Analysis on the Constituents of Branches,Berries,and Leaves of Hippophae rhamnoides L. by UHPLC-ESI-QTOF-MS and Their Anti-Inflammatory Activities

Abstract

Sea buckthorn (Hippophae rhamnoides L.) is a medicinal plant widely distributed in Asia and Europe, containing plentiful bioactive substances. Our research aimed to promote the comprehensive utilization of the branches, leaves, and berries of sea buckthorn. Qualitative analysis of chemical constituents in branches, leaves, and berries of sea buckthorn was conducted by ultra-high performance liquid chromatography accurate mass quadrupole time-of-flight mass spectrometry with electrospray ionization (UHPLC-ESI-QTOF-MS). As a result, the branch, leaf, and berry samples could be clearly separated in principal component analysis scores plot, and 24 significant markers were found to distinguish these parts by partial least squares regression discrimination analysis in Mass Profiler Professional software. Meanwhile, the compositional similarity of sea buckthorn leaves and branches was higher than that of leaves and berries. In addition, the inhibition of nitric oxide (NO) production of different parts in macrophage RAW 264.7 cells was carried out. At the concentration of 10 μg/mL, sea buckthorn extracts showed good anti-inflammatory activities with NO inhibition values from 73% to 98%.

Keywords

sea buckthorn UHPLC-ESI-QTOF-MS qualitative analysis RAW 264.7 cells anti-inflammatory activity

Sea buckthorn (Hippophae rhamnoides L.) is a kind of hardy deciduous plant belonging to family Elaeagnaceae, mainly distributed in countries along the Silk Road. As an important economic plant with potential health benefits and high commercial interests, sea buckthorn has drawn worldwide attention. Sea buckthorn has significant pharmacological effects, such as antioxidant,^1,2 antimicrobial,³ antiviral,⁴ anti-inflammatory,^5
-7 and anticancer⁴ activities. Sea buckthorn contains plentiful bioactive substances, such as ellagitannins,⁸ flavonol glycosides,^8,9 proanthocyanidins,^10,11 carotenoids,¹² sterols,¹³ and tocopherols.¹³

The UHPLC-ESI-QTOF-MS method is widely used to analyze and identify various chemical components with excellent sensitivity and resolution. Phenolic compounds in sea buckthorn have been analyzed by liquid chromatography - mass spectrometry (LC-MS).^8,9,11,14-16 It has been demonstrated that ellagitannins are the predominant phenolic compounds in leaves,¹⁵ and flavonol glycosides are the main phenolic compounds in berries.¹⁵ Moreover, the content of flavonol glycosides in leaves is higher than that of berries.^9,14 Isorhamnetin and quercetin are the major aglycones in berries^14,17; casuarinin and stachyurin are the most abundant ellagitannins in leaves.⁸ Phenolic compounds possess a broad range of health-related properties, such as anti-inflammatory,^18,19 antioxidant,³ and antimicrobial²⁰ effects. To our knowledge, the researches on the branch of sea buckthorn are still very rare. In order to promote the comprehensive utilization of sea buckthorn, we carried out the analysis on the components and markers of different parts (berry, leaf, and branch) of sea buckthorn by UHPLC-ESI-QTOF-MS. The anti-inflammatory activities of the branches, leaves, and berries of sea buckthorn were determined by measuring the nitric oxide (NO) production in RAW 264.7 cells.

Identification of Compounds in Sea Buckthorn Branch, Leaf, and Berry

Information of all the sea buckthorn samples was listed in Table 1. As shown in Figure 1 and Table 2, a total of 44 peaks were detected in the branch, leaf, and berry of 3 representative samples S7, S8, and S9 by UHPLC-ESI-QTOF-MS. These peaks were identified or tentatively identified by comparing the fragmentation patterns in mass spectra to those of standard compounds or references. The secondary mass spectrometry (MS/MS) spectra of 44 peaks were presented in Supplemental Figure S1.

Figure 1

Representative UHPLC-ESI-QTOF-MS total ion chromatograms in negative ion mode of leaf, sample S8 (a); berry, sample S9 (b); and branch, sample S7 (c).

Table 1

Information of Sea Buckthorn Samples.

