Myrtinols A–F: New Anti-Inflammatory Peltogynoid Flavonoid Derivatives from the Leaves of Australian Indigenous Plant Backhousia myrtifolia

Abstract

Our in-house ethnopharmacological knowledge directed our anti-inflammatory investigation into the leaves of Backhousia mytifolia. Bioassay guided isolation of the Australian indigenous plant Backhousia myrtifolia led to the isolation of six new rare peltogynoid derivatives named myrtinols A–F (1–6) along with three known compounds 4-O-methylcedrusin (7), 7-O-methylcedrusin (8) and 8-demethylsideroxylin (9). The chemical structures of all the compounds were elucidated by detailed spectroscopic data analysis, and absolute configuration was established using X-ray crystallography analysis. All compounds were evaluated for their anti-inflammatory activity by assessing the inhibition of nitric oxide (NO) production and tumor necrosis factor- α (TNF-α) in lipopolysaccharide (LPS) and interferon (IFN)-γ activated RAW 264.7 macrophages. A structure activity relationship was also established between compounds (1–6), noting promising anti-inflammatory potential by compounds 5 and 9 with an IC₅₀ value of 8.51 ± 0.47 and 8.30 ± 0.96 µg/mL for NO inhibition and 17.21 ± 0.22 and 46.79 ± 5.87 µg/mL for TNF-α inhibition, respectively.

1. Introduction

Inflammation is the body’s natural defense response to various harmful stimuli including pathogens, heat, toxic chemicals, and injuries [1]. During the initial stage of trauma or infection, body initiates various cellular and molecular events which include the secretion of many proinflammatory cytokines and chemokines such as interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) as well as reactive oxygen species (ROS) and nitric oxide (NO) to restore tissue homeostasis and resolve acute inflammation [2,3]. The pro-inflammatory cytokines and/or bacterial lipopolysaccharides (LPS) can activate inducible nitric oxide synthase (iNOS) to produce continuously high concentrations of NO, which can then further induce injury at inflammatory sites [4]. Therefore, suppressing the overproduction of NO appears to be a promising strategy for the control of inflammatory diseases.

Backhousia myrtifolia (Myrtaceae) which is commonly referred to as carrol, neverbreak, iron wood, grey myrtle or cinnamon myrtle is a small rainforest tree species that grows in subtropical rainforests regions of Eastern Australia [5,6,7]. It was first discovered and subsequently used by the Indigenous communities of Australia, where the leaves were used to treat colic babies. B. myrtifolia is also known to harbor oils that have cinnamon-like aroma displaying both anti-bacterial and anti-fungal properties [8].

Our ongoing search to discover new anti-inflammatory molecules led us to explore the leaves of B. myrtifolia resulting in the isolation and identification of six new peltogynoid type flavonoids, myrtinols A–F (1–6), along with three known compounds, 4-O-methylcedrusin (7), 7-O-methylcedrusin (8), and 8-demethylsideroxylin (9) (Figure 1).

2. Structural Elucidation

Myrtinol A (1) was obtained as a green sticky mass. HRESI (−) MS analysis displayed a [M − H]⁻ deprotonated molecular ion peak at m/z 357.0959, corresponding to a molecular formula of C₁₉H₁₇O₇ requiring 11 double bond equivalents (DBEs). NMR data (600 MHz, CD₃OD) (

Table 1. ¹H NMR data for compounds 1–6.

