GC/MS analysis and potential synergistic effect of mandarin and marjoram oils on Helicobacter pylori

Figures & data

Figure 1. GC-MS Chromatogram of mandarin oil.

Figure 2. GC-MS Chromatogram of marjoram oil.

Table 1. The essential oil composition of mandarin leaves oil.

Peak no.	Components	Molecular formula	Retention time (min)	RIcal	RILit	% Composition	Method of identification
1.	p-Cymene	C10H14	10.105	1015	1018	0.72	RI, MS
2.	Limonene	C10H16	10.228	1019	1022	1.35	RI, MS
3.	γ-Terpinene	C10H16	11.198	1050	1055	6.25	RI, MS
4.	Methyl-N-methyl anthranilate	C9H11NO2	21.753	1406	1402	89.93	RI, MS
5.	β-Caryophyllene	C15H24	21.876	1411	1418	1.11	RI, MS
6.	Caryophyllene oxide	C15H24O	26.104	1576	1572	0.63	RI, MS
	Total identified					100

Major compounds are in bold.

Table 2. The essential oil composition of marjoram oil.

Peak no.	Components	Molecular formula	Retention time (min)	RIcal	RILit	% Composition	Method of identification
1.	Camphene	C10H16	8.530	959	953	0.65	RI, MS
2.	p-Cymene	C10H14	10.114	1015	1018	3.18	RI, MS
3.	γ-Terpinene	C10H16	11.452	1058	1059	3.51	RI, MS
4.	trans-Sabinene hydrate	C10H18O	12.506	1092	1098	36.11	RI, MS
5.	Dehydro Sabinene ketone	C9H12O	13.137	1112	1117	1.12	RI, MS
6.	3-Isothujenol	C10H18O	13.703	1130	1134	0.82	RI, MS
7.	Terpinen-4-ol	C10H18O	14.914	1169	1174	17.97	RI, MS
8.	α-Terpineol	C10H18O	15.335	1183	1186	6.17	RI, MS
9.	Linalyl acetate	C12H20O2	17.163	1245	1254	9.18	RI, MS
10.	U.I.	–	17.659	1263	–	2.55	–
11.	Bornyl acetate	C15H18O2	18.199	1281	1280	1.24	RI, MS
12.	3-Thujanyl acetate	C12H20O2	18.485	1291	1295	0.53	RI, MS
13	U.I.	–	18.560	1294	–	2.10	–
14	U.I.	–	20.200	1351	–	1.94	–
15	U.I.	–	23.502	1475	–	2.45	–
16	Spathulenol	C15H24O	25.961	1571	1577	2.24	RI, MS
17	Caryophyllene oxide	C15H24O	26.103	1576	1581	8.25	RI, MS
	Total identified					93.52

U.I.: Unidentified, major compounds are in bold.

Figure 3. The MIC₉₀ & MIC graphs of Anti-Helicobacter pylori activity of mandarin oil leaves (A), marjoram oil (B), equal amounts of both oils(C), and Clarithromycin (D) as standard. All determinations were carried out in a triplicate manner and values are expressed as the mean ± SD.

Figure 4. 2D and 3D-binding affinities of caryophyllene oxide (A,D), linalyl acetate (B,E), methyl-N-methyl anthranilate (C,F) and concomitant interactions of seven major compounds identified in marjoram and mandarin oils with the active sites of H. pylori urease domain.

Figure 5. 2D and 3D-binding affinities of caryophyllene oxide (A,D), linalyl acetate (B,E), methyl-N-methyl anthranilate (C,F) and concomitant interactions of seven major compounds identified in marjoram and mandarin oils with the active sites of H. pylori CagA domain.

Table 3. Docking scores of marjoram and mandarin major oil compounds on H. pylori virulent factors domains (urease and CagA).

Compound name	Urease (1e9y)	Cag A (4dvy)
Caryophyllene oxide	−11.4	−9.5
Linalyl acetate	−9.1	−8.3
Methyl-N-methyl anthranilate	−8.9	−8.2
α- Terpineol	−7.6	−7.4
trans-Sabinene hydrate	−7.4	−6.9
Terpine-4-ol	−7.2	−7.1
γ-Terpinene	−6.6	−5.4

Table 4. The predicted toxicity of marjoram and mandarin major oil compounds.

Compound name	Any potential toxicity	Predicted LD50
trans-Sabinene hydrate	2000 mg/kg	None
Terpine-4-ol	1016 mg/kg	None
Linalyl acetate	12000 mg/kg	None
Caryophyllene oxide	5000 mg/kg	None
α-Terpineol	2830 mg/kg	None
Methyl-N-methyl anthranilate	2910 mg/kg	None
γ-Terpinene	2500 mg/kg	None

Figure 6. The predicted physicochemical properties for selected compounds, such as linalyl acetate (A) and trans-sabinene hydrate (B).

Table 5. The predicted pharmacokinetics of marjoram and mandarin major oil compounds.

Parameter	trans-Sapinene hydrate	Terpine-4-ol	Linalyl acetate	Caryophyllene oxide	α-Terpineol	Methyl-N methyl anthranilate	γ-Terpinene
GIA	High	High	High	High	High	High	High
BBB	Yes	Yes	Yes	Yes	Yes	Yes	Yes
P-gP substrate	No	No	No	No	No	No	No
CYP1A2 inhibitor	No	No	No	No	No	Yes	No
CYP2C19 inhibitor	No	No	No	Yes	No	No	No
CYP2C9 inhibitor	No	No	No	Yes	No	No	No
CYP2D6 inhibitor	No	No	No	No	No	No	No
CYP3A4 inhibitor	No	No	No	No	No	No	No
Lipinski Violations	No	No	No	No	No	No	No
PAINS	No	No	No	No	No	No	No

GIA (gastrointestinal absorption), BBB (Blood Brain Barrier), PgP (P-glyco pro- tein transporter), CYP1A2, CYP2C19, CYP2C9, CYP2D6 and CYP3A4 are the five forms of cytochromes P450 (CYP). PAINS (Pan Assay Interference).