Sample	Origins	Parts	Collection time
S1	Aletai, Xinjiang, China	Branch	2018-10
S2	Aletai, Xinjiang, China	Leaf	2018-10
S3	Aletai, Xinjiang, China	Berry	2018-10
S4	Kashi, Xinjiang, China	Branch	2017-09
S5	Kashi, Xinjiang, China	Leaf	2017-09
S6	Kashi, Xinjiang, China	Berry	2017-09
S7	Youyv, Shanxi, China	Branch	2018-10
S8	Youyv, Shanxi, China	Leaf	2018-10
S9	Youyv, Shanxi, China	Berry	2018-10
S10	Lvliang, Shanxi, China	Branch	2018-10
S11	Lvliang, Shanxi, China	Leaf	2018-10
S12	Lvliang, Shanxi, China	Berry	2018-10
S13	Dingxi, Gansu, China	Branch	2015-10
S14	Dingxi, Gansu, China	Leaf	2015-10
S15	Dingxi, Gansu, China	Berry	2018-10
S16	Gilgit-Baltistan, Pakistan	Berry	2017-09
S17	Gilgit-Baltistan, Pakistan	Leaf	2017-09
S18	Gorno-Badakhshan, Tajikistan	Leaf	2017-09

Table 2

Mass Data of Compounds Detected by UHPLC-ESI-QTOF-MS of Sea Buckthorn in Negative Ion Mode.

