Abstract
The cultivation of ginseng in fields is time-consuming and
labor-intensive. Thus, culturing adventitious ginseng root in vitro
constitutes an effective approach to accumulating ginsenosides. In this
study, we employed UPLC-QTOF-MS to analyze the composition of the
cultured adventitious root (cAR) of ginseng, identifying 60 chemical
ingredients. We also investigated the immunomodulatory effect of cAR
extract using various mouse models. The results demonstrated that the
cAR extract showed significant activity in enhancing the immune
response in mice. The mechanism underlying the immunomodulatory effect
of cAR was analyzed through network pharmacology analysis, revealing
potential ‘key protein targets’, namely TNF, AKT1, IL-6, VEGFA, and
IL-1β, affected by potential ‘key components’, namely the ginsenosides
PPT, F1, Rh2, CK, and 20(S)-Rg3. The signaling pathways PI3K–Akt,
AGE–RAGE, and MAPK may play a vital role in this process.
Keywords: cultured adventitious root of ginseng, ginsenosides,
UPLC-QTOF-MS, immunomodulatory, network pharmacology analysis
1. Introduction
Ginseng (Panax ginseng C. A. Meyer), known as ‘the King of Herbs’, has
demonstrated biological activities in heart protection
[[38]1,[39]2,[40]3], as well as anti-tumor [[41]4,[42]5,[43]6],
anti-inflammation [[44]7,[45]8], and anti-oxidation [[46]9,[47]10]
activities, among other benefits. The root of ginseng contains the
majority of its saponins, which are the predominant active ingredients
in ginseng. However, the cultivation of ginseng in fields usually
requires 5–7 years before it can be harvested, and it is a
labor-intensive process. The yield is highly susceptible to
environmental factors, including climate, soil, pathogens, and pests,
which limits the commercial usage of ginseng at a low cost. The in
vitro culturing of ginseng adventitious root (AR) has shown great
potential as an alternative method of producing ginsenosides [[48]11].
Adventitious root culturing is an effective approach to the
accumulation of ginseng biomass. It only takes weeks before the roots
are ready for harvesting, and the culture conditions are highly
controllable in a bioreactor [[49]12]. Most importantly, it has been
demonstrated that the composition of an adventitious root can be
adjusted by modifying the ingredients of the culture media
[[50]13,[51]14,[52]15]. A study on the influence of temperature and
light on the culture of hairy roots has reported an optimal condition
of 20 °C/13 °C over a day (12 h)/night (8 h) cycle [[53]13]. The
production of ginsenosides can be significantly affected via the
addition of methyl jasmonate in a ginseng–echinacea co-culture system
[[54]14]. Similarly, organic germanium can also improve the biomass and
accumulation of ginsenosides in cultured adventitious roots (cARs)
[[55]15]. Thus, it is practical to evaluate the chemical compositions
of ginseng cultured from a specific culturing protocol. Recently, we
established an optimized cAR protocol that has been shown to achieve
higher biomass and production in large-scale bioreactors. In this
study, we employed UPLC-QTOF-MS coupled with UNIFI to analyze the
ingredients of ginseng cAR. UPLC-QTOF-MS is a fast and accurate
technique with high sensitivity that generates mass spectrometric
fragmentation signals for the simultaneous determination of multiple
components. It has been widely used in analyzing the chemical
ingredients of ginseng root, leaf, berry, stem, etc. [[56]16,[57]17].
The UNIFI Scientific Information System is an informatics platform with
an embedded Traditional Medicine Library that enables the rapid,
comprehensive, and accurate identification and analysis of ingredients
in cAR. In this study, we analyzed components with molecular weights
ranging from 100 to 1500 Da in ginseng cAR.
Ginseng has been widely reported as an immune system modulator
[[58]18,[59]19,[60]20]. Various parts of ginseng can maintain immune
homeostasis and enhance the immune response to microbial attacks. There
are various types of cells in the immune system, and they respond
differently to ginseng treatment. Ginseng extract can enhance the
phagocytic activity of macrophages [[61]21,[62]22], drive the
maturation of dendritic cells [[63]23], enhance natural killer cell
functions [[64]18,[65]24,[66]25], induce antigen-specific antibody
responses [[67]26,[68]27], control proinflammatory cytokine responses
[[69]28,[70]29,[71]30], etc. However, there have been no studies on the
immunomodulatory effect of cAR of ginseng. In this study, we evaluated
how cAR extract influenced the immune system using multiple mouse
models.
2. Results
2.1. The Total Saponin and Total Polysaccharide Content
For calculating the total saponin content, the regression equation was
Y = 0.00627x − 0.00633 (r = 0.9996), and the linear range was 10 μg to
100 μg. The total saponin content of the cultured adventitious roots
(cAR) was 11%.
For calculating the total polysaccharide content, the regression
equation was Y = 4.5879X + 0.0523 (r = 0.9994), and the linear range
was 0.02 mg to 0.16 mg. The total polysaccharide content of the cAR was
1.07 g/100 g.
2.2. Identification of Components from the Cultured Adventitious Root of
Ginseng
The components of ginseng cAR were identified using UPLC-Q/TOF-MS. The
base peak intensity (BPI) chromatograms were measured in ESI^− modes
([72]Figure 1). Through an analysis based on their mass, retention time
(tR), and fragmentation, a total of 60 components were identified from
cAR extracts in ESI^− modes, comprising 50 saponins, 1 steroid, 4 fatty
acids, 3 phenolic acids, 1 amino acid, and 1 sugar ([73]Table 1). Among
the compounds, 32 were confirmed using chemical standards, namely
sucrose, quinic acid, tryptophan, the notoginsenosides R1, Rg1, Re, Rf,
F5, and Rb1, the notoginsenosides R2, Rb2, 20(R)-Rg2, 20(S)-Rh1, Ro,
Rb3, Rs1, F1, Rc, Rd, and Rs2, the gypenosides XVII and Rd2, and the
notoginsenosides Fd, 20(S)-Rg3, F4, 20(R)-Rg3, 20(S)-Protopanaxatriol,
Rh2, CK, linolenic acid, linoleic acid, and 9-octadecenoic acid. In
total, 24 components were putatively identified by comparing the tR and
characteristic MS fragments with published results, and a total of four
compounds were compared with CFM-ID 4.0. The structures of these
compounds are shown in [74]Figure S1.
Figure 1.
[75]Figure 1
[76]Open in a new tab
Representative BPI chromatograms of ginseng cultured adventitious root
(cAR) extract in negative mode.
Table 1.
Compounds identified from ginseng cAR extract via UPLC-QTOF-MS^E.
