New Rare Ent-Clerodane Diterpene Peroxides from Egyptian Mountain Tea (Qourtom) and Its Chemosystem as Herbal Remedies and Phytonutrients Agents.

Genus Stachys, the largest genera of the family Lamiaceae, and its species are frequently used as herbal teas due to their essential oils. Tubers of some Stachys species are also consumed as important nutrients for humans and animals due to their carbohydrate contents. Three new neo-clerodane diterpene peroxides, named stachaegyptin F-H (1, 2, and 4), together with two known compounds, stachysperoxide (3) and stachaegyptin A (5), were isolated from Stachys aegyptiaca aerial parts. Their structures were determined using a combination of spectroscopic techniques, including HR-FAB-MS and extensive 1D and 2D NMR (1H, 13C NMR, DEPT, 1H-1H COSY, HMQC, HMBC and NOESY) analyses. Additionally, a biosynthetic pathway for the isolated compounds (1-5) was discussed. The chemotaxonomic significance of the isolated diterpenoids of S. aegyptiaca in comparison to the previous reported ones from other Stachys species was also studied.

1. Introduction

The genus Stachys (woundwort) has about 300 species growing wild in the temperate and tropical regions throughout the world except the continent of Australia and New Zealand [1]. In the Mediterranean region and Iran, Stachys species are known as mountain tea with great medicinal and nutritional values due to their traditional uses as food additives, herbal teas, and medicinal supplements [2,3,4,5]. The tubers of some species are used as phytonutrients rich in carbohydrates, particularly in some parts of Europe and China [6]. In folk medicine, the infusions, decoctions, and ointments made from flowers and leaves of these herbs have been used in the treatment of some disorders such as skin infections, inflammation, wounds, digestive problems, cough, ulcers, and stomach ache, and applied as antispasmodic, sedative, and diuretic agents, and cardiac tonic [3,5,7,8,9,10], and recently administrated for genital tumours, sclerosis of the spleen, and inflammatory cancerous ulcers [11,12,13]. Phenolic extracts and essential oils of Stachys species showed a number of important biological activities such as antioxidant [14,15,16,17,18], anti-inflammatory [16,19], antiangiogenic [20], anti-nociceptive [21,22], antimicrobial [3,4,23,24], cytotoxic, and anticancer [25,26,27,28,29,30]. Additionally, the genus Stachys is rich with flavonoids and phenolic [17,31,32,33,34,35,36], diterpenoids [10,21,27,37,38,39,40,41,42], iridoids [20,43,44,45], and phenylethanoid glycosides [46,47] metabolites.

Stachys aegyptiaca Pers., a member of this genus, is a perennial aromatic plant growing wild in Sinai Peninsula, Egypt, and is called “Qourtom”. Previous phytochemical investigations on this species led to the isolation of diterpenes [27,40,41,48], flavonoids [40,49,50,51,52]), and essential oils [53,54]. In our previous work on this species, we isolated five new diterpenes of the neo-clerodane type, stachaegyptin A-E, in addition to seven known flavonoids from the aerial parts [27,40].

Herein, we report the isolation and structural determination of further three new ent-neo-clerodane diterpene peroxides, named stachaegyptin F-H (1, 2, 4), as well as two known compounds, stachysperoxide (3) and stachaegyptin A (5) (Figure 1), from the aerial parts of this species using extensive 1D and 2D NMR and HR-FAB-MS analyses. Additionally, a biosynthetic pathway of the isolated metabolites (1–5) as well as the chemotaxonomic significance of the isolated diterpenoids from S. aegyptiaca were studied.

Figure 1

Structures of the isolated diterpenes from Stachys aegyptiaca.

2. Results and Discussion

The CH2Cl2:MeOH (1:1) extract of S. aegyptiaca aerial parts afforded three new ent-neo-clerodane diterpenoids, named stachaegyptin F (1), stachaegyptin G (2), and stachaegyptin H (4), together with two known compounds, stachysperoxide (3) and stachaegyptin A (5) (Figure 1), using chromatographic techniques. Their structures were established using extensive 1D [1H (Table 1), 13C NMR (Table 2)], and 2D NMR (1H-1H COSY, HMQC, HMBC and NOESY) analyses(the details in Supplementary Materials).