Peak no.	Rt (min)	[M−H]⁻ (m/z)	MS/MS	Formula	Compounds identification
1	2.309	783.0710	300.9992, 302.0025, 481.0622	C₃₄H₂₄O₂₂	Pedunculagin^a
2	3.545	289.0719	125.0239, 179.0331, 203.0712, 245.0805	C₁₅H₁₄O₆	Catechin^b
3	3.863	451.2197	113.0237,149.0977, 167.1082, 209.1208, 249.0072, 273.0031, 300.9987		Unknown
4	4.309	633.0752	169.0146, 275.0197, 300.9999, 463.0537	C₂₇H₂₂O₁₈	Galloyl-HHDP-hexoside^b
5	5.036	935.0827	275.0194, 633.0741, 917.0701	C₄₁H₂₈O₂₆	Stachyurin^a
6	5.136	625.1435	301.0351, 463.0887	C₂₇H₃₀O₁₇	Quercetin-dihexoside^b
7	5.354	1103.0889	633.0748, 783.0731, 935.0807,1059.0980	C₄₈H₃₂O₃₁	Hippophaenin B^a
8	5.485	1153.2657	287.0540, 423.0727, 575.1197,739.1723, 865.2022, 1001.2121		Unknown
9	5.930	289.0721	125.0243, 179.0345, 203.0716, 245.0820	C₁₅H₁₄O₆	Epicatechin^b
10	6.127	635.0912	313.0568, 465.0685, 466.0713, 467.0744, 483.0793	C₂₇H₂₄O₁₈	1,2,6-Tri-O-galloyl-β-d-glucopyranose^a
11	6.035	427.1844	101.0245, 113.0235, 161.0445, 223.0624, 249.1353, 250.1380, 381.1784		Unknown
12	6.308	755.2073	285.0400, 431.1002, 609.1480	C₃₃H₄₀O₂₀	Kaempferol-sophoroside-rhamnoside^b
13	6.349	463.0538	300.9988	C₂₁H₂₀O₁₂	Ellagic acid-hexoside^b
14	7.000	609.1488	301.0350, 447.0935, 463.0903	C₂₇H₃₀O₁₆	Quercetin-hexoside-rhamnoside I^b
15	7.317	785.0881	275.0197, 300.9989, 483.0707, 633.0735	C₅₂H₁₈O₉	Digalloyl-HHDP-hexoside^b
16	7.318	609.1480	301.0368, 447.094, 463.0899	C₂₇H₃₀O₁₆	Quercetin-hexoside-rhamnoside II^b
17	7.818	542.0353	169.0139, 275.0186, 300.0251, 315.0148, 343.0100, 365.0305, 383.0394, 399.0366, 425.0158, 450.9951		Unknown
18	7.885	433.0427	300.9991	C₁₉H₁₄O₁₂	Ellagic acid-pentoside^b
19	8.226	977.2617	301.0358, 447.0859, 625.1402, 771.1978, 831.2044	C₄₄H₄₉O₂₅	Quercetin-sinapoyl-sophoroside-rhamnoside^b
20	8.353	300.9985	185.0241, 229.0140, 257.0101, 283.9956	C₁₄H₆O₈	Ellagic acid^a
21	8.392	624.9894	300.9991	C₂₇H₃₀O₁₇	Ellagic acid-sophoroside^b
22	8.499	623.1645	315.0536, 461.1088, 477.1058	C₂₈H₃₂O₁₆	Isorhamnetin-3-O-glucoside-7-O-rhamnoside^a
23	8.536	769.2230	315.0511, 623.1623	C₃₄H₄₂O₂₀	Isorhamnetin-rutinoside-rhamnoside^b
24	8.762	609.1488	151.0037, 178.9997, 301.0351	C₂₇H₃₀O₁₆	Quercetin-3-O-rutinoside^a
25	8.898	623.1629	300.0226, 315.0507, 461.1103, 477.1044	C₂₈H₃₂O₁₆	Isorhamnetin-hexoside-rhamnoside^b
26	9.074	593.1534	300.0256, 315.0511, 447.0933, 461.1092	C₂₇H₃₀O₁₅	Isorhamnetin-pentoside-rhamnoside^b
27	9.081	463.0879	151.0036, 301.0348	C₂₁H₂₀O₁₂	Quercetin-3-O-glucoside^a
28	9.670	419.2315	133.9272, 151.9241, 277.3203, 230.9140, 289.9360, 373.0963		Unknown
29	9.879	907.3274	301.0004, 583.2143, 699.2594, 745.2671	C₃₆H₆₀O₂₆	Unknown
30	10.216	593.1543	285.0406	C₂₇H₃₀O₁₅	Kaempferol-rutinoside^b
31	10.534	623.1640	300.0275, 315.0496	C₂₈H₃₂O₁₆	Isorhamnetin-3-O-rutinoside^a
32	10.543	447.0951	151.0030, 229.0505, 257.0454, 285.0392	C₂₁H₂₀O₁₁	Kaempferol-hexoside^b
33	10.761	477.1062	151.0042, 300.0256, 315.0490	C₂₂H₂₂O₁₂	Isorhamnetin-3-O-glucoside^a
34	10.838	739.1752	300.0255, 315.052, 623.1634	C₃₂H₃₅O₂₀	Isorhamnetin-malyl-rutinoside^b
35	11.534	711.3999	207.0496, 441.3390, 503.3401, 665.3911		Unknown
36	11.943	593.1317	151.0042, 163.0411, 257.0485, 285.0412, 447.0931	C₂₇H₃₀O₁₅	Kaempferol-hexoside-rhamnoside^b
37	11.988	301.0364	107.0127, 151.0032, 178.9977	C₁₅H₁₀O₇	Quercetin^a
38	12.452	280.9831	130.9961, 141.9092, 161.0938, 236.9566		Unknown
39	12.724	315.0524	107.0147, 151.0033, 272.0302, 283.0242, 300.0276, 301.0303	C₁₆H₁₂O₇	Isorhamnetin^a
40	13.179	394.9764	118.9919, 180.9901, 330.9792		Unknown
41	13.396	426.9672	282.9811, 338.9884, 386.9554, 406.9628		Unknown
42	13.761	444.9736	101.7807, 118.9920, 168.9892, 380.9775		Unknown
43	6.627	785.2187	315.0508, 460.1023, 639.1597	C₃₄H₄₂O₂₁	Isorhamnetin-3-O-sophoroside-7-O-rhamnoside^a
44	3.341	577.1366	125.0245, 289.0719,407.0788, 425.0876	C₃₀H₂₆O₁₂	Dimeric procyanidin B^b

HHDP, hexahydroxydiphenoyl; Rt, retention time.

^aIdentified by standard compounds. ^bPutatively annotated.