No. t[R] (min) Formula Theoretical Mass (Da) Calculated Mass (Da) Mass
Error (ppm) MS^E Fragmentation Identification Reference
1 0.59 C[12]H[22]O[11] 342.1162 342.1155 –2.15 387.1137 [M+HCOO]^−,
341.1084 [M−H]^−,
179.0562 [M−H−Glu]^− Sucrose s
2 0.65 C[7]H[12]O[6] 192.0634 192.0656 0.65 191.0584 [M−H]^−,
173.0460 [M−H−H[2]O]^−,
127.0407 [M−H−H[2]O−HCOOH]^− Quinic acid s
3 1.68 C[11]H[12]N[2]O[2] 204.0899 204.0905 3.04 203.0832 [M−H]^−,
159.0938 [M−H−CO[2]]^− Tryptophan s
4 2.50 C[16]H[18]O[9] 354.0951 354.0944 −1.93 353.0871 [M−H]^−,
191.0562 [M−H−C[9]H[6]O[3]]^−,
173.0463 [M−H−C[9]H[8]O[4]]^− 1-O-caffeoylquinic acid [[77]31]
5 4.31 C[16]H[18]O[8] 338.1002 338.0992 −2.95 337.0920 [M−H]^−,
191.0559 [M−H−C[9]H[6]O[2]]^−,
145.0300 [M−H−C[7]H[12]O[6]]^− 3-O-p-Coumaroylquinic acid [[78]32]
6 5.02 C[17]H[20]O[9] 368.1107 368.1098 –2.44 367.1026 [M−H]^−,
179.0356 [M−H−C[8]H[12]O[5]]^−,
135.0455 [M−H−C[9]H[12]O[7]]^− Methyl 4-caffeoylquinate [[79]33]
7 5.94 C[48]H[82]O[19] 962.5450 962.5436 –1.50 1007.5418 [M+HCOO]^−,
961.5354 [M−H]^−,
799.4800 [M−H−Glu]^− Notoginsenoside N [[80]34]
8 5.94 C[48]H[82]O[19] 962.5450 962.5436 −1.43 1007.5418 [M+HCOO]^−,
781.4742 [M−H−Glu]^− Majoroside F6 [[81]35]
9 6.61 C[30]H[54]O[5] 494.3971 494.3958 −2.49 539.3940 [M+HCOO]^−,
347.2520 [M−C[8]H[19]O[2]]^− Dammar-3β, 6α, 12β, 20R, 25-pentaol CFM-ID
10 7.26 C[48]H[82]O[19] 962.5450 962.5428 −2.25 961.5325[M−H]^−,
621.4401[M−H−H[2]O−2Glu]^− Notoginsenoside R6 [[82]36]
11 7.72 C[48]H[82]O[19] 962.5450 962.5414 −3.62 1007.5393 [M+HCOO]^−,
799.4836 [M−H−Glu]^− 20-O-D-glucopyranosyl-ginsenoside Rf [[83]37]
12 7.74 C[47]H[80]O[18] 932.5345 932.5318 –2.84 977.5300 [M+HCOO]^−,
799.4836 [M−H−Xyl]^− Notoginsenoside R1 s
13 8.17 C[42]H[72]O[14] 800.4922 800.4882 –5.04 845.4864 [M+HCOO]^−,
799.6775 [M−H]^−,
637.4304 [M−H−Glu]^−,
476.3833 [M−H−2Glu]^− Ginsenoside Rg1 s
14 8.21 C[48]H[82]O[18] 946.5501 946.5454 –4.98 991.5436 [M+HCOO]^−,
945.5383 [M−H]^−,
783.4896 [M−H−Glu]^−,
476.3833 [M−H−2Glu−Rha]^− Ginsenoside Re s
15 8.52 C[44]H[74]O[15] 842.5028 842.5006 −2.58 841.4933 [M−H]^−,
799.4831 [M−Ac]^−,
679.4391 [M−Rha]^−,
637.4319 [M−Ac−Rha]^− Vinaginsenoside R1 [[84]38]
16 8.80 C[51]H[84]O[21] 1032.5505 1032.5474 –3.00 1031.5401 [M−H]^−,
987.5516 [M−H−Ac]^− Malonyl-ginsenoside Re [[85]39]
17 9.20 C[48]H[82]O[19] 962.5450 962.5419 –3.25 1007.5401 [M+HCOO]^−,
961.5376 [M−H]^−,
799.4810 [M−H−Glc]^− Ginsenoside Re3 [[86]40]
18 9.41 C[48]H[82]O[19] 962.5450 962.5426 –2.57 1007.5408 [M+HCOO]^−,
961.5353 [M−H]^−,
946.5444 [M−H−CH[3]]^−,
800.4871 [M−H−Glu]^− Majoroside F1 [[87]34]
19 10.23 C[42]H[72]O[14] 800.4922 800.4875 –5.83 845.4857 [M+HCOO]^−,
799.4807 [M−H]^−,
637.4313 [M−H−Glu]^−,
475.3780 [M−H−2Glu]^− Ginsenoside Rf s
20 10.51 C[41]H[70]O[13] 770.4816 770.4783 –4.38 815.4765 [M+HCOO]^−,
769.4706 [M−H]^−,
637.4313 [M−H−Ara]^− Ginsenoside F5 s
21 10.59 C[54]H[92]O[23] 1108.6029 1108.5960 –6.22 1153.5942
[M+HCOO]^−,
1107.5890 [M−H]^−,
945.5387 [M−H−Glu]^−,
765.4771 [M−H−2Glu]^− Ginsenoside Rb1 s
22 10.69 C[57]H[94]O[26] 1194.6033 1194.5973 –5.05 1193.5900 [M−H]^−,
1089.5804 [M−H−mal]^−,
927.5304 [M−H−mal−Glu]^− Malonyl-ginsenoside Rb1 [[88]34]
23 10.72 C[41]H[70]O[13] 770.4816 770.4791 –3.34 815.4773 [M+HCOO]^−,
769.4724 [M−H]^−,
637.4290 [M−H−Ara]^−,
475.3764 [M−H−Ara−Glu]^− Notoginsenoside R2 s
24 10.79 C[53]H[90]O[22] 1078.5924 1078.5870 –4.96 1123.5852
[M+HCOO]^−,
915.5283 [M−H−Glu]^−,
765.4773 [M−H−Glu−Ara]^−,
621.4376 [M−H−2Glu−Ara]^− Ginsenoside Rb2 s
25 10.82 C[42]H[72]O[13] 784.4973 784.4936 –4.77 829.4918 [M+HCOO]^−,
637.4306 [M−H−Rha]^−,
475.3793 [M−H−Glu−Rha]^− 20(R)-Ginsenoside Rg2 s
26 10.86 C[36]H[62]O[9] 638.4394 638.4374 –3.07 683.4356 [M+HCOO]^−,
637.4293 [M−H]^−,
475.3793 [M−H−Glu]^− 20(S)-Ginsenoside Rh1 s
27 10.91 C[56]H[92]O[25] 1164.5928 1164.5875 –4.53 1163.5802 [M−H]^−,
1119.5900 [M−H−CO[2]]^−,
1059.5708 [M−H−Mal]^−,
1031.5398 [M−H−Ara]^−,
945.5390 [M−H−Ara−Mal]^− Malonyl-ginsenoside Rc [[89]34]
28 10.97 C[48]H[76]O[19] 956.4981 956.4938 –4.49 955.4865 [M−H]^−,
793.4361 [M−H−Glc]^− Ginsenoside Ro s
29 11.03 C[53]H[90]O[22] 1078.5924 1078.5856 –6.26 1123.5852
[M+HCOO]^−,
1077.5802 [M−H]^−,
915.5301 [M−H−Glu]^−,
783.4883[M−H−Glu−Xyl]^−,
621.4378 [M−H−2Glu−Xyl]^− Ginsenoside Rb3 s
30 11.20 C[56]H[92]O[25] 1164.5928 1164.5875 –4.53 1163.5802 [M−H]^−,
1060.4652 [M−H−Mal]^−,
928.5332 [M−H−Ara−Mal]^−,