Table 1

The 1H NMR data assignments for compounds 1–4 (600 MHz, in CDCl3) a.

Position	1	2	3 a	4
1α	2.41 dd, (17.0, 14.0)	2.41 dd (17.0, 14.4)	2.52 dd (17.0, 14.0)	2.41 m *
1β	2.29 dd (17.0, 3.4)	2.60 dd (17.0, 2.8)	2.32 dd (17.0, 3.4)	2.80 dd (17.0, 3.4)
2	---	---	---	---
3	5.68 br s	5.68 br s	5.69 br s	5.69 br s
4	---	---	---	---
5	---	---	---	---
6α	2.20 dd (14.0, 2.7)	2.22 dd (14.0, 2.7)	2.19 dd (14.0, 2.7)	2.17 dd (14.0, 2.7)
6β	1.60 dd (14.0, 3.4)	1.63 dd (14.0, 3.4)	1.57 dd (14.0, 3.4)	1.57 dd (14.0, 3.4)
7	4.09 br d (3.4)	4.11 br d (2.4)	4.07 m	4.11 br d (2.7)
8	1.90 m *	1.71 m	2.06 m	1.69 m *
9	---	---	---	---
10	2.14 dd (14.0, 3.4)	2.25 dd (14.0, 2.8)	2.11 dd (14.0, 3.4)	2.41 m *
11a	1.64 dd (16.5, 7.5)	1.62 dd (16.5, 7.5)	1.91 dd (14.0, 10.5)	1.96 dd (16.5, 10.3)
11b	1.50 dd (16.5, 2.0)	1.52 dd (16.5, 2.0)	1.44 m *	1.42 m *
12	4.66 dd (7.5, 2.7)	4.66 d (8.2)	4.18 br d (10.5)	4.18 d (10.3)
13	---	---	---	---
14	6.29 dd (17.0, 11.0)	6.31 dd (17.0, 11.0)	5.58 br s	5.57 br d (2.5)
15a	5.49 d (17.0)	5.45 d (17.0)	4.61 br d (14.0)	4.61 br dd (14.4, 2.5)
15b	5.17 d (11.0)	5.15 d (11.0)	4.28 br d (14.0)	4.29 br d (14.4)
16a	5.23 s	5.23 s	1.73 s	1.71 s
16b	5.13 s	5.18 s	---	---
17	1.09 d (7.0)	0.99 d (7.5)	1.13 d (7.0)	1.06 d (7.0)
18	1.92 s	1.91 s	1.91 s	1.88 s
19	1.39 s	1.39 s	1.42 s	1.39 s
20	1.02 s	1.01 s	1.07 s	1.07 s

a Data are given for comparison with the new compound 4. * Overlapping signals.

Table 2

The 13C NMR data assignments for compounds 1-4 (150 MHz, in CDCl3) a.

C	1	2		3 a	4
	δC	δC	DEPT	δC	δC	DEPT
1	35.3	35.5	CH2	35.4	35.3	CH2
2	199.8	200.9	C=O	199.8	200.7	C=O
3	125.1	125.2	CH	125.0	125.5	CH
4	172.9	172.7	C	173.1	172.2	C
5	39.6	39.0	C	38.8	38.8	C
6	41.9	42.0	CH2	41.2	41.4	CH2
7	73.2	73.2	CH	73.3	73.3	CH
8	39.8	39.6	CH	39.7	38.8	CH
9	39.6	39.5	C	39.6	39.2	C
10	46.4	46.6	CH	45.9	46.4	CH
11	41.2	41.3	CH2	38.0	38.0	CH2
12	83.7	82.6	CH	79.2	79.0	CH
13	146.3	146.9	C	134.7	134.2	C
14	134.8	135.3	CH	118.7	119.1	CH
15	116.4 *	115.5	CH2	69.9	69.8	CH2
16	116.5 *	115.6	CH2	19.1	19.0	CH3
17	12.8	12.7	CH3	12.5	12.6	CH3
18	19.4	19.2	CH3	19.7	19.4	CH3
19	20.2	20.4	CH3	20.3	20.6	CH3
20	19.1	19.1	CH3	19.4	19.3	CH3

a Data are given for comparison with the new compound 4. * Overlapping signals.

