Chemical Constituents of Murraya tetramera Huang and Their Repellent Activity against Tribolium castaneum

Sixteen compounds were isolated from the leaves and stems of Murraya tetramera Huang. Based on the NMR and MS spectral results, the structures were determined. It was confirmed that the isolated compounds included three new compounds (9, 10 and 13) and one new natural product (8), which were identified asmurratetra A (9), murratetra B (10), murratetra C (13) and [2-(7-methoxy-2-oxochromen-8-yl)-3-methylbut-2-enyl]3-methylbut-2-enoate (8), respectively. Meanwhile, the repellent activity against Tribolium castaneum was investigated for 13 of these isolated compounds. The results showed that the tested compounds had various levels of repellent activity against T. castaneum. Among them, compounds 1 (4(15)-eudesmene-1β,6α-diol), 11 (isoferulic acid) and 16 (2,3-dihydroxypropyl hexadecanoate) showed fair repellent activity against T. castaneum. They might be considered as potential leading compounds for the development of natural repellents.


Introduction
The red flour beetle (Tribolium castaneum) is one of the most destructive pests in stored products and often causes serious losses of stored goods in warehouses of grains, foods or traditional Chinese medicinal materials [1,2]. In an infestation, T. castaneum not only consumes stored materials, but also leads to an accelerated growth of molds in elevated temperatures and humid environments [3]. Nowadays, synthetic insecticides are mainly used to control the insects in a warehousing system. However, widespread use of these chemicals has resulted in a series of problems such as pesticide residue, health hazards to humans, environmental pollution and insect resistance [4]. Today, the attention of more and more researchers has been focused on seeking plant-derived materials which could be used as effective natural anti-insect agents or leading compounds for the control of insects. Some successful substances have been found. For example, the commercial botanical insecticide pyrethrum is a natural mixture product extracted from Chrysanthemum cinerariifolium, and it has been widely used in the world [5,6].
The genus Murraya comprises about 12 species all over the world, and nine of them are widely distributed in the south China [7]. The plants possess a special aroma, and insect pests rarely appear The genus Murraya comprises about 12 species all over the world, and nine of them are widely distributed in the south China [7]. The plants possess a special aroma, and insect pests rarely appear on Murraya species. In practice, it has been reported that some plants of the genus possess anti-insect properties [8][9][10]. The resources of genus Murraya are abundant in our country; however, the utilization of genus Murraya resources is still inadequate. Except a few parts of plants such as Murraya exotica, Murraya paniculata being used as medicine, most of the plants still have not been used properly. If the active constituents for control of the stored product insects are found from the waste plant resources, as it was expected, it would provide a new pesticide and the resources of genus Murraya would be further exploited and used as well.
In our previous works, it was found that the methanol extract of Murrayate tramera Huang showed significant repellent activity. In this work, compounds with significant repellent activity were expected to be obtained from the methanol extract of M. tetramera. Here, three new compounds, one new natural product and 12 known compounds were isolated from the methanol extract of M. tetramera and their repellent activity was evaluated.
Compound 13 was obtained as a yellow oil. The molecular formula was assigned as C 14  337.0894). This was verified by 14 carbon signals in the 13 C NMR. The 13 C-NMR data revealed one carbonyl carbon at δ C 157.3, six olefinic carbons, one of them bearing a methoxy group at δ C 167.2, six characteristic peaks of a glucose, and one methoxy group. The 1 H-NMR spectrum displayed one ortho-disubstituted phenyl moieties at δ H 7.78 (1H, d, J = 8.0 Hz), δ H 7.56 (1H, t, J = 8.0 Hz), δ H 7.42 (1H, d, J = 8.0 Hz), δ H 7.15 (1H, t, J = 8.0 Hz), as well as the seven characteristic peaks of a glucose, including the terminal proton at δ H 4.91 (1H, d, J = 7.5 Hz), and one methoxy group. The HMBC spectrum showed correlations arising from H-6 (δ H 7.78) to C-7' (δ C 157. 3 The HMBC correlations are presented in Figure 2. On the basis of the results and literature [27], the structure of compound 13 was identified as 2-methoxy-benzoyl-β-D-glucopyranoside.  Figure 2. On the basis of the results and literature [27], the structure of compound 13 was identified as 2-methoxy-benzoyl-β-D-glucopyranoside.