619.4217 [M−H−2Glu−Ara−Mal]^− Malonyl-ginsenoside Rb2 [[90]34]
31 11.29 C[47]H[74]O[18] 926.4875 926.4863 −1.34 925.4790 [M−H]^−,
569.3833 [M−H−Ara−Glu−HCOOH]^− Chikusetsu saponin I[b] [[91]41]
32 11.45 C[48]H[82]O[17] 930.5552 930.5524 –3.02 873.4846 [M+HCOO]^−,
784.4798 [M−H−COCH[3]]^−,
695.2912 [M−H−Xyl]^−,
491.4938 [M−H−Xyl−Glu−Ac]^−, 455.2535 [M−H−Xyl−Glu−Ac−2H[2]O]^−
Gypenoside XI [[92]34]
33 11.49 C[55]H[92]O[23] 1120.6029 1120.5987 –3.75 1119.5915 [M−H]^−,
1077.5815 [M−H−Ac]^−,
915.5313 [M−H−Ac−Glu]^−,
781.4724 [M−H−Ac−Ara−Glu]^− Ginsenoside Rs1 s
34 11.49 C[36]H[62]O[9] 638.4394 638.4381 –2.01 683.4363 [M+HCOO]^−,
637.4310 [M−H]^−,
475.3796 [M−H−Glu]^− Ginsenoside F1 s
35 11.50 C[53]H[90]O[22] 1078.5924 1078.5896 –2.55 1123.5878
[M+HCOO]^−,
1077.5646 [M−H]^−,
915.5313 [M−H−Glu]^−,
781.4724 [M−H−Ara−Glu]^−,
576.4474 [M−H−Ara−2Glu]^− Ginsenoside Rc s
36 11.65 C[48]H[82]O[18] 946.5501 946.5448 –5.65 991.5430 [M+HCOO]^−,
945.5379 [M−H]^−,
783.4878 [M−H−Glu]^−,
621.4353 [M−H−2Glu]^− Ginsenoside Rd s
37 11.76 C[51]H[84]O[21] 1032.5505 1032.5457 –4.65 1031.5384 [M−H]^−,
987.5494 [M−H−CO[2]]^−,
927.5294 [M−H−mal]^−,
765.4797 [M−H−mal−Glu]^− Malonyl-ginsenoside Rd [[93]34]
38 11.86 C[55]H[92]O[23] 1120.6029 1120.5991 –3.45 1165.5973
[M+HCOO]^−,
1159.5869 [M−H]^−,
985.5371 [M−H−Ara]^−,
915.5299 [M−H−Glu−Ac]^− Ginsenoside Rs2 s
39 12.08 C[48]H[82]O[18] 946.5501 946.5456 –4.81 991.5438 [M+HCOO]^−,
945.5389 [M−H]^−,
783.4880 [M−H−Glu]^−,
621.4338 [M−H−2Glu]^− Gypenoside XVII s
40 12.25 C[47]H[80]O[17] 916.5396 916.5367 –3.16 961.5349 [M+HCOO]^−,
915.5290 [M−H]^−,
783.4881 [M−H−Ara]^− Notoginsenoside Fe [[94]34]
41 12.43 C[47]H[80]O[17] 916.5396 916.5342 –5.87 961.5324 [M+HCOO]^−,
915.5276 [M−H]^−,
783.4874 [M−H−Ara]^−,
621.4362 [M−H−Ara−Glu]^− Ginsenoside Rd2 s
42 12.59 C[47]H[80]O[17] 916.5396 916.5356 –4.28 961.5338 [M+HCOO]^−,
915.5283 [M−H]^−,
621.4359 [M−H−Glu−Ara]^− Notoginsenoside Fd s
43 12.70 C[47]H[80]O[17] 916.5396 916.5343 −5.48 961.7556 [M+HCOO]^−,
915.7454 [M−H]^−,
765.4754 [M−H−Xyl]^− Chikusetsu saponin Ⅲ [[95]42]
44 13.34 C[48]H[82]O[17] 930.5552 930.5524 −2.80 975.5507 [M+HCOO]^−,
765.4807 [M−H−Rha]^− Gypenoside X [[96]43]
45 13.56 C[42]H[72]O[13] 784.4973 784.4933 –5.10 829.4915 [M+HCOO]^−,
783.4892 [M−H]^−,
621.4357 [M−H−Glu]^−,
459.4833 [M−H−2Glu]^− Ginsenoside F2 [[97]34]
46 14.05 C[42]H[66]O[14] 794.4453 794.4407 –5.74 839.4409 [M+HCOO]^−,
795.4334 [M−H]^−,
613.3729 [M−H−Glu]^−,
569.3844 [M−H−Glu−H[2]O−Ac]^− Chikusetsusaponin IVA [[98]34]
47 14.52 C[42]H[72]O[13] 784.4973 784.4927 –5.85 829.4909 [M+HCOO]^−,
783.4880 [M−H]^−,
621.4374 [M−H−Glc]^− 20(S)-Ginsenoside Rg3 s
48 14.60 C[42]H[70]O[12] 766.4867 766.4840 –3.58 811.4822 [M+HCOO]^−,
765.4780 [M−H]^−,
747.4664 [M+H−H[2]O]^−,
619.4188 [M−H−Rha]^− Ginsenoside F4 s
49 14.67 C[42]H[72]O[13] 784.4973 784.4945 –3.62 829.4921 [M+HCOO]^−,
783.4902 [M−H]^−,
621.4396 [M−H−Glu]^− 20(R)-Ginsenoside Rg3 s
50 15.01 C[41]H[64]O[13] 764.4347 764.4325 −2.93 763.4252 [M−H]^−,
613.3720 [M−H−Xyl]^−,
569.3848 [M−H−Xyl−CO[2]]^− Pseudoginsenoside Rp1 CFM-ID
51 15.28 C[41]H[70]O[12] 754.4867 754.4847 −2.50 799.4829 [M+HCOO]^−,
621.4355 [M+H−H[2]O−Xyl]^−, Chikusetsusaponin Ia CFM-ID
52 16.27 C[30]H[52]O[4] 476.3865 476.3860 −0.89 521.3843 [M+HCOO]^−,
475.3791 [M−H]^− 20(S)-Protopanaxatriol s
53 16.75 C[36]H[62]O[8] 622.4445 622.4425 –3.22 667.4407 [M+HCOO]^−,
621.4346 [M−H]^−,
459.3818 [M−H−Glu]^− Ginsenoside Rh2 s
54 16.82 C[42]H[70]O[12] 766.4867 766.4854 −1.69 811.4837 [M+HCOO]^−,
765.6676 [M−H]^− Ginsenoside Rg4 [[99]44]
55 17.05 C[42]H[70]O[12] 766.4867 766.4855 –1.64 811.4837 [M+HCOO]^−,
765.4777 [M−H]^−,
603.4244 [M−H−Glu]^− Ginsenoside Rg5 [[100]45]
56 17.35 C[36]H[62]O[8] 622.4445 622.4431 –2.13 667.4413 [M+HCOO]^−,
621.4354 [M−H]^−,
459.3826 [M−H−Glu]^− Ginsenoside CK s
57 21.34 C[18]H[30]O[2] 278.2246 278.2244 –0.82 277.2244 [M−H]^−,
259.2146 [M−H−H[2]O]^−,
135.1178 [M−H−C[8]H[14]O[2]]^− Linolenic acid s
58 22.80 C[18]H[32]O[2] 280.2402 280.2398 –1.54 325.2387 [M+HCOO]^−,
279.2325 [M−H]^−,
261.2225 [M−H−H[2]O]^− Linoleic acid s
59 24.49 C[18]H[34]O[2] 282.2559 282.2552 –2.34 327.2541 [M+HCOO]^−,
281.2479 [M−H]^−,
236.2496 [M−H−COOH] 9-Octadecenoic acid s
60 27.95 C[35]H[60]O[6] 576.4390 576.4377 −2.03 621.4359 [M+HCOO]^−,
575.3045 [M−H]^− β-daucosterol CFM-ID
[101]Open in a new tab
s: identified with the standard. CFM-ID: compared with CFM-ID 4.0.