Compound 1 was isolated as a colorless oil with an optical rotation of +30 (c, 0.001, MeOH). Its molecular formula C20H30O4 was determined from the high-resolution FAB-MS analysis with a molecular ion peak [M + Na]+ at m/z 357.2045 (calcd. for C20H30O4Na, 357.2044), indicating six degrees of unsaturation. The 13C NMR spectrum revealed the presence of 20 carbon resonances (Table 2), which was in agreement with the molecular formula. Their multiplicities were deduced from the results of 13C DEPT NMR analyses as four methyls, five methylenes (two olefinic), six methines (two olefinic and two oxygenated at δC 73.2 and δC 83.7), and five quaternary carbons (two olefinic and one keto at δC 199.7) (Table 2). With 20 carbons and six degrees of unsaturation; one of them was assigned as a keto group (δC 199.8) and three were attributed to double bonds, therefore, compound 1 is apparently a bicyclic diterpene. The 1H NMR analysis of 1 (Table 1) displayed typical signals for two tertiary methyls at δH 1.02 and 1.39 (each 3H, s), a secondary methyl at δH 1.09 (3H, d, J = 7.0 Hz) and an olefinic methyl at δH 1.92 (3H, s), which showed a correlation in the Double Quantum Filtered COSY (DQF-COSY) spectrum with an olefinic proton signal at δH 5.68 (1H, br s), indicating the presence of a trisubstituted double bond. The spectrum also showed two oxomehine protons at δH 4.09 (1H, br d, J = 3.4) and δH 4.66 (1H, dd, J = 7.5 and 2.7 Hz), an ABX spin system at δH 5.17 (1H, d, J = 11.0 Hz), δH 5.49 (1H, d, J = 17.0 Hz) and δH 6.29 (1H, dd, J = 17.0, 11.0 Hz), and two terminal olefinic protons at δH 5.23 and 5.13 (each 1H, s). The COSY spectrum exhibited four spin systems coupled with ring A, ring B, and the side chain (Figure 2). All these accumulated data are regular with the plain skeleton of neo-clerodane diterpenes formerly isolated from this genus [27,40,55].

Figure 2

Observed 1H-1H-COSY and HMBC correlations for 1 and 4.

Interpretation of the 2D NMR data, including DQF-COSY, HMQC and HMBC, clearly indicated that we are dealing with a structure similar to that of stachaegyptin A (5), previously isolated from this species, and its structure was confirmed by X-ray crystallography [40]. The distinct difference observed in the 1H NMR spectrum of 1 was the additional oxymethine proton at δH 4.66 (1H, dd, J = 7.5 and 2.7 Hz) (H-12), which showed couplings in the DQF-COSY spectrum with H2-11 at δH 1.64 (1H, dd, J = 16.5, 7.5 Hz) (H-11a) and δH 1.50 (1H, dd, J = 16.5, 2.7 Hz) (H-11b), while in the HMQC spectrum this proton showed a correlation with the oxymethine carbon at δC 83.7. The 13C NMR data of 1 also revealed similarities with those of stachaegyptin A (5) except that the methylene carbon C-12 in 5 was replaced by the oxomethine carbon at δC 83.7 in 1. The HMBC experiment (Figure 2) confirmed the presence of 12-oxymethine in 1 by the HMBC connections from H-12 (δH 4.66) to C-9 (δC 39.6), C-11 (δC 41.2), C-14 (δC 134.8) and C-16 (δC 116.5). With four oxygen atoms in 1 (C20H30O4, HR-FAB-MS), three of them were assigned from the 13C NMR data as two oxomethine carbons [δC 73.2 (C-7) and δC 83.7 (C-12)] and one keto group at δC 199.8 (C-2). Additionally, and due to the lack of an additional oxymethine signal, the remaining oxygen should, therefore, be a part of a hydroperoxyl group instead of a hydroxyl group.

This was supported by the positive TLC spray test for hydroperoxides (N,N-dimethyl-1,4-phenylenediammonium chloride) [56] as well as from the unusual downfield chemical shift of 12-oxymethine at δC 83.6, which was very similar to those reported for related 12-hydroperoxy diterpenes [56,57]. Related 12-hydroxy diterpenes, by contrast, showed a 12-oxymethine between δC 62.0–64.0 [58,59,60]. Comprehensive assignment of 1 was established from the results of DQF-COSY, HMQC, and HMBC NMR experiments. Therefore, 1 could be elucidated as 12-hydroperoxy derivative of 5.