Position	δH (J in Hz) 1 a	δH (J in Hz) 2 a	δH (J in Hz) 3 a	δH (J in Hz) 4 a	δH (J in Hz) 5 a	δH (J in Hz) 6 b
2	4.53, d (9.0)	4.57, d (9.5)	4.74, d (9.9)	4.56, d (9.6)	4.58, d (9.1)	4.67, d (9.5)
3	3.64, m	3.67, ddd (10.7, 9.5, 6.1)	3.67, dd (9.9, 8.3)	3.71, ddd (10.5, 9.6, 6.1)	3.67, ddd (10.6, 9.1, 5.9)	3.74, m
4a 4b	3.11, dd (15.5, 6.0) 2.63, dd (15.3, 10.7)	3.15, dd (15.4, 6.1) 2.66, dd (15.4, 10.7)	5.01, d (8.3)	3.10, dd (15.4, 6.1) 2.57, dd (15.4, 10.5)	3.14, dd (15.4, 5.9) 2.65, dd (15.4, 10.6)	3.23, dd (15.3, 6.0) 2.71, dd (15.3, 10.6)
8	6.21, s	6.23, s	6.15, s	6.19, s	6.36, s	6.36, s
5-OMe	3.71, s	3.72, s			3.73, s	3.73, s
6-Me	2.05, s	2.07, s	1.98, s	2.02, s	2.06, s	2.09, s
7-OMe			3.77, s	3.76, s	3.80, s	3.80, s
2′	6.66, s	6.78, s	6.79, s	6.83, s	6.72, s	6.84, s
7′a	4.89, d (15.2)	4.88, (15.3)	4.92, d (15.1)	4.89, d (15.1)	4.91, d (15.2)	4.96, d (15.4)
7′b	4.73, d (15.2)	4.73, (15.3)	4.72, d (15.1)	4.74, d (15.1)	4.76, d (15.2)	4.81, d (15.4)
8′	5.92, dd (15.3)				5.93, d (14.0)
3′-OMe		3.88, s	3.89, s	3.89, s		3.95, s
4′-OMe		3.79, s	3.81, s	3.79, s		3.91, s

^a Recorded in CD₃OD; ^b Recorded in CDCl₃.

, Figure 2) revealed the presence of one olefinic methyl (δ_H 2.05), one methoxy (δ_H 3.71), three distinct methylenes, including a methylene dioxy, H₂-8’ (δ_H 5.91, δ_C 101.9) and an oxy-methylene, H₂-7’ (δ_H 4.89, 4.73, δ_C 65.8) and a more traditional downfield methylene H₂-4 (δ_H 3.11, 2.63, δ_C 28.1). The presence of a downfield oxy-methine H-2 (δ_H 4.53, δ_C 74.6) and a traditional oxy methine H-3 (δ_H 3.64, δ_C 73.1) was also evident. The presence of two aromatic resonances (δ_H 6.21) and (δ_H 6.66) both as singlets showed several HMBC correlations to different quaternary carbons and oxy-carbons suggesting the presence of two separate penta-substituted benzene systems. The aromatic proton H-8 (δ_H 6.21) from the first of the benzene system (ring A) showed HMBC correlations to C-6 (δ_C 110.5), C-7 (δ_C 155.2), C-9 (δ_C 152.9) and C-10 (δ_C 105.1). The olefinic methyl (δ_H 2.05, δ_C 8.6) was assigned to be attached at C-6 in ring A based on having HMBC correlations to C-5 (157.6), C-6 and C-7 (Figure 1). The only methoxy (δ_H 3.71) in the structure was also assigned to ring A based on a HMBC correlation to C-5 (Figure 1). The aromatic proton H-2’ (δ_H 6.66) from the second benzene system (ring B) showed HMBC correlations to C-1’ (δ_C 117.7), C-3’ (δ_C 133.4), C-4’ (δ_C 147.6) and C-2 (δ_C 74.6). These correlations indicated the existence of another heavily substituted benzene ring. HMBC correlations from the methylene dioxy protons H₂-8’ (δ_H 5.91, δ_C 101.9) to C-3’ and C-4’ confirmed the attachment of this ring system (ring E) onto the benzene ring generating a 1,3-benzodioxole moiety. COSY correlations revealed a single isolated spin system consisting of two oxymethines (H-2 (δ_H 4.53, δ_C 74.6), H-3 (δ_H 3.64, δ_C 73.1) and a downfield methylene H₂-4 (δ_H 3.11, 2.63, δ_C 28.1). With the current structural fragments accounting for 9 out of the 11 DBEs, the situation required making room for two additional ring systems to be incorporated in the structure (Figure 1). Key diagnostic HMBC correlations from H₂-4 to C-9 and C-10 and H₂-7’ to C-1’, C-5’, C-6’ and C-3 confirmed the existence of two heterocyclic ring systems (rings C and D) where ring D was connected to ring B via an oxymethylene bridge representative of the peltogynoid type flavonoid system.