2.3. Body Weights and the Organ/Body Weight Ratio
We administered cAR extracts to mice through oral gavage for 30 days
([102]Figure 2A). The body weights of mice in all five groups, namely 0
(negative control), 21, 42, 83, and 125 mg/kg of body weight (BW) of
cAR, were recorded once per week for four weeks ([103]Figure 2B,
[104]Table S1). There was no significant body weight loss after the cAR
treatment. The thymus/body weight ratio and the spleen/body weight
ratio of the mice were also measured ([105]Figure 2C, [106]Table S1),
showing no significant changes after the 30-day cAR treatment compared
to the negative control. We also examined the appearance of the thymus
and the spleen in each group. We did not observe obvious differences
between the negative control group and the cAR treatment groups. Thus,
we consider the maximum dose of cAR (125 mg/kg BW) to be non-toxic.
Figure 2.
[107]Figure 2
[108]Open in a new tab
(A) Experimental design of the biological assays in this study. ‘n’
represents the number of animals in each group in a specific assay. (B)
The influence of cAR extract on the average weekly body weight (BW) of
the mice in each group (n = 15). (C) The thymus/BW and spleen/BW ratios
of each experimental group (n = 10).
2.4. Spleen Lymphocyte Proliferation
Lymphocytes and their subgroups are vital to the immune response
process. They recognize pathogens, respond to eliminate antigenic
substances, maintain the stability of the body’s environment, and
protect the body. Lymphocytes can assist in cell−mediated immunity,
indirectly boosting immune function. We assessed how cAR extracts can
modulate cell-mediated immunity by measuring spleen lymphocyte
proliferation. T lymphocytes require external stimulation to
differentiate and expand from a resting state. Concanavalin A (Con A)
is a mitogen that often serves as a substitute stimulant, rather than
antigens. In T cell stimulation, Con A irreversibly binds to
glycoproteins on the cell surface, inducing the proliferation of T
lymphocytes. The MTT proliferation assay showed that T lymphocytes
proliferated significantly after the administration of cAR ([109]Table
2, [110]Figure 3A). However, the lymphocyte proliferation did not occur
in a dose−dependent manner.
Table 2.
Results of five assays evaluating the immunomodulatory effect of cAR
extracts, including spleen lymphocyte proliferation, the quantitative
hemolysis of SRBC (QHS), a hemolysis assay, the phagocytic function of
the peritoneal macrophages, and natural killer cell activities. The
results are presented as means ± SDs.
Lymphocyte Proliferation QHS Assay Hemolysis Assay Phagocytic Function
NK Cell Activity
Stimulation Index OD HC[50] U/mL Phagocytosis Rate % Cell Activity %
Control 0.231 ± 0.038 0.297 ± 0.010 56.031 ± 5.465 25.30 ± 1.64 11.94 ±
3.92
21 mg/kg BW 0.396 ± 0.023 0.346 ± 0.007 71.531 ± 13.578 33.20 ± 2.90
35.29 ± 12.25
42 mg/kg BW 0.358 ± 0.033 0.345 ± 0.008 81.651 ± 21.554 42.40 ± 4.22
39.64 ± 3.65
83 mg/kg BW 0.317 ± 0.037 0.325 ± 0.005 91.397 ± 48.498 48.90 ± 3.07
50.18 ± 1.49
125 mg/kg BW 0.336 ± 0.050 0.387 ± 0.032 91.248 ± 40.844 72.00 ± 3.92
38.89 ± 6.59
[111]Open in a new tab
Figure 3.
[112]Figure 3
[113]Open in a new tab
Evaluation of ginseng cAR extract in terms of enhancing the immune
response during multiple biological tests in mice. (A) Spleen
lymphocyte proliferation with MTT assay (n = 15); (B) quantitative
hemolysis of SRBC (QHS) (n = 15); (C) hemolysis assay (n = 15); (D) the
phagocytic function of the peritoneal macrophages (n = 10); (E) natural
killer cell activity (n = 15). * p < 0.05 vs. the control and ** p <
0.01.
2.5. Quantitative Hemolysis of SRBC (QHS) Assay
Humoral immunity is another aspect of immune function, and it refers to
the formation of effector B cells and memory cells generated by B cells
after stimulation via antigens. Effector B cells secrete antibodies to
clear antigens, while long-lived memory cells are produced to
continuously surveil the same antigen in the blood and lymph for future
immune responses. The enhancement of humoral immunity can be evaluated
through antibody formation or serum hemolysis. In this study, we
employed a quantitative hemolysis of SRBC assay, measuring the optical
densities at 413 nm. After the treatment with cAR, the formation of
lymphoid cell antibodies significantly increased ([114]Figure 3B,
[115]Table 2).
2.6. Hemolysis Assay
The hemolysis assay assesses the extent to which red blood cells (RBCs)
are lysed by measuring the released hemoglobin in the surrounding
fluid. In this study, sheep RBCs, as exogenous cells, were used for
immunization in experimental mice. Thus, the immune cells of mice would
recognize SRBC and lyse them. By measuring the OD of the released
oxidized hemoglobin, the hemolysis reactions were assessed, and the
immunomodulatory effects of cAR on humoral immunity were evaluated. In
this study, all cAR treatment groups showed significantly increased
HC[50] values ([116]Figure 3C, [117]Table 2).
2.7. Phagocytic Function of Peritoneal Macrophages
The mononuclear–phagocyte system consists of mononuclear cells in the
blood and macrophages in tissues, both of which have phagocytic
functions to eliminate foreign substances. The mononuclear–phagocyte
system can also secrete protective substances such as interleukin-1
(IL-1), interferons, and complement. Thus, the phagocytic function of
macrophages is another indicator used to evaluate the immune function
of the body. We employed the well-characterized method of the
phagocytosis of chicken RBCs via mouse peritoneal macrophages to
measure the phagocytic function in this study. [118]Figure 3D shows
that cAR can significantly increase the phagocytic function in mice in
a dose-dependent manner.
2.8. Natural Killer Cell Activity
NK cells are another important type of immune cell in the body, and
they are distinct from T and B cells. Instead of recognizing antigens,
they distinguish abnormal tissue cells within the body from normal
self-tissue cells. Activated NK cells can secrete various cytokines,
regulate immune and hematopoietic functions, and directly kill target
cells. Thus, an improvement in NK cell activity means an improvement in
immune function. To assess the activity of NK cells after the cAR
treatment, a lactate dehydrogenase (LDH) assay was used. Compared to
the negative control group, all groups treated with cAR showed improved
NK cell activity ([119]Figure 3E, [120]Table 2).
2.9. Network Pharmacology Analysis
We have demonstrated that ginseng cAR performs immunomodulatory
activities. In this section, we employed network pharmacology analysis
to study the corresponding protein targets and related pathways of the
biological activity of cAR. We queried multiple databases and
identified 398 protein targets of the 60 components of cAR. In total,
1944 protein targets related to immunodeficiency were cross-compared
with the cAR targets ([121]Figure 4A). In total, 121 intersection
proteins were considered potential targets responsible for the
immunomodulatory effects of cAR. These intersection proteins were
connected by 114 nodes and 1252 edges ([122]Figure 4B). Of the
intersection proteins, 38 were enzymes, constituting 31.4% of the
intersection proteins. The rest comprised 26 receptors, 23 kinases, and
34 other proteins. The top five key targets, selected based on the
degree value from the PPI network, were tumor necrosis factor (TNF),
RAC-alpha serine/threonine-protein kinase (AKT1), interleukin-6 (IL-6),
vascular endothelial growth factor A (VEGFA), and interleukin 1 beta
(IL-8β).