Repellent Activity
The repellent activity against T. castaneum was investigated for 13 isolated compounds including the new compound (10), using DEET (N,N-diethyl-3-methylbenzamide) as a positive control sample. The results are presented in Table 1.  [28]. ** Means in the same column followed by the different letters differ significantly (p < 0.05) in ANOVA and Tukey's tests. PR was subjected to an arcsine square-root transformation before ANOVA and Tukey's tests.
The results showed that the isolated compounds exhibited various levels of repellent activity against T. castaneum.

Discussion
There were not many research works with the chemical components of M. tetramera and bioactivities reported. However, some papers have revealed some interesting chemical information about M. tetramera. For example, two new carbazole dimers, two novel heterodimers and three known analogues of carbazole were isolated from M. tetramera and some of them exhibited inhibition effects on NO production in BV-2 microglial cells [29,30], and You reported that two sesquiterpenes and three coumarins isolated from M. tetramera showed fair cytotoxic effects [31].
In this report, three new compounds and one new natural product were isolated from the methanol extract of M. tetramera, along with 12 other known compounds. The structures of three new compounds were determined with modern spectral techniques and they belonged to amide, triterpenoid and glucoside, respectively. The above work enriches the chemical information on M. tetramera. Although the plant of M. tetramera was historically used for insect prevention only few papers have been published about the chemical basis of insect prevention. Here the repellent activity against T. castaneum was primarily investigated for the chemical components isolated from M. tetramera. The results indicated that the variety of repellent activities between the compounds was obviously affected by the testing concentrations and the exposure duration. By comparing the repellent levels of five different concentrations, we found the recommended concentration of compounds 1 and 11 against T. castaneum was 78.63 µg/cm 2 , whereas 15.73 µg/cm 2 is the recommended concentration of compound 16 against T. castaneum. The above findings indicated that the compounds 1, 11 and 16 have the potential for further development as possible natural repellents for the control of insects in stored products. However, to develop a practical application for the pure compounds as novel repellents, further investigations that focus on the efficiency and safety of the compounds should be conducted. Moreover, additional studies on the development of formulations are also needed to improve the efficacy and stability.
The various repellent activities were related to the different chemical structures. Among the 13 compounds, compound 1 showed the most potent repellent activity against T. castaneum. The significant repellent activity of the sesquiterpenoid was related to its volatility. Some sesquiterpenoids in the literature also indicated fair repellent activity against T. castaneum [32][33][34]. Coumarins 3-6 shared the same basic skeleton with different substitution patterns, yet their repellent activities varied greatly. Interestingly, the results showed that the compounds possessing carbonyls on 8-substituents had weak repellent activity. However, compounds possessing double bonds on 8-substituents had better repellent activity. Thus, we conjectured the carbonyls and double bonds on 8-substituents of coumarins were related to the repellent activity. Compounds 3-6 shared a similar basic skeleton, yet their repellent activities varied greatly. The results indicated the repellent activity of ester compounds decreased after ring closing and a longer alkyl-substituent contributed to the repellent activity. Further study is needed to investigate the structure-active relationships. The yields of pure compounds were low, so the application was limited. The structure modification and synthesis of the compounds are considerable methods.
In addition, the plant species had already been tested with the essential oils [33]. Compared with the methanol extract, the essential oil exhibited stronger repellency against T. castaneum. The PR value averages were assigned to different classes (0 to V) in the literature using the following scale [35]. Class, PR value%: 0, 0.01-0.1; I, 0.1-20.0; II, 20.1-40.0; III, 40.1-60.0; IV,60.1-80.0; and V, 80.1-100. At the concentration of 78.63 nL/cm 2 , the PR value of the essential oil was 98% (class V) at 2 h after exposure and 100% (class V) at 4 h after exposure. At the concentration of 15.73 nL/cm 2 , the PR value of the essential oil was 94% class V) at 2 h after exposure and 100% (class V) at 4 h after exposure. At the concentration of 314.54 nL/cm 2 , the PR value of the methanol extract was 83% at 2 h after exposure and 93% (class V) at 4 h after exposure. At the concentration of 62.91 nL/cm 2 , the PR value of the methanol extract was 67% (class IV) at 2 h after exposure and 80% (class IV) at 4 h after exposure. The compounds isolated from the essential oil exhibited significant repellent activity against T. castaneum.
The PR values of α-terpinene, β-caryophyllene, spathulenol, α-caryophyllene were greater than 80.1% (class V), and the PR values of alloaromadendrene, β-eudesmol were 80% (class IV) at the testing concentration of 78.63 nL/cm 2 and 2 h after the insects were exposed. However, compounds 1, 11, 16 isolated from the methanol extract also exhibited strong repellent activity against T. castaneum. The PR values of the three compounds were greater than 80.1% (class V) at the testing concentration of 78.63 µg/cm 2 and 2 h after exposure. The essential oil would be the better substance in terms of pest control. However, some compounds isolated from the methanol extract are also a choice for pest control.