Myrtinol B (2) was obtained as a green solid. HRESI (+) MS analysis revealed a pseudomolecular ion [M + Na]⁺ ion peak at m/z 397.1247 ([M + Na]⁺, calcd 397.1263) in accordance with the molecular formula of C₂₀H₂₂O₇Na requiring ten double bond equivalents. It was evident early on from the NMR spectrum and data that (2) was a close structural analogue of myrtinol A (1), and comparison of the spectroscopic data of 1 with 2 showed that 2 retained the ring system A–D. The only difference was the presence of two additional methoxy resonances at (δ_H 3.88 and δ_H 3.79) exhibiting HMBC correlations to C-3′ (δ_C 136.2) and C-4′ (δ_C 153.2), respectively, with the absence of the methylene dioxy resonance which supported the opening up of ring E (Table 1,

Table 2. ¹³C NMR data for compounds 1–6.

Position	* δC 1 a	* δC 2 a	* δC 3 a	* δC 4 a	* δC 5 a	* δC 6 b
2	74.6	74.4	72.7	74.2	74.6	74.3
3	73.1	72.9	78.3	73.3	72.7	72.8
4	28.2	28.1	70.6	28.6	28.0	28.4
5	157.6	158.8	156.8	158.8	158.3	157.4
6	110.5	111.6	106.8	106.5	112.8	112.6
7	155.2	156.2	160.0	158.5	158.8	157.8
8	99.7	99.4	92.1	92.4	101.9	99.6
9	152.9	154.0	154.0	154.2	154.8	153.1
10	105.1	106.8	103.8	102.8	108.0	106.8
5-OMe	60.3	60.2			60.1	61.1
6-Me	8.6	8.4	7.7	8.1	8.7	9.4
7-OMe			60.8	55.8	55.7	56.7
1′	117.7	116.0	116.1	116.3	118.4	114.2
2′	98.4	101.3	101.4	101.0	98.6	102.4
3′	133.4	136.2	135.5	136.2	134.9	134.4
4′	147.6	153.2	153.2	153.3	148.8	147.7
5′	135.7	146.4	147.0	147.1	137.5
6′	126.9	129.7	129.4	130.1	128.4	128.7
7′	65.8	65.5	65.6	65.5	65.8	65.7
8′	101.9	101.3			101.9
3′-OMe		56.3	56.2	56.2		57.1
4′-OMe		60.9	60.8	60.9		62.2

^a Recorded in CD₃OD; * assignments supported by HSQC and HMBC; ^b Recorded in CDCl₃.

and Table S3 and Figure 3).

In addition to this, we were successful in generating a crystal for compound 2, which was then subjected to X-ray crystallography and the data obtained assisted in confirmation of the structure (Figure 4, Table S1) and helped resolve the absolute stereochemistry for the chiral centers C-2 and C-3 as R and S. From the NMR data alone, a large coupling of J_H-2/H-3 (9.5 Hz) also suggested a trans confirmation.