The relative stereochemistry of 1 was determined by the coupling constants, the NOESY experiments (Figure 3) with inspection of the 3D molecular model, and the biogenetic correlation with stachaegyptin A (5), where its structure and stereochemistry were confirmed by X-ray crystallography [40]. The hydroxyl group configuration at C-7 was assigned to be α (axial), conferring the small coupling constants of H-7 (3.4 Hz), which was similar to those reported for 5 and other neo-clerodane diterpenes [27,40]. The NOESY connections between H-7 (δH 4.09) and H-8 (δH 1.90) indicated that these protons are on β-configuration of the B ring. The NOESY correlations observed between CH3-17 (δH 1.09) and CH3-20 (δH 1.02) and between CH3-20 and CH3-19 (δH 1.39) indicated that these methyl groups are all on the same side in an α-configuration. The absence of a NOESY correlation between CH3-19α and H-10 revealed that the A/B ring system was trans-diaxially oriented, and the orientation of H-10 was β. All of previous results were well matched with the biogenetic precedent and formerly reported NMR chemical shift data for stachaegyptin 5 and related neo-clerodane diterpenes with the same configurations [27,40]. The C-12 configuration was determined by the NOESY analysis with inspection of the 3D molecular model (Figure 3). The observed correlations between H-12 (δH 4.66), H-1β (δH 2.29), and H-10 (δH 2.14) implied that these protons were in closeness and confirmed that the C-12 stereo center had the R configuration as those reported for (12R) 12-hydroperoxy and 12-hydroxy diterpenes [56,57,58,59,60,61,62]. Therefore, the structure of 1 was established as 12(R)-12-hydroperoxy-7α-hydroxy-neo-cleroda-3,13(16),14-triene-2-one, and was named stachaegyptin F.

Figure 3

Stereo configurations based on NOESY correlations and 3D molecular model for 1–4.

Compound 2 was isolated as a colorless oil with an optical rotation of 29 (c, 0.005, MeOH). The FAB-MS spectrum of 2 exhibited the base peak at m/z 357 [M + Na]+, consistent with a molecular formula C20H30O4, which was established by a molecular ion peak at m/z 357.2042[M + Na]+ (calcd. for C20H30O4Na, 357.2044) in the HR-FAB-MS analysis. This formula was the same as that reported for 1. The positive reaction on TLC with N,N-dimethyl-1,4-phenylenediammonium chloride) [60] also revealed the presence of a hydroperoxid as in 1. The 1H and 13C NMR spectra of 2 (Table 1 and Table 2) were almost identical with those reported for 1, except for the upfield chemical shifts of CH3-17 (δH 0.99) as well as H-8 (δH 1.71), in addition to the downfield shift of H-1β (δH 2.60) in 2 comparing with those of 1. The 2D NMR experiments including the DQF-COSY, HMQC, and HMBC exhibited an identical planar structure to that of 1. Additionally, combined NOESY and coupling contacts analysis clearly indicated that 2 is matching the relative stereochemistry of 1 in the bicyclic system. All the above data and differences between 1 and 2 established that 2 should be an epimer of 1 at C-12 (S configuration) as previously shown in related compounds [57,60,61,62]. This was supported by the NOESY experiment with inspection of the 3D-molecular model (Figure 3). The strong correlations between H-12, H-10β, and H-8β, together with the absence of a NOESY correlation between H-12 and H-1β, confirmed the S configuration at C-12 in 2 instead of 12R as in 1.

Further confirmation was given by the relative downfield shift of H-1β at δH 2.60 in 2, instead of that at δH 2.29 in 1, which was attributed to the presence of H-1β in a close proximity to the hydroperoxyl group. By contrast, H-8β and CH3-17 were slightly shifted at higher-field (δH 1.71 and δH 0.99, respectively), than those of 1 at δH 1.90 (H-8β) and δH 1.09 (CH3-17) [57,59,61,62]. Accordingly, the structure of 2 was established as 12(S)-12-hydroperoxy-7α-hydroxy-neo-cleroda-3,13(16),14-triene-2-one, and was named stachaegyptin G. Both epimers 1 and 2 have 6 stereocenters, and only one center (C-12) was inverted from 12R to 12S. Therefore, 1 and 2 are diastereomers.