Thirteen peaks, pedunculagin (1, Supplemental Figures S1), stachyurin (5, Supplemental Figures S1-S5), hippophaenin B (7, Supplemental Figures S1-S7), 1,2,6-tri-O-galloyl-β-d-glucopyranose (10, Supplemental Figures S1-S10), ellagic acid (20, Supplemental Figures S1-S20), isorhamnetin-3-O-glucoside-7-O-rhamnoside (22, Supplemental Figures S1-S22), quercetin-3-O-rutinoside (24, Supplemental Figures S1-S24), quercetin-3-O-glucoside (27, Supplemental Figures S1-S27), isorhamnetin-3-O-rutinoside (31, Supplemental Figures S1-S31), isorhamnetin-3-O-glucoside (33, Supplemental Figures S1-S33), quercetin (37, Supplemental Figures S1-S37) isorhamnetin (39, Supplemental Figures S1-S39), and isorhamnetin-3-O-sophoroside-7-O-rhamnoside (43, Supplemental Figures S1-S43), were confirmed unambiguously with standard compounds.

In the negative ion mode, mass fragment at m/z 301 was an indicator of the presence of quercetin aglycone.^8,21,22 Compound 6 (Supplemental Figures S1-S6) was identified as quercetin-dihexoside based on the molecular ion [M−H]⁻ at m/z 625, and 2 major fragments at m/z 463 [M−H-162]⁻ and 301 [M−H-162-162]⁻.²³ Two compounds (14 and 16, Supplemental Figures S1-S14, S16) showed the same molecular ion [M−H]⁻ at m/z 609 and fragment ions at m/z 463 [M−H-146]⁻, 447 [M−H-162]⁻, and 301 [M−H-146-162]⁻, which were identified as quercetin-hexoside-rhamnoside.²³ Compound 19 (Supplemental Figures S1-S19) was tentatively identified as quercetin-sinapoyl-sophoroside-rhamnoside due to the molecular ion [M−H]⁻ at m/z 977, and fragments at m/z 831 [M−H-146]⁻, 771 [M−H-206]⁻, 625 [M−H-146-206]⁻, 447 [M−H-206-324]⁻, and 301 [M−H-206-324-206]⁻.²⁴

The fragment ion at m/z 285 was an indicator of the presence of kaempferol aglycone.^8,21 Compound 12 (Supplemental Figures S1-S12) showed the molecular ion [M−H]⁻ at m/z 755, and characteristic fragment ions at m/z 609 [M−H-146]⁻, 431 [M−H-324]⁻, and 285 [M−H-146-324]⁻, which was identified as kaempferol-sophoroside-rhamnoside.²⁵ Compound 30 (Supplemental Figures S1-S30) was identified as kaempferol-rutinoside based on the molecular ion [M−H]⁻ at m/z 593 and the exclusively fragment ion at m/z 285 [M−H-308]⁻.²¹ Compound 32 (Supplemental Figures S1-S32) gave the [M−H]⁻ signal at m/z 447 and fragment ion at m/z 285 [M−H-162]⁻, which was identified as kaempferol-hexoside.²³ MS/MS fragmentation of compound 36 (Supplemental Figures S1-S36) ([M−H]⁻ at m/z 593) produced ions at m/z 447 [M−H-146]⁻ and 285 [M−H-146-162]⁻, which suggested that the compound was kaempferol-hexoside-rhamnoside.⁸

The fragment ion at m/z 315 was an indicator of the presence of isorhamnetin aglycone.⁸ Compound 23 (Supplemental Figures S1-S23) was identified as isorhamnetin-rutinoside-rhamnoside based on the molecular ion [M−H]⁻ at m/z 769 and fragment ions at m/z 623 [M−H-146]⁻ and 315 [M−H-146-308]⁻. Compound 25 (Supplemental Figures S1-S25) gave the [M−H]⁻ signal at m/z 623, fragments at m/z 477 [M−H-146]⁻, 461 [M−H-162]⁻, 315 [M−H-146-162]⁻, and fragment at m/z 300 obtained by the loss of CH₃, which was identified as isorhamnetin-hexoside-rhamnoside.⁸ Compound 26 (Supplemental Figures S1-S26) provided a molecular ion [M−H]⁻ at m/z 593 and fragment ions at m/z 315 [M−H-132-146]⁻, 447 [M−H-146]⁻, and 461 [M−H-132]⁻, which was identified as isorhamnetin-pentoside-rhamnoside.⁸ Compound 34 (Supplemental Figures S1-S34) was preliminarily identified as isorhamnetin-malyl-rutinoside due to the molecular ion [M−H]⁻ at m/z 739, and fragments at m/z 623 [M−H-116]⁻, 315 [M−H-116-308]⁻, and 300 [M−H-116-308-15]⁻.²⁴