Myrtinol C (3) was obtained as a white solid. Its molecular formula was determined as C₂₀ H₂₁ O₈ from HRESI (−) MS ion analysis at m/z (389.1246 ([M − H]⁻, calcd 389.1236). A close comparison of the spectroscopic data of 3 with 2 confirmed that myrtinol C possessed the same ring system as myrtinol B, with the only exceptions being the positional change of the hydroxy and methoxy groups present in the ring A and the introduction of a hydroxy functionality C-4 (δ_H 5.01, δ_C 70.6). This above structural changes were supported by HMBC correlation from H-4 (δ_H 5.01) to C-3 (δ_C 78.3), C- 9 (δ_C 154.0) and C-10 (δ_C 103.8), from H-8 (δ_H 6.15) to C- 6 (δ_C 106.8), C- 7 (δ_C 160.0), C-9 and C-10, from H-6 (δ_H 1.98) to C-5, C-6, C-7, and from H-7 (δ_H 3.77) to C-7 (Table 1, Table 2 and Table S4 and Figure 3). A large coupling of J_H-3/H-4 (8.3 Hz) was suggestive of a trans confirmation, placing the absolute stereochemistry of C-4 to be R.

Myrtinol D (4) was obtained as a white solid. The molecular formula was revealed as C₂₀H₂₁O₇ based on HRESI (−) MS ion analysis at m/z 373.1284 ([M − H]⁻, calcd 373.1287), which was identical to myrtinol B (2). Myrtinol D (4) was identified as a structural isomer of (2) with the only difference being the positional change between the meta coupled hydroxy and methoxy functionalities in ring A of the benzene unit. This was supported by HMBC correlations from 7-OMe (δ_H 3.76) to C-7 (δ_C 158.5) and H-8 (6.19) to C-7.

Myrtinol E (5) was obtained as a yellow solid. The molecular formula was revealed as C₂₀H₁₉ O₇ from HRESI (−) MS ion analysis at m/z 371.1122 ([M − H]⁻, calcd 371.1131. Myrtinol E (5) showed the closest similarity to its analogue myrtinol A (1). The only difference in the NMR data and spectra was the detection of an additional methoxy resonance, which was confirmed to be attached at C-7 based on HMBC correlations of 7-OMe (δ_H 3.80) to C-7 (δ_C 158.8) and H-8 (δ_H 6.36) to C-7.

Myrtinol F (6) was obtained as a green sticky mass. The molecular formula was revealed as C₂₁H₂₃O₇ from HRESI (−) MS ion analysis at m/z 387.1454 ([M − H]⁻, calcd 387.1444. Comparison of the spectroscopic data of 2 and 6 showed that myrtinol F possessed the same ring system as myrtinol B, with the only change being the replacement of hydroxy substituent at C-7 by a methoxy group, which was supported by HMBC correlation from H-8 (δH 6.36) C- 7(δ_C 157.8), and 7-OMe (δ_H 3.80) to C-7.

We also take into account that the level of purity of myrtinol F was not 100%, with the possibility of a terpene like impurity present in this fraction (Figure S35). Low yields and the level of difficulty experienced separating this two-compound mixture prevented us from obtaining an absolutely pure sample of myrtinol F. However, the HRMS data and the clear key NMR resonances attributed to myrtinol F allowed for its complete structural assignment.

The absolute stereochemistry for the chiral centers C-2 and C-3 for the remaining myrtinol analogues was assigned as R and S, respectively, based on the large coupling constant between J_H-2/H-3 which was suggestive of a trans configuration along with the consideration of a likely biosynthetic relationship to myrtinol B (2) (Table 1 and Table 2) [9,10].

In addition to the discovery of the new myrtinols, three known compounds 4-O-methylcedrusin (7), 7-O-methylcedrusin (8) and 8-demethylsideroxylin (9) were identified by interpretation of their spectroscopic data (Figures S37, S39 and S41) and a close comparison with published data [11,12,13,14].

3. Anti-Inflammatory Activity

All compounds were assessed for their anti-inflammatory activity by evaluating the inhibition of NO production and TNF-α in LPS plus interferon (IFN)-γ activated RAW 264.7 macrophages. All compounds were also evaluated for their cytotoxicity using the Alamar blue assay (

Table 3. In vitro anti-inflammatory activity of compounds 1–9 in LPS + IFN-γ activated RAW 264.7 macrophages.