Compound 4 was isolated as a colorless oil with an optical rotation of -10 (c, 0.005, MeOH). The molecular formula C20H30O4 was recognized from the HR-FAB-MS analysis, which exhibited a molecular ion peak at m/z 357.2044 [M + Na]+ (calcd. for C20H31O4Na, 357.2042), demonstrating six degrees of unsaturation in agreement with the 13C NMR spectrum of 4 (Table 2), which displayed 20 carbon resonances. Their multiplicities were determined from DEPT analysis as five methyls, four methylenes (one oxygenated at δC 69.8), six methines (two olefinic and two oxygenated at δ 73.3 and 79.0), and five quaternary carbons (two olefinic and one keto at δ 200.7). The 1H NMR spectrum of 1 (Table 1) exhibited characteristic signals for two tertiary methyls at δH 1.07 and 1.39 (each 3H, s), a secondary methyl at δH 1.06 (3H, d, J = 7.0 Hz), and two olefinic methyls at δH 1.71 and 1.88 (each 3H, s), which showed correlations in the DQF-COSY spectrum with two olefinic protons at δH 5.57 (1H, d, J = 2.5 Hz) and 5.69 (1H, br s), respectively, indicating the presence of two trisubstituted double bonds. The spectrum also showed two oxomehine protons at δH 4.11 (1H, br d, J =2.7) and δH 4.18 (1H, br d, J = 10.3 Hz), as well as two protons of an oxymethylen at δH 4.61 (1H br dd, J = 16.5, 10.3) and δH 4.29 (1H, br d, J = 14.4 Hz). The COSY spectrum exhibited four spin systems associated with ring A, ring B, and the side chain (Figure 2).

The 1H and 13C NMR spectra as well as the 2D NMR data, including DQF-COSY, HMQC and HMBC (Figure 2), clearly established that we are dealing with a structure almost identical to that of stachyaegyptin C (3), previously isolated from this species [41]. The distinct differences observed in the 1H NMR spectrum of 4 showed a slightly higher-field position chemical shift of CH3-17 (δH 1.06) in 4 than that in 3 (δH 1.13), also H-8 was shifted at higher field (δH 1.69) in 4 than that of 3 (δH 2.06). In contrary, the chemical shift of H-1β was at lower field value (δH 2.80) in 4 than 3 (δH 2.32). The results of the 2D NMR experiments achieved an indistinguishable planar structure to that of 3. The NOESY and coupling contacts analysis clearly indicated that 4 had identical relative stereochemistry with 3 in the bicyclic system. All the above data and differences between 4 and 3 established that compound 4 should be an isomer of 3 epimerized at C-12 (S configuration). This result was supported by the NOESY experiment with inspection of the 3D molecular model (Figure 3).

The strong correlations of H-12 with H-10β, H-8β, and CH3-16, and the correlation between CH3-17 with H-11a (1.42) and CH3-16, as well as the absence of a NOESY correlation between H-12 and H-1β, confirmed the S configuration at C-12 instead of 12R in 3. Further confirmation was given by the relative downfield shift of H-1β at δH 2.80 in 4, instead of that at δH 2.11 in 3, which was attributed to the presence of H-1β in a close proximity to the cyclic peroxide ring. On the other hand, H-8β and CH3-17 were slightly shifted at higher field (δH 1.69 and δH 1.06, respectively) than those of 3 at δH 2.32 (H-8β) and δH 1.13 (CH3-17) [61,63,64,65,66]. Accordingly, the structure of 4 was established as 12(S)-12,15-peroxy-7α-hydroxy-neo-cleroda-3,13-diene-2-one, and was named as stachaegyptin H. Compounds 3 and 4 have 6 stereocenters, and only one center (C-12) was inverted from 12R to 12S. Accordingly, 3 and 4 are diastereomers.

To the best of our knowledge, these new diterpenes hydroperoxides (1 and 2) and the cyclic peroxide (4) are rare secondary metabolites.