Figure 4.
[123]Figure 4
[124]Open in a new tab
Results of the network pharmacological analysis. (A) Intersection
proteins of cAR and immunodeficiency targets; (B) classification of
intersection proteins; (C) cAR components–core targets network.
Next, we constructed the ‘cAR components—core targets’ network to
identify the potential key components of cAR extract corresponding to
its immune-enhancing activity ([125]Figure 4C). This network contained
180 nodes and 702 edges. Eighty-three percent of the components were
ginsenosides, indicating the importance of ginsenosides. The
top-10-ranking key components were selected based on their degree
values, and they are listed in [126]Table 3.
Table 3.
Top-10-ranking cAR components with the highest degree values.
No. Compound Name Degree
1 20(S)-Protopanaxatriol 37
2 Ginsenoside F1 35
3 Ginsenoside Rh2 34
4 Ginsenoside CK 32
5 20(S)-Ginsenoside Rg3 25
6 Ginsenoside Rg5 16
7 20(E)-Ginsenoside F4 12
8 Ginsenoside Rg4 12
9 20(S)-Ginsenoside Rh1 12
10 Ginsenoside Rg1 12
[127]Open in a new tab
The core targets were also analyzed using GO enrichment and KEGG
signaling pathway enrichment ([128]Figure 5). The GO analysis revealed
55 GO entries comprising 26 biological processes, 17 cellular
components, and 12 molecular functions ([129]Figure 5A). We selected 10
of the 181 enriched KEGG pathways based on their p-values and published
results ([130]Figure 5B). The p-value is represented by color in the
figure, while the number of genes related to the specific pathway is
proportional to the size. Thus, the size and color of the bubbles
illustrate the significance of these signaling pathways in the
immunomodulatory activity of cAR. Three pathways, PI3K-Akt, AGE–RAGE,
and MAPK, had larger bubbles with darker colors. As shown in
[131]Figure 5C, the ‘cAR components-core targets-key pathway’ network
was established. This network had 190 nodes and 840 edges. The degree
value of the PI3K-Akt, AGE–RAGE, and MAPK pathways was greater than
that of others, which was consistent with the KEGG enrichment results.
This further confirmed the significance of these three pathways in cAR
activities.
Figure 5.
[132]Figure 5
[133]Open in a new tab
Results of the network pharmacological analysis. (A) GO enrichment, GO
terms are in descending order; (B) bubble chart of KEGG enrichment
(Sig.Path. represents signaling pathway); (C) cAR components–core
targets–key pathway.
3. Discussion
Chromatography-coupled mass spectroscopy methods have been widely used
in studying the molecular compositions of different species of
field-cultivated Panax ginseng. Researchers have discovered over 600
types of ginsenosides. However, to date, there has been no
comprehensive assessment of the ingredients of cultured ginseng. Most
studies have focused on evaluating the accumulation of ginsenosides
using extraction and HPLC isolation, as described by Yu [[134]46]. In
this study, UPLC-QTOF-MS was employed to perform an unbiased screening
of cAR components. We identified 60 components in the ESI^− mode, of
which 32 were confirmed using chemical standards, 24 were putatively
identified by comparing the retention times and characteristic MS
fragments with published results, and 4 were compared with CFM-ID 4.0.
It is worth mentioning that a quantitative study of cAR ingredients was
begun in our lab. A comprehensive comparison of cAR and cultivated
ginseng in fields will be performed as soon as we collect these data.
The immunomodulatory effects of field-cultivated ginseng have been
extensively reported [[135]18,[136]21,[137]47,[138]48,[139]49]. Kang
and Min reviewed over a hundred published works before 2012 on the
‘immune boost’ activities of ginseng in relation to innate immunity,
acquired immunity, and cytokines [[140]47]. Kim recently reviewed the
immunomodulatory effects of different types of ginseng, including white
ginseng, red ginseng, and individual ginsenosides [[141]48]. Although
there are no published results on how cAR influences the immune system,
we expect the immune-enhancing activity of cAR due to its compositional
similarities to field-cultivated ginseng.
To evaluate the immunomodulatory effect of cAR of ginseng, we also
tested the delayed-type hypersensitivity (DTH) response and the carbon
clearance assay. These two assays are also classical tests that measure
the immune response in vivo. DTH measures the increased volume of each
hind footpad as an indicator of enhanced cell-mediated immunity, while
the carbon clearance assay determines the phagocytic activities of
macrophages. As shown in previous results, the cAR extract was shown to
perform significant activities in enhancing the immune system of mice
in assays measuring isolated immune cells from mice. However, the cAR
extract did not show a positive immunomodulatory effect during these
assays that directly monitored the mice. We will further explore the
pharmacological behavior of cAR in mice.
The results of the network pharmacology analysis suggested that the
main chemical components of cAR, such as the ginsenosides PPT, F1, Rh2,
CK, and 20(S)-Rg3, potentially act on key targets including TNF, AKT-1,
IL-6, VEGFA, and IL-1β. These results are consistent with some previous
publications. The ginsenoside Rh2 has been reported for its activities
in improving IL-2 production in vitro [[142]50] and increasing the
number of T cells in a melanoma mouse model [[143]51]. Rg3 is another
ginsenoside that has been well studied for its anti-tumor activity. It
was also shown to perform activities in enhancing the phagocytosis in
macrophages [[144]52], as well as regulating cytokines and
transcription factors [[145]53]. We also identified key signaling
pathways, namely the PI3K–Akt pathway, AGE–RAGE signaling pathway, and
MAPK signaling pathway, which cAR may affect. These pathways have also
been reported as the key targets of ginsenosides [[146]54,[147]55].
This information provides some valuable hypothetical points for further
investigation. It is imperative to validate these findings
experimentally.
4. Materials and Methods
4.1. Materials and Reagents
Fresh adventitious roots were provided by Tonghua Herbal Biotechnology,
Co., Ltd. (Tonghua, China). The fresh adventitious roots were
air-dried, ground, and sieved with a Chinese National Standard Sieve 3
(R40/3 Series). The homogeneous powder obtained was refluxed with 40%
ethanol three times (for 2 h, 2 h, and 1 h each time). Then, the
extracts were combined, concentrated, and evaporated until their
relative density was 1.08 to 1.12 (measured at 70 °C). After that, the
concentrated liquid was dried via spray-drying to obtain the final
cultured adventitious root sample (cAR). The cAR powder was dissolved
in 70% methanol, and after being filtered, the methanolic solution was
injected directly into a UPLC system.
The ginsenosides Rf, F2, Ro, Rb1, Rb2, Rb3, Rc, Re, Rg1, 20(S)-Rg3, and
PPT were provided by the School of Chemistry at Jilin University. The
notoginsenosides Fe, Fd, Rd2, and Rg5 were purchased from Chengdu
Desite Bio-Technology company (Chengdu, China). The notoginsenosides
R2, Rg2, F1, Rd, CK, and F4 were obtained from Chengdu Push
Bio-technology Co., Ltd. (Chengdu, China). UPLC-MS-grade methanol and
acetonitrile were purchased from Thermo Fisher Scientific Inc.
(Waltham, MA, USA), while MS-grade formic acid was purchased from
Sigma-Aldrich. Leucine enkephalin and sodium formate were purchased
from Waters Technologies Corporation (Milford, MA, USA). Deionized
water was obtained from Guangzhou Watson’s Food & Beverage Co., Ltd.