Compounds	Inhibition of NO Production (µg/mL), N = 6	Cell Viability (µg/mL), N = 6	Inhibition of TNF-α Production (µg/mL), N = 6
1	11.47 ± 0.14	18.76 ± 0.39	24.54 ± 0.28
2	16.25 ± 0.77	37.22 ± 2.09	52.35 ± 7.47
3	>100	>100	>100
4	29.31 ± 10.95	71.46 ± 14.74	91.26 ± 7.14
5	8.51 ± 0.47	15.52 ± 0.42	17.21 ± 0.22
6	12.62 ± 0.26	19.79 ± 0.86	30.55 ± 5.01
7	44.11 ± 13.39	>100	>100
8	>100	>100	>100
9	8.30 ± 0.96	20.45	46.79 ± 5.87
curcumin (+ve control)_	4.64 ± 1.5	10.86 ± 1.3	4.19 ± 2.6

).

4. Discussion

As shown in Table 3, the varying anti-inflammatory activity depended mostly on the functional groups attached to rings A, B and C. Based on the slight structural variations around the tetracyclic backbone of myrtinols A–F (1–6), we have attempted to evaluate the observed structural-activity relationship (SAR) among them. A SAR was observed between compounds 1 and 5 (both having a 1,3-benzodioxole moiety attached to ring B) which showed promising inhibition of NO production and TNF-α production, with a IC₅₀ values of 11.47 ± 0.14, 24.54 ± 0.28 and 8.51 ± 0.47, 17.21 ± 0.22 µg/mL, respectively. In comparison, compounds 2 and 6 showed NO production inhibition with an IC₅₀ value of 16.25 ± 0.77 and 12.62 ± 0.26 µg/mL, and TNF α production inhibition with an IC₅₀ value 52.35 ± 7.47, and 30.55 ± 5.01 µg/mL, respectively, suggesting that the presence of 1,3-benzodioxole moiety (ring E) and methoxy group at C-5 and C-7 might play an important role in their anti-inflammatory activity. Interestingly, when comparing the SAR between compounds 3 and 4, the only change between them being the introduction of a hydroxyl group at C-4 in 3, this rendered the molecule inactive compared to the baseline activity observed for compound 4 where the NO inhibition was determined to be 29.31 ± 10.95 µg/mL.

We are mindful of the fact that the experimental NO inhibition values obtained for myrtinols may not be a true reflection of their anti-inflammatory activity profile mainly due to the fact that they mostly have a low therapeutic index associated with them. This suggests that future investigations on assessing their cytotoxic activity need to be performed in order to re-evaluate their potential as cytotoxic agents.

Among the known compounds, Compound 9 displayed interesting activity with an IC₅₀ value 8.30 ± 0.96 µg/mL and IC₅₀ value of 46.79 ± 5.87 µg/mL, whereas 7 and 8 did not show good anti-inflammatory activity (Table 3).

Peltogynoids are a rare type of cyclized flavonoid, where a carbon atom is attached between 3-OH and C-2′ of ring B [9,15]. Robinson and Robinson isolated the first peltogynoid type flavonoid, peltogynol in 1935 [16]. Phytochemical studies of Australian and South African Acacia species including A. carnei by Roux and co-workers identified additional peltogynoid derivatives, (+)-2,3-trans-3,4- trans- peltogynols, (+)-2,3-trans-3,4-cis-peltogynol, (+)-2,3-trans-peltogynone, carnein, peltogynin, β-photomethylquercitin [17]. Peltogynoid derivates have been reported to be isolated from different genera and species including Goniorrhachis marginatu Taub [18], Pltogyne caringae, P. confertiflora; P. paniculate [19], Caesalpinia pulcherrima [20], Acacia nilotica (L.) Delile [21], Phytolacca icosandra [22], Bougainvillea spectabilis [23]. The new peltogynoid derivatives reported in this paper bear the closest resemblance to the reported pubeschin analogues [24].

