University, Kaohsiung, Taiwan.

Conclusions
In this study, one new carboline alkaloid, demethoxydaibucarboline A (1), two new eudesmanolidetype sesquiterpenes, methylneolitacumone A (2), neolitacumone E (3), and twelve known compounds (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) were isolated from the root of N. acuminatissima. These compounds were investigated by using an anti-eβG assay. The results indicated that two isolated compounds, demethoxydaibucarboline A (1) and quercetin (8) showed significant anti-eβG activity with an inhibition ratio of approximately 80%, respectively. Methylneolitacumone A (2) exhibited a moderate inhibitory effect and eβG activity was less than 45%. Compounds 1 at 1 mM and 8 at a lower concentration of 0.3 mM exhibited specific inhibition of eβG activity but not hβG, suggesting that active secondary metabolites of N. acuminatissima are potential β-Glucuronidase inhibitors. These secondary metabolites will be potential β-Glucuronidase inhibitors that protect intestines from injury and thus relieve chemotherapy-induced diarrhea (CID). Although the detailed mechanism of action of these compounds remains to be determined, the results confirmed that N. acuminatissima is a valuable source from which natural product-based supplements and medicinal products can be derived.