General Information
NMR spectra were recorded on a Bruker Avance III NMR spectrometer with the magnetic field of 11.74 Tesla. HR-ESI-MS were acquired on a Bruker Q-TOF mass spectrometer. Preparative HPLC was performed on a Waters Delta Prep 4000 system equipped with a Waters 2487 dual λ absorbance detector. A Rainbow Kromasil-C 18 (10 mm × 250 mm, 10 µm) column was selected for preparative HPLC. Column chromatography was performed on silica gel (160-200 mesh) and TLC analysis was carried on silica gel G plates (Qingdao Marine Chemical Plant, China). Sephadex LH-20 was purchased from Amersham Pharmacia Biotech (Beijing, China). MCI GELCHP20P (75-150 µm) was supplied by the Kaiteki Company (Tokyo, Japan). Analytical grade solvents were produced by Beijing Chemical Factory (Beijing, China).The deuterated solvents (CDCl 3 , CD 3 OD, DMSO-d 6 , deuterated ratio, 99.8%) with TMS as the internal referent were produced by Cambridge Isotope Labo-ratories, Inc. (Andover, MA, USA).

Plant Material
The

Insects
Laboratory cultures of the red flour beetles were maintained in the dark cabinet of an incubator at 28-30 • C and 70-80% relative humidity. The insects were reared in glass containers (0.5 L) containing wheat flour at 12-13% moisture content mixed with yeast (10:1, w/w). Insects used in the experiments were about seven-day-old adults.

Repellency Tests
The repellent activity of isolated compounds from M. tetramera against T. castaneum adults was tested using the area preference method [36]. The compounds were dissolved in ethanol to prepare testing solutions of five concentrations (78.63, 15.73, 3.15, 0.63, and 0.13 µg/cm 2 , respectively). The filter paper (9 cm in diameter) was cut into two equal pieces. One piece was uniformly treated with

Repellency Tests
The repellent activity of isolated compounds from M. tetramera against T. castaneum adults was tested using the area preference method [36]. The compounds were dissolved in ethanol to prepare testing solutions of five concentrations (78.63, 15.73, 3.15, 0.63, and 0.13 µg/cm 2 , respectively). The filter paper (9 cm in diameter) was cut into two equal pieces. One piece was uniformly treated with 500 µL of testing solution as a testing part, and the other piece was dealt with 500 µL of ethanol as a control part. The filter papers were air dried for about 8 min to evaporate the solvent completely, and then the two pieces of filter paper were tightly fixed on the bottom of a Petri dish side by side with solid glue. For each test, 20 insects were released at the center of the filter paper disc which was then covered with a lid. Five replicates were performed for each tested concentration and the experiment was repeated three times. The number of insects presented on the control (N c ) and the testing (N t ) parts of the filter paper wasrecorded after 2 and 4 h, respectively. A commercial repellent, N,N-diethyl-3-methylbenzamide (DEET, product of Dr. Ehrenstorfer, Germany), was used as a positive control. The value of percent repellency (PR) was calculated as follows: Analysis of variance (One-Way ANOVA) and Tukey's test were conducted by using SPSS 20.0 (IBM Corp., Armonk, NY, USA) for Windows 2016 [37]. Percentage was subjected to an arcsine square-root transformation before variance and Tukey's tests.

Conclusions
In this work, we investigated the chemical constituents of Murraya tetramera Huang. Three new compounds (9, 10, 13), one new natural product (8) and 12 known compounds (1-7, 11, 12, 14-16) were isolated from the leaves and stems of M. tetramera Huang for the first time. The repellent activity of 13 compounds which were isolated from the M. tetramera was measured. Comparable to that of the positive control, DEET, compounds 1, 11 and 16 exhibited significant repellent activity against T. castaneum. The results indicated that compounds 1, 11 and 16 have potential for development into novel repellents to supply or substitute the heavy application of conventional repellents. They might be considered as potential leading compounds for the development of natural repellents. Further investigations that focus on the efficiency, safety and application of the compounds are also necessary.