5. Experimental Section

5.1. General Experimental Procedures

UV spectra were recorded on an Agilent Carry UV-Vis Multicell Peltier spectrometer. NMR spectroscopic data were recorded on a Bruker Avance 600 MHz spectrometer (Bruker Biospin GmbH, Germany). HRMS (High Resolution Mass Spectrometry) was carried out using a Waters SYNAPT G2-Si mass spectrometer operating in the positive and negative ESI mode.

5.2. Plant Material

The leaves of B. myrtifolia were collected from the Australian Botanic Garden at Mount Annan (NSW, Australia). A voucher specimen (2005-0104) has been deposited at the Australian Botanic Gardens, at Mount Annan, NSW, Australia.

5.3. Extraction and Bioactivity-Directed Isolation

The fresh leaves of B. myrtifolia (300 g) were crushed using a hand blender and extracted sequentially using organic solvents based on their polarity (n-hexane, dichloromethane (DCM), ethyl acetate (EtOAc), ethanol (EtOH), methanol (MeOH), and finally, water) using a Buchi-811 Soxhlet Extraction system. Immediately after the initial stages of sequential fractionation, each corresponding fraction was subjected to anti-inflammatory screening using the inhibition of NO in LPS plus IFN-γ treated RAW 264.7 macrophages following the Griess test. The most active extract (DCM) (Table S8) was resuspended in EtOH and was then later subjected to semi-preparative HPLC (Agilent 1260 Infinity II series) using an Agilent C₁₈ column (5 µm, 250 × 9.4 mm) eluting at 1.8 mL/min from 10% MeCN/H₂O to 100% MeCN (with a constant 0.01% FA (formic acid) modifier) over 60 mins and held for a further 6 mins and then equilibrated back to 10% MeCN/H₂O in 1 min and maintained at 10% MeCN/H₂O for an additional 3 mins, to give 17 fractions (Fr. 1–17) which included four pure fractions (Fr. 4 (7), Fr.8 (1), Fr.9 (2), and Fr.15 (8), corresponding to the following compounds: 7 (6.6 mg, t_R 24.7 min), 1 (5.4 mg, t_R 39.1 min), 2 (4.2 mg, t_R 41.7 min), and 8 (2.5 mg, t_R 50.3 min).

Fr.11 was re-purified by SB-C3 semipreparative column, (250 × 9.4 mm) using a gradient system of 50–60% MeCN/H₂O with a flow rate of 2mL/min (with a constant 0.01% FA modifier) over 30 min to afford compounds 3 (1.4 mg, t_R 22.4 min), and 4 (2.0 mg, t_R 23.1 min). Fr.16 was purified using SB-Phenyl semipreparative column, (250 × 9.4 mm) with a gradient system of 10–80% MeCN/H₂O over 15 mins followed by a change from 80–100% MeCN/H₂O over 9 mins with a constant flow rate of 2 mL, and then held at 100% MeCN for 2 mins and equilibrated back to 10% MeCN/ H₂O in 1 min and maintained at this gradient for an additional 2 min, to afford compound 5 (1.9 mg, t_R 21.8 min). Fr.17 was repurified using a semi-preparative Agilent C₁₈ column (5 µm, 9.4 × 250 mm) by employing a gradient system of 10–90% MeCN/H₂O over 15 mins followed by a gradient change of 90–100% MeCN/ H₂O for an additional 5 min, and then held at 100% MeCN for an additional 2 min followed by an equilibration to 10% MeCN/H₂O in 3 mins, affording compound 6 (6.4 mg, t_R 18.3 min). Compound 9 (4 mg, t_R 13.4 min) was acquired from Fr.5 using SB-Phenyl semipreparative column, (250 × 9.4 mm) eluting with 10–55% MeCN/H₂O (with a constant 0.01% FA modifier) over 8 min at 2 mL/min, followed by a gradient change from 55–70% MeCN/H₂O for an additional 7 min, and finally with a 70–100% MeCN/H₂O (with a constant 0.01% FA modifier) over 2 min and then equilibrated back to 10% MeCN within 1 min and maintained 10% MeCN/H₂O for an additional 2 min.