3. Proposed Biosynthetic Pathway of the Isolated Compounds

Biosynthetically, diterpenoids classes in plant catalyze a proton-initiated cationic cycloisomerization of geranylgeranyl diphosphate (GGPP), generating a labdane-type intermediate [63]. Subsequently, labdane as precursor can undergo a stepwise migration process of methyl and hydride shift, yielding a halimane-type intermediate, which can then progress to either cis or trans clerodanes [31]. Compound 5 is proposed to go through simply enzymatic hydroxylation and oxidation of clerodane-type intermediate [64]. Based on Capon’s model for biosynthesis of endoperoxides, compound 5 is subjected to enzymatic hydroperoxidation at C-12 to generate compound 1, which then undergoes oxa-Michael cyclization to produce compound 3 [65]. In addition, both compound 1 and 3 can generate their corresponding epimers 2 and 4, respectively, by further rearrangement and isomerization reactions (Figure 4).

Figure 4

Proposed scheme for the biosynthesis pathway of the isolated metabolites (1–5).

4. Chemosystematic Significance

Different diterpenoids types of ent-clerodane, kaurane, labdane, and rosane were isolated from about 27 species of Stachys including the present one that is known to produce around 35 compounds/classes of terpenes. The kaurane, labdane, ent-labdane, and rosane types of diterpenoids were rare, while only the neo-clerodane ones were common. The 2,7 di-substituted neo-clerodane derivatives were reported as annuanone, which was isolated from three species, S. annua, S. inflate, and S. Sylvatica [66]; stachysolone from S. recta [37], S. annua [66], and S. lavandulifolia [67]; 7-mono-acetyl-stachysolone in S. recta [37] and S. annua [66]; diacetyl-stachysolone from S. aegyptiaca [41]; stachone and stachylone in S. inflate, S. atherocalyx, S. annua, and S. palustris [66]. The 2,3,4 tri-substituted neo-clerodane as reseostetrol was isolated from S. rosea [68] and 3α,4α-epoxy rosestachenol from in S. glutinosa besides the mono-substituted neo-clerodanes as roseostachone and roseostachenol in S. rosea [55]. However, the kaurane-type diterpenoids were represented only in peroxide form as stachyperoxide from S. aegyptiaca [41].

In addition, four hydroxylated kaurane derivatives, i.e., 3α,19-dihydroxy-ent-kaur-16-ene, 3α-hydroxyl-19-kaur-16-en-oic acid from S. lanata, and 6β-hydroxyl-ent-kaur-16-ene, and 6β,18-dihydroxy ent-kaur-16-ene from S. sylvatica [64] were isolated. Rare labdane diterpenoids were found only in one species as (+)-13-epi-Jabugodiol, (+)-6-deoxy-andalusol, and (+)-plumosol from S. plumose [42]. Also, only two ent-labdane diterpenoids, namely ribenone and ribenol in S. mucronata [39], as well as only three rosane diterpenoids, were reported from S. paraviflora as stachyrosane, stachyrosane 1, and 2 [38,69]. In the present study, five neo-clerodane diterpenoids including four ent-neo-clerodane peroxides were isolated from S. aegyptiaca. The comparative study of previous data revealed that S. aegyptiaca is characterized by having the capability to produce neo-clerodane peroxides, which are different than other reported diterpenoids from other Stachys species. This proved that the S. aegyptiaca has a unique biosynthetic pathway to generate neo-clerodane peroxides recognized as rare types of clerodanes. Those are known for their significant biological activities as anticancer, antimitotic, and antifungal [70,71] and used in treatment of various inflammation and metabolic disorders [72].