The mass fragment at m/z 301 (300.999) in the negative ion mode was an indicator of the presence of ellagic acid derivatives, and the hexahydroxydiphenoyl (HHDP) group was lactonized spontaneously to ellagic acid.^8,21,22 Compound 4 (Supplemental Figures S1-S4) was identified as galloyl-HHDP-hexoside with molecular ion [M−H]⁻ at m/z 633 and characteristic fragment ions at m/z 275, 301 [M−H-170-162]⁻ and 463 [M−H-170]⁻.²⁶ MS/MS fragmentation of compound 13 (Supplemental Figures S1-S13) ([M−H]⁻ at m/z 463) produced ion at m/z 301, which suggested that the compound was ellagic acid-hexoside.²⁶ Compound 15 (Supplemental Figures S1-S15) showed the molecular ion [M−H]⁻ at m/z 785 and yielded characteristic fragment ions at m/z 275, 301, 483, and 633, which was identified as digalloyl-HHDP-hexoside.²⁶ Compound 18 (Supplemental Figures S1-S18) was identified as ellagic acid-pentoside based on the molecular ion [M−H]⁻ at m/z 433 and fragment ion at m/z 301.²⁶ Compound 21 (Supplemental Figures S1-S21) showed the deprotonated molecular ion [M−H]⁻ at m/z 625 and yielded the exclusively fragment ion at m/z 301, which were identified as ellagic acid-sophoroside.

Two compounds (2 and 9, Supplemental Figures S1-S2, and S9) showed the same deprotonated molecular ion [M−H]⁻ at m/z 289 and yielded the same characteristic fragment ions at m/z 125, 179, 203, and 245, which were identified as catechin and epicatechin.²⁷ Compound 44 (Supplemental Figures S1-S44) was identified as dimeric procyanidin B with molecular ion [M−H]⁻ at m/z 577 and characteristic fragment ions at m/z 125, 289, 407, and 425.^27,28 However, 11 compounds (3, 8, 11, 17, 28, 29, 35, 38, 40, 41, and 42, Supplemental Figures S1-S3, S8, S11, S17, S28, S29, S35, S38, and S40-S42) have not been identified by their mass spectra.

Comparison of Different Parts of Sea Buckthorn

Unsupervised principal component analysis (PCA) was applied to observe the differences and similarities in chemical constituents between different parts of sea buckthorn (Figure 2). The PCA score plot showed that the branch, leaf, and berry samples were well separated. The first 2 principal components of the PCA model explained 56.10% of the total variance.

Figure 2

The score plot of principal component analysis for different parts of sea buckthorn in negative ion mode (leaf, berry, and branch).

The hierarchical cluster analysis (HCA) of sea buckthorn samples was shown in Figure 3. We can clearly see that all samples of sea buckthorn were classified into 3 significant clusters of berry, leaf, and branch. Cluster I (S3, S6, S9, S12, S15, and S16) consisting of all berry samples showed higher compositional similarity of sea buckthorn berries in Kashi, Aletai, and Gilgit-Baltistan. Cluster I also indicated that the composition of sea buckthorn berries in Youyv, Lvliang, and Dingxi had a certain degree of similarity. Cluster II (S2, S5, S8, S11, S14, and S18) forming from all leaf samples showed higher compositional similarity of sea buckthorn leaves in Aletai and Dingxi. Cluster III (S1, S4, S7, S10, and S13) consisting of all branch samples showed higher compositional similarity of sea buckthorn branches in Youyv and Dingxi. Based on 3 clusters shown in HCA, we can speculate that the compositional similarity of sea buckthorn leaves and branches was higher than that of leaves and berries.

Figure 3

Hierarchical clustering analysis of different parts of sea buckthorn tested in negative ion mode.

A better separation between 3 groups was observed in the supervised partial least squares regression discrimination analysis (PLS-DA) score plot (Figure 4). Cross-validation was used to estimate the predictive abilities of the constructed model. The number of components, folds, and repeats of the model was set to 3, 7, and 200, respectively. Validation matrix accuracy was 100%. The parameters for the model were 0.904 (R ² Y) and 0.709 (Q ² Y). Hence, the established model had good fitness and prediction. We work on finding the chemical markers in different parts of sea buckthorn with variable importance in projection (VIP) >1.5 and P < 0.05.