Myrtinol A (1): Greenish sticky mass [α]²⁵_D +172.9 (c 0.001, MeOH); UV-Vis λ_max (MeOH) nm (log ε) 208 (5.31), 232 (5.37) and 280 (5.44); 1D and 2D NMR (600 MHz, CD₃OD) data (see Table 1, Table 2 and Table S2); HRESI (−) MS m/z 357.0959 [M − H]⁻ (calcd for C₁₉H₁₇O₇⁻; 357.0974).

Myrtinol B (2): Green crystal (DCM:MeOH) [α]²⁵_D +178.3 (c 0.001, MeOH); UV-Vis λ_max (MeOH) nm (log ε) 208 (5.31), 233 (5.37) and 281 (5.45); 1D and 2D NMR (600 MHz, CD₃OD) data (see Table 1, Table 2 and Table S3); HRESI (+) MS m/z 397.1247 [M + Na]⁺ (calcd for C₂₀H₂₂O₇Na; 397.1263).

Myrtinol C (3): White solid [α]²⁵_D +176.1 (c 0.0003, MeOH); UV-Vis λ_max (MeOH) nm (log ε) 208 (5.31), 233 (5.37) and 280 (5.44); 1D and 2D NMR (600 MHz, CD₃OD) data (see Table 1, Table 2 and Table S4); HRESI (−) MS m/z 389.1246 [M − H]⁻, (calcd for C₂₀ H₂₁ O₈ 389.1236).

Myrtinol D (4): White solid [α]²⁵_D +174.6 (c 0.0005, MeOH); UV-Vis λ_max (MeOH) nm (log ε) 208 (5.31), 232 (5.37) and 280 (5.44); 1D and 2D NMR (600 MHz, CD₃OD) data (see Table 1, Table 2 and Table S5); HRESI (−) MS m/z 373.1284 [M − H]⁻, (calcd for C₂₀H₂₁O₇⁻; 373.1287).

Myrtinol E (5): Yellow solid [α]²⁵_D +171.7 (c 0.0005, MeOH); UV-Vis λ_max (MeOH) nm (log ε) 208 (5.31), 232 (5.37) and 280 (5.44); 1D and 2D NMR (600 MHz, CD₃OD) data (see Table 1, Table 2 and Table S6); HRESI (−) MS m/z 371.1122 [M − H]⁻, (calcd for C₂₀ H₁₉ O₇⁻; 371.1131).

Myrtinol F (6): Green sticky mass [α]²⁵_D +178.2 (c 0.001, MeOH); UV-Vis λ_max (MeOH) nm (log ε) 208 (5.31), 233 (5.37) and 280 (5.44); 1D and 2D NMR (600 MHz, CD₃OD) data (see Table 1, Table 2 and Table S7); HRESI (−) MS m/z 387.1454 [M − H]⁻ (calcd for C₂₁H₂₃O₇⁻; 387.1444).

5.4. X-ray Crystallographic Analysis of 2

Crystals were obtained by slow cooled evaporation from MeOH: DCM (1:1) solution; suitable crystals were selected for X-ray crystallographic analysis using an MX1 beamline at the Australian Synchrotron, using silicon double crystal monochromated radiation (λ = 0.71073 Å) at 100 K [25]. The XDS software package [26] was used on site for data integration, processing, and scaling. SADABS [27] was used to apply an empirical absorption correction. Shelxt [28] was applied to solve the structure by the intrinsic phasing method, and a suite of SHELX programs [28,29] were used for refinement, via the Olex2 graphical interface [30]. Crystallographic data of 2 (CCDC number: 2236594) was deposited at the Cambridge Crystallographic Data Centre. Additional crystallographic information is available in the Supporting Information (Table S1).