5. Materials and Methods

5.1. General Procedures

The 1H NMR (600 MHz, CDCl3), 13C NMR (150 MHz, CDCl3), and the 2D NMR spectra were recorded on a JEOL JNM-ECA 600 spectrometer (JEOL Ltd., Tokyo, Japan). All chemical shifts (δ) are given in ppm units with reference to TMS as an internal standard, and coupling constants (J) are reported in Hz. The IR spectra were taken on a Shimadzu FT-IR-8100 spectrometer. Specific rotations were measured on a Horiba SEPA-300 digital polarimeter (l = 5 cm). FAB-MS and HR-FAB-MS were recorded on a JEOL JMS-GC-MATE mass spectrometer. For chromatographic separations COSMOSIL-Pack type (C18-MS-II) (Inc., Cambridge, MA 02138, USA, 250 × 4.6 mm i.d.) and (250 × 20 mm i.d.) columns were used for analytical and preparative separations, respectively, with compound detection via a Shimadzu RID-10 A refractive index detector. For open silica gel column separations, normal-phase column chromatography employed BW-200 (Fuji Silysia, Aichi, Japan, 150–350 mesh) and reversed-phase column chromatography employed Chromatorex ODS DM1020 T (Fuji Silysia, Aichi, Japan, 100–200 mesh). TLC separations used precoated plates with silica gel 60 F254 (Merck, Pfizer, Sanofi, 0.25 mm) (ordinary phase) or reversed-phase precoated plates with silica gel RP-18 WF254S (Merck, Pfizer, Sanofi, 0.25 mm) with compounds observed by spraying with H2SO4-MeOH (1:9) followed by heating.

5.2. Plant Material

The aerial parts of S. aegyptiaca were collected from Southern Sinai in Egypt during May 2016. A voucher specimen (SK-1055) has been deposited in the Herbarium of Saint Katherine protectorate, Egypt, with collection permission granted for scientific purposes by the Saint Katherine protectorate.

5.3. Extraction and Isolation

Extraction and fractionation of the air-dried aerial parts of S. aegyptiaca (1.5 kg) were previously described [40]. The n-hexane-CH2Cl2 (1:3) fraction (14.0 g) and 100% CH2Cl2 (7.0 g) were added together due to same chromatographic system then chromatographed on a ODS column (3 × 90 cm) eluted with 80%, 90% (MeOH:H2O) then washed with 100% MeOH. Fractions were obtained as two main portions: A (6.0 g) and B (7.0 g). Subfraction A was re-purified by reversed-phase HPLC using MeOH/H2O (65–35% 500 mL) to afford 5 (20 mg). Subfraction B was re-purified by reversed-phase HPLC using MeOH:H2O (70:30%, 1000 mL) to afford 3 (10 mg) and 4 (12 mg). The 5% MeOH fraction (8.5 g) was chromatographed on ODS column (3 × 90 cm) eluted with 80%, 90% (MeOH:H2O) then washed with MeOH. Fractions were obtained as one main portion (2.5 g), which was re-purified by reversed-phase HPLC using MeOH:H2O (80:20%, 1000 mL) to afford 2 (9 mg) and 3 (11 mg).

The 12(R)-12-hydroperoxy-7α-hydroxy-neo-cleroda-3,13(16),14-triene-2-one (stachaegyptin F, 1). Colorless oil, +30 (c, 0.001, MeOH), 1H (CDCl3, 600 MHz), and 13C (CDCl3, 150 MHz) NMR, see Table 1 and Table 2; FAB-MS m/z 335 [M + H]+ HR-FAB-MS m/z 357.2045 (calcd. for C20H30O4Na, 357.2044); IR (νmax cm−1): 3445, 1665 and 1615 cm−1.

The 12(S)-12-Hydroperoxy-7α-Hydroxy-neo-cleroda-3,13(16),14-triene-2-one (stachaegyptin G, 2). Colorless oil, -29 (c, 0.005, MeOH), 1H (CDCl3, 600 MHz), and 13C (CDCl3, 150 MHz) NMR, see Table 1 and Table 2; FAB-MS m/z 335 [M + H]+ HR-FAB-MS m/z 357.2042 (calcd. for C20H30O4, 357.2044); and m/z 357.2044 (calcd. for C20H30O4Na, 335.2042); IR (νmax cm−1): 3445, 1665, and 1615 cm−1.

The 12(S)-12,15-peroxy-7α-Hydroxy-neo-cleroda-3,13-diene-2-one (stachaegyptin H, 4).

Colorless oil, -10 (c, 0.005, MeOH), 1H (CDCl3, 600 MHz), and 13C (CDCl3, 150 MHz) NMR, see Table 1 and Table 2; FAB-MS m/z 335 [M + H]+ HR-FAB-MS m/z 357.2044 (calcd. for C20H30O4Na, 357.2042); IR (νmax cm−1): 3450, 1660, and 1620 cm−1.