Figure 4

The score plot of 3D partial least squares regression discrimination analysis model in negative ion mode (leaf, berry, and branch).

As a result, a total of 24 significant ions were selected as the chemical markers (M1-M24) for distinguishing branch, leaf, and berry samples (Table 3). The chemical markers were preliminarily identified by MS and MS/MS spectra, combining with references. M9, M10, and M15 were identified as ellagic acid-sophoroside, isorhamnetin-pentoside-rhamnoside, and isorhamnetin-malyl-rutinoside, respectively. The other markers could not be identified under our current condition, and we mark them as “unknown.”

Table 3

Mass Data of Chemical Markers in Different Parts of Sea Buckthorn in Negative Ion Mode.

No.	Rt (min)	Mass	Proposed compound	Proposed formula	Detected mass (m/z)	MS/MS
M1	5.37	2246.1470	Unknown		1122.0653 [M−2H]⁻²
M2	5.381	2230.1730	Unknown		1114.0793 [M−2H]⁻²
M3	7.22	1124.3267	Unknown	C₅₀H₆₀O₂₉	1123.3188 [M−H]⁻
M4	7.841	908.0921	Unknown	C₄₀H₂₈O₂₅	889.0737 [M−H−H₂O]⁻
M5	8.259	1130.0511	Unknown	C₅₂H₂₆O₃₀	1129.0437 [M−H]⁻
M6	8.272	1138.9980	Unknown	C₅₆H₁₉O₂₈	1137.9915 [M−H]⁻
M7	8.275	1108.0685	Unknown	C₅₀H₂₈O₃₀	1107.0598 [M−H]⁻
M8	8.392	656.9259	Unknown	C₂₅H₅O₂₂	655.9186 [M−H]⁻
M9	8.392	625.9962	Ellagic acid-sophoroside	C₂₇H₃₀O₁₇	624.9894 [M−H]⁻	300.9991
M10	9.0741	594.1615	Isorhamnetin-pentoside-rhamnoside	C₂₇H₃₀O₁₅	593.1534 [M−H]⁻	300.0256, 315.0511, 447.0933, 461.1092
M11	9.879	908.3349	Unknown	C₃₆H₆₀O₂₆	907.3274 [M−H]⁻	301.0004, 583.2143, 699.2594, 745.2671
M12	10.032	908.3344	Unknown	C₃₆H₆₀O₂₆	907.3274 [M−H]⁻
M13	10.273	962.1046	Unknown	C₄₃H₃₀O₂₆	961.0962 [M−H]⁻
M14	10.517	1270.3237	Unknown		1269.3167 [M−H]⁻
M15	10.838	740.1824	Isorhamnetin-malyl-rutinoside	C₃₂H₃₅O₂₀	739.1752 [M−H]⁻	300.0255, 315.052, 623.1634
M16	11.364	1188.2790	Unknown	C₆₀H₅₂O₂₆	1187.2712 [M−H]⁻
M17	11.761	952.3255	Unknown	C₃₇H₆₀O₂₈	951.3184 [M−H]⁻
M18	12.266	613.2810	Unknown	C₃₇H₄₁O₈	612.2737 [M−H]⁻	149.0635, 300.9980, 315.8816, 316.1673, 462.2037, 463.2093, 493.2186
M19	12.322	583.2706	Unknown	C₃₆H₃₉O₇	642.2866 [M+CH₃COO]⁻	149.0628, 175.0384, 342.1472, 372.1595, 492.2158, 493.2204, 506.2357, 522.2277
M20	12.381	673.3024	Unknown	C₃₉H₄₅O₁₀	672.2952 [M−H]⁻	149.0617, 175.0393, 346.1699, 372.1591, 522.228
M21	12.617	1418.7126	Unknown		1417.7054 [M−H]⁻
M22	12.947	1460.7230	Unknown		1459.7166 [M−H]⁻
M23	13.062	1314.6671	Unknown		1313.6590 [M−H]⁻
M24	14.517	652.3699	Unknown	C₃₁H₅₆O₁₄	651.3627 [M−H]⁻

Rt, retention time.