5.5. Crystal Data for 2

C₂₀H₂₁O₇ (M = 373.37 g/mol); triclinic, 0.2 × 0.1 × 0.1 mm³, space group P1_, V = 872.6(3) ų, Z = 2, D_c = 1.421 g/cm³, F(000) = 394.0, Mo Kα radiation, λ = 0.71073 Å, T = 100 K, μ = 0.108 mm⁻¹; 2θ_range = 3.552 to 57.202°, 20,884 reflections collected, 6838 unique (R_int = 0.0191); final GooF = 1.048, R1 = 0.0383 [I > 2σ(I)], wR2 = 0.1031; absolute structure parameter = 0.06(13).

5.6. Maintenance of RAW 264.7 Macrophages

Cells were grown in 75 cm² flasks on Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) that was supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL) and L-glutamine (2 mM). The cell line was maintained in 5% CO₂ at 37 °C, with media being replaced every 3–4 days. Once cells had grown to confluence in the culture flask, they were harvested using a rubber policeman, as opposed to using trypsin, which can remove membrane-bound receptors.

5.7. Pro-Inflammatory Activation of Cells

RAW 264.7 cells (1 × 10⁶ cells/mL) were seeded in 96 well plates (Corning^® Costar^®, Sigma, Sydney, Australia) overnight until confluency. When the cells were confluent, each compound was serially diluted from a starting concentration of 100 μg mL⁻¹ to construct a dose response curve (i.e., 100, 50, 25, 12.5, 6.25, and 3.13 μg mL⁻¹) and co-incubated with cells for 1 hr prior to the addition of 1 μg mL⁻¹ LPS and 10 U mL⁻¹ (1 unit = 0.1 ng/mL) IFN-γ. After activation, the cells were incubated for another 24 hrs at 37 °C. The supernatant was then collected for NO, and TNF-α assays. The cells were subjected to cell viability measurement using the Alamar Blue assay. Non-activated cells (exposed to media only) were used as negative control and activated cells were positive control.

5.8. Determination of Nitrite by the Griess Assay

Nitric oxide was determined by the Griess reagent as described in previous studies [31]. Griess reagent was freshly made up of equal volumes of 1% sulfanilamide in 5% phosphoric acid and 0.1% N-1-naphthylethylenediamine dihydrochloride in Milli-Q water. From each well, 50 µL of supernatant was transferred to a fresh 96-well plate and mixed with 50 µL of Griess reagent. The production of nitrite as an indicator of NO was measured at 540 nm in a POLARstar Omega microplate reader (BMG Labtech, Mornington, Australia).

5.9. Determination of TNF-α by ELISA

The stored supernatants were analyzed for TNF-α synthesis using a commercial ELISA kit (Peprotech, Brisbane, Australia) according to the manufacturer’s instructions. The absorbance was measured at 410 nm [32]. The concentrations of TNF-α in the experimental samples were extrapolated from a standard curve.

5.10. Determination of Cell Viability by the Alamar Blue Assay

After various treatments and the stimulation by LPS and IFN-γ overnight, 100 µL of Alamar Blue solution [10% Alamar Blue (resazurin) in DMEM media] was added to cells and incubated at 37 °C for 2 hrs. The fluorescence intensity was measured with excitation at 530 nm and emission at 590 nm using a microplate reader. The results were expressed as a percentage of the intensity to that of control cells (non-activated cells).

5.11. Statistical Analysis

Data analysis was carried using GraphPad Prism 9.3.1. Calculations were performed using MS-Excel version 16.61.1. IC₅₀ values were obtained by using the sigmoidal dose–response function in GraphPad Prism. The results were expressed as mean ± standard deviation (SD).

6. Conclusions

In conclusion, we have isolated and characterized six new rare peltogynoid flavonoids from the leaves of Backhousia myrtifolia. Myrtinols exhibited promising anti-inflammatory activity; however, their low therapeutic index warrants further cytotoxic evaluation as part of a future investigation.