(Guangzhou, China). The other chemicals were of analytical grade.
Calf serum (Invitrogen) was bought from Beijing Bioway (Beijing,
China), while Hank’s solution was obtained from Beijing Solarbio
Sciences & Technology Co., Ltd. (Beijing, China). PBS buffer (pH
7.2–7.4) was purchased from Thermo Fisher (Waltham, MA, USA). Chicken
blood red cells, Indian ink, and Na[2]CO[3] were purchased from
Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Sheep red
blood cells (SRBCs) were bought from Beijing Hancheng Bio-Technology
Co., Ltd. (Beijing, China).
The instruments employed in this study included the following: a Waters
Xevo G2-S Q-TOF mass spectrometer (Waters Co., Milford, MA, USA), an
ACQUITY UPLC, MassLynx™ V4.1 workstation and UNIFI^® v1.7 (Waters
Technologies Corporation, Milford, MA, USA), an N-A35 nitrogen
generator (Shanghai Jinlang Technology Co., Ltd. Shanghai, China), a
KQ-250B ultrasonic cleaner (Jiangsu Kunshan Ultrasonic Instrument
Corporation, Kunshan, China), a TGL-16aR super speed centrifuge
(Shanghai Anting Scientific Instrument Factory, Shanghai, China),a
PTX-FA2105 electronic balance (Fujian Huazhi Electronic Technology Co,.
Ltd. Fuzhou, China), an Automatic Biochemical Analyzer Chemray 240
(Shenzhen Leadman Biochemistry Co., Ltd. Shenzhen, China), an Epoch
microplate reader (BioTek, Santa Clara, CA, USA), a TU-1810PC UV-VIS
spectrophotometer (Beijing Puxi Instrument Co., Ltd., Beijing, China),
and an Agilent 8453 UV-VIS spectrophotometer (Agilent, Santa Clara, CA,
USA).
UPLC-Q/TOF-MS-coupled UNIFI analysis. The chemical ingredients of cAR
were determined via UPLC-QTOF-MS-coupled UNIFI analysis.
Chromatographic separation was performed using a Waters ACQUITYUPLC BEH
C[18] column (100 mm × 2.1 mm, 1.7 μm, Waters Co., Milford, MA, USA).
The temperatures of the UPLC column and autosampler were set to 30 °C
and 15 °C, respectively. Mobile phase A consisted of 0.1% formic acid
in water (v/v), while mobile phase B applied 0.1% formic acid in
acetonitrile (v/v). The gradient elution was as follows: 0–2 min, 10%
B; 2–26 min, 10% → 100% B; 26–29 min, 100% B; 29–29.1 min 100% → 10% B;
and 29.1–32 min, 10% B with a flow rate of 0.4 mL/min. For a weak or
strong wash solvent, 10% or 90% acetonitrile/water (v/v) was used,
respectively.
The MS^E system working conditions were as follows: electrospray ion
source (ESI) temperature: 150 °C; desolvation temperature: 400 °C;
desolvation gas flow: 800 L/h; cone voltage: 40 V; and cone gas flow:
50 L/h. The capillary voltages were 2.2 kV for the negative mode. The
low-energy function was 6 V, while the high-energy function was 20–40
V. The masses recorded were in the range of 100 to 1500 Da. Leucine
enkephalin (5 μL) was injected at a rate of 15 μL/min as an external
reference with an m/z of 554.2615 in the negative mode. Continuum Mode
was used to record the MassLynx data.
The components of ginseng cAR were then analyzed. The raw MS data were
compressed using the Waters Compression and Archival Tool (v1.10) and
screened using the streamlined workflow of the UNIFI 1.7.0 software
[[148]44,[149]56]. The minimum peak area was set to 200 for
two-dimensional (2D) peak detection, while the peak intensities of low
and high energy were set to greater than 1000 and 200 counts,
respectively. The negative adducts were -H and +COOH. The mass error
was set to ±5 ppm. A self-built database was established by inquiring
about the composition of ginseng in PubMed, ChemSpider, Web of Science,
and Medline and imported into the Waters Traditional Medicine Library
module of UNIFI for MS data analysis. The identified components were
further confirmed by comparing the retention times and characteristic
MS fragments with published results.
4.2. Quantitative Analysis of Total Saponins and Total Polysaccharides
Total saponins. (i) Solution preparation: A standard stock solution of
the ginsenoside Re (1 mg/mL) was prepared in methanol. An 8% vanillin
solution was prepared by dissolving 0.8 g of vanillin in anhydrous
ethanol (10 mL). A 72% sulfuric acid solution was prepared by adding 72
mL of sulfuric acid to an appropriate amount of water, cooling it to
room temperature, and diluting it to 100 mL with water. (ii)
Preparation of the test solution: An accurately weighed quantity of 1 g
cAR was wrapped with neutral filter paper and placed in a Soxhlet
extractor. It was then extracted with ether and soaked in methanol
overnight. Afterward, it was refluxed with methanol six times, and the
methanol was combined, vacuum-evaporated, and dried in a water bath.
The resulting extraction was dissolved in water (30 mL to 40 mL) and
extracted with water-saturated n-butanol (30 mL) four times. The upper
liquid was evaporated to dryness, dissolved, and diluted with methanol
to 5.0 mL, creating the test solution. (iii) Drawing the standard
curve: 10 μL, 20 μL, 30 μL, 40 μL, 60 μL, 80 μL, and 100 μL of the Re
reference solution were accurately measured, respectively, into tubes.
The methanol was evaporated in a water bath (<90 °C). Then, 0.5 mL of
the 8% vanillin solution and 5 mL of the 72% sulfuric acid solution
were added to each tube. The tubes’ contents were mixed well and heated
at 60 °C for 10 min; then, they were immediately cooled in ice water
for 10 min. The absorbance was measured at 544 nm, the standard curve
was drawn, and the cAR content was calculated using the linear
regression equation. (iv) Determination of the total saponin content:
The steps in (iii) were repeated using the substance being examined.
Next, 10 μL to 40 μL of the test solution was accurately measured
instead of the reference solutions. The total saponin content was
calculated according to Equation (1) (the total saponin content (%);
[CONC], the mass of the test sample calculated using the regression
equation (μg); V[1], the constant volume (mL); V[2], the sample volume
(μL); m, the weighed mass of the test sample (mg)).
[MATH:
X=[CONC]V<
mn>2×V1
m×100 :MATH]
(1)
Total polysaccharides. (i) Solution preparation: An accurately weighed
quantity of 1.0000 g of glucose standard (dried at 105 °C to a constant
weight) was dissolved and diluted with distilled water to 100 mL,
yielding a standard stock solution of glucose (10 mg/mL). Subsequently,
it was further diluted to 0.1 mg/mL. A phenol solution (50 g/L) was
prepared by dissolving 5.0 g of phenol in 100 mL of distilled water.
(ii) Preparation of the test solution: An accurately weighed quantity
of 2.00 g of cAR was extracted with 70 mL of distilled water using
ultrasonic for 30 min. It was then extracted for 4 h at 100 °C in a
water bath, cooled to room temperature, and diluted to 100 mL. Next, 5
mL of this solution was taken and mixed with 15 mL of an 80% ethanol
solution. After centrifuging at 10,000 r/min for 10 min, the
supernatant was discarded, and the residual was dissolved in 5 mL of an
80% ethanol solution and centrifuged. The supernatant was again
discarded, and the residual was dissolved and diluted to 100 mL with
distilled water, serving as the test solution. (iii) Drawing the
standard curve: 0 mL, 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, 1.0 mL,
1.2 mL, 1.4 mL, and 1.6 mL of the glucose reference solution were
accurately measured, respectively, into a 25 mL tube with a stopper.