Detailed information of 24 marker compounds was clearly shown in the heatmap (Figure 5). The heatmap indicated that the content of M21, M22, M23, and M24 in the berries and M1, M2, M5, and M6 in the leaves was significantly higher than that of other parts, and the content of M8, M9 (ellagic acid-sophoroside), M18, and M20 in the berries and M10 (isorhamnetin-pentoside-rhamnoside), M14, and M17 in the branches was significantly lower than that of other parts.

Figure 5

Heatmap of marker compounds in different parts of sea buckthorn in negative ion mode.

Anti-Inflammatory Activities of Sea Buckthorn

As shown in Table 4, the anti-inflammatory activities of sea buckthorn branches, leaves, and berries were measured by the NO production. In addition, at the concentration of 10 µg/mL, sea buckthorn extracts showed NO inhibition rates from 73% to 98%, and cytotoxic effects have not been observed in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays (Supplementary Table S1), indicating that sea buckthorn extracts displayed good anti-inflammatory activities on RAW 264.7 macrophages.

Table 4

The Nitric Oxide Inhibition Rate of Sea Buckthorn (mean ± SD, N = 3, 10 µg/mL).

Sample no.	Inhibition rate (%)
S1	74.68 ± 0.017
S2	78.26 ± 0.011
S3	76.18 ± 0.010
S4	76.95 ± 0.004
S5	75.80 ± 0.003
S6	77.52 ± 0.017
S7	74.25 ± 0.018
S8	73.69 ± 0.033
S9	76.02 ± 0.037
S10	76.01 ± 0.016
S11	74.98 ± 0.037
S12	73.67 ± 0.024
S13	85.12 ± 0.029
S14	85.40 ± 0.043
S15	91.54 ± 0.063
S16	95.21 ± 0.026
S17	97.36 ± 0.017
Quercetin	64.43 ± 0.014

The NO inhibition rates of S13, S14, S15, S16, and S17 were significantly higher than those of other samples, which showed that the branches, leaves, and berries in Dingxi and leaves and berries in Gilgit-Baltistan have better anti-inflammatory activities. As the samples of Gilgit-Baltistan were taken from the town of Sost (75°25′E, 36°50′N), with an average altitude of 4700 m. The higher elevation may enhance the anti-inflammatory activity of sea buckthorn. In future, we need to collect more samples for the further research.

Experimental

Chemicals and Reagents

The berries, leaves, and branches of sea buckthorn were collected from China, Pakistan, and Tajikistan. All samples were identified by the author of Dr Zhigang Yang and dried under shade until the study began.

Reference compounds of isorhamnetin-3-O-glucoside-7-O-rhamnoside, isorhamnetin-3-O-sophoroside-7-O-rhamnoside, isorhamnetin-3-O-glucoside, and quercetin-3-O-rutinoside were isolated from sea buckthorn berries in our lab. Isorhamnetin, quercetin, ellagic acid, stachyurin, pedunculagin, hippophaenin B, and 1,2,6-tri-O-galloyl-β-d-glucopyranose were isolated from sea buckthorn leaves by the author. Isorhamnetin-3-O-rutinoside and quercetin-3-O-glucoside were purchased from Chengdu Pufeide Biotech Co., Ltd. (China). LC-MS grade methanol and acetonitrile were purchased from Mreda Technology Inc. (United States). LC-MS grade water and formic acid were purchased from Merck Company (Germany).

Dulbecco’s Modified Eagle’s Medium (DMEM), dimethyl sulfoxide, and MTT were purchased from Sigma-Aldrich (United States). Fetal bovine serum (FBS) and antibiotics (penicillin and streptomycin) were purchased from Gibco BRL (Grand Island, NY, United States). Lipopolysaccharide (LPS) was purchased from Beijing LabLead Biotech Co., Ltd. (China). Nitric Oxide Colorimetric Assay Kit (NO kit) was purchased from Beijing BioDee Biotech Co., Ltd. (China).

Sample Preparation

The sea buckthorn samples were milled into powder. Each powdered sample (0.2 g) was precisely weighed and extracted by ultrasonic extraction with 20 mL 70% LC-MS grade methanol, followed by centrifugation with centrifugal filter units. A mixture of stock standard solution containing 13 reference compounds was prepared. All collected sample solutions and standard solutions were stored at 4°C prior to injection.

