Neochlorogenic acid: an anti-HIV active compound identified by screening of Cortex Mori [Morus Alba L. (Moraceae)]

Figures & data

Table 1. RT-PCR primers and probes.

Target DNA	Primer or probe	Name and sequence
Total RT (R/U5)	Early RT forward	MH535: 5′-AACTAGGGAACCCACTGCTTAAG-3′
	Early RT reverse	Early 2: 5′-CTGCTAGAGATTTTCCACAC-3′
	Early RT probe	MH603: 5′-(FAM)-ACACTACTTGAAGCACTCAAGGCAAGCTTT-(BHQ)-3′
Late RT (flank PBS)	Late RT forward	MH531: 5′-TGTGTGCCCGTCTGTTGTGT-3′
	Late RT reverse	MH532: 5′-GAGTCCTGCGTCGAGAGAGC-3′
	Late RT probe	LRT-P: 5′-(FAM)-CAGTGGCGCCCGAACAGGGA-(BHQ)-3′
Integrated DNA (Alu-LTR)	First-round Alu-LTR PCR
	LTR forward	GSTLTR: 5′-AGACAGATAGGGCCGTTAAACTAGGGAACCCACTGCTTAAG-3′
	Alu reverse	Alu 2: 5′-GCCTCCCAAAGTGCTGGGATTACAG-3′
	Second round real-time PCR
	GST forward	GST: 5′-AGACAGATAGGGCCGTTAAAC-3′
	LTR reverse	Early 2: 5′-CTGCTAGAGATTTTCCACAC-3′
	Probe	MH603: 5′-(FAM)-ACACTACTTGAAGCACTCAAGGCAAGCTTT-(BHQ)-3′
mtDNA (10,620–10,710)	mtDNA forward	MH533: 5′-ACCCACTCCCTCTTAGCCAATATT-3′
	mtDNA reverse	MH534: 5′-GTAGGGCTAGGCCCACCG-3′
	mtDNA probe	mito-probe: 5′-(FAM)-CTAGTCTTTGCCGCCTGCGAAGCA-(BHQ)-3′

Figure 1. Vector map and sequencing results. (A) pLenti-P2A Vector. (B) pLenti-P2B Vector. (C) pHIV-lus-ZsGreen Vector. (D) Sequence alignment results.

Figure 2. Plasmid transfection and viral packaging. (A,B) Transfection of plasmids into 293 T cells, (C,D) 786-O cells infected by packaged viruses.

Figure 3. Screening results of eight Chinese medicinal compositions. (A) Effects of eight Chinese medicinal compositions on cell survival rates. (B) Effects of eight Chinese medicinal compositions on viral inhibition rates. 1: G. uralensis; 2: R. japonica; 3: N. cataria; 4: L. erythrorhizon; 5: S. flavescens; 6: C. cassia; 7: E. japonica; 8: Cortex Mori.

Figure 4. Effects of Cortex Mori on the expression of RT enzyme products at different stages. (A) Effects of Cortex Mori on the expression of total RT enzyme products. (B) Effects of Cortex Mori on the expression of late RT enzyme products. (C) Effects of Cortex Mori on the expression of integrated DNA enzyme products. CM group: DMSO, HIV, and Cortex Mori were added to the cells; AZT group: only AZT and DMSO were added to the cells; HIV group: only HIV and DMSO were added to the cells; DMSO group: only DMSO was added to the cells; control group: the cells did not receive any medicinal treatments. Data are expressed as the mean ± SEM. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5. Chromatogram of liquid phase mass spectrum peaks for separation and identification of Cortex Mori granules.

Table 2. Chromatographic peak parameters corresponding to the chromatograms of 10 Chinese medicine monomers contained in Mori Cortex.

Item	Peak number	Name	Retention time	Area	High
1	15	Mulberroside A	10.457	2,824,281	378,595
2	20	Chlorogenic acid	11.658	178,434	22,963
3	21	Chlorogenic acid	12.213	3288	496
4	24	Neochlorogenic acid	13.212	116,590	15,164
5	33	Palmitic acid	17.393	32,853	3500
6	38	Astragalin	19.988	20,374	1741
7	56	β-Sitosterol	43.795	21,922	3192
8	57	Emodin	44.436	1623	393
9	62	Ursolic Acid	47.045	24,769	3121
10	63	Morusin	48.084	48,665	5922
11	66	Lupeol	51.958	50,306	7128

Figure 6. Effects of five active monomer compounds in Cortex Mori on the expression of products at different stages of HIV infection. (A) Effects of five active monomer compounds in Cortex Mori on the expression of total RT enzyme products. (B) Effects of five active monomer compounds in Cortex Mori on the expression of late RT enzyme products. (C) Effects of five active monomer compounds in Cortex Mori on the expression of integrated DNA products. Data are expressed as the mean ± SEM. ***p < 0.001, ****p < 0.0001.

Figure 7. Analysis of the target of neochlorogenic acid. (A) GO enrichment analysis. (B) Pathway analysis. (C) PPI analysis.

Figure 8. Target analysis of neochlorogenic acid acting on HIV. (A) HIV-related pathway. (B) The intersection of HIV-related genes and new chlorogenic acid targets. (C) Intersection gene enrichment results. (D) The structural formula of neochlorogenic acid. (E) Combination model of neochlorogenic acid and 1MU2.

Data availability statement

The relevant data have been uploaded as supplementary and can also be obtained from the corresponding author.