The solution was diluted to 2.0 mL with water, and then 1.0 mL of a 5%
phenol solution was added. The contents were mixed well, and 5 mL of
the sulfuric acid solution was quickly added. The contents were shaken
for 5 min, allowed to stand for 10 min, heated in a boiling water bath
for 20 min, cooled to room temperature, and measured for absorbance at
486 nm. (iv) Determination of the total polysaccharide content: The
operation in (iii) was repeated using the substance being examined.
Then, 2 mL of the test solution was accurately measured instead of the
reference solutions, and the total polysaccharide content was
calculated according to Equation (2) (X, the total polysaccharide
content (g/100 g); C1, the mass of the test sample calculated using the
regression equation (μg/mL); f, the dilution ratio of the solution; V,
the constant volume (mL); m, the weighed mass of the test sample (g)):
[MATH: X=C1×f×Vm×1000×1000 :MATH]
(2)
4.3. Immunomodulatory Activity of Cultured Adventitious Ginseng Root
Animals and groups. Three-week-old male KM breeding mice (body weight:
18–22 g) were purchased from SPF (Beijing) Biotechnology Co., Ltd.
(Beijing, China) The mice had an acclimation period of 7 days before
they were used in the experiments. Throughout the experiment, the mice
had free access to food, and a 12-h light/12-h dark cycle was
maintained. The mice were euthanized through cervical dislocation. All
animal experiments were approved by the Experimental Animal Ethics
Committee of the Academic Committee at Beijing University of Chinese
Medicine, Protocol No. BUCM-4-2022092904-3120. The number of mice used
is indicated in the main text.
The mice were divided into 5 groups based on the dosage of cAR tested,
that is, 0 (the negative control), 21, 42, 83, and 125 mg/kg of body
weight (BW) of the mice. Each group contained 10–15 mice. The cAR
extract was delivered through oral gavage (10 mL/kg of body weight)
once per day for 30 days. For immunization, the mice were administered
0.2 mL of 2% SRBC through intraperitoneal (i.p.) injection from day 25
once per day for 5 days. The mice’s body weights were monitored once
per week from the first day of the cAR treatment. The thymus/BW ratio
and the spleen/BW ratio of the mice were measured after dosing with cAR
for 30 days via oral gavage.
Serum preparation. After 30 days of cAR treatment, the mice’s eyeballs
were dissected, and their blood was collected. The blood was
centrifuged at 3000 rpm at 4 °C for 15 min to separate the serum for
further use.
Spleen cell suspension preparation. The spleen was dissected and placed
in Hank’s solution under sterile conditions. The spleen was gently
minced using forceps and passed through a 200-mesh sieve or four-layer
gauze to obtain a single-cell suspension. The suspension was
centrifuged at 1000 rpm for 10 min and further washed twice with Hank’s
solution. The spleen cells were then suspended in 1 mL of culture
medium. Cell viability was determined with trypan blue staining and was
required to be above 95%. The cell concentration was adjusted to 3 ×
10^6 cells/mL. After 5 days of SRBC immunization, a spleen cell
suspension of SRBC-immunized mice was prepared following the same
procedure as described above, and the concentration was adjusted to 5 ×
10^6 cells/mL in Hank’s solution.
Spleen lymphocyte proliferation. We employed an MTT-based assay to
evaluate the proliferation of spleen lymphocyte with modifications
[[150]57]. After 30 days of cAR dosing, the mouse spleens were
collected, and single-cell suspensions were prepared as described
above. Each sample was added to two wells of a 24-well plate (1
mL/well). These wells were further supplied with 75 μL of concanavalin
A (Con A) or left blank for the negative control. The plate was
incubated under 5% CO[2] and 37 °C for 72 h. Four hours before the end
of the culture, 0.7 mL of the supernatant from each well was gently
replaced with 0.7 mL of fetal-bovine-serum-free RPMI1640.
Simultaneously, 50 μL of MTT (5 mg/mL) was added to each well. After
the completion of the culture, 1 mL of acidic isopropanol was added to
each well and mixed gently to completely dissolve the purple crystals.
Then, each sample was added to three wells of a 96-well plate to
measure the optical density (OD) at a 570 nm wavelength. The OD
difference between the samples with and without Con A represented the
spleen lymphocyte proliferation.
Quantitative hemolysis of the sheep red blood cell (QHS) assay. The
antibody-producing capabilities of splenic cells were assessed using
the QHS method described by Simpson with modifications [[151]58]. The
mice were administered cAR for 30 days, while SRBC was used for
immunization from day 25. The mice were then euthanized through
cervical dislocation, and their spleens were dissected to prepare a
cell suspension as described above. The splenic cell suspension was
mixed with Tris-NH[4]Cl (pH 7.2, 0.017 M Tris, and 0.75% NH[4]Cl) at
room temperature for 10 min to lyse the red blood cells and then
centrifuged at 2000 rpm for 5 min, and the supernatant was discarded.
The cells were washed twice with 5 mL of PBS. The cells were
centrifuged at 2000 rpm for 5 min, and the supernatant was discarded.
Next, 1 mL of PBS was added for cell counting, and the concentration
was adjusted to 2 × 10^7 cells/mL. Then, 0.2 mL of the spleen cell
suspension, 0.2 mL of SRBC, and 0.2 of mL guinea pig blood were added
together as the experimental group, while the spleen cell suspension
was replaced with 0.2 of mL PBS in the control group. The samples were
mixed and incubated at 37 °C for 1 h. Then, they were centrifuged at
3000 rpm for 5 min. The supernatant was added to 96-well plates with
0.1 mL/well for three replicates of each sample. The OD at 413 nm was
measured.
Serum hemolysis assay (HC[50]). The serum hemolysis assay was performed
based on the description by Jiang with modifications [[152]59]. The
mice were administered cAR for 30 days, while SRBC was used for
immunization from day 25. The mice were then sacrificed through
cervical dislocation, and serum from their eyeballs was prepared as
described above. The serum sample was diluted 500-fold with SA buffer.
To 50 μL of diluted serum, 25 μL of 10% (v/v) SRBC and 50 μL of
complement (1:8 diluted with SA) were added for the experimental group,
while the serum was replaced with an SA buffer in the control group.
The samples were incubated at 37 °C for 30 min. The reactions were
terminated by placing the plate on ice. The samples were centrifuged at
1500 rpm for 10 min. Then, 50 μL of the sample supernatant was
transferred to a 96-well plate, and 150 μL of hemoglobin oxidizing
agent was added. For the 50% hemolysis sample, 12.5 μL of 10% (v/v)
SRBC was used, and a hemoglobin oxidizing agent was added to a total
volume of 200 μL. It was mixed thoroughly, and the samples were left to
sit for 10 min. The OD at 540 nm was measured. HC[50] was calculated as
follows (Equation (3)):
[MATH:
HC50=sample OD5
0% hemol
ysis OD<
/mi>×dilution ratio :MATH]
(3)
The phagocytic function of peritoneal macrophages. The phagocytic
function of the peritoneal macrophages was measured according to the
method described by Okimura with modifications [[153]60]. The mice were
administered cAR for 30 days, and 0.2 mL of SRBC (2%) was used for
immunization through i.p. injection from day 25. The mice were then
euthanized through cervical dislocation, and 4 mL of calf serum
containing Hank’s solution was injected into the peritoneal cavity. The
abdomen was gently massaged 20 times to thoroughly wash out the
peritoneal macrophages. Next, 2 mL of peritoneal lavage fluid was
collected from a small incision in the abdominal wall. Then, 1 mL of
peritoneal fluid was mixed with 0.5 mL of chicken red blood cells (1%
CRBC). A 0.5 mL mixture was added within the gelatin ring on a glass
slide and incubated at 37 °C for 15–20 min. After incubation, the
non-adherent cells were quickly rinsed off with saline. The adherent
cells were fixed in methanol for 1 min and stained with Giemsa solution
for 15 min. The slide was then washed with distilled water and
air-dried. The number of macrophages was counted (100 per slide). The
phagocytosis rates were calculated to evaluate the phagocytic function.
The phagocytosis rate (Equation (4)) is the percentage of macrophages
that engulfed CRBCs out of every 100 macrophages.
[MATH:
phagocytosis
rate=macrophages eng
ulfed C<
mi>RBC100 macrophages×100% :MATH]
(4)
Natural killer cell activity. The activities of natural killer cells
were evaluated according to the method described by Lv [[154]61]. The
mice were treated with cAR for 30 days via oral gavage. The mice were
sacrificed through cervical dislocation on day 31, their spleens were
collected, and a single-cell suspension was prepared as described
above. The cell concentration was adjusted to 2 × 10^7 cells/mL. Then,
100 μL of effector and target cells (50:1) were added to a U-shaped,
96-well cell culture plate. A culture medium was used in place of
effector cells for the natural release group, while 1% NP40 or 2.5%
Triton was used in place of effector cells for the maximum release
group. Three replicate experiments were conducted for each group. The
plates were incubated at 37 °C with 5% CO[2] for 4 h. After
centrifuging at 1500 rpm for 5 min, 100 µL of the supernatant was
transferred to a flat 96-well plate, and 100 μL of LDH was added for
3–10 min incubation at room temperature. The reaction was stopped by
adding 1 M of HCl (30 μL/well). The OD was measured at a 490 nm
wavelength. The NK cell activity was calculated as follows (Equation
(5)):
[MATH:
NK activity=Experimental−spontane
ous re<
mi>lease maximum
−sponta<
mi>neous release×100% :MATH]
(5)
Statistical analysis. All data were analyzed with SPSS 2.0. Statistical
significance was assessed using a one-way ANOVA. The least significant
difference (LSD) was used for the homogeneity of variances, while
Tamhane’s T2 was used for the heterogeneity of variances. For
non-normal distributed data, the Kruskal–Wallis test was used. The
values are presented as means ± SDs, and p < 0.05 was considered
statistically significant.
4.4. Network Pharmacology Analysis
The components of ginseng cAR determined via UPLC-MS were further
analyzed through network pharmacology analysis to explain the
underlying mechanism of their immunomodulatory effect. The key
components, related proteins, and pathways were analyzed using
Cytoscape 3.10.0 (Cytoscape Consortium, San Diego, CA, USA).
SwissTargetPrediction and Symmap were employed to predict the protein
target of the cAR components. The protein targets related to
immunodeficiency were identified through Malacards and DisGeNET using
the keywords ‘Immunodeficiency disease’, ‘Acquired Immunodeficiency
Syndrome’, or ‘Immune System Disease’. (1) For ‘key protein targets’,
the cAR protein target and immunodeficiency-related proteins were
cross-compared, and the intersection targets formed the ‘core targets’.
A protein–protein interaction (PPI) network was then generated by
importing the ‘core targets’ list into the String database
([155]http://string-db.org/cgi/input/pl, accessed on 1 March 2023 to 25
May 2023). The highest confidence was set to 0.4, and the discrete
nodes were selected ‘hiding’. The ‘key protein targets’ with the
highest degree values were identified by analyzing the PPI network
through Cytoscape 3.10.0. (2) For the ‘key cAR components’, all 60 cAR
components and 121 ‘core targets’ were considered in constructing a
‘components-core targets’ network through Cytoscape 3.10.0. The key
components were identified based on the degree values. (3) For the ‘key
signaling pathway’, the ‘core targets’ were analyzed for gene ontology
(GO) enrichment via Gprofiler
([156]https://biit.cs.ut.ee/gpprofiler/convert, accessed on 1 March
2023 to 25 May 2023) and Kyoto Encyclopedia of Genes and Genomes (KEGG)
pathway enrichment analysis through DAVID
([157]https://david.ncifcrf.gov/, accessed on 1 March 2023 to 25 May
2023). Omicshare ([158]https://auth.lifemapsc.com, accessed on 1 March
2023 to 25 May 2023) was employed for data visualization. The
top-10-ranked enriched signaling pathways were used to construct a
‘components-targets-pathways’ network.
5. Conclusions
Ginseng has been widely used and studied due to its highly valuable
pharmacological potency. However, it is essential to increase the
production of ginsenosides, which are the main biologically active
components in ginseng. The culturing of adventitious ginseng root in
vitro has shown great potential. In this study, we identified 60
ingredients from the cAR of ginseng extracts using UPLC-QTOF-MS. The
extracts showed positive results in improving spleen lymphocyte
proliferation, enhancing the hemolysis actions of splenic cells and
serum, promoting the phagocytic function of macrophages, and increasing
NK cell activities. Network pharmacology analysis suggested that the
immunomodulatory effect of cAR may work through (1) protein targets
including TNF, AKT1, IL-6, VEGFA, and IL-1β, (2) ginsenosides including
PPT, F1, Rh2, CK, and 20(S)-Rg3, or (3) the PI3K–Akt, AGE–RAGE, and
MAPK signaling pathways.
Supplementary Materials
The following supporting information can be downloaded at:
[159]https://www.mdpi.com/article/10.3390/molecules29010111/s1, Figure
S1: Chemical structures of compounds identified in cAR of ginseng
extract; Table S1: Body weights and organ/body weight ratio of mice in
each group. Results are in average ± SD. Body weight is measured in
gram.
[160]Click here for additional data file.^ (803KB, zip)
Author Contributions
Conceptualization, H.C. (Hong Chen) and P.L.; methodology, H.C. (Hong
Chen), H.F. and P.L.; investigation, X.L., H.C. (Hang Chi), Q.W. and
C.W.; data analysis, X.L., Z.L. and C.W.; writing, H.C. (Hong Chen) and
Z.L.; review, P.L. All authors have read and agreed to the published
version of the manuscript.
Institutional Review Board Statement
All animal experiments were approved by the Experimental Animal Ethics
Committee of the Academic Committee at Beijing University of Chinese
Medicine, Protocol No. BUCM-4-2022092904-3120.
Informed Consent Statement
Not applicable.
Data Availability Statement
Data are contained within the article and [161]Supplementary Materials.
Conflicts of Interest
The authors declare that this study received funding from Tonghua
Herbal Biotechnology, Co., Ltd. for APF. The authors Hong Chen,
Xiangzhu Li, and Hang Chi are employees of this funder. This funder
provided the cultured adventitious ginseng roots but was not involved
in the study design, data collection and interpretation, or the writing
and submitting of this work for publication. The other authors declare
no conflicts of interest.
Funding Statement
This research was funded by the Jilin Provincial Department of Science
and Technology (Funder): Preclinical Study on PPT Dropping Pill, a
Class 1 Innovative Chemical Drug for the Treatment of Cerebral
Thrombosis and Nerve Injury (Funding No. 20220204025YY).
Footnotes
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References