UHPLC-ESI-QTOF-MS Analysis

Chromatographic analysis was performed on an Agilent 1290 series UHPLC system (Agilent Technologies, United States). The sea buckthorn samples were loaded onto a Waters CORTECS UHPLC C18 analytical column (2.1 mm × 100 mm, 1.6 µm) at temperature of 36°C. The injection volume was 2 µL and the flow rate was 0.3 mL/min. Mobile phase consisted of water (A) and acetonitrile (B) (both containing 0.1% formic acid), and the following gradient program was used: 0 to 2 minutes, 7% B; 2 to 3 minutes, 7% to 12% B; 3 to 8 minutes, 12% to 18% B; 8 to 12 minutes, 18% to 60% B; 12 to 13 minutes, 60% to 100% B; and 13 to 14 minutes, 100% B.

Mass spectrometry analysis was performed on an Agilent 6560 Ion Mobility QTOF MS system (Agilent Technologies, United States) equipped with an electrospray ionization (ESI) source. Mass spectrometry parameters in negative ion mode were as follows: drying gas (N₂) flow rate 5 mL/min; drying gas temperature 300°C; nebulizer pressure 35 psig; capillary voltage 3500 V; nozzle voltage 1000 V; fragmentor 400 V; and fixed collision energies 20 eV. The mass range was obtained by scanning ions from m/z 100 to 1700.

Statistical Analysis

All the raw data in negative ion mode collected from the UHPLC-ESI-QTOF-MS were imported into Profinder software (version B.08.00), grouping samples by branches, leaves, and berries. The parameters of Profinder were set as follows: retention time tolerance 0.25 minutes, mass tolerance 10.00 ppm ±2.00 mDa, score filter 90, height filter 5000 counts, compounds minimum filter 100% of file in at least 1 sample group.

Then, the data files in CEF format generated in Profinder were then imported into Agilent Mass Profiler Professional software (MPP, version 14.9) for analysis. All files were grouped into branches, leaves, and berries, and the filter parameter was set to retain the entities that appear in at least 80.0% of samples in at least 1 condition. Principal component analysis, HCA, and PLS-DA were performed in MPP software. The chemical markers in different parts of sea buckthorn with VIP > 1.5 and P < 0.05 were filtered out. The analysis of all compounds was performed by Agilent MassHunter Qualitative Analysis (B.08.00) software.

Anti-Inflammatory Assays

RAW 264.7 macrophages were cultured in DMEM supplemented with 10% FBS and with 1% antibiotics (penicillin and streptomycin). The cells (1 × 10⁵) were added and inoculated in 96-well plates in an incubator with a 5% CO₂ atmosphere at 37°C for 24 hours. Then, the culture solution was discarded, and 100 µL of DMEM containing the mixture of sea buckthorn extracts and LPS (1 µg/mL) was added into the plate. A positive control of quercetin (10 µg/mL) was set up. After 24 hours incubation, the cell suspension was taken to determine NO inhibitory according to the instructions of the NO kit. In addition, MTT assays were used to evaluate cytotoxic activity.

Supplemental Material

Supplementary material - Supplemental material for Analysis on the Constituents of Branches, Berries, and Leaves of Hippophae rhamnoides L. by UHPLC-ESI-QTOF-MS and Their Anti-Inflammatory Activities

Supplemental material, Supplementary material, for Analysis on the Constituents of Branches, Berries, and Leaves of Hippophae rhamnoides L. by UHPLC-ESI-QTOF-MS and Their Anti-Inflammatory Activities by Wen-Hui Zheng, Hai-Ying Bai, Shu Han, Fang Bao, Kai-Xue Zhang, Li-Li Sun, Hong Du, and Zhi-Gang Yang in Natural Product Communications

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the grant from the Major International S&T Cooperation Project, Ministry of Science and Technology of the People’s Republic of China (2016YFE0129000); the Key Program for International S&T Cooperation Projects of Gansu Province (18YF1WA115); Research project of Gansu Provincial Administration of Traditional Chinese Medicine (GZK-2015-21); and the Fundamental Research Funds for the Central Universities (lzujbky-2017-k26 and lzujbky-2017